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The decline of the performance of the human muscle with aging is out of discussion. The rate of decline can be very well drawn from the decline of the world records of the master athletes in various track and field disciplines. Actually all track and field events are power tests and the performance of the athletes can be transformed into dimensionless parameter proportional to the power developed in carrying out the events. These are astonishingly linear in behavior and points to 0 power at 110 years of age.1 For some of them (running events) the processing of the athletic results into power parameters associated with the age of the master athletes is simple; for the throwing events the dimensionless parameters are calculated with a two steps process; for the jumping events the processing is more problematic. In this area we are concentrating our present work because the jumping results (high jump) are not proportional to the power developed: this is proportional to the raising of the center of gravity of the athletes., There is something in our genome that dictates life expectancy and there is nothing that can be done to avoid this; indeed, there is not yet any record of a person who has cheated death. Our physical prowess can vacillate substantially in our lifetime according to our activity levels and nutritional status and we may fight aging, but we will inevitably lose.1 Premature or accelerated aging of muscle may occur as the result of many chronic diseases. One extreme case is provided by irreversible damage of the Conus and Cauda Equina, a spinal cord injury (SCI) sequela in which the human leg muscles may be completely and permanently disconnected from the nervous system with the almost complete disappearance of muscle fibers within 3-5 years from SCI.2 In cases of this extreme example of muscle degeneration, we have used 2D and 3D Muscle Color CT to gather data supporting the idea that electrical stimulation of denervated muscles can retain and even regain muscle.2-5 We show also that, if people are compliant, atrophy can be reversed, but if the home FES is discontinued, muscle degeneration occurs again.3-5 Here we would like to show that it is possible to extend the CM-CT approach to the cases of disuse muscle atrophy in incomplete denervation of muscle tissue, or just in aging.6 The tissue characterization process is based on using CT data and special software tools to segment and analyze the different tissues within a region of interest. In the process of assessing muscle quality in the lower limbs we discriminate the soft tissues dividing them in: subcutaneous fat, intramuscular fat, low density muscle, normal muscle and fibrous-dense connective tissues. The first step in the segmentation process is to establish a threshold, which discriminates the tissue of interest from the rest by grey value (CT number or Hounsfield value in CT modality). The second segmentation tool which typically follows thresholding is region growing. Region growing is an image segmentation approach in which neighboring pixels of the current region’s boundaries are examined and added to the region class if no edges are detected (or more generally some inclusion criteria is met). The Muscle Color Computed Tomography allows to: 1. Quantify the percent contents of the different soft tissues (muscle, fat and other connective tissues, either loose or fibrous) and 2. the volume of anatomically defined muscles (here, rectus femoris or tibialis anterior). The methods are used to measure in a patient (male, 53 years, ASIA 1, that had suffered with a trauma to the spinal cord 15 years before the MC-CT) the residual asymmetric innervation of the leg muscles and the effects on muscles of a 6-week rehabilitation regime that allowed the patient to perform short walks without crouches after years of supported walking and many hours a day of bed resting. Further, a young active male (35 years old) and a typical sedentary senior (male of 72-years) are also analyzed., Even in the absence of overt diseases, our physical capacity decays with age, fluctuating substantially in our lifetime according to activity levels and nutritional status. We may indeed fight aging by appropriate counter-measures, but we will inevitably lose. Further, we have presented evidence that the atrophy which accompanies aging is to some extent related to a background loss of innervation.1 Comparing muscle biopsies of sedentary seniors to those elderly with a lifelong high level of sport activity, we showed that these groups indeed have a different distribution of muscle fiber types and of their diameter. The senior sportsmen have many more slow fiber-type groupings than the sedentary people providing strong evidence of denervation-reinnervation of muscle fibers. It appears that activity maintains the motoneurons and the muscle fibers. Further, comparing muscle morphometry data of groups of sedentary (untrained) subjects to those obtained by the analyses of muscle biopsies from age-matched sedentary seniors at T0 there are no statistical significant differences, but after 9 weeks of training with either electrical stimulation (ES) or leg press (LP), interesting findings were observed.2,3 At T1, that is after 9 weeks of training in ES and LP groups, significant increases in the overall and fast type mean myofiber diameters were observed between the three groups (one way ANOVA p < 0.05). Further, the Bonferroni post-hoc test shows that these differences are statistically significant between the ES (p, Physical activity plays an important role in preventing chronic disease and muscle degeneration in adults and the elderly. Voluntary physical exercise is not always feasible and other therapies should be applied such as Functional Electrical Stimulation (FES) and/or Leg Press (LP) training.1-7 The key process of Calcium (Ca2+) storage uptake and release is essential in muscle adaptation. This study shows the effects of physical training (LP) and FES in situ in human Vastus Lateralis (VL) muscle. Through immunofluorescence analysis of muscle cryo-sections, a huge increment of NFAT positive nuclei, was found after both treatments (from 3% to 60%); moreover after both trainings an increment of P-CamkII was observed by western blotting analysis. These findings indicate that both trainings activate the CaM-dependent phosphatase signaling (known to be involved in muscle plasticity). Muscle total homogenates obtained from biopsies performed before and after completing a nine weeks FES treatment on a group of volunteers and Calsequestrin (CASQ), SERCA, Sarcalumenin, protein expression were determined by Western blot. After FES significant increase of SERCA2 and Sarcalumenin and decrease of CASQ1 were observed, while LP did not show any variations of proteins levels. Immunofluorescence analysis were also performed to localize in situ MHCII/SERCA2 co-expressing muscle fibers, an interesting tool to identify subpopulation of muscle fibers involved in muscle adaptation. The overall results indicate that the applied FES protocol, simulating a motoneuron slow-type firing pattern, potentiates Ca2+ uptake and storage in a peculiar class of skeletal muscle fibers, and further validates, at molecular level, the FES strategy.5-11, Damage to the recurrent laryngeal nerve (RLN) causes: 1. severe dyspnea because of bilateral paralyzed vocal cords and 2. An impaired voice in cases of unilateral damage of vocal cords because of reduced ability to bring the vocal fold on the damaged side to the midline. The same symptom is caused by reduced firing of the recurrent laryngeal nerve in aged related to disuse atrophy. Damage of the facial nerve leads to a loss of muscle tone and the soft tissues of the face. Voluntary motor movement is lost, and mimic muscles can no longer be moved. The inability to close the eyelid indirectly leads to vision disorders and the eye may dry out. Lack of mouth movement limits speaking and eating. Axonal collateral sprouting leads clinically to simultaneous movement of several target muscles (a condition called synkinesis) - involuntary lid closure while moving the mouth, e.g., when eating. Simultaneous movement of antagonist muscles leads to the autoparalytic syndrome: muscle forces cancel each other out and no movement is observed clinically despite innervation. Aging in the facial region typically means a loss of muscle tone, muscle volume and/or a loss or reduction of connective and fat tissue, e.g., below the eyes and/or at the cheek. As a result of the aging muscles, facial muscles and/or other facial tissues may diminish or atrophy. FES may be an effective method of providing training therapy to human subjects weakened or denervated muscles in order to strengthen a weakened voice or face. United States Patent Applications: Applicant: MED-EL Elektromedizinische Geraete GmbH, Innsbruck, AT, One of the main determinants of the overall size of neural implantable pulse generators is usually the size of the battery. Engineers are facing the challenge of designing devices that are small in volume whilst fulfilling their stimulation task as long as possible. Therefore efficient stimulation methods are crucial for their success. Wongsarnpigoon 1 pointed out three different types of stimulation efficiency. A “charge-efficient” stimulation could have the positive effect of reducing tissue damage. As the battery size is directly proportional to the maximal instantaneous power required – a “power-efficient” stimulation could reduce battery-size and therefore the overall size of an implant. An “energy- efficient” stimulation on the other hand, could be advantageous with regard to the battery lifetime. The aim of our project is to compare different rectangular waveforms according to their “energyefficiency”. 5 different rectangular waveforms (monophasic, biphasic, biphasic with interphase gap, asymmetric biphasic, asymmetric biphasic with interphase gap) that have shown potentially useful effects in other studies2-6 have been investigated. Bipolar stainless-steel loop electrodes were placed under the common peroneal nerve of rats under buprenorphine/isofluorane anaesthesia. The isometric force produced by the extensor-digitorum-longus muscle was measured using a load-cell. Our results did not support several of the claims made in the literature. Introducing an interphase-gap generally increased the stimulating currents necessary to reach a certain force-threshold. This was also the case for asymmetric waveforms. The standard symmetric biphasic pulse was the most energy-efficient, out of all tested waveforms. The differences between this experiment and other studies reflect different stimulation methods (mostly monopolar), stimulation patterns (mostly pulse trains) or stimulation sites (e.g. cochlear stimulation, epiretinal stimulation). But they emphasise that the specific relationship between the means of the injection of stimulation energy and the measured outcome need to be considered when efficiency or efficacy of stimulation is discussed. The results from this experiments were used to propose an algorithmic approach for finding stimulation parameters that yield a particular energy-minimum for a certain targeted force-level. These parameters provide a starting point for further parameter fitting sessions in clinical settings where electrical stimulation is applied., According to PubMed roughly 10% of the annually added publications are describing findings from the animal model. Half of these studies are done in mice and rats. It can be assumed that there is a need for implantable electrical stimulators which are flexible, reliable and small enough (~1cm3) that even mice can tolerate it and move freely. The MiniVStim 12A is a battery powered implant with an outer diameter of 15 mm and a volume of 1.2 cm3. It can be preprogramed according to the experimental protocol and controlled by resetting it with a magnet. It can deliver constant current monophasic pulses up to 2 mA and 1 ms pulse width (@ 1 kOhm). MiniVStim 12B has the same mechanical dimensions and electrical characteristics but can be fully programed via a wireless bidirectional data link. Both types of implants are already successfully used in studies. The latest generation of implants is the MiniVStim18B. It is slightly larger (22 mm outer diameter) than the 12B but offers the 8 fold battery life time. Moreover, it can deliver biphasic pulses and extends the stimulation parameter range up to 8 mA at a maximum output voltage of 10 V and pulse width of 5 ms (@ 1 kOhm) for monophasic and 2×5 ms for biphasic pulses. This extended parameter range gives the opportunity to perform long term studies on denervated muscles in small animals., Age related atrophy of the vocalis muscle and its adjacent structures affects the voice and may lead to presbyphonia, a condition affecting more and more people in aging western societies. So far therapy modalities comprised conservative speech therapy as well as phonosurgical approaches. Chronic electrical stimulation of the afferent nerve (recurrent laryngeal nerve, RLN) is a completely new therapeutic option that has not been tested before. 18 male Wistar rats were implanted with a unilateral nerve stimulator. One week after implantation stimulation protocol was initiated over eight weeks, twice daily. Changes were observed on the muscular level histologically (cross section area, number of muscle fibers etc.) as well as on the cellular level (immuno-histochemically and qPCR). All animals tolerated the stimulation procedures well and showed normal feeding. We could not identify differences in cross-section area, number of muscle fibers, or satellite cells. We identified a trend towards increased number of neuro-muscular junctions which failed slightly statistical significance. Our pilot study proved the technical feasibility of the implantation of a RLN pacemaker system in small rodents. We could not identify significant changes after eight weeks of stimulation in any parameter. We assume that this is due to the young age of the test animals. The next step will be to test these protocols in aged rats., Muscle atrophy is a hallmark of the ageing process and as such also affects the larynx, where it constitutes the major cause of presbyphonia, i.e. a considerable glottic gap with massive loss of air during phonation. Patients suffer from a highly hoarse voice and rapid vocal fatigue which leads to social withdrawal. Current treatment options are mainly conservative (e.g. speech therapy) and far from being satisfactory. Thus, novel approaches for promoting hypertrophy of aged laryngeal muscles are in demand. In this respect, electrical stimulation of motor neurons constitutes a promising strategy. Using aged sheep as an animal model, we recently completed a first efficacy study in which chronic electrical stimulation of laryngeal muscles was accomplished via a mini-electrode that targeted the right recurrent laryngeal nerve (RLN; unilateral stimulation). Based on preceding experiments, functional electrical stimulation (FES) implants were programmed to deliver a pattern able to evoke supramaximal muscle stimulation over a period of 29 days. Surgical and post-surgical interventions were well tolerated by all animals, FES implants remained functional throughout the whole study, and only relatively mild increases in the necessary threshold for muscular stimulation were recorded. At the end of the study, vocalis and posterior crico-arytenoid muscles were prepared to obtain samples for molecular and histological analyses. To quantify the expression levels of genes related to distinct muscle fiber types, a real-time PCR (RT-qPCR) analysis pipeline was newly established which will also be central to subsequent studies. First results of the analysis “at transcript level” will be discussed in the talk. Moreover, an outline of the planned further analyses will be presented., Stroke is a leading cause of adult disability and it is constantly increasing due to growing life expectancy and unhealthy lifestyles. Fibrinolysis is the main therapy for ischemic stroke, but it has a narrow therapeutic window. Neuroprotective treatment strategies that may have the potential to reduce damage or to enhance regenerative aspects are under investigations in many experimental models. The aim of this study was to evaluate the neuromodulation effect induced by vagus nerve stimulation (VNS) in an animal model of focal cerebral ischemia, using a novel stimulator device. Ischemia was achieved by transient proximal middle cerebral artery occlusion in XX rats with a nylon monofilament introduced in the internal carotid artery. The vagus nerve stimulator was housed subcutaneously in the abdomen and tunnelled up to the cervical part of the nerve. All rats were clinically examined and half of them were stimulated everyday for 2 weeks. At post-mortem brains were fixed to perform histological, immunohistochemical and molecular analyses. The neurological examination revealed an impairment in all treated rats. The damage seemed to be more consistent in not stimulated rats. Histological examinations did not show classic signs of CNS ischemia, whilst immunohistochemical analyses revealed higher GFAP and IBA1 expression in treated ischemic rats in comparison to sham animals. Molecular microarray analysis revealed differences in mRNA expression in many cellular pathways (neuroactive ligand-receptor interaction for example) between controls and ischaemic groups, although the investigations are still underway. Our preliminary data indicate that 2 weeks of VNS is feasible without any side effects, although the behavioral improvement needs to be studied deeply and with a greater number of animals. To sum up, our brain ischemia animal model should be improved since histological evidence of necrosis has not been achieved yet; however, immunohistochemical and molecular results highlight a putative neuroprotective action of the applied VNS device., Rehabilitation of patients suffering of muscle denervation is still a challenge for clinicians. In past years the treatment consisted essentially in prescription of orthotics and/or walking aids. This approach is still useful but recently, electrical stimulation for denervated muscle showed the possibility to evoke muscular tonic contractions and recover muscle trophism also in long term denervation. This offers the chance to include electrical stimulation in diagnostics, treatment and follow up evaluations of this condition. In this presentation, based on an example case of neuralgic amyotrophy, we discuss the rehabilitative strategies and propose our clinical approach to this kind of patients., The neuralgic amyotrophy (NA) o Parsonage-Turner syndrome is a rare disease of the peripheral nervous system characterized by sudden onset of severe pain in the upper limbs, followed by rapid weakness, atrophy and motor slow remission, which can last months to years. It has an idiopathic and hereditary form. The NA occurs at any age, but is most common in the 3-7 decade of life and among men. Some patients experience a relapsing/remitting course with symptom-free intervals while others have an incomplete recovery with persisting neurologic deficit. In 50% of cases, the NA is associated with a point mutation or duplication of the susceptibility gene on chromosome 17q25.3 SEPT9. Diagnosis is based on typical clinical signs and exclusion of other diseases by laboratory testing, electromyography, imaging of the cervical spine and brachial plexus. Pain control using a combination of long-acting opioids and NSAIDs associated to oral prednisone during the first weeks of an attack and accelerate recovery. Rehabilitation therapy is also important in NA. We present a case of 54 years old man with NA. When he was 49 years old, he started complaining of sudden neck pain radiating to the left shoulder and subsequent weakness of the limb. The patient presented a hypothrophy and hypotonia of flexor-extensor of the left shoulder muscles, associate a lack of tactile sensitivity of the lateral surface of the left arm, consequently the patient decide to suspend the driving. He performed laboratory tests, which were normal, an EMG of the upper limb which confirmed the presence of a peripheral nervous denervation and CT of the neck and chest that was normal. The patient was treated with oral cortisone without benefits. Due to the worsening symptoms immune globulin and cortisone were administered and the patient began physiotherapy which was later associated with muscle electrostimulation (ES), massage and physiotherapy obtaining a reduction in pain after 6-7 months. Patient had initially two and a half hours of ES once per day to muscle affected with exponential monophasic currents by “Siemens Neuroton 626 “device. One year and a half after, the patient was treated with “Miostim” device applying biphasic currents and associating massage, employs assisted exercises, exercises against resistance, and dynamic splints for finger). Currently the patient has only occasionally or no pain. He returned to work and restarted to drive. He has mild weakness bilaterally on extensors of the wrists (4/5), more to the left arm. On the left the patient has also a deficit of the flexor muscles (4/5) and the drt are hyporeflexive. The prognosis of NA is variable: usually patients recover 70-90% of their previous motor function after 1-2 years. In general, the result in our patient is satisfactory. This case shows a successful treatment including physical therapy and ES in patients with NA., The Vienna Strategies for Functional Electrical Stimulation (FES) of human muscles in neuromuscular disorders and aging were inspired by conventional protocols of neuromuscular stimulation (usually called NMES, Neuro Muscular Electrical Stimulation), but succeed to be applied to the extreme case of degenerating muscles due to long term irreversible denervation when some of the dogma of NMES/FES were violated.1-4 Long term denervated muscle undergo several stage of excitatory and contraction disorders that call for specialized stimulation protocols, in particular long stimuli in the range of 100-300 msec, that is hundreds of time long impulses. However, when excitability is recovered, shortening of the impulse allow to reach the most desirable sustained contraction by train of impulses delivered for 1-2 sec at 30Hz and thus Functional movements of the legs. That is achieved only if the inter-pulse OFF time is shortened to 10 msec and the pulse to 30-40 msec. We would like to stress that such a pattern of stimulation is very different from the conventional stimulation pattern of innervated muscle (that is, < 1 millisec at 50-100 Hz) and thus it can be used to demonstrate that the responding muscle are NOT reinnervated. Anyhow, protocols of progressive loading of the muscles can be thereafter prescribed to increase muscle resistance and strength. As to the stimulation of human muscle in aging,5-8 it is here enough to say that the conventional NMES strategies for muscle resistance and strength are applied, but increasing the safety measure to avoid tendon, muscle and joint lesions.

Neuromuscular electrical stimulation (NMES) usually involves the application of intermittent stimuli over the muscle with the aim to produce strong contractions through the activation of intramuscular nerve branches. The main physiological uniqueness of these electrically-evoked contractions is that motor unit recruitment is different from a voluntary action, as it has been shown to be spatially fixed, temporally synchronous, mainly superficial and non-selective.1 Indeed, NMES leads to the activation of both slow and fast motor units even at relatively low force levels. This specific motor units activation pattern has been associated with an exaggerated metabolic demand and a greater muscle fatigue as compared with voluntary exercise performed at the same intensity,1 thereby limiting the widespread utilization of NMES in clinical settings. It has been recently highlighted that NMES can also induce significant muscle damage as illustrated by major histological alterations such as z-lines disruption and macrophage infiltration as well as by the prolonged decrease in voluntary force production capacities.2 In the first part of the presentation, we will provide an overview of the main physiological consequences of the peculiar motor unit recruitment associated with NMES and provide some recommendations for limiting or preventing the corresponding “adverse” effects of NMES. Over the last two decades, chronic NMES application has been used as an effective way of improving muscle strength in both healthy humans and athletes. The magnitude of the strength gains has been related to the level of electrically evoked force. Given that the subject’s tolerance of the electric current determines the force evoked by NMES, there is a large inter-individual variability in NMES response. Of interest, the time course of neuromuscular adaptations to NMES training appears similar to that taking place in response to voluntary strength training programs. Indeed, adaptations within the central nervous system occurred in the early phase of NMES training as illustrated by the increased electromyographic activity and neural activation,3 enhanced V-wave amplitudes,3 and significant cross-education effects.4 These findings clearly indicated that NMES does not actually bypass the central nervous system due to the activation of both muscle and cutaneous afferent fibers. In addition, long-term NMES training programs (i.e., >6-8 weeks) may further induce muscle hypertrophy, improve muscle oxidative capacity and result in a fast-to-slow muscle fiber type transition.5 Surprisingly, the relevance of such phenotypic adaptations for the translation to endurance performance that is particularly important for sport and daily activities remains to be demonstrated. The second part of the presentation will address how and to what extent NMES-induced neural and muscle adaptations might be relevant in a clinical context. We will also suggest potential directions for future implementation of NMES in inactive patients with advanced disease., In people with spinal cord injuries (SCI) autonomic dysfunction is related with several conditions which increase cardiovascular risk: abnormalities in blood pressure, heart rate variability, arrhythmias and an altered cardiovascular response to exercise. If all these factors limit the performance in physical activity in the SCI population, several evidences in literature show that physical inactivity is the main independent risk factor for the development of cardiovascular diseases.1-3 Aims of the study was the monitoring of cardiovascular performance parameters, respiratory parameters and muscular working capacity of a population of disabled athletes with complete spinal cord injury in chronic phase. 29 athletes, performing agonist sport were evaluated. The characteristics of the population are: a complete spinal cord injury classified as ASIA A (13 persons had a neurological level above Th6 and 16 a neurological level below Th6), 25 males and 4 females; age 42.24 ± 12.40 years; BMI 23.20 ± 3.26; time to the lesion (the spinal cord injury) 17.14 ± 12.30 years. Assessments (clinical evaluation, blood tests, spirometric test, incremental test at the crank ergometer with monitoring of cardio-respiratory parameters) were carried out in 2008 (t0) and after 6 year in 2014 (t1). By multiple regression we analyzed at t0 and t1 the contribution on maximum oxygen consumption parameters (VO2max) of variables as age, Body Mas Index (BMI), lesional level, years to the injury and weekly hours of training. At t0 the contribution on VO2max parameters of the other variables taken into account was statistically significant (p = 0.0075) for the lesional level. The correlation between VO2max and the lesional level was confirmed by analysis of variance (ANOVA) (p = 0.096). This means that the lower the lesional level the higher the VO2max in subjects who practice sports. At t1 we achieved a statistically significant correlation between VO2max parameters and weekly training hours (p = 0.0091), therefore in the long term in our subjects an increase in VO2max is related to the increase in weekly training hours. We also checked at t1 a statistically significant correlation between VO2max and BMI, with an increase in VO2 max correlated with a reduction in BMI (p = 0.005) of our athletes. The continued practice of physical activity is critical in improving cardiovascular performance in people with spinal cord injuries, especially in most affected persons. In the SCI population in chronic phase, hours of practice in sports activities and maintaining an adequate BMI are extremely important for saving cardiovascular fitness., Long standing lower motor neuron denervation of skeletal muscle is known to end in fibrotic degeneration of muscle tissue.1 However, long term survival of a subset of skeletal myofibers also occurs.2,3 We performed transverse and longitudinal studies of SCI patients suffering with complete Conus and Cauda Equina Syndrome and of sedentary and active seniors which included analyses of muscle biopsies from the quadriceps muscle. Surprisingly, we discovered that human denervated myofibers survive years of denervation after full and irreversible disconnection from their motor neurons.1 Open is, however, the extent of contribution of muscle fiber regeneration to these observations.4 We found that atrophic myofibers could be rescued by home-based Functional Electrical Stimulation (h-bFES), using purpose developed stimulators and electrodes.5,6 Although denervated myofibers quickly lose the ability to sustain high-frequency contractions, they continue to respond to single, very long impulses (up to 200 millisec) that are able to recover enough muscle excitability to allow for re-emergence of tetanic contractions. A description of the very early changes in humans are hampered by a paucity of patients suffering complete Conus and Cauda Equina Syndrome, but the cohort enrolled in the EU RISE Project has shown that even five years after SCI, severe atrophic myofibers, with a peculiar cluster reorganization of myonuclei,3 are present in human muscles and respond to h-bFES.5,6 Thus, human myofibers survive permanent denervation much longer than generally accepted and they maintain the capacity to respond to h-bFES beyond the stage of simple atrophy. Furthermore, long-term denervation/reinnervation events occur in elderly,7 and is part of the mechanisms responsible for muscle aging and again h-bFES was beneficial in delaying aging decay.8,9 Indeed, physical exercise is known to have beneficial effects on muscle trophism and force production modulating signaling pathways involved in fiber type plasticity, muscle growth and mitochondria respiratory efficiency. It has been shown that the decrease of muscle mass and strength observed in aging is linked to intracellular and extracellular abnormalities, that is, sarcoplasmic reticulum-to-mitochondria malfunctions and extracellular matrix metabolism, respectively. When healthy seniors are exposed to regular neuromuscular ES training for a period of 9 weeks outcomes are an increase in muscle strength and muscle fibers and, most importantly, an increase of fast fibers, the more powerful of skeletal muscle motor units.8,9 Electron microscopy analyses show remodelling of mitochondrial apparatus as a consequence of fusion phenomena that are consistent with adaptation to physical exercise. Altogether the results indicate that the ES-dependent beneficial effects on muscle mass and force are associated with changes in mitochondrial-related proteins involved in Ca2+ homeostasis, providing new targets to develop therapeutic strategies to promote healthy aging., Spinal cord injury causes paralysis and subsequent muscle wasting and loss of muscle function, which are especially severe after complete and permanent damage to lower motor neurons. However, long term survival of a subset of skeletal myofibers also occurs.1 We performed transverse and longitudinal studies of SCI patients suffering with complete Conus and Cauda Equina Syndrome2 and found that atrophic myofibers could be rescued by home-based Functional Electrical Stimulation (h-bFES), using purpose developed stimulators and electrodes.3 The recommended parameters and time intervals are suggestions based on the EU project RISE and our clinical experience.2,3 They should be adapted to personal needs of patients in respect to time span of denervation, and condition of muscle and function. Patient training should start with single twitch stimulation with an impulse duration (ID) of 150ms and an impulse pause (IP) of 500ms for the first 2 months (can be reduced if the time of denervation is under 6 months) and 120ms ID, 400ms IP, after 2 months to excite denervated muscle fibers still hard to activate. After eliciting sufficient muscle reaction the next training phase implements tetanic bursts of a stimulation duration (SD) of 3s and a stimulation pause (SP) of 3s with impulses of 40ms ID and 10ms IP after 2 months of stimulation – in addition to the single twitch program - to increase muscle fiber diameter, muscle mass, density and force with leg extensions (after 2-5 months) with and without additional weights on the subjects ankle. If a good condition is achieved (depending not only from the training also from the time span of denervation) the strength training can be replaced with stand-up, stepping and walking exercises in parallel bars performed with continuous stimulation controlled by an external switch. In conclusion, human myofibers survive permanent denervation much longer than generally accepted1-5, and they maintain the capacity to respond to h-bFES beyond the stage of simple atrophy2,3., The Stimulette den2x is a high performance 2-channel electrotherapy stimulator, specialized to be used for activating flaccid paralyzed denervated muscles. Damages of the lower motor neuron in conus-cauda-lesion or peripheral nerve injury cause dramatic changes in the affected muscle. With adequate stimulation parameters those changes can be stopped or even reversed.1,2 This bridges the time gap until reinnervation occurs in nerve injury. In conus-cauda-lesion, where we usually see severe muscle atrophy, it preserves/recovers muscles mass improving its trophic state, thus helping to prevent pressure sores. In this workshop the changes due to denervation, and the constrains for results of adequate electrical stimulation will be discussed. Furthermore the practical application of the stimulation device Stimulette den2x, now commercially available, will be fully demonstrated with the help of voluntary persons and patients. The EU Project RISE demonstrated that home based FES of denervated muscles is a secure and effective home therapy. Benefits of stimulating denervated muscles are: 1. Recovery of tetanic contractility; 2. Restoration of muscle fibre structure; 3. Recovery of fibre size and muscle mass; 4. Better skin condition; 5. Reduced risk of pressure sores; 6. Improved cosmetic appearance of lower extremities; 7. Increased self-esteem. Furthermore, if standing upright is accomplished: 8. Improved cardiovascular fitness; 9. Unloading of seating surface. The conclusion of the RISE project was that a commercial electrotherapy device for home based FES was a priority. The Stimulette den2x by Dr. Schuhfried is the first device that delivers the needed power and technical requirements to fullfil the clinical requests. The following parameters are programmable: Impulse amplitude: max +/- 300 mA; Impulse waveform: rectangular / ramp shaped (3 different waveforms); Impulse duration ID: 10 ms—200 ms; Impulse pause IP: 1 ms-2 s; Surge duration: 100 ms—11 s; Rise: 5 % - 100 % surge duration; Decay: 5 % – 100 % surge duration; Surge interval: 0 ms—11 s; Treatment duration: 1—59 min; all currents are biphasic. The Switchbox: The Switchbox has been developed to enable flaccid paraplegic patients to practice standing, stepping and a type of „walking“ at the parallel bars. Functional Electrical Stimulation of denervated muscles — a novel therapeutic option after peripheral nerve lesion is a realistic option. In conclusion, the Stimulette den2x represents a major breakthrough in FES., Rehabilitation treatment is still a challenge for clinicians in patient suffering from muscle atrophy following spinal cord Injury and/or peripheral neuropathies. Electrical Stimulation (ES) is a discussed option, but it plays in our opinion an important a role at least to maintain muscle trophism of denervated muscles and recover from atrophic innervated muscles.1-3 In our hospital, functional and electrical stimulation tests are part of the standard evaluation in patients treated with electrical stimulation for denervated muscle after peripheral nerve injury. However, to better explain the effects of ES and verify the efficacy of the treatment, muscle imaging could help clinician for the follow up of this kind of patients. In this presentation we discuss the usefulness and use of different type of muscle imaging (MRI, CT, dynamic echomyography) to assess muscle tissue health in clinical rehabilitation perspectives.4-6 We will present case reports to offer the opportunity to discuss rehabilitative pathwaies for diagnostics and rehabilitation of patients suffering of peripheral denervation, a condition that is still a challenge for clinicians. In particular we would like to evaluate the opportunities of the Quantitative Muscle Color Computed Tomography (QMC-CT), a quantitative imaging analysis introduced by our group to monitor skeletal muscle. Validation of QMC-CT will provide physicians an improved quantitative tool to diagnose the condition of skeletal muscle during rehabilitation of mobility-impaired persons, so that managements can be better prescribed, evaluated and altered where needed., The cellular basis of age-related tissue deterioration remains largely obscure. The ability to activate compensatory mechanisms in response to environmental stress is an important factor for survival and maintenance of cellular functions. Autophagy is activated both under short and prolonged stress and is required to clear the cell of dysfunctional organelles and altered proteins. We report that autophagy in muscles declines with ageing and its inhibition correlates with age-dependent muscle loss and weakness. Specific autophagy inhibition in muscle has a major impact on neuromuscular synaptic function and, consequently, on muscle strength, ultimately affecting the lifespan of animals. Inhibition of autophagy also exacerbates aging phenotypes in muscle, such as mitochondrial dysfunction, oxidative stress, and profound weakness. Mitochondrial dysfunction and oxidative stress directly affect acto-myosin interaction and force generation but show a limited effect on stability of neuromuscular synapses. Mitochondria shape is also a critical factor for sarcopenia and for systemic ageing. Mechanistically, mitochondria control a cascade of signalling events that induce muscle secretion of myokines that cause systemic ageing and premature death.1-4, Physical medicine therapies are first line of intervention, with pharmacologic prior to surgical treatments for several musculoskeletal diseases, such as low back pain. Herbal cataplasms containing a rubefacient substance, (Cayenne pepper, CP) are directly applied to the skin at the site of the painful areas provoking a hyperemic response, that involves both epidermis and muscle tissue nociceptor fibers, with beneficial analgesic effects. Capsaicin is the most abundant capsaicinoid present in the Cayenne pepper and it is an agonist of Transient Receptor Potential Vanilloid 1 (TRPV1). This treatment is generally well tolerated, but data on its possible side effects and secondary targets are missing. We tested 20-min application of 5% Cayenne pepper cataplasm (CPC) on healthy subjects, monitoring its effects on serum levels (before and 0.5, 1, 3, 6, 24 hrs after application) of general Laboratory parameters (hemogram, CRP, sedimentation, CK, albumin, cortisol), pro-and antiinflammatory cytokines (TNF-alpha, IL-1β, IL-6, TGF-β1) biomarkers specific for blood vessels damage (leukotriene B4, E-selectin, P-selectin, VCAM-1), and a panel of selected miRNAs possibly implicated in the cellular processes modulated by Caspaicin topical treatment.1-3 Specifically, we analysed miRNA regulating TRPV1 transcription (miR-199a, and miR-199b), those mediators of inflammation (miR-155, miR-21, miR-146a), intracellular Ca2+ homeostasis (miR-25), endothelial cell damage (miR-126), cardiac and skeletal muscle homeostasis (miR-1, miR-133, and miR-206). No significant changes in the serum levels of tested cyokines or Laboratory parameters have been observed over the analysed time period. Interestingly, changes of the plasma levels of c-miRNA regulating Th1>Th2 inflammatory response and TRPV1 (specific pharmacologic target of Capsaicin) were detected. These results suggest that 5% Munari cataplasm seems to be a safe treatment targeting specific receptor responsible for pain sensation. In addition, circulating miRNAs are novel good candidate biomarkers for testing and monitoring treatment’s effects in patients affected with Low Back Pain. Further studies are needed to investigate the immediate and long-term effects of repeated CPC applications as well as to understand the intersecting underlying mechanisms activated by Capsaicin and other identified factors, in order to further validate them for physical medicine therapies., Endocannabinoids are endogenous lipid mediators with wide range of biological effects similar to those of marijuana. They exert their biological effects via two main G-protein-coupled cannabinoid receptors, the CB1 (cannabinoid receptor 1) and CB2 (cannabinoid receptor 2). Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies gave evidence for the presence of cannabinoid receptors in different types of tissues.1,2 Their presence was supposed in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points, suppressing proinflammatory cytokines such as IL-1beta e TNF-alpha and increasing anti-inflammatory cytokines.3,4 However, until now the expression of CB1 and CB2 in fasciae and in fascial fibroblasts has not yet been established. In this work small samples of fascia were collected from volunteers patients: for each sample were done a fibroblast cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies) and real time RT-PCR to detect the expression of CB1 and CB2 and evaluation of gene expression of CB1 and CB2 receptors after fibroblasts mechanical stimulation. The immunostaining results demonstrate the expression of CB1 and CB2 on fascial fibroblasts and fascial tissue. In the tissue not all the fibroblasts are positive, whereas the isolated and expanded cells are homogeneous. These results are confirmed by the real time PCR where the specificity of the reaction on fibroblasts and fascial tissue is the same, but the amount of expression in the tissue is lower, for both CB1 and CB2. The mechanical stimulation has shown that there is an increase of CB2 expression on fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. These results could represent a new target for drugs to care fascial fibrosis and inflammation. The presence of the endocannabinoid system in the fascial fibroblasts can also explain the efficacy of cannabis to care myofascial pain and the observation that a mechanical stimulation has given an increase of receptor gene expression could explain the possible stimulation during manipulative treatments and exercises.5 More studies about the interactions between fibroblasts, extracellular matrix and CB1 and CB2 receptors could help to understand the role of these receptors on myofascial pain., Skeletal muscle repair goes through a modulation of several stages, which are mainly accomplished through changes in the activation profile of macrophages. This process results in changes in the phenotype and function of involved cells and macrophages, which play a key role in this progression and are considered the targets for therapeutic intervention.1,2 Mitochondria also exert a crucial modulatory effect on inflammatory macrophages pathways, leading to the production of cytokines (Mitogen Activated Protein Kinases and Nuclear Factor-Kappa β) pathways. When an inflammatory stimulus triggers macrophage activation, the mitochondria amplify these pathways, resulting in increased production of cytokines and inflammatory mediators. Over the last ten years, many studies demonstrate that the employment of the laser therapy modulates many biochemical processes, especially the decrease of muscle injures, the increase in mitochondrial respiration and ATP synthesis, crucial to accelerate the healing process. However, nowadays there is no consensus over the best laser protocol to employ in the clinical practice in order to obtain the most efficient biological response. For this reason, many in vitro studies focus their attention on the highest effect on mitochondria by laser light. Among the most clinical employed wavelengths, it is already known that red and infrared laser lights stimulate photochemical and photophysical events in mitochondria, thus resulting in increased mitochondrial membrane potential and higher enzyme activity in the respiratory chain. It is possible to observe structural changes, such as the formation of giant mitochondria through the merging of membranes of smaller and neighbouring mitochondria, which lead to higher levels of respiration and ATP to cells. It has also been demonstrated that laser therapy improves enzyme activity of the complex IV (cytochrome c oxidase) in skeletal muscle mitochondria. This effect is crucial since the oxidative capacity of muscle fibres is related to the density of mitochondria, able to oxidize glucose, fatty acids and proteins for ATP synthesis during muscle contraction. To optimize muscle recovery, when adding laser therapy to low intensity exercises, it is possible to foster this mechanism working on mitochondrial biogenesis, both to favour aerobic metabolism and to reduce muscle fatigue from metabolic origin.3-6 MTT assay on myocytes assesses an increased mitochondrial activity and cell activation after laser treatment. In addition, it is possible to observe a clear reduction in Tumor Necrosis Factor-a production 24 hours after the irradiation of activated macrophages. So, thanks to laser therapy muscle performance could be increased reducing its fatigue; the most accredited and studied mechanisms to this specific behaviour are: i) enhance mitochondrial activity, ii) phosphocreatine resynthesis and iii) mitochondria lactate oxidation. Although in vitro studies offer the possibility to standardize the obtained results, thanks to their cellular and molecular highly reproducible models, the results of such studies cannot be directly correlated with clinical outcomes. Nevertheless, the knowledge of the effect of laser therapy on the mitochondria contained in different muscle cell types is of paramount importance for the design of in vivo protocols that can exert more effective modulation of the muscle repair process., Chronic low back pain (CLBP) is a disabling condition affecting a majority of people of the western countries. It deeply affects the quality of life as it is often linked to multidimensional disturbances such as poor sleep, mood disorders, chronic fatigue and joint pain. There is no other condition with higher social and economic costs. It has been reported that only a minority of patients with gut inflammation suffers from intestinal symptoms. In a previous paper it was proposed that gastrointestinal disturbances, beyond mechanical issues, could be overlooked in the management of these patients. Dietary changes were successful in the positive resolution of the described clinical case. In this paper we further test this hypothesis. We measured on 5 subjects specific parameters related to gastrointestinal and digestive physiology that have been associated with metabolic and immune related pathological conditions. Specifically we tested the levels of zonuline (related to intestinal permeability) the presence of undigested substances, ph and the colonization of specific bacteria (symbiotic vs pathoghenous). Inflammation in the gut can lead to altered mucosa permeability indeed. The entrance in the blood stream of abnormal molecules activates the immune system in a cascade of events affecting remote systems and possibly the integrity of structures like the neuromuscolar junction or the pathways of energy production. Conditions that are currently managed by orthopaedists, reumatologists or neurologists could benefit from a screening of the gastrointestinal functionality., It is well known that repeated bouts of exercise (i.e. exercise training) lead to an elevated content of mitochondria within muscle. This adaptation confers metabolic advantages during exercise, such as an increase in the aerobic metabolism of lipids, reduced glycogen usage, and diminished lactate production. The molecular basis for this increase in organelle content involves the activation of PGC-1α along with numerous transcription factors which increase the expression of nuclear genes encoding mitochondrial proteins. Among these are Tfam, the transcription factor which mediates mtDNA replication and transcription, in an effort to coordinate the nuclear and mitochondrial genomic responses to the exercise signals. These organelle synthesis processes (termed biogenesis) have been well-studied, and reviewed recently.1 On the other hand, it is also recognized that the steady state mitochondrial content of muscle is determined not only by rates of synthesis, but rather by organelle turnover, represented by a balance between synthesis and degradation. The degradation process is termed mitophagy. In contrast to biogenesis, our understanding of mitophagy in muscle is in its infancy. Mitophagy involves the activation of the general autophagy pathway within the cell, where the ultimate target for degradation is the dysfunctional mitochondrion. Targeting mitochondria involves tagging the organelle for degradation by ubiquitination, followed by its engulfment within an autophagosome for fusion to a lysosome, and subsequent proteolysis. We have previously shown that a single bout of exercise initiates mitophagy flux signaling, measured as the activation of kinases which trigger autophagy, along with localization of LC3-II and p62 on the surface of the organelle. We found that the degree of mitophagy flux enhanced by exercise was PGC-1α-dependent, such that the absence of the coactivator led to reduced mitophagic responses to exercise. Thus, PGC-1α is involved not only in organelle biogenesis, but also in its degradation.2 In contrast to the enhanced mitochondrial content in muscle in response to exercise, aging is a progressive condition in which mitochondrial content and function, along with the level of PGC-1α, are reduced in muscle, contributing to altered metabolism and decrements in muscle mass.3 In addition, while muscle adaptations are certainly possible in response to exercise, the biogenesis adaptations to standardized workloads is not as robust with age, as it is in younger subjects.4 Thus, while previous work has documented blunted stages of biogenesis in aged muscle, no research has documented the degree of change in mitophagy. The prevailing dogma suggests that mitophagy is decreased in aging muscle, however limitations in methodologies preclude this conclusion. Furthermore, how chronic exercise may affect mitophagy in aged muscle remains unexplored. Thus, we have examined the effect of aging and chronic exercise on mitophagy flux using 6 and 36 month old Fisher 344 Brown Norway rats that serve as an excellent model of aging skeletal muscle. To invoke comparable levels of chronic exercise, the animals were implanted with a stimulator to activate the peroneal nerve which innervates the tibialis anterior muscle to induce chronic contractile activity (CCA; 3hrs/day, 9 days). The contralateral limb served as control. Colchicine is a microtubule inhibitor which interferes with the transport of the autophagosome to the lysosome for degradation. Thus, administering this drug for 3 days (0.4 mg/kg/day) allowed us to measure mitophagic flux when levels of p62 and LC3-II are compared to vehicle-treated animals. To evaluate mitophagy, intermyofibrillar mitochondria were isolated from the TA muscle and protein localization was assessed by immunoblotting. As expected, aged animals exhibited reduced mitochondrial content and an attenuated adaptation to CCA in agreement with previous work.4 Colchicine successfully inhibited autophagy in our model and allowed for the quantification of mitophagy flux. In young animals following the mitochondrial adaptations to 9 days of CCA we observed decreased mitophagy,5 consistent with the idea that improved mitochondrial content or function after CCA obligates lower organelle degradation rates. In contract, mitophagy flux was higher in muscle of aged animals,5 in contrast to suggestions from the literature, and the attenuation of mitophagy as a result of CCA was less pronounced. These high rates of mitophagy may contribute to the age-related loss of mitochondrial content, but when combined with a reduced capacity for biogenesis, this pattern of organelle turnover within aged muscle is insufficient to maintain the high quality of mitochondria compared to muscle from younger animals. Our data also fortify the concept that exercise is a useful therapy to modify mitochondrial turnover rates, in an effort to sustain, or enhance, the healthiest mitochondrial pool within skeletal muscle., Preserving mobility is central to maintaining a high quality of life and participation in activities to be fully independent in the community.1 Unfortunately, aging is associated with a progressive decline in mobility, as well as cognitive and physical function, leading to a loss of independence. As diverse as the etiologies of physical disability are, a growing body of evidence strongly implicates chronic low-grade systemic inflammation as playing a significant role in contributing to sarcopenia and associated functional decline.2,3 A variety of endogenous factors (e.g., adiposity) and exogenous factors (e.g., lifestyle habits) appear to contribute to the rise in systemic levels of inflammation seen with aging.4 To date, few therapeutic approaches have been specifically identified to reduce chronic systemic inflammation with the goal of reducing pain levels and improving functional performance in seniors. There are, however, a number of promising approaches that have emerged during the past decade that appear capable of targeting chronic systemic inflammation. Given the increasing number of older adults with elevated levels of systemic inflammation who are at risk for functional decline, new therapies are urgently needed to reduce systemic inflammation levels and improve or maintain functional ability in this high risk population. Thus, the purpose of this presentation is to provide an overview of promising therapeutic approaches, including lifestyle interventions, hormonal replacement, natural compounds, and pharmaceutical agents, to avert levels of chronic systemic inflammation during aging and preserve function in older adults., Iron dyshomeostasis (high cellular and low systemic levels) are strong risk factors in the development of disease, disability and premature death. Systemic iron deficiency (anemia with old age) impairs oxygen carrying capacity, while in contrast increased cellular levels can increase DNA lesions. Disturbances of iron metabolism including uptake, export, and storage have shown to play a causal role in cellular and mitochondrial dysfunctions with age and disease. Iron is found in several forms: heme iron (i.e., haemoglobin, myoglobin) and non-heme iron (i.e., Ferritin). A distinct fraction of chelatable non-heme iron is referred to as the labile iron pool, which comprises less than 5% of total cellular iron. Labile iron consists of Fe2+ and Fe3+ ions associated with a variety of small molecules, including organic anions, polypeptides, and phospholipids. Labile iron can participate in Fenton reactions, producing highly destructive hydroxyl radicals, which are thought to be a major contributor to the formation of DNA mutatons. Cellular iron acquisition occurs through iron import proteins such as transferrin receptor (TfR1), divalent metal transporter-1 (DMT1), and Zip14, whereas cellular iron export is mediated by ferroportin (FPN), the only known iron exporter in mammals. The mitochondria contain mitoferrin (Mt iron importer), iron storage proteins such as frataxin and Mt ferritin (MtF) (which binds with iron), and ABCB7 (a heme export protein), all known to play an important role in the storage and regulation of Mt iron. We and others have found that in animals and humans, labile iron and non-heme iron increases with age and is associated with elevated expression of ferritin. In contrast, transferrin receptor 1 (TfR1; cellular iron import protein) showed a dramatic down regulation with age. In addition, mitochondrial iron levels effect Mt permeability transition pore opening susceptibility (i.e., Ca2+ retention capacity) in mitochondria from old animals. Further studies to better understand iron metabolism with aging are warranted to design interventions to reduce DNA lesions., Depletion of calcium (Ca2+) from intracellular stores triggers store-operated Ca2+ entry (SOCE), a ubiquitous mechanism that allows recovery of Ca2+ ions from the extracellular space. To date, the subcellular location for SOCE in skeletal muscle fibers has not been unequivocally identified. Here we show by electron microscopy (EM) that 1 hour of incremental treadmill running of mice (from 5 m/min to 25 m/min) drives a striking remodeling of the existing sarcotubular system in skeletal fibers leading to formation of previously unidentified junctions between sarcoplasmic reticulum (SR) and transverse-tubules (TTs). In addition, using immunohistochemistry, immunogold labeling for EM, and western blot analyses we demonstrate that these new SR-TT junctions contain the molecular machinery that mediate SOCE: a) stromal interaction molecule-1 (STIM1), which functions as Ca2+ sensor in the SR, and b) Ca2+ permeable Orai1 channels in TTs. Finally, we used a stimulation protocol (30 x 1s-60Hz pulses every 5 seconds) to compare susceptibility to in vitro muscle fatigue of EDL muscles from either control or exercised mice. EDL muscles from exercised mice exhibited an increased capability of maintaining contractile force in presence of 2.5 mM extracellular Ca2+, that was abolished by either the presence of SOCE inhibitors (BTP-2 and 2-APB) or by equimolar replacement of extracellular Ca2+ with Mg2+. We propose that exercised-induced formation of newly formed SR-TT junctions containing STIM1 and Orai1 proteins function as Ca2+ Entry Units (CEUs), structures that provide a pathway to rapidly recover Ca2+ ions from the extracellular space during repetitive muscle activity.., The ERG1 potassium channel is known to participate in repolarization of the cardiac action potential.1 However, we reported detection of this protein in the Gastrocnemius muscle of mice experiencing atrophy as a result of both disuse (i.e., unweighting) and cancer cachexia while it was not detected in the Gastrocnemius muscles of appropriate control animals.2 In subsequent studies, we showed that ERG1 participates in muscle degradation by enhancing ubiquitin proteolysis through increased abundance of the E3 ligase, MuRF1.3,4 However, to our knowledge, ERG1 has not been reported in human skeletal muscle. Here we have used immunohistochemistry and confocal microscopy to image ERG1 protein with a fluorescent marker and report detection of ERG1 immunofluorescence in the Rectus abdominis (RA) muscle of adult humans. Interestingly, we detect statistically greater immunofluorescence (67.0%; p≤0.01) in the RA muscle of people having cancer cachexia (n=6) than in the same muscle of age-matched healthy adults (n=7). We detect ERG1 immunofluorescence at low levels only in the RA muscle of young adults (n=4); however, our results show that the signal trends toward greater fluorescence (11.0%) in the RA muscle of healthy aged adults than in that of the younger ones. Although the difference in ERG1 immunofluorescence in the healthy aged and young adult RA muscle is not statistically significant, Power analysis of the data demonstrates that an increase in sample size to 46 (23 each group) from the current size of 11 people would produce a significant difference in the data. Indeed, our data suggest that ERG1 may be related to the skeletal muscle loss that occurs with cachexia and aging in humans., Tubular aggregates (TAs), ordered arrays of sarcoplasmic reticulum (SR) tubes, form in ageing fast twitch fibers of mice, preferentially in males. TAs are also the main morphological alteration in biopsies from patients affected by TA Myopathy (TAM). TAM has been linked to mutations in the genes encoding for STIM1 and Orai1, the two proteins that mediate store-operated Ca2+ entry (SOCE), a mechanism that allows recovery of extracellular Ca2+ when the SR is depleted. We have previously shown that: i) TAs contain SERCA1 and CASQ1, two proteins involved in reuptake and storage of Ca2+ in the SR; ii) tubes of TAs appear linked by small bridges. Here, we combined different experimental approaches - electron and confocal microscopy (EM and CM), western blots (WB), and ex-vivo stimulation protocol (30 x 1s - 60 Hz pulses every five seconds) performed in inctact EDL muscles - to study localization and function of STIM1 and Orai1 in muscle containing TAs. In EDL muscles from mice of 4 and 24 months of age: i) ageing causes STIM1 and Orai1 to accumulate in TAs; ii) the expression levels of both STIM1 splicing variants increase with age (STIM1S = 0.44±0.03 vs 0.66±0.08 A.U.; STIM1L = 0.38±0.05 vs 0.56±0.05 A.U. respectively for adult and aged mice); iii) EDL muscles from aged mice exhibit a decreased capability to maintain contractile force compared to adult mice (relative force after 10 tetani: 61.6±3.0%, and 52.7±4.3% respectively for adult and aged EDL muscles). Our findings suggest that accumulation of STIM1 and Orai1 in TAs, is dysfunctional as Ca2+ entry during repetitive stimulation is impaired in aged EDL muscles., The sarcomere is a highly organized structure that represents the functional unit of the contractile apparatus of striated muscles. The maintenance of both sarcomere integrity and the correct reciprocal arrangement between myofibrils and organelles, like nuclei and sarcoplasmic reticulum, costameres, etc., represent a crucial requirement that striated fibers must fulfill to efficiently accomplish repeated cycles of contraction and relaxation. Obscurin is a giant sarcomeric protein mainly localized at the M-band and, with minor distribution, at the Z-disk. The structural layout of Obscurin, which is based on the presence of different modular binding, adhesion and signaling motifs, allows the simultaneous interaction with sarcomeric and non-sarcomeric proteins, thus placing Obscurin in a key molecular crossroad to contribute to the overall muscle fiber architecture. Indeed, binding of Obscurin to Titin, Myomesin and OBSl1 provides an important structural support to sarcomere integrity and stability at the level of the M-band. In addition, the ability of Obscurin to interact with distinct members of the ankyrin family contributes to establish multiple molecular contacts between the contractile apparatus and sarcoplasmic reticulum, microtubules and costameres.1 We have recently reported studies with Obscurin KO mice suggesting a role of Obscurin in supporting fiber integrity following heavy exercise.2 These results will be presented and discussed also in relation to the recent identification of mutations in the Obscurin gene in patients with cardiac and skeletal muscle diseases.3, Loss of skeletal muscle mass and force aggravates age-related sarcopenia and numerous pathologies, like cancer and diabetes. The AKT-mTORC1 pathway plays a major role in stimulating adult muscle growth, however, the functional role of its downstream mediators in vivo is unknown. Here we show that simultaneous inhibition of mTOR signaling to both S6K1 and 4E-BP1 is sufficient to reduce AKT-induced muscle growth and render it insensitive to the mTORC1-inhibitor rapamycin. Surprisingly, lack of mTOR signaling to 4E-BP1 only, or deletion of S6K1 alone, is not sufficient to reduce muscle hypertrophy or alter its sensitivity to rapamycin. However, while not required for muscle growth, we report that S6K1 is essential for maintaining muscle structure and force production. Hypertrophy in the absence of S6K1 is characterized by a compromised ribosome biogenesis and the formation of p62-positive protein aggregates. These findings identify S6K1 as a crucial player for maintaining muscle function during hypertrophy., This work outlines the methods and applications of X-ray Computed Tomography imaging to analyze soft tissue and skeletal muscle density and volume in the context of modern challenges in the field of translational myology. The approaches described here use medical imaging processing techniques and computational methods to: quantify muscle morphology, illustrate changes with 3D models, develop numerical profiles specific for each individual, and assess muscle changes due to targeted medical treatment. Applications of these methodologies are employed: to depict subject specific muscle profiling associated with age, to illustrate and quantify muscle degeneration and its partial reversal via Functional Electrical Stimulation (FES), and to highlight recovery following total hip arthroplasty.1-5, The functional recovery from severe atrophy of long-term denervated muscle by h-bFES of DDM is a fact standing on sound foundations.1 Among them, a new quantitative muscle color computed tomography (QMC-CT)2,3 adds to functional evidence and muscle biopsy analyses, the results based on 2D (left panels) and 3D (right panel) clinical imaging analysis. We are extending the methods to managements of severe atrophy in oldest persons, which need simplified methods of evaluation, and safe, easy to performe rehabilitations at home.4 A major problem is to convince subjects to maintain volitional exercise at home. We are confident that strong evidence of structural improvements of muscles could motivate reluctant older persons to take home anti-aging full-body in-bed gym5 and functional electrical stimulation (FES) for mobility compromised elderly persons.4, Myotonic Dystrophy (DM1) is the most common form of adult-onset muscular dystrophy, but is missing circulating biomarkers as well as an effective rehabilitation protocol. In our work we aim to propose a clinical-molecular protocol to monitor rehabilitation therapy versus standard care in this common inherited muscle disorder. For all DM1 patients the maximum standard of care was achieved through special medical attention and locomotor study, cardio-respiratory and nutritional care, interview for psychological problems, quality of life, we investigated the role of serum MicroRNAs as biomarkers of the disease in order to correlate their levels with disease severity, multiorgan involvement and possibly the efficacy of physical rehabilitation program. We aimed to explore the cellular action of micro-RNAs that are non-coding-RNAs modulating gene expression, whose expression is dysregulated in DM1. In order to investigate the micro-RNA origin a initial aim was to measure the levels of muscle-specific myo-miRNAs (miR-1, miR-133a/b, miR-206) in muscle of 12 DM1 patients.1 Muscle fiber morphometry with a new grading of histopathological severity score were used to compare specific myo-miRNA level and fiber atrophy. We found that the levels of miR-1 and miR-133a/b were significantly decreased, while miR-206 was significantly increased as compared to controls. The histopathological score did not significantly correlate with the levels of myo-miRNAs, even if the lowest levels of miRNA-1 and miRNA-133a/b, and the highest levels of miRNA-206 were observed in patients with either severe histopathological scores or long disease duration. The histopathological score was inversely correlated with disease duration. Nowadays DM1 muscle biopsies are scanty, since patients are usually diagnosed by genetic analysis, our study offers a unique opportunity to present miRNA expression profiles in muscle and correlate them to muscle morphology in this rare multisystem disorder. Our molecular and morphologic data suggest a post-transcriptional regulatory action of myo-miRNA in DM1, highlighting their potential role as biomarkers of muscle plasticity. We explored in 10 patients (9 male and 1 female) during our new rehabilitative protocol we developed.2 Serum microRNAs appeared as biomarkers to monitor DM1 patients while in a protocol of aerobic lower extremity Functional Electrical Stimulation lower aerobic rehabilitation.2 We observed improvement of our patients during this exercise protocol and all microRNas decreased during rehabilitation (Figure). This study validate clinical use of microRNAs after the first discovery in MD1.3 In our investigations in muscle and serum, some microRNA (miR-1, miR-133a, miR-133b, miR-206) appeared promising in detecting changes in DM1 in natural history and during rehabilitation to correlate with functional outcomes, we found that reversal of muscle atrophy and onset of muscle regeneration in DM1 might be revealed by decreased microRNA levels. These circulating biomarkers were validated in this study in twelve DM1 cases., Several epidemiological studies have repeatedly shown a statistical association between life-long physical exercise and better preserved cognition later in life. This association was based on self-reports coded as variables which do not retain much quantitative variability. Some studies have used metabolic conversion to give a biological flavour to their findings. A few recent experimental studies have identified physical activity as a protective factor for cognitive decline. The role of physical activity as a protective factor has received more attention than other popular ways of stimulating the brain, e.g. cognitive stimulation. Studies have focused on discovering the biological mechanisms behind this effects and attention has been given to mitochondrial activity and the pathways by which ATP is produced, with a specific focus on aerobic exercise. Research studies have also compared the effects of acute vs chronic exercise. Experimental work has been carried out on acute exercise (i.e. single sessions) to explore the mechanisms involved and shed light on the biological underpinning of the beneficial effects of physical activity on cognition. This research has often involved young adults because of the opportunity to implement better manipulation of variables such as intensity and duration of exercise. Brain activity has been measured with Near Infrared Spectroscopy to study how brain function changes during acute exercise in an attempt to infer the mechanisms behind the long term effect of exercise. Because chronic exercise is associated with long term effects, there is a clinical interest to clarify the mechanisms that are involved in short and long term benefits due to exercise. Many studies have used exercise in combination with mixed interventions (e.g. diet and exercise, or cognitive stimulation and exercise), however. More recent experimental approaches have put forward possible explanations about the basis of the beneficial effects of exercise and suggested that physical activity triggers an improvement of cardiovascular fitness and improvement in cognition, but it is still unknown whether the two are causally linked. The implication is that cognitive benefits are the indirect outcome of cerebrovascular improvements. Other studies have suggested that physical activity increases neuroplastic mechanisms in humans, by fostering hippocampal neurogenesis, by regulating cortisol and BDNF and by enhancing motor-cortical plasticity as elicited by the TMS-based technique “cerebellar inhibition”. A crucial modulating factor appears to be played by individual genetic profiles, such as that for the ApoE gene. There is experimental evidence that suggests that the long term beneficial effects of exercise might be the result of optimisation of prefrontal resources via continuous exercise dependent hypofrontality. Overall, better designed trials with more sophisticated outcome measures are necessary to test experimentally the extent to which physical activity might be an effective form of intervention to prevent cognitive decline in ageing and neurodegeneration. There is, however, some recent evidence that the regular practice of walking improves cognition in Alzheimer’s disease, while strength training is particularly more effective for improving postural and motor function, and reducing the risk of developing Alzheimer’s disease, since it improves muscle mass and strength, shown to be affected in this disease., The neuromuscular system is subject to many kinds of damage, from traumatic nerve injury to slowly progressive neuropathies. The emerging field of electroceuticals aims to intervene by recording, processing and normalising neural activity to enhance the function of failing organ systems. Electrical activation has the potential both to maintain muscle mass and to promote neural growth after peripheral neural trauma.1,2 But interaction with the musculoskeletal system must take into account the changes in that system that affect the requirements for artificial activation. The most obvious example is that denervated muscles require much greater current to flow in their membranes to activate release of calcium and contraction than do innervated muscles, whose activation is based on the electrochemical generation of action potentials in the muscle fibre membrane beneath the motor end plates. Similarly, if we are to use stimulation therapy to treat diabetes by neuronal stimulation, then we must take into account that diabetes is often associated with altered neuronal function. The need to inject current from implanted electrodes brings its own risks of tissue damage, tissue heating, and electrolysis of electrode materials. A target denervated muscle may be situated among other innervated muscles, or adjacent to sensory structures. Thus the selection of electrode material, shape and size is important to the outcome. This presentation will review theoretical and practical design criteria to achieve safe and efficient activation of musculoskeletal structures, with some examples.3, Age related changes of the muscle and its adjacent structures also affect the larynx.1 Muscular atrophy leads to an incomplete closure of the vocal folds, leading to a hoarse and breathy voice. The consequences are reduced quality of life and reduced working capacity of persons who are depending on their voices professionally (teachers, policemen etc.). Chronic electrical stimulation of the afferent nerve (recurrent laryngeal nerve) is a completely new therapeutic option that has not been tested before. In a preliminary study we could show that electrical stimulation of the recurrent laryngeal nerve led to an increase of mean muscle fiber diameter in aged sheep, even with a very conservative pattern of two minutes tetanic contraction daily over a period of 29 days.2 Here we present data of an ongoing sheep trial where the electrode was implanted unilaterally adjacent to the terminal branch of the inferior laryngeal nerve. This surgical approach is already close to a clinical setting in humans., The access to different structures in the larynx - especially to the intrinsic muscles in vivo - is limited. Additionally the volumetric quantification is problematic due to their covering with mucosa. Nevertheless it is necessary to generate accurate models of these structures for the purpose of answering muscle-specific issues. Nowadays this is possible with modern imaging procedures such as micro-CT scanning. This technology has advantages over MRI in terms of better resolution and the samples are not destroyed during the imaging process as in histologic sampling. To differentiate the muscles from soft tissue and cartilage, the samples are fixed and preserved in neutral buffered formalin (NBF) and stained with iodine potassium iodide (I2KI) to enhance contrast in the CT-scan.1 The purpose of this study is to generate 3D-models of the laryngeal frameworks and the intrinsic laryngeal muscles by segmentation and finite-element generation using the 3D-analysis-software Avizo®. This modeling technique will be used in ongoing experiments in the field of muscle stimulation for analysis of the results, especially muscle volumes, surfaces and structure. Additionally, phonation experiments on the same subjects were performed to find out correlations between functional parameters and morphometric measurement parameters.2 Phonation analysis included aerodynamic parameters such as the subglottal pressure or the laryngeal flow resistance and acoustic parameters such as the sound pressure level or the fundamental frequencies. Furthermore, high-speed recordings have been performed to visually assess the vocal fold vibrations.3,4, Vocal fold paralysis is a pathological motion impairment of the vocal fold, mostly caused by damage of the N. vagus or the N. laryngeus. If the vocal fold does not reinnervate, paralysis occurs due to denervation of the M. posticus1 Patients with unilateral vocal cord paralysis suffer from hoarseness due to additional atrophy of the M. vocalis with glottal closure insufficiency during phonation. Today’s standard treatment of unilateral paralysis includes surgical medialization through either injection augmentation or laryngeal framework surgery.2 In combination with voice therapy also electrical stimulation of laryngeal muscles has already been used in order to achieve muscle hypertrophy.3 Furthermore research with functional electrical stimulation of patients with long-term denervated limb muscles showed very promising results.4 The selective stimulation of denervated muscles has been investigated in rabbits with unilateral paresis of the recurrent laryngeal nerve. It could be shown that with triangular ramping and very long pulses (> 200ms) afferent and efferent nerve fibers where not reacting at intensity level that already stimulated denervated muscle, with change in muscle fibers confirmed through histology. 5,6 Combining these facts led to the following investigations: Investigating a screening possibility using surface electrodes onto the neck to selectively stimulate the denervated muscle fibers of the vocalis avoiding pain or excitation of sensory nerve fibers or the activation of innervated muscles was the goal of several test stimulations. First results applying long triangular ramping pulses (>200ms) using surface electrodes are surprising. The position and size of electrodes used in the trials were improved continuously. Success could be reported only in the non-awake patient, whereas reasons have to be identified., Facial nerve paralysis as a peripheral nerve injury results in neuromuscular atrophy or in a combination of muscle atrophy and false reinnervation of facial muscles. The symptoms include significant aesthetic, functional and often life-altering consequences. Several procedures such as nerve grafting, facial reanimation by muscle transfer and rehabilitation physiotherapy have been developed to treat functional and cosmetic aspects of this disease.1 Nerve grafting is a sophisticated surgery, that requires experience but offers promising results. Although cable grafting is state of the art, the method suffers the disadvantage of long nerve regrowth time.2 Facial pacing systems show promising results to treat facial paralysis.3,4Former research showed good results stimulating denervated extremity muscles using functional electrical stimulation (FES).5 Nevertheless this field of research has been neglected so far for facial muscles and is lacking optimal stimulation settings to selectively recruit denervated atrophic or simply age-related atrophic facial muscles under non painful conditions. To analyze first optimal FES setting will be the prerequisite to establish FES as a screening tool to select patients for facial pacing. Several ES devices were considered to investigate optimal stimulation settings in patients with chronic facial palsy. To encourage noninvasive screening methods for facial pacing, surface electrodes were used to estimate the optimal settings for stimulations. The use of surface electrodes need for optimized electrode positioning, which was also investigated. Martin et al.6 showed that recruitment of denervated muscles requires exponentially shaped pulses with long phase durations (>200ms). The outcome of our investigation confirmed these findings as well, showing best performance when recruiting paralyzed facial human muscles with biphasic long-duration impulses. It is crucial to position the surface electrodes appropriately in order to avoid stimulation of neighboring muscles not affected by facial palsy, for instance the masseter muscle. Surface electrodes, combined with the optimal stimulation settings, offer a screening possibility for facial pacing but also a therapeutic option to prevent atrophy. Since muscles affected by age-related atrophy could be recruited too, further research is necessary to show effectiveness of training using the determined exponential patterns., Recent studies have correlated physical activity with a better prognosis in cachectic patients, although the underlying mechanisms are not yet understood. In addition, diets enriched with n-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to exert a positive effect on diseased muscle. Muscle diseases as different as cachexia and dystrophy are characterized but reduced or absence of dystrophin expression, latent or overt muscle damage and impaired regeneration, thus sharing several patophysiological features, such as muscle wasting, loss of muscle mass and function. With the aim to test in preclinical models and in human patients the efficacy of physical, pharmacological and nutritional interventions against muscle wasting and disease, we exploited two different rodent models of cachexia and muscular dystrophy and validated part of these findings in human patients. Part 1. Cancer cachexia. Since we previously found that satellite cells (SC) impairment, due to Pax7 over-expression, contributes to cachexia,1 we studied the effects of voluntary exercise on these cell in colon carcinoma (C26)-bearing mice. We found that endurance exercise rescues Pax7 expression to physiological levels, suggesting that this could be a mechanism underlying its beneficial effects in this condition.2 Moderate exercise training protocols induced muscle adaptation in both control and C26-bearing mice, which are mediated by PPARgamma in a Hsp60-dependent way.3 Indeed, voluntary exercise prevented loss of muscle mass and function, ultimately increasing survival of C26-bearing mice. We found that the exercise mimetic AICAR, rapamycin and exercise equally affect the autophagic system and counteract cachexia.4 We believe autophagy-triggering drugs may be exploited to treat cachexia, especially in conditions in which exercise cannot be prescribed, since cancer patients show abnormal expression of autophagy markers, suggesting that the autophagic flux is blocked in cachexia, thus contributing to muscle wasting. Part 2. Muscle dystrophy. Since flaxseed is one of the richest sources of the n-3 PUFA acid α-linolenic acid (ALA), we assessed the effects of flaxseed and ALA in models of skeletal muscle degeneration characterized by high levels of Tumor Necrosis Factor-α (TNF) and exhaustion of SC myogenic potential. Our study was carried out on dystrophic hamsters and differentiating C2C12 myoblasts treated with TNF, both in the absence or presence of flaxseed diet or ALA treatment, respectively.5 The flaxseed-enriched diet protected the dystrophic muscle from apoptosis and preserved muscle myogenesis both in vivo and in vitro, indicating that flaxseed may exert potent beneficial effects by preserving skeletal muscle regeneration and homeostasis partly through an ALA-mediated action. In conclusion, physical activity, pharmacological treatment (exercise mimetics such as AICAR) and nutritional supplementation (such as ALA) are beneficial for muscle mass preservation and life span increase in the presence of cancer cachexia or muscle dystrophy and should be considered when planning multimodal therapies for muscle diseases., Cachexia is a multifactorial syndrome characterized by body weight loss, muscle wasting, and metabolic abnormalities, that occurs in 50 to 80% of cancer patients and is considered as a predictor of reduced survival accounting for more than 20% of cancer-related deaths.1 Cachexia was defined also as an energy-wasting syndrome, in which mitochondria play a central role as the main energy source. Indeed, mitochondrial alterations and an upregulation of mitophagy markers have been found in the skeletal muscle of cachectic animals.2 In addition to the effects exerted by the tumor, also anti-cancer treatment may contribute to muscle wasting.3 Some years ago, exercise has been proposed as a therapeutic tool to counteract cachexia and the related metabolic alterations,4 including autophagy dysregulation, mitochondrial dysfunction and oxidative capacity reduction.2,5 The present study aimed at evaluating the effects of moderate exercise training on muscle wasting in C26-bearing mice treated with chemotherapy (oxaliplatin+5-fluorouracil; OXFU), focusing on both alterations of muscle autophagy/mitophagy and mitochondrial function. OXFU administration was able to extend the lifespan of the C26-bearing mice (100% survival at 28 days after tumor implantation), but also resulted in exacerbated cachexia. In C26 OXFU mice, exercise partially protected from muscle mass loss and associated with an improvement of muscle function. Chemotherapy further dysregulated cancer-induced autophagy, increasing the levels of Beclin-1 and LC3I. Exercised C26 OXFU mice showed a lower content of Beclin-1 and of both LC3B isoforms compared to sedentary mice. Focusing on mitochondria, the levels of cytochrome c, used as a measure of mitochondrial content, decreased in sedentary C26 OXFU mice, associated with a reduction of SDH protein levels and enzymatic activity. Sedentary C26 OXFU mice showed also increased levels of Bnip-3 and PINK-1, two proteins involved in mitophagy. In C26 OXFU mice, exercise increased the levels of cytochrome c, PGC1α and both SDH content and activity, decreasing also the levels of PINK-1. The alterations seen in C26 OXFU animals were associated with a strong reduction in protein synthesis, that was not improved by exercise. In conclusion, chemotherapy exacerbated tumor-associated muscle wasting and metabolic alterations. Moderate exercise training was able to partially counteract muscle loss and recover muscle function, increasing mitochondrial content, autophagy and damaged-mitochondria clearance, and rescuing muscle oxidative capacity. Therefore, exercise exerts beneficial effects potentially exploitable in the management of cancer patients receiving chemotherapy., Neutral Lipid Storage Disease with Myopathy (NLSDM) is a very rare disorder characterized by a defect in the degradation of cytoplasmic neutral lipids and their accumulation in the lipid droplets (LDs). This neutral lipid metabolism deficiency is associated with mutations of PNPLA2 gene, which encodes adipose triglyceride lipase (ATGL).1-2 ATGL leads to the breakdown of triacylglycerols (TAGs), releasing free fatty acids. NLSDM patients may develop progressive myopathy (100%), cardiomyopathy (44%), diabetes (24%), hepatomegaly (20%), chronic pancreatitis (14%) and short stature (15%). No specific therapy is available today.3-4 Fibroblasts cell lines from two patients and one healthy subject have been reprogrammed into induced pluripotent stem cells (iPSCs). iPSCs are a new technology which can provide an unlimited number of human disease-affected stem cells from different somatic cell lines.5 The first NLSDM patient was homozygous for the c.541_542delAC PNPLA2 mutation that causes the production of a truncated protein lacking the LD-binding domain.3 The second patient was homozygous for the c.662G>C PNPLA2 mutation, determining the p.R221P amino-acid change; this mutation leads to the production of ATGL protein with decreased lipase activity, but able to bind to LDs.2 After about 4 weeks from the Senday infection, karyogram showed a normal karyotype of controls and NLSDM-iPSCs; moreover genomic sequencing analysis confirmed that NLSDM-iPSC lines still contained the disease-specific mutations of PNPLA2 gene. We tested the pluripotency properties of NLSDM-iPSCs evaluating the expression of TRA-1-81, SSEA4 and OCT4 by immunostaining and of SOX2, NANOG, ZFP42, OCT4, hTERT, LIN28, DPPA2 and TDGF1 by qRT-PCR analysis. NLSDM-iPSCs were also able to differentiate into three-germ layers, as revealed by β-III tubulin (ectoderm), α-smooth muscle actin (mesoderm), and FOXA2 (endoderm) expression. Finally, we demonstrated that NLSDM-iPSCs showed an higher storage of TAGs in comparison with control iPSCs, exactly as it could be observed in NLSDM original fibroblasts when compared with control fibroblasts. Indeed, after 3 days in culture, cells were stained with Nile Red and the LD number and dimension were analysed by immunofluorescence analysis; compared to control cells, the NLSDM-iPSCs had 20 times more LDs and almost 5 larger LDs, similar to fibroblasts obtained from the patients. Moreover, oleic acid pulse-chase experiments were performed to confirm that lipase activity was impaired in NLSDM-iPSCs compared to control cells. Collectively, data from this study consistently show that NLSDM-iPSCs recapitulate the disease phenotype of interest. The perspective to differentiate iPSCs into striatum/cardiac muscle lineages will allow us to define a disease model to investigate the pathogenetic mechanisms and to evaluate specific approaches for new pharmacological treatments., How does one choose a pattern of electrical stimulation for therapeutic effect? Often there is a useful guide from normal physiology, and many therapeutic strategies try to mimic or replace a natural activation pattern. Another strategy is to try to generate a numerical model of the excitable tissue to be stimulated so that trials can be achieved in silico.1 Many optimised activation strategies are based on such simulations. We have tested some of the conclusions of studies that have investigated the charge efficiency of activation.2-7 We have used the simple experimental model of a single motor nerve trunk activated by two electrodes placed near to the nerve (common peroneal in rats). The degree of activation has been monitored indirectly by measuring the isometric force of the edl muscle because it has discrete proximal and distal tendons and can thus be mechanically isolated between a proximal clamp and a distal load sensor. We are in a process of critically analysing this data because some of our initial results appeared surprising. We will present results that compare the actual electrode current against the anticipated current based on the use of a voltage-to-current converter. We will also present further analysis of the linearization method that we used to select optimal parameters for the various pulse shapes that we tested. We find that the opportunities to improve energy efficiency are more relevant to monopolar stimulation with one remote electrode far from the nerve than to bipolar stimulation, in which the current field is created between two electrodes both near to the nerve. Such fine differences are important when designing low energy implanted stimulators such as may be used in retinal stimulation or brain stimulation or activation of fine autonomic nerves., In the development of implantable prosthetic devices, much effort has been put into finding optimal anatomical targets for different nerve stimulation techniques. Little work however, has been done to improve the efficiency of nerve stimulation by using analytically driven designs and configurations of the stimulating electrodes. Namely, an electrode geometry can affect the effective impedance, spatial distribution of the electric field in tissue, and consequently the pattern of neural excitation. One approach to enhance the efficiency of neural stimulation is to increase the irregularity of the surface current profile. In this relation, it has been shown, that adequately optimized electrode geometries and surfaces that increase the variation of current density on the electrode surface enable also an increase of the efficiency of neural stimulation. In this relation, a variety of mechanical adaptations, such as geometry and surface roughness of the electrodes, have been investigated and implemented. The purpose of the study was therefore to assess "in vitro" the electrochemical performance of two stimulating electrodes (WEs) with different surface structures obtained by treating the surface with smooth and rough sand paper. To craft the stimulating electrodes, 0.03-mm-thick cold-rolled platinum foil strips with 99.99 wt.% purity and dynamic annealing in an argon atmosphere were used. The obtained final dimensions of the electrodes exposed to the physiological solution were: width 0.66 mm, length 3 mm and surface area 2 mm2. For adaptations of two investigated WEs via increase their real surface, two differently grained sand papers (Waterproof Silica Carbide Paper FEPA P#500 and FEPA 4000, Struers ApS, Pederstrupvej 84, 2750 Ballerup, Denmark) were used. A surface of the WE1 was enlarged using rough sand paper FEPA P#500 while WE2 was enlarged using fine-grained sand paper FEPA P#4000. For the purpose of spot welding of the stainless-steel wire and the platinum foil, a custom-designed, capacitive-discharge, research-spot-welding device, providing a standard single pulse, was developed. The welding energy for both electrodes is defined experimentally. To analyse any failure and to reveal the microstructure of the weld, and consequently to set up optimum welding conditions, scanning electron microscopy was used. The results provide evidence that the welds between the stainless-steel wire and the platinum foil do not show any typical welding defects, such as oxide films, oxide inclusions, gas bubbles or shrinkage porosity. Obtained results also show that an impedance of WE1 is lower than impedance of WE2. Accordingly, the WE1 is more suitable for safe stimulation than WE2., Transcutaneous spinal cord stimulation (tSCS) has been shown to abbreviate spasticity in lower limbs in people with incomplete spinal cord injury (SCI) people.1,2 Therefore tSCS is a therapy of choice for SCI in our clinic. It is also known that SCI modulates the organisation of the brain in the way that it decreases the areas allocated for the control of the not connected extremity part.3 Therefore we hypothesize that the tSCS treatment can influence the plasticity of the brain as well. In this work the footprint of the tSCS in the EEG is sought in order to verify that the stimulating signals are transmitted to the brain. In this first approach one healthy subject for control and one Cerebral palsy (CP) patient participated. Cortical somatosensory evoked potentials (SEP) where recorded during tibial nerve stimulation and during tSCS. The recording of SEP during tibial nerve is well documented so it serves as a proof of method. Then SEP was also recorded during voluntary ankle dorsiflexion and analyzed for event-related (de-)synchronization (ERD/ERS).4 SEP is clearly to be seen in the sensorimotor cortex during tSCS. It is though different in form from the SEP during tibial nerve stimulation. As expected the ERD/ERS were focused over the Cz electrode as documented in the literature.4 After movement by the CP subject the synchronisation was limited and therefore different to a healthy subject. But no significant changes where found after treatment. As the tSCS modifies the SEP the hypothesis that the treatment could influence the brains plasticity is supported. The difference in SEP between tSCS and tibial nerve stimulation suggests that different fibres in the spinal cord are stimulated. ERD/ERS patterns are changed in CP compared to a healthy subject., Aging of the human skeletal muscles results from decline of both muscles strength and power.1 The athletic world records of the Master athletes at ages ranging between 35 to 100 years are an excellent proof of such decline in all competitions. The world record performances can be transformed into dimensionless parameters proportional to the power developed in the trials. Such parameters range from 1 for the Senior world record (i.e. the maximum human performance) through medium values for the Master athletes to reach 0 for a null performance.1 Therefore, the decline of the power parameter with relation to human aging can be analysed and compared as follow: the trend-lines start to decline very close to the age of 30 years and arrive to 0 around the age of 110 years for each athletic discipline. There are no reasons, for each one of us, to decline differently from the world record-men, provided that each of us remains in a stable fitness condition without disabling pathologies. On the other hand, the methods to evaluate decline in the older olds need to be adapted to the extent of decay (as it is very commonly done in pathology). This is particularly important after 70 years of age and according to sex difference in power. We have adapted clinical methods,2,3 to evaluate dexterity and mobility in normal older olds introducing 5 simplified Tests. Patients are assessed with the Timed Up and Go Test (TUGT), Five Chair Rise Test (5xCRT), and Jug Test (JT).3,4 The Timed Up and Go Test has been validated as a useful indicator of leg muscle performance in numerous populations, including patients with neuromuscular diseases. Additionally, maximal isometric torque of quadriceps muscle on a force measurement chair is determined as [Nm/s] and the time which a subject needs to rise from a chair with arms folded across the chest 5 times (i.e., Five Chair Rise Test, 5xCRT) is measured.3 The “jug test” (floor-to-table jug test, JT) provides information on the behaviour of arm, shoulder and trunk muscles. Specifically, participants move five 1-gallon jugs (≈3.9 kg) from the floor to a normal 75 cm high table level - as quickly as possible.4 This action is quite like the everyday activity of lifting a shopping bag from ground to table. The weight of the jug varies according to age and gender of subjects as indicated in the following template of Functional Test Report. Further, every day mobility is assessed by providing a pedometer (Nakosite, USA). The participants hold it 24 hours a day, for two weeks with break periods of three months. All functional results are correlated to 3D false color computed tomography of skeletal muscles.5, MicroRNAs (miRNAs) are small non-coding RNAs that have been shown to modulate a wide range of biological functions under various pathophysiological conditions. miRNAs are 17-27 nucleotides long molecules that regulate post-transcriptional mRNA expression, typically by binding to the 3’-untranslated region of the complementary mRNA sequence, and resulting in translational repression and gene silencing. Therefore, an increase in a specific miRNA results in a decreased expression of the corresponding protein product. Several studies have shown that there are thousands of different human miRNA sequences that control the expression of 20-30% of protein-coding genes, indicating that miRNAs are “master regulators” of many important biological processes. MiRNAs are known to be secreted by various cell types and, unlike most mRNAs, they are markedly stable in circulating body fluids due to proteic protection from ribonucleases. Because of these properties, miRNAs have recently gained attention for their potential as minimally invasive and cost-effective disease biomarkers. Because of their stability in plasma and serum, they can be reliably detected even at low concentration and used not only as markers of disease, but also of disease staging, and possibly to quantitatively measure the effectiveness of novel drug therapies. These miRNAs (miR-206, miR-133a, miR-133b, miR-1) are called “myo-miRNA” and are considered as markers of muscle regeneration, myogenesis, fiber type differentiation, degeneration, injury and might represent indicators of residual muscle mass consequent to a chronic atrophy of muscle. Myo-miRNAs are variably expressed in several muscle processes, including myogenesis, and muscle regeneration.1-3 We explored their function beside in several conditions with severe muscular atrophy, including Amyotrophic Lateral Sclerosis (ALS). ALS is a rare, progressive, neurodegenerative disorder caused by degeneration of upper and lower motoneurons. The effects of exercise and rehabilitation in patients with ALS are still debated. A moderate and regular exercise is supported in the treatment of many neuromuscular diseases. We previously conducted microRNAs studies in ALS patients and we observed differences in myomiRNAs levels in spinal versus bulbar onset (4). In this study we analysed the role of circulating myomiRNAs after physical rehabilitation. We measured muscle specific microRNAs (miR-1,miR-206,miR-133a,miR-133b) by Real Time PCR in 19 ALS patients (12 male,7 female). We analysed the levels of these microRNAs in serum collected before (T0) and after (T1) a period of 6-8 weeks of rehabilitation. We observed a general down-regulation of all miRNAs studied after rehabilitation. In our population myomiRNAs decreased in a similar manner in male and female patients, therefore no gender effect was found. On the contrary the age of patients under study was found to be relevant: patients under 55 years old have a more marked decrease in myomiRNAs levels than patients with older age. We have found that microRNAs are an important tool to monitor rehabilitation in ALS patients and suggests a positive effect of the treatment. There seems to be a more pronounced decrease in myomiRNA levels in patients with younger age in this motoneuron disease after physical rehabilitation. Further studies are needed to correlate circulating microRNAs with muscle atrophy and to confirm age differences., Within a study which eventually demonstrated the efficacy of peri-patellar injections of high molecular weight Hyaluronic Acid (HA) in the maintenance of the tendon structure during detraining in the rats1,2, a transcriptomic study using Next Generation Sequencing was carried out in rat hearts in order to evaluate training-and detraining-associated adaptations in gene expression. While the comparison between trained and untrained hearts yielded 593 differentially expressed (p≤0.05) genes, as many as 762 genes were found to be differentially expressed in the comparison between the hearts of detrained rats receiving either HA or saline peri-patellar injections. Differentially expressed genes were assigned to functional categories and to KEGG pathways by using the FatiGO software. By and large, gene expression analysis suggested that HA injections at a distant site appear to support the ability of the heart to repair injuries and to enforce differentiative pathways. HA has a well-known role in cardiac differentiation, by activating the ERK 1/2 and pathways3 and modulating the WNT/β-catenin and Smad signaling.4,5 The experimental use of HA in in vivo recovery from ischemia/reperfusion injuries has been so far limited to animal studies, owing to the concept that, in order to be effective, HA-containing hydrogels should be applied on the site of injury, a very delicate and potentially harmful procedure. Should the present transcriptomic study be validated by ongoing proteomic studies, these serendipitous results may pave the way for the validation of HA administration at distant sites and even orally, in the therapy of infarcted patients and even in the prevention of cardiovascular diseases in subjects at risk and in the elderly. This work has been partly supported by a grant awarded by FIDIA, Friedreich’s Ataxia (FRDA, OMIM #229300) is a severe neurodegenerative disease due to an autosomal recessive mutation and characterized by progressive impairment of voluntary movements. In most patients, FRDA is associated with hypertrophic dilated cardiomyopathy, which is the more frequent cause of death. The underlying mutation in FRDA causes a marked reduction of a small protein, frataxin, which is involved in iron handling, mostly, but not exclusively, in mitochondria; its main role is the assistance in the formation of iron-sulphur containing protein complexes. Patients affected by FRDA show iron inclusions in cardiomyocytes and iron aggregates in the cardiac tissue1. We therefore devised to study the iron homeostasis in iPSC-derived cardiomyocytes obtained from a patient affected by FRDA, which were compared to iPSC-derived cardiomyocytes obtained from a healthy subject. Induced Pluripotent Stem Cells were obtained from skin fibroblasts according to the Yamanaka procedure, differentiated following the GiWi protocol2, and thoroughly characterized. The gene expression of Hepcidin, Ferroportin, Transferrin Receptor 1 and Ferritin was studied in basal conditions; their change following an iron load is the object of a study presently being carried out in our lab. Messenger RNA levels for Hepcidin were found to be increased in cardiomyocytes from the FRDA patient, while the amounts of Ferroportin and Transferrin Receptor 1 mRNAs were decreased with respect to cardiomyocytes from a control subject. These data will be discussed in the light of the role played by the proteins coded by the above mentioned genes in iron homeostasis and of their expression in different experimental models. This work has been partly supported by AISA ONLUS (Associazione Italiana per le Sinromi Atassiche), All progressive muscle contractile impairments need permanent managements, including aging-related muscle-strength decline . Frail elderly persons due to advanced age or associated diseases are often hospitalized for long periods of time. There, their already modest amount of daily physical activity is reduced, contributing to limit their independence up to force them to the bed. Immobility is associated with neuromuscular weakness, functional limitations, thromboembolism and high costs.1-3 Beside the eventual pharmacology therapy, a home-based physical exercise approach is helpful. Awaiting development of electroceuticals, as effective as pace-makers or cochlear implants, education of hospitalized patients to take-home physical exercise managements is an effective low cost alternative. Inspired by the proven capability to recover skeletal muscle strength by home-based Functional Electrical Stimulation even in the worse cases of neuromuscular traumatic injuries,3-4 but, guided by common sense, we suggest a brief (15-20 minutes) daily routine of twelve easy-to-be-done physical exercises that are performed in bed (Full-body In-Bed Gym).5 Full-body Inbed Gym is an extension to all body muscles of well-established physiotherapy approaches of in-bed cardio-circulation-ventilation workouts. If sedentary borderline persons challenge, without stress, them-self, in hospital Full-body In-Bed Gym may increase muscle strength, fatigue resistance and independence in daily life activities. In surgical units this will grant standing of patients soon after operation, a mandatory measure to prevent risk of thromboembolism. Full-body In-Bed Gym helps also to mitigate the bad mood that accompanies mobility limitations, strengthening patients’ confidence in recovering partial or total independence. Full-body In-Bed Gym may also mitigate eventual arterial hypertension, a major risk factor in elderly persons. Continued regularly, Full-body In-Bed Gym may help to maintain the independence of frail older people and to reduce the risks of the possible serious consequences of accidental falls. Simplified Functional Tests may be used to follow-up the suggested approaches. Take home messages: It is never too early, it is never too late to start anti-aging Full-body In-Bed Gym and FES to help older olds and change lazy, depressed person into active seniors. There are no needs of personal trainers or demanding devices. Secure to your self, please, a better life-style watching the video of Full-body In-Bed Gym.5 http://www.bio.unipd.it/bam/video/InterviewCarraro-tutorial.mp4, MicroRNAs are small non coding RNAs that are associated to stress granules, mitochondria and other subcellular organelles in muscle. Few studies have explored microRNAs role in muscle atrophy in Amyotrophic lateral sclerosis(ALS). We previously observed that there is different serum microRNA profile in spinal versus bulbar ALS. We have investigated muscle biopsies in a series of ALS cases both sporadic and genetic. We studied, in EI Escorial proven ALS cases muscle biopsies obtained for diagnostic reasons, myomicroRNAs (MiR-1;MiR-206; MiR-133a; MiR-133b; MiR-27a) and inflammatory microRNAs (MiR-155; MiR-146a; MiR-221; MiR-149*) by qRT-PCR.ALS cases were divided according to gender and age of onset. Atrophy factors were calculated in muscle fibers according to Dubowitz. Two cases had mutation of SOD and c9orf. Morphometric analysis of muscle fiber size was done to correlate muscle atrophy with molecular parameters. All microRNAs studied were strongly up-regulated in muscle biopsies of ALS patients versus controls with the exception of miR-149*. Significant overexpression of miRNAs was present in genetic versus sporadic and in male versus female gender. Morphometric analysis confirmed a muscle fibre atrophy in ALS patients compared to controls. Two genetic ALS (SOD, C9ORF) were atrophic with high fiber CSA variability in agreement with the up-regulation we found of myomiRNAs that directly correlates with the degree of atrophy. In conclusion, these results provide evidence on molecular role of microRNAs in correlation to muscle atrophy. In addiction we observed an increased expression of microRNAs in genetic ALS and dysregulation of inflammatory microRNA., We report muscle histopathological, ultrastructural and radiological features of a large Italian-Spanish family with autosomal dominant LGMD, previously mapped to 7q32.2-32.2 (LGMD1F). We collected the DNA, clinical history, muscle biopsies histopathology of one LGMD1F kindship. Biopsy of two affected patients mother and daughter was studied (in the daughter two consecutive biopsies at 9 and 28 years and in the mother at 48 years). In LGMD1F patients the age of onset varied from 2 to 35 years, weakness occurred either in upper or in lower girdle; in 14 cases there was hypotropy both in proximal upper and lower extremities in calf muscles. Muscles MRI showed hyperintensity in proximal limb muscles. The daughter has a severe clinical course and the fiber atrophy was more prominent in the second biopsy at 28 years. The mother has a relatively compromised histopathology and many small muscle fibers, and autophagic changes by acid-phosphates stain. Immunofluorescence against desmin, myotilin, p62 and LC3 showed accumulation of myofibrils, ubiquitin binding proteins aggregates and autophagosomes. Ultrastructural analysis revealed myofibrillar disarray, vacuolar changes, granular material and dense subsarcolemmal bodies deriving from cytoskeleton-myofibrillar proteins. We hypotize that the pathogenetic mechanism in LGMD1F might lead to disarrangement of desmin-associated cytoskeletal network. Transportin-3 (TPNO3), which was found by NGS to be the causative gene in LGMD1F, is suggested to mediate the nuclear inport-export. The non-stop mutation identified in this family encodes for a longer protein which is expected to be unable to move to the nucleus. Clinical phenotype penetrance in this family correlates at 92% with mutation presence. MRI imaging is a powerful tool for the follow up in the evolution of this dominant LGMD and demonstrated atrophy of lower girdle., Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial biogenesis. In skeletal muscle, PGC-1α expression is induced by exercise.1 Along this line, mice overexpressing PGC-1α specifically in the skeletal muscle are characterized by enhanced exercise performance in comparison with wild-type animals; this is mainly due to increased myofiber mitochondrial content that results in markedly improved energy metabolism. In addition to an increased proportion of oxidative fibers vs glycolytic ones,2 the histological analysis of muscle overexpressing PGC-1α revealed a high number of fibers with centrally located nuclei, which is indicative of muscle regeneration. Starting from this unexpected observation, the aim of the study was to investigate the effects on myogenesis exerted by PGC-1α overexpression. Myogenic stem cells are more abundant in transgenic mice compared to wild-type animals. When cultured in differentiating medium, cells isolated from PGC-1α mice form myotubes larger than those generated by cells derived from wild-type animals. To understand if such improved in vitro myogenic capacity also occurs in vivo, both wild-type and PGC-1α transgenic mice received an intramuscular injection of BaCl2 in order to induce muscle regeneration. While 14 days after muscle injury myofiber cross sectional area was not different in wild-type and transgenic mice, at day 8 from BaCl2injection the number of central nuclei was higher in the latter than in the former. On the whole, these results suggest that overexpression of PGC-1α might favor both myogenic differentiation and regeneration when mild damage occurs, such as during exercise, but it is not able to accelerate muscle recovery when acute damage is inflicted, despite the high propension to myogenesis shown in vitro., Skeletal muscle atrophy is the loss of muscle size and strength which occurs with neural and skeletal muscle injuries, prolonged bed rest, space flight, normal aging, and diseases such as sepsis cachexia, diabetes, etc. If unabated, skeletal muscle atrophy can be extremely debilitating, increasing mortality and morbidity in affected people. Current strategies for diagnosis and evaluation of skeletal muscle are not adequate to evaluate fully the condition of this tissue. Thus, proper diagnosis and treatment are often delayed, resulting in unnecessary human discomfort and down time. Quantitative Muscle Color Computed Tomography (QMC-CT) is a highly sensitive quantitative imaging analysis recently introduced by our group to monitor skeletal muscle condition. Despite its powerful potential, this technique is not widely known. Therefore, the objective of this project is to validate QMC-CT as a superior Muscle Imaging technique for evaluating skeletal muscle. This project addresses the “Barriers to Successful Therapy Outcomes” option within the Rehabilitation Focus Area of the DOD Peer Reviewed Orthopaedic Research Program because it will explore the sensitivity of QMC-CT and thus validate its use as an improved method for monitoring skeletal muscle health and recovery. Validation of QMC-CT will provide physicians an improved tool to quantitate skeletal muscle before and during rehabilitation so that therapy for mobility-impaired persons can be better prescribed, evaluated and altered where needed. Benefit to Military Service Members and Veterans: A recent report from the U.S. Army describes injuries as an “epidemic” which has become the “number one health threat” to the U.S. military.1 This document reports that the majority of injuries occurring at Army garrisons were musculoskeletal injuries to the ankle, knee, lower back or shoulders. Further, it has been reported that non-combat injuries have resulted in more medical air evacuations from Iraq and Afghanistan than combat injuries.2 These injuries result in physical discomfort and potential mental duress in addition to some degree of personnel down time. The more serious injuries can result in life long issues. QMC-CT will provide medical personnel with a superior technique for imaging skeletal muscle and surrounding tissues. In the short term, the use of QMC-CT will enhance the speed and accuracy of patient evaluation, thus improving diagnosis, treatment and patient morale. In the long term, the improved initial treatments will reduce patient treatment time, personnel down time and enduring negative injury-related issues. Because the technology has the potential to improve medical treatment in both military and non-military facilities, the method has the potential to improve health care for soldiers, veterans and the population at large., The most severe forms of muscular dystrophies (MD) occur due to mutations in the components of the dystrophin-glycoprotein complex (DGC), a molecular scaffold which is localized to sarcolemma and provides mechanical stability to striated muscle. Studies have shown that loss of DGC proteins results in the activation of several pathological cascades1. Dystrophic muscle is characterized by chronic inflammation, fibrosis and progressive myofiber loss. No effective treatment is currently able to counteract MD pathological cascades. Plant-derived nutritional compounds exhibit ability to modulate several pathological pathways in various degenerative diseases2. Our studies have been demonstrated that a Plant-based diet enriched of flaxseed (FS-diet), is able to stimulate multiple protective and regenerative mechanisms on skeletal muscles of dystrophic hamster, affected by a deletion in the δ-sarcoglycan gene. The FS-diet modulates lipid membrane composition preserving expression of key-role signaling proteins, such as caveolin-3, α-dystroglycan, and sarcoglycans, therefore repairing the sarcolemma damage, which is the primary consequence of gene mutation. The FS-diet prevents inflammation, fibrosis and skeletal muscle degeneration in dystrophic hamster, extending the animals’ lifespan3. The mechanisms involved include modulation of various pathways such as the TNF, PI3K/Akt, TGF-β, and Bax/Bcl-2 signaling pathways. Because flaxseed is one of the richest sources of omega-3 fatty acid, a-linolenic acid (ALA) a further step of “in vitro” experiments were performed on ALA-treated differentiating myoblasts3,4. ALA prevents the TNF-induced inhibition of myogenesis and reduces apoptosis in C2C12 cells by regulating key proteins involved in balancing survival/death in skeletal muscle such as caveolin-3, caspase-3 and Bcl-2. These findings indicate that flaxseed may exert pleiotropic beneficial effects on the dystrophic skeletal muscle partly through an ALA-mediated action. As a nutraceutical that exerts multifaceted effects, the omega-3 fatty acid ALA, as well as others compounds contained in flaxseed, should be clinically developed further for use in the prevention and treatment of the muscular dystrophies., Understanding the underlying mechanisms involved in maintenance, increase and loss of muscle mass remains an interesting and challenging field with potential applications not only in bodybuilding, but also with respect to counteract the decline in muscular function caused by disease or ageing. Muscular activity and loading are essential parameters controlling the equilibrium between protein synthesis and degradation. Various animal models have been used in the past to investigate hypertrophy in rodents by increasing the average loading of particular muscles.1-9 Some models demonstrated the effect of compensatory hypertrophy by removal or denervation of antagonists producing a constant overload.1-3 Others established training modalities including squats,3 weight lifting,4,5 jumping for a food reward, and treadmill or ladder climbing,6 sometimes with added weights,7 to increase the muscular effort. Our recent study tests the effect of programmed resistance training of the tibialis anterior (TA) muscle by means of electrical stimulation, on muscular hypertrophy. In the rat hind limb, the dorsiflexor muscles that lift the foot are supplied by the common peroneal nerve (CPN) whereas the plantarflexor muscles are supplied by the tibial nerve. In preliminary force measurements we investigated the loading experienced by the TA muscle for unloaded concentric contractions and isometric contractions for the fully recruited CPN. Further measurements were performed in which part of the antagonistic plantarflexors was simultaneously activated with the fully recruited TA muscle. This was achieved with a single channel pulse generator by placing the cathode under the CPN and the anode under the tibial nerve, further referred to as “SpillOver” stimulation of the plantarflexors, because the amount of activation of the tibial nerve can be controlled by adjusting stimulus amplitude above the level that produces supramaximal activation of the CPN. The results of these force measurements suggest that unloaded contractions, even with full activation of the CPN might not provide a sufficient stimulus to induce muscular hypertrophy. To test this hypothesis we performed experimental trials on 10 animals comparing the hypertrophic response of unloaded concentrations elicited by stimulation of the CPN (n=5) versus antagonistic co-contraction using the proposed SpillOver stimulation (n=5). A stimulation pattern of one session per day consisting of 5 sets of 10 repetitions at 100Hz (2s ON 2s OFF) and 2.5 minutes between sets, was applied for a duration of 4 weeks by small implantable pulse generators (MiniVStim 12B, Center of Medical Physics and Biomedical Engineering, Medical University of Vienna, Austria). After the experiments the TA muscles were harvested, weighed and snap-frozen for further histometric analysis. The wet weight of the TA muscle showed an increase of +5.4 % ± 2.5 % (MEAN ± SEM) for unloaded contractions while antagonistic co-contraction revealed an increase +13.9 % ± 1.3 %. The average differences of the median fibre cross-sectional-area were +12.8 % ± 6.4 % and +33.3 % ± 16.5 % for unloaded contractions and co-contractions, respectively. We will use this model to investigate further the sensitivity to hypertrophy of the various fibre types and the cellular pathways that are activated in this response.

Physical exercise is known to have beneficial effects on muscle trophism and force production modulating signaling pathways involved in fiber type plasticity, muscle growth and mitochondria respiratory efficiency.1 It has been shown that the decrease of muscle mass and strength observed in aging is linked to intracellular and extracellular abnormalities, that is, sarcoplasmic reticulum-to-mitochondria malfunctions and extracellular matrix metabolism, respectively. Lifelong, high-level physical activity delays the medium and long term effects of aging. Furthermore, when healthy seniors are exposed to regular neuromuscular Functional Electrical Stimulation (FES) training for a period of 9 weeks outcomes are an increase in muscle strength and muscle fiber size and, most importantly, an increase of fast fibers, the more powerful of skeletal muscle motor units. Electron microscopy analysis of aging muscle show remodeling of mitochondrial apparatus as a consequence of fusion phenomena that are consistent with adaptation to physical exercise. Altogether these results indicate that the FES-dependent beneficial effects on muscle force and mass are associated with changes in mitochondrial- and sarcoplasmic reticulum-related proteins involved in Ca2+ homeostasis, providing new targets to develop therapeutic strategies to promote healthy aging., All permanent or progressive muscle contractility impairments (including age-related muscle power decline) need permanent managements. Beside eventual pharmacology therapy, a home-based physical exercise approach is helpful, in particular for bed-rested or bed-ridden patients. Awaiting development of implantable devices for muscle stimulation, i.e., of electroceuticals as effective as pace-makers for cardiac arrhythmias or cochlear implants for hearing loss, education of hospitalized patients to take-home physical exercise managements is an effective low cost alternative. Frail elderly due to advanced age or associated diseases are often hospitalized for long periods of time. There, their already modest amount of daily physical activity is reduced, contributing to limit their independence up to force them to the bed. Inspired by the proven capability to recover skeletal muscle contractility and strength by home-based Functional Electrical Stimulation (h-bFES) even in the worse cases of neuromuscular traumatic injuries,3-5 but, mainly guided by common sense, we suggest a short (15-20 minutes) daily sequence of fifteen easy volitional physical exercises that are performed in bed (In-Bed Gym). If sedentary borderline persons challenge, but not stress, them-self, in a few days in hospital In-Bed Gym may increase muscle strength, fatigue resistance and independence in daily life activities. In surgical units this will grant standing of patients soon after operation, a mandatory measure to prevent thromboembolism risk. In-Bed Gym helps also to mitigate the bad mood that accompanies mobility limitations, strengthening patients’ confidence in recovering partial or total independence Continued regularly, In-Bed Gym may help to maintain the independence of frail older people and to reduce the risk of the possible serious consequences of accidental falls. In-Bed Gym may also mitigate eventual arterial hypertension, a major risk factor in elderly people.7, Disruption of circadian rhythms in humans and rodents has implicated a fundamental role for circadian rhythms in aging and the development of many chronic diseases including diabetes, cardiovascular disease, depression and cancer. The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop with Bmal1 encoding a core molecular clock transcription factor. Germline Bmal1 knockout (Bmal1 KO) mice have a shortened lifespan, show features of advanced aging and exhibit significant weakness with decreased maximum specific tension at the whole muscle and single fiber levels.1 We tested the role of the molecular clock in adult skeletal muscle by generating mice that allow for the inducible skeletal muscle-specific deletion of Bmal1 (iMSBmal1),2 Here we show that disruption of the molecular clock, specifically in adult skeletal muscle is associated with a muscle phenotype including reductions in specific tension, increased oxidative fiber type, and increased muscle fibrosis similar to that seen in the Bmal1 KO mouse. Remarkably, the phenotype observed in the iMSBmal1-/- mice was not limited to changes in muscle. Similar to the germline Bmal1 KO mice, we observed significant bone and cartilage changes throughout the body suggesting a role for the skeletal muscle molecular clock in both the skeletal muscle niche and the systemic milieu.3 This emerging area of circadian rhythms and the molecular clock in skeletal muscle holds potential to provide significant insight into intrinsic mechanisms of the maintenance of muscle quality and function as well as identifies a novel crosstalk between skeletal muscle, cartilage and bone., Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. We compared the circadian transcriptomes of two mouse hind-limb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific *Bmal1* KO). In addition, we compared the effect of denervation on muscle circadian gene expression. We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. Our studies suggest that a major physiological role of the skeletal muscle clock is to prepare the muscle for the transition from the light/inactive/fasting phase to the dark/active/feeding phase, in anticipation of periodic fluctuations in fuel supply and demand., Muscle wasting in intensive care unit (ICU) patients may be related to the primary disease, but there is heterogeneity of underlying disease and pharmacological treatment among patients exhibiting similar outcomes. Thus, it is highly likely that a common component of ICU treatment per se is directly involved in the progressive impairment of muscle function and muscle wasting during long-term ICU treatment. The specific mechanisms underlying the muscle wasting and impaired muscle function associated with the ICU intervention are poorly understood in the clinical setting.1 This is in part due to heterogeneity in pharmacological treatment, underlying disease, clinical history etc. There is, accordingly, compelling need for experimental animal models closely mimicking the ICU condition, including long-term exposure to mechanical ventilation and immobilization (lack of weight bearing and activation of contractile proteins, i.e., “mechanical silencing”). In this project, the muscle dysfunction, which by far exceeds the loss in muscle mass in limb and respiratory muscles in patients with CIM2,3 and VIDD,4 have been investigated in detail at the cellular and molecular levels in a rodent experimental ICU model, allowing detailed studies in an immobilized and a mechanically ventilated rat for long durations (up to weeks-months). The long-term scientific goals of the research are to: (a) define the causative agents, (b) develop sensitive, quick and accurate diagnostic tools and monitoring devices, and (c) develop efficient intervention strategies. This project, which focuses on the mechanisms underlying the severely impaired limb and respiratory muscle function (CIM and VIDD) in response to long-term mechanical ventilation and immobilization and the introduction of specific intervention strategies, constitutes a significant component of the attempt to achieve these long-term goals., The diaphragm is the most important inspiratory muscle. It has a thin, dome-shaped structure, and separates the thoracic and abdominal cavities, mechanically interacting with the surrounding organs during its contractile function. Moreover, its muscle fibers have a complicated geometry, connecting to several ribs, lumbar vertebral bodies and to the central tendon. All these peculiar features make it a very challenging organ to be modeled and analyzed by means of finite element methods. We present our work aimed at creating a finite element model of the human diaphragm. A computational model based on a realistic, potentially patient specific, geometry, may help in the understanding of diaphragm related pathologies, such as chronic obstructive pulmonary disease (COPD), mechanical ventilation (MV) induced diaphragm inactivity, amyotrophic lateral sclerosis (ALS) or even for respiratory tumor motion tracking to reduce radiation treatment side effects. On the other side, several diaphragm pathologies are related to relevant modifications of the single fiber properties, such as contractile weakness, atrophy, or reduced cross sectional area. Moreover different fiber isoforms differently affect the whole muscle behavior. In the design of our model, we adopted a bottom-up approach, trying to explain the whole muscle behavior starting from the single fiber characterization. Therefore, we first developed a model for a single fiber based on a three elements Hill’s model which is able to reproduce the classic protocols for the force velocity curve (isometric contraction followed by an isotonic contraction at different external forces) as well as the slack-test protocols (isometric contraction followed by a rapid shortening imposed to the fiber. We then characterized the parameters for both fast and slow human skeletal fibers, based on the original experimental data obtained in our lab, taking into account the effects of temperatures as single fiber mechanics is generally studied at low temperature (12-20°C). The characteristic width of the elements in the mesh of the diaphragm is several tens of microns, which means that each element likely include several slow and fast fibers. Then a characterization of the mixed fiber bundles is needed. We therefore characterized a mesh describing a bundle of fast and slow fibers to reproduce the mixing effect on the force velocity curve. The transversal connections linking each other adjacent fibers were taken into consideration. The mesh for the bundles was based on histological cross sections of diaphragm, which can be found in the literature [1]. Finally, we included our results in a 3-D mesh representing the human diaphragm. The bottom-up approach based on the real behavior of single bundles of fibers, is particularly useful to predict how different pathologies at the fiber level can influence the whole muscle performance., All skeletal muscles undergo an age-dependent loss of mass and functional deterioration. As follow-up of the functional impairment of lower limb muscles, the elderly experience limitations in locomotion and eventually lose the possibility of independent life. Accordingly, an impairment in respiratory function is the expected effect of the age-dependent decline of respiratory muscles. In spite of its essential role in breathing the age-dependent modifications of diaphragm muscle (DIAm), which is the main inspiratory muscle, have received less attention than limb muscles (Greising et al 2013, Elliott et al 2015). In this study we aimed to investigate the age-related changes of the mouse diaphragm and the possible impact of moderate endurance training. The respiratory function in mice is very different compared to humans. Breathing in mice as in other small mammals is characterized by very high frequency (400-500/min) and low tidal volume (, Denervated left emi-diaphragm of adult rats is well known to paradoxically increase in weight during the first ten days after denervation, then it atrophy as any other denervated muscle.1 Increased activity of eIF-2 initiation factor accounts at least in part for the enhancement of protein synthesis in 2-day denervated emi-diaphragm of adult rats.2 Nothing is known about the behavior of the denervated emi-diaphragm in oldest (30-month) rats. We compared in two groups of four animals (3-month adults vs 30-month oldest male rats) the effect on functional and structural properties of 7-day denervation in the diaphragm and in denervated EDL and Soleus leg muscles. At 30-month the body weight of the rats is high significantly heavier than that of the 3-month animals (gr 289+/-44 vs 401+/-38, mean+/-SD, p< 0.001). Despite the increase in body weight the weight of the innervated EDL, and SOL muscles are lower in the oldest rats. Equal or slightly lower are also sizes of muscle fibers in 30-month innervated EDL, SOL and diaphragm. Contrastingly, the response to 7-day denervation is muscle related: EDL and SOL leg muscles show a 15% decrease in muscle fiber size, while the muscle fiber of denervated left emi-diaphragm are indistinguishable from contralateral innervated muscle in 3-month and 30-month rats. Of note is the fact, that in the 7-day denervated left emi-diaphragm of the oldest rats (30-month) slow-type muscle fiber present a 5.7% highly significantly increase in fiber size, and that in two out of the four rats total muscle fiber size analyzed in semi-thin section after fixation for electron microscopy present highly significant increase (+ 14.5 %) when compared with the contralateral innervated emi-diaphragm. On the other hand, muscle fibers of 7-day denervated EDL and SOL of the same rats show the expected decrease in size (-18.7 %). In conclusion, both young and old diaphragms appear to be unresponsive to 7-day denervation atrophy., Neuromuscular Junction (NMJ) is the synapse that connects motor-neuron with skeletal muscle and its stability is critical for muscle contraction. Inherited or acquired conditions that perturb the components of this unit lead to weakness, denervation and, ultimately, paralysis.1,2 The signals that control such complex/critical structure in adulthood are largely unknown. Here we show that a retrograde signal is released from adult skeletal muscles to maintain a stable and functional NMJ. An efficient autophagy system is required for a functional NMJ because it controls MuSK clustering3 Impairment of autophagy or inhibition of MuSK leads to NMJ dismantle and denervation. These results identify MuSK-autophagy axis as a retrograde signal that is required for NMJ maintenance and function in adulthood., Endolysosomal carriers containing nicotinic acetylcholine receptors (CHRN) are either recycled (1) or degraded via autophagy during atrophic conditions (2, 5). However, regulatory processes underlying their processing have remained elusive. We have recently shown that internalized CHRN are accompanied by TRIM63 (MuRF1) and SH3GLB1 (3, 4). Here we further dissected the role of SH3GLB1 in regulating the autophagic flux of CHRN. Upon sciatic denervation, we found a tight regulation of SH3GLB1/phosphorylated-T145 (p-T145) SH3GLB1 protein amounts. While p-T145 SH3GLB1 levels remained constant upon induction of muscle atrophy, the non-phosphorylated SH3GLB1 protein was increased. Overexpression of a T145 phosphomimetic mutant (T145E) of SH3GLB1 slowed down the processing of CHRN endolysosomes while as T145 phosphodeficient mutant (T145A) of SH3GLB1 strongly augmented it. The slow processing of CHRN endolysosomes brought about by T145E was rescued upon co-expression of the early endosomal orchestrator RAB5, suggesting a role of SH3GLB1 in regulating CHRN endocytic trafficking at steps upstream of autophagosome formation., Protein kinase CK2, a pleiotropic serine/threonine kinase, plays an important role in many different biological processes inside of cells.1 Conditional muscle-specific CK2 mutant mice lack grip strength and show muscle fatigability.2 We identified the role of CK2 in skeletal muscle cells as a regulator of neuromuscular junction maintenance by phosphorylation of different protein members of the postsynaptic apparatus.2,3 Moreover, CK2 is involved in ensuring proper mitochondrial homeostasis in skeletal muscle fibers by fine-tuning mitochondrial protein import through the translocase of the mitochondrial outer membrane. In absence of CK2-dependent phosphorylation of a mitochondrial outer membrane translocase protein, muscle fibers undergo accelerated mitophagy, as demonstrated by an up-regulated PINK/Parkin/p62 pathway., Mammalian neuromuscular junctions (NMJs) undergo a postnatal topological transformation from a simple oval plaque to a complex branch-shaped structure often called a “pretzel”. Although abnormalities in NMJ maturation and/or maintenance are frequently observed in neuromuscular disorders, such as congenital myasthenic syndromes (CMSs), the mechanisms that govern synaptic developmental remodeling are poorly understood. It was reported that myotubes, when cultured aneurally on laminin-coated surfaces, form complex postsynaptic machinery, which resembles that at the NMJ. Interestingly, these assemblies of postsynaptic machinery undergo similar stages in developmental remodeling from “plaques” to “pretzels” as those formed in vivo. We have recently demonstrated that podosomes, actin-rich adhesive organelles, promote the remodeling process in cultured myotubes and showed a key role of one podosome component, Amotl2. We now provide evidence that several other known podosome-associated proteins are present at the NMJ in vivo and are located to the sites of synaptic remodeling. Additionally, we identified proteins that interact with Amotl2 in muscle cells. We show that two of them: Rassf8 and Homer1, together with other podosome components, are concentrated at postsynaptic areas of NMJs in the indentations between the AChR-rich branches. Our results provide further support for the hypothesis that podosome-like organelles are involved in synapse remodeling and that Rassf8 and Homer1 may regulate this process. This research was supported by grants 2012/05/E/NZ3/00487, 2013/09/B/NZ3/03524 and 2014/13/B/NZ3/00909 from the National Science Centre (NCN)., The neuromuscular junction is one of the few human tissues capable of complete regeneration after major damages. We have set up a reliable model of acute degeneration of the motor axon terminals followed by complete recovery of function.2 We have found that alarmins released by mitochondria of the degenerating nerve terminal are key factor that act on the perisynaptic cells and muscle fibre.2 These cells are activated and release signals that act retrogradely on the nerve terminal inducing its regeneration. Some of these signals are currently being investigated by imaging and transcriptomics methods., Two fundamental differences exist between voluntary muscle contractions and those induced by electrical stimulation . During voluntary movements motor units are activated asynchronously and a strict hierarchical order of recruitment is always maintained where the smallest motor units are activated first followed by contractions of larger units. Thus during voluntary movement the largest motor units are least active and are used only during maximal effort. This order of recruitment is cancelled when electrical stimulation of the muscle is used; indeed due to the biophysical properties of the axons that innervate the muscle the largest motor units are activated preferentially and therefore the parts of the muscle that are usually used rarely are active most frequently. Thus during electrical stimulation it is the motor units that are normally least active that experience the biggest increase in their use and consequently the biggest change in their characteristic properties. Thus electrical stimulation by bypassing the hierarchical order of recruitment, indeed by reversing it, is able to activate those motor units and muscle fibres that can only be activated during most strenuous exercise. It can therefore exploit the adaptive potential of muscle more efficiently then exercise and maintain much higher levels of activity over time then exercise. This enhanced activity is restricted to specific target muscles and is unlikely to have unwanted systemic effects. Finally, high amounts of activity can be imposed on a muscle from the beginning, since the CNS, cardiovascular and other systems will not interfere or limit the amount of activity carried out by the muscle, as is the case during exercise (see for review Pette and Vrbová, 1999).1 On the other hand, there are several functions that electrical stimulation of individual muscle groups cannot accomplish and that are unique to exercise induced activity. During exercise-induced activity coordinated movement is carried out and it is therefore likely that the individual’s skills in carrying out movement of this kind will improve. Thus, while exercise can improve coordination, electrical stimulation is unlikely to do so. In addition the flexibility of joints and lengthening of muscles can be improved by exercise but not by electrical stimulation. Particular exercise regimes such as Pilates and yoga are particularly effective in achieving these goals. Improvement of the cardiovascular system is also easier achieved by exercise. Nevertheless, it can be argued that having muscles that are less fatiguable than usual, an advantage that is readily achieved by electrical stimulation, enables the individual to exercise more efficiently and achieve all the goals regarding fitness more readily and in a shorter time., Regular exercise and neuromuscular electrical stimulation (NMES) have been used in a variety of settings for different populations. Various modes of exercise (eccentric, concentric, resistance and aerobic) and NMES training regimes (localization, intensity, duration, frequency) exist for healthy subjects and athletes, patients in a variety of rehabilitation and preventive settings, either partially or in totally immobilized subjects. Both standard exercise interventions and NMES have been shown to be effective in preventing the decrease in muscle strength, muscle mass and the loss in oxidative capacity of skeletal muscles following multiple types of surgical (orthopedic) procedures. However, it is still not entirely clear whether biological adaptations are similar and the duration differences in their adaptation duration. We will discuss potential biological differences in adaption at the neuromuscular junction and potential differences in bioenergetics adaptation. Future research needs to determine potential molecular differences and beneficial post adaptation differences., Duchenne muscular dystrophy (DMD) is caused by loss of the force transmitting and membrane complex organizing protein, dystrophin, and is characterized by progressive muscle deterioration with failed regeneration and replacement with a fatty-fibrous matrix.1 Dystrophin replacement therapies to date have shown only limited ability to slow the disease process, and thus therapeutics targeting other aspects of the disease, which can be used in combination, are needed. One potential target is nuclear factor κB (NFkB, which is upregulated in the DMD muscles.2 We examined a novel class of NFkB inhibitors in mdx mouse and golden retriever muscular dystrophy (GRMD) dog models of DMD. These orally bioavailable compounds improved the phenotype of voluntarily run mdx mice, in terms of amount of activity, muscle mass and function, inflammation, and fibrosis. Surprisingly, the muscles were also more resistant to contraction-induced damage, which we demonstrated was significant increases in dysferlin, a protein required for membrane damage repair. We also evaluated the cardiac impact of a phosphodiesterase 5 (PDE5) inhibitor, tadalafil, which has been shown to improve blood flow in exercising skeletal muscles in mdx mice and human DMD patients.3 Cardiomyopathy is a leading cause of mortality among DMD patients and is well modeled by the golden retriever muscular dystrophy (GRMD) dog model of DMD. Prophylactic use of the PDE5 inhibitor, tadalafil, improved GRMD histopathological features of the hearts, decreased levels of the pathogenic cation channel TRPC6, increased phosphorylation of TRPC6, decreased m-calpain levels and indicators of calpain target proteolysis, and elevated levels of the dystrophin ortholog, utrophin. The progressive loss of cardiac function was significantly slowed by these effects. These data demonstrate that prophylactic use of tadalafil delays the onset of dystrophic cardiomyopathy, which is likely attributed to modulation of TRPC6 levels and permeability and inhibition of protease content and activity, which results in higher levels of the protective protein, utrophin. Thus PDE5 inhibition and NF-kB inhibition are potential therapeutics to consider in developing a combinatorial approach to the treatment of DMD., IGF-I and insulin are intrinsically connected through their actions on the IGF-I and insulin receptors to regulate blood glucose (1). Reduced circulating IGF-I can be compensated by heightened insulin, but chronically elevated insulin can lead to insulin resistance and ultimately diabetes . Further, increased circulating or local muscle IGF-I may enhance glucose uptake. If IGF-I from muscle and liver is equivalent, then loss of muscle IGF-I should result in a similar pathologic diabetic state (2). By extension, if muscle IGF-I is elevated, it may serve a protective role in glucose homeostasis, either through increased muscle mass providing a greater glucose sink (3), or through increased hybrid receptor activation by IGF-I (4). To address the impact that these factors have on metabolism, we elevated IGF-I by local AAV-IGF-I injections into both hindlimbs of adult male mice (5), and reduced muscle IGF-I through an inducible muscle specific deletion of Igf1. Mice were subjected to tests for body composition, glucose uptake, and energy expenditure compared to age-matched controls. It was not surprising that the hindlimb injections boosting IGF-I levels only in a small group of muscles did not alter the whole animal body composition. Further, when mice were subjected to treadmill running for 60 minutes, there were no significant changes in blood levels of glucose or lactate pre- or post-exercise. While increased muscle mass did not appear to alter basal glucose uptake, increased IGF-I content altered contraction induced glucose uptake in muscle when normalized to mass. To understand the consequences of diminished muscle IGF-I production, we generated mice with inducible muscle specific IGF-I deletion, with induction in adult mice. In mice with muscle specific deletion of Igf1, glucose levels following uphill treadmill running increased by 10%, in contrast to controls where blood glucose decreased by ~40%, supporting that glucose clearance is mediated in part through muscle IGF-I. Further, a marked impairment of glucose clearance occurred following a simple glucose tolerance test. Based on these results, we assert that the IGF-I produced by the muscle has an endocrine function, and like IGF-I produced by the liver, modulation of muscle levels of IGF-I will lead to changes in glucose homeostasis., Sarcopenia is the age-related loss of muscle mass, strength and function leading to loss of muscle power, which in the end results in frailty and disability. At molecular level, sarcopenia is a complex condition characterized by insufficient antioxidant defense mechanism, increased oxidative stress and altered function of respiratory chain (Fulle S., 2005). It has been hypothesized that the accumulation of oxidative stress is also related to an impaired regeneration cooperating to the atrophic state that characterizes muscle ageing (Beccafico S., 2007). To the purpose, we investigated the myogenic process in satellite cells, the skeletal muscle stem cells, as myoblasts and myotubes collected by human Vastus Lateralis skeletal muscle of young and old subjects through needle-biopsies (Pietrangelo T., 2011). NBT and H2DCF-DA assays were used to measure O2•- and ROS production, respectively. Data revealed that oxidant species are more concentrated in elderly myoblasts compared to young ones. To evaluate if mitochondria are affected by ROS using JC-1 assay we found that in elderly myoblasts mitochondrial transmembrane potential decreases much more than in young ones probably due to their lower endogenous antioxidant abilities. Furthermore, MitoSOX™ Red reagent was used to measure directly O2•- in mitochondria. We found that in elderly myoblasts O2•- production is increased respect to young ones and the result is worsened in myotubes. Gene expression analysis revealed that genes involved in atrophic and ubiquitin-proteasome pathways were upregulation together with the dysregulation of the proliferative one suggesting an alteration at gene expression level in elderly myoblasts vs young ones (Pietrangelo T., 2009). In an attempt to ameliorate muscle regeneration in elderly mitochondrion-specific liposomes carrying antioxidant were synthetized. The toxicity of liposomes were tested on human satellite cells and C2C12 cells, a murine skeletal muscle cell line. Preliminary results demonstrated that liposomes made using DPPC 97.5%/BOLA 2.5% gave the lowest toxicity at 24-48-72 hours. Overall, if we need more data and further analysis, up to day our data suggest that oxidative stress impairs muscle regeneration in elderly subjects., The sarcoplasmic reticulum (SR) is organized in longitudinal and junctional SR (j-SR). In skeletal muscle, this latter domain together with the T-tubules form specific junctions called triads, where proteins regulating the excitation-contraction coupling mechanism assemble. Junctophilins (JPs) are directly involved in the formation and maintenance of triads. Basically, they are anchored to the SR via their C-terminal transmembrane domain (TMD), while their N-terminus contains eight MORN motifs, which associate with the phospholipids of the T-tubules. Nevertheless, how JPs are targeted to triads is not known. The roles of the N-terminal and the C-terminal regions of JP1 in this process were investigated. Expression in primary myotubes and/or muscle fibers of JP1 deletion mutants lacking the TMD resulted in protein distribution at both the surface sarcolemma and the T-tubules, confirming that MORN motifs are involved in JP1 interaction with the sarcolemma, but are not sufficient to restrict its localization at the T-tubules. On the other hand, progressive deletion of the eight MORN motifs or even of the entire cytosolic region, did no affect JP1 localization at triads, indicating that the presence of the TMD is sufficient for JP1 localization at the j-SR. FRAP analysis performed on a GFP-TMD fusion protein expressed in myotubes indicated that this protein has a high mobility, suggesting the absence of strong protein-protein interactions occurring at the j-SR. Further work is needed to better understand the molecular mechanisms driving TMD-mediated JP1 localization at triads., Physical Calsequestrin (CASQ) is the major protein of the sarcoplasmic reticulum of striated muscle that binds Ca2+ with high capacity and moderate affinity. CASQ exist as a monomer and polymers, depending on Ca2+ concentration. CASQ switches from an unfolded state to a folded monomer when the ionic strength increases allowing the formation of front-to-front first and then back-to-back interactions in higher Ca2+ concentrations. Recently we reported one mutation in the skeletal CASQ1 gene in a group of patients with a vacuolar myopathy characterized by the presence of inclusions containing CASQ1 and other SR proteins. The CASQ1 mutation (CASQ1D244G) affects one of the high-affinity Ca2+- binding sites of the protein and alters the kinetics of Ca2+ release in muscle fibers from patients. Expression of the CASQ1D244G in myotubes and in mouse fibers causes the appearance of SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in patients. Studies of Ca2+ release showed an increase in Ca2+ storage in CASQ1WT COS-7 transfected cells whereas no increase was observed in CASQ1D244G. Moreover both CASQ1WT and CASQ1D244G were expressed in bacteria, purified and analysed for their ability to polymerize at increasing Ca2+ concentrations. The results obtained indicate that the CASQ1D244G protein polymerizes at lower Ca2+ levels and more rapidly than CASQ1WT. These results suggest that the CASQ1D244G mutation interferes with the correct process of Ca2+-dependent protein polymerization causing altered intracellular calcium storage and the formation of protein aggregates., Store-operated Ca2+ entry (SOCE), also referred to as capacitative Ca2+ entry, plays an important role in intracellular Ca2+ regulation. SOCE is a ubiquitous Ca2+ entry mechanism, first described in non-excitable cells, that is triggered by depletion of intracellular Ca2+ stores (endoplasmic/sarcoplasmic reticulum, respectively ER and SR). SOCE is coordinated by the interaction of stromal interaction molecule 1 (STIM1), which acts as the Ca2+ sensor in the ER lumen,1 and Orai1, the Ca2+-permeable channel in the plasma membrane (PM).2 Specific Gap of Knowledge. SOCE is also well-documented in skeletal muscle,3 where it limits muscle fatigue during repetitive fatiguing stimulation.4 Several studies suggest that STIM1-Orai1 coupling occurs within the pre-formed SR-TT junctions of the triad,5 also known as Ca2+ release units (CRUs), the sites of excitation-contraction (EC) coupling. However, the precise subcellular location of STIM1-Orai1 SOCE complexes in skeletal muscle has not yet been unequivocally identified. Recent breakthroughs. Here we show by electron microscopy (EM) that prolonged muscle activity drives the formation of previously unidentified intracellular junctions between the SR and extensions of the TTs membrane. The activity-dependent formation of these unique SR-TT junctions reflects a striking and unexpected remodeling of the existing sarcotubular system at the I-band of the sarcomere. Using immunohistochemistry and immunogold labeling for EM we demonstrate that these junctions contain the molecular machinery known to mediate SOCE: STIM1 in the SR and Orai1 channels, which move into the I band as a result of the elongation of existing TTs. Thus, these newly formed junctions are referred to as Ca2+ Entry Units (CEUs), the first new, molecularly-defined subcellular structure in skeletal muscle in over 30 years. We propose that CEUs: a) play a fundamental role in coordinating STIM1-Orai1 coupling in muscle, b) represent the structural framework for SOCE providing an ideal Ca2+ entry pathway to refill SR stores, and c) plays a key role during repetitive stimulation., Deep brain stimulation (DBS) of the subthalamic nucleus (STN) or globus pallidus internus (GPi) is now an established, safe and effective treatment option for Parkinson’s disease (PD), used by more than 125,000 patients worldwide. Solid scientific evidence indicates that it can significantly alleviate motor disability, levodopa-induced complications and improve a patient’s overall quality of life. The indications for DBS have expanded in recent years, including earlier application in Parkinson patients, use in other motor disorders such as dystonia and essential tremor, and the potential for use in intractable epilepsy and psychiatric disorders, for example. In Parkinson patients DBS produces a marked improvement in motor fluctuations and dyskinesias even if evidence suggests that the reduction in motor disability is greater with medications than with STN-DBS. Benefits associated with DBS persists for many years, although disability still progresses, reflecting degeneration in non-dopaminergic sites. It is noteworthy, however, that the use of DBS is limited not only by several restrictive exclusion criteria, but also by the comparatively high risk of severe complications, such as neuropsychiatric morbidity, intracranial bleeding (which can occur in 2-8% of patients undergoing stereotactic neurosurgery), and in some cases increased mortality., EC coupling in muscle links the transverse(T)-tubule depolarization to release of Ca2+ from the sarcoplasmic reticulum (SR).1-2 These membranes communicate in specialized structures, i.e. calcium release units (CRUs), thanks to a cross-talk between voltage-dependent Ca2+ channels CaV1.1 (or dihydropyridine receptors, DHPRs) in the T-tubule and Ca2+ release channels, or ryanodine receptors type-1 (RYR1), in the SR.3-5 Mutations in the gene encoding for RYR1), the SR Ca2+ release channel, underlie debilitating, life-threatening muscle disorders such as central core disease (CCD) and malignant hyperthermia (MH). To date, MH is only seen as a clinical syndrome in which genetically predisposed individuals respond to volatile anesthetics in the operating room with potentially lethal episodes characterized by elevations in body temperature and rhabdomyolysis of skeletal muscle fibers. However, virtually identical over-heating episodes have been reported in individuals also after exposure to environmental heat, physical exertion, or even during febrile illness. The life-threatening nature of EHS underscore the critical need for a deeper mechanistic understanding of these syndromes and for the development of new and effective treatments. Specific Gaps of Knowledge. A) Mutations in RYR1 have been found in many, but not all, MH cases suggesting the potential involvement of additional genes in the pathogenesis of this syndrome. B) The relationship between classic MH and overheating episodes triggered by different stressors (heat, exertion, fever, etc.) is not yet widely recognized. C) The cascade of molecular mechanisms that from SR Ca2+ leak leads to rhabdomyolysis of muscle fibers are still unclear and needs to be fully elucidated. Recent breakthroughs. In the last years, thanks to the support of Telethon (GGP08153 and GGP13213), we have moved significant steps forward. We have demonstrated in animal models that: A) MH episodes can result not only from mutations in RYR1, but also from mutations in proteins that interact with RYR1 (such as Calsequestrin-1, CASQ1); B) the mechanisms underlying hyperthermic episodes triggered by anesthetics and by heat and exertion are virtually identical, suggesting that these syndromes could be possibly treated/prevented using similar treatments; C) during lethal MH/EHS crises Ca2+ leak from intracellular stores results in a feedforward mechanism mediated by excessive production of oxidative species of oxygen and nitrogen (ROS and RNS), which eventually will lead to depletion of the SR and to massive activation of Store Operated Ca2+ Entry (SOCE)., A crucial system severely affected in different pathological conditions is the antioxidative defense, leading to accumulation of ROS. The discovery that the anti-oxidant status decreases with age and it is affected in several pathological conditions, such as disuses, chronic fatigue syndrome, cancer, muscular dystrophy and amyotrophic lateral sclerosis (ALS), has placed oxidative stress as a central mechanism in the pathogenesis of these diseases. A critical aspect underlying the mechanisms of age-related muscle loss is the reduction in the number of nerve terminals, fragmentation of the neuromuscular junctions (NMJs), and a decrease in neurotransmitter release. However, considering that skeletal muscles in one of the tissues that generate considerable ROS, an important issue to address is whether a selective alteration of oxidative stress balance in skeletal muscle is sufficient to induce these alterations. Preliminary results demonstrate that muscle specific accumulation of oxidative stress induces mitochondria dysfunction/alterations and triggers NMJs dismantlement, associated with higher rate of Acetilcholine Receptor (AchR) turnover and morphological alterations of the pre-synaptic neuromuscular endplate. We also defined the potential molecular mechanisms that mediate the toxic effects of oxidative stress and NMJs dismantlement., Physical exercise is known to have beneficial effects on muscle trophism and force production modulating signaling pathways involved in fiber type and muscle growth also via intracellular Ca2+ and inducing specific mitochondrial adaptations. Several evidences both in vitro and in vivo, have demonstrated that during muscle contraction, Ca2+ concentration in the mitochondrial matrix is increased. Importantly, alterations of mitochondrial Ca2+ homeostasis controlled by mitochondrial calcium uniporter (MCU) has been recently shown to regulate muscle mass in vivo in mice. In skeletal muscle, mitochondria exist as dynamic network that is continuously remodeled through fusion and fission phenomena that are important for maintenance of functional mitochondria. In particular, fission occurs upon the recruitment of dynamin-related protein 1 (DRP1), while fusion is controlled by mitofusins (MFN) 1 and 2 and by optic atrophy 1 (OPA1). OPA1 also regulates mitochondrial adaptations to bioenergetic conditions at the level of inner membrane ultrastructure and remodeling of mitochondrial cristae, controlling muscle atrophy and mitochondria respiratory efficiency. It has been shown that the decrease of muscle mass and strength observed in age-related Sarcopenia is linked to abnormalities of mitochondrial morphology, number and function. In the present study we intended to investigate the effects of 9 weeks strength training by neuromuscular electrical stimulation (ES) in comparison to voluntary leg press (LP) in sedentary 70yrs old subjects on mitochondria dynamics, MCU and mitochondria respiratory chain enzymes modulation. Our results show that ES mediated muscle structural and functional improvements are linked to signaling pathways related to muscle mass regulation and enhanced MCU, and COX IV respiratory chain enzyme. Electron Microscopy ultrastructural analyses showed remodelling of mitochondrial apparatus as a consequence of fusion phenomena that is consistent with adaptation to physical exercise and with increased OPA1 protein expression levels as documented by WB analyses. LP training showed moderate effects both at structural and functional level, with no impact on mitochondrial dynamics, that are consistent with a milder protocol of training in comparison to neuromuscular ES. Altogether these results indicate that the ES-dependent beneficial effects on muscle mass and force are associated with changes in mitochondrial-related proteins involved in Ca2+ homeostasis and mitochondrial shape, providing new targets to develop therapeutic strategies counteracting Sarcopenia and promoting healthy ageing., Skeletal muscle atrophy is a debilitating loss of muscle mass (resulting from decreased myofiber size) and strength that normally occurs during aging, muscle disuse / inactivity, neuropathies / myopathies and with other pathological diseases. The ether-a-gogo related gene (ERG1a) is a K+ channel that is up-regulated in atrophying gastrocnemius muscles of both unweighted and cachectic mice.1 Ectopic expression of mouse erg1a (Merg1a) in mouse muscle increases ubiquitin proteasome activity by up-regulation of at least one known E3 ligase, MuRF1.2 Because Murf1 expression is up-regulated by increased NFκB activity3, we hypothesized that ectopic expression of Merg1a would increase NFκB activity and lead to increased Murf1. To test this, we electro-transferred plasmid encoding an NFκB firefly luciferase (FFL) reporter and a second plasmid encoding Renilla luciferase (Ren) into mouse gastrocnemius muscles. We added a Merg1a plasmid in the left leg while adding a control plasmid in the right. We then harvested muscle on days 0-7 and assayed for both FFL and Ren activities and used the FFL to Ren ratio as a measure of NFκB activity (to correct for differences in transfection efficiency). Surprisingly, the muscles expressing Merg1a showed a decreased NFκB activity when compared to the controls. Thus we hypothesized that there may be a factor present in a physiological model of atrophy that would cause MERG1a to modulate NFκB activity differently. Because sciatic nerve transection does not produce an increase in Merg1a expression, but it increases NFκB activity3, we repeated our electro-transfer with mice and then denervated both legs 18-24 hours later. We harvested muscles at days 0, 2, 4, 7 and 10 post-denervation. The Merg1a treated muscle still experienced a decrease in NFκB activity. Thus, we conclude that ectopic expression of Merg1a modulates NFκB activity in both innervated and denervated skeletal muscle. Future studies will include efforts to determine if this finding is truly physiological and, if so, then by what mechanism does MERG1a affect NFκB activity., Physical activity plays an important role in preventing chronic disease and muscle degeneration in adults and the elderly persons.1 Age-related changes in skeletal muscle innervation, independent of patent peripheral neuropathies, are known to contribute to the decline in quality of life often reported in older population.2 The changes and the mechanism(s) by which they occur are not well understood. We had the opportunity to examine the effects of lifelong high-level physical activity comparing cohorts of young adults and septuagenarians either sedentary or recreational sportsmen, collecting what is, in our opinion, strong evidence that aging atrophy is, at least in part, a result of progressive denervation that can be counter-acted by lifelong high-level exercise.3 We used immunolabeling methods to analyze the fiber type composition of muscle biopsies. Our main results demonstrate that biopsies from: 1. young men seldom contain denervated (0.2±0.5 %), or transforming muscle fibers (0.5±0.6 %); 2. sedentary seniors contain both denervated (2.6±1.9 %), coexpressing myofibers (1.8±1.7 %) and a few reinnervated clustered myofibers of the fast type (3.0±4.7 %); and 3. senior sportsmen present with a larger percentage of healthy, slow myofibers (up to 68.5±14.1 %,) that appear mainly clustered in slow fiber-type groupings (7.9±7.4 %).4,5 Data analysis reveals that there was no difference between the athletic and sedentary senior groups in terms of their very low percentages of muscle fibers co-expressing fast and slow MHCs (0.6±0.6 %), suggesting that lifelong exercise does not induce motor unit transformation. On the other hand the recreational sportsmen had both considerably higher percentages of slow-type myofibers and greater numbers of slow fiber-type groupings, providing sound evidence that lifelong cycles of denervation/reinnervation occurred. It appears that lifelong exercise protects muscle function by saving otherwise lost muscle fibers through reinnervation by different, mainly slow, motor axons.6 On the other hand, volitional exercise is not always feasible or people are reluctant to do it and other strategies should be applied such as Functional Electrical Stimulation (FES).7 This study shows the effects in situ of FES in human Vastus Lateralis (VL) muscle of sedentary elderly people, in particular on the key process of Ca2+ uptake and release and related control mechanisms that are essential in muscle adaptation. Through immunofluorescence analysis of muscle cryosections, a huge increment of NFAT positive nuclei was found after treatment (from 3% to 60%); moreover an increment of P-CamkII was observed by western blotting analysis. These findings indicate that FES activate the CaM-dependent phosphatase signaling (known to be involved in muscle plasticity). Muscle total homogenates were obtained from biopsies performed before and after completing a nine weeks FES treatment on a group of volunteers and Calsequestrin (CASQ), SERCA, Sarcalumenin, protein expression was determined by Western blot. After FES significant increase of SERCA2 and Sarcalumenin and decrease of CASQ1 were observed. Immunofluorescence analysis were also performed to localize in situ MHCII/SERCA2 co-expressing muscle fibers, an interesting tool to identify subpopulation of muscle fibers involved in muscle adaptation. The overall results indicate that the applied FES protocol, simulating a motoneuron slow-type firing pattern, potentiates Ca2+ uptake and storage in skeletal muscle fibers validating at molecular level the FES strategy as a safe and effective rehabilitation strategy in elderly persons., The Tat protein is able to translocate through the plasma membrane and when it is fused with other peptides may act as a protein transduction system. This ability appears particularly interesting to induce tissue-specific differentiation when the Tat protein is associated to transcription factors. In the present work the potential of the complex Tat-MyoD in inducing equine peripheral blood mesenchymal stromal cells (PB-MSCs) towards the myogenic fate, was evaluated. Results showed that the internalization process of Tat-MyoD needs the absence of serum and the nuclear localization of the fused complex is observed after 15 hours of incubation. However, the supplement of Tat-MyoD only was not sufficient to induce myogenesis and, therefore, in order to achieve the myogenic differentiation of PB-MSCs, conditioned medium was added. The latter was obtained coculturing PB-MSCs with C2C12 without a direct contact. These results suggest that TAT-mediated protein transduction system, if supported by conditioned medium, might represents a useful methodology to induce myoblasts differentiation., Both strength and power developed by human skeletal muscles decline with increasing age.1,2 The athletic world records of the Master athletes of age ranging from 35 years to 100 years are an excellent proof of such decline in every track and field competition. The world record performances of running, jumping and throwing events can be transformed into dimensionless parameters proportional to the power developed in the trials. Such parameter ranges from 1 for the Senior world record (i.e. the maximum human performance) to lower values for the Master athletes of increasing age down to 0 for a null performance.3,4 With this procedure the declines of the power parameter with increasing age can be analysed and compared: the trend-lines start to decline very close to the age of 30 years and arrive to 0 around the age of 110 years for every athletic discipline (running, jumping and throwing).5 The comparison of the various trend-lines show significantly different rates of decline. Each declining trend-line reaches a “critical” threshold at different ages for the running, jumping and throwing activities. Such thresholds indicate different age limits for most of everyday tasks: walking, climbing stairs and lifting weights above a table. The decline of the Master world records, transformed into a dimensionless power parameter declining from 1 toward 0 with increasing age is the decline of the power developed by each one of us, starting from 1 in our youthful age and declining toward lower values with increasing age. There are no reason, for each one of us, to decline differently from the world record-men, provided that each of us remains in stable fitness condition without disabling pathologies., We present a case report of atypical amyotrophic neuralgia of the suprascapular nerve with isolated denervation atrophy of rotator cuff muscles and related biomechanics impairments of the shoulder. The patient, lamenting yearly-long unsatisfactory results of standard clinical physiotherapy, after a baseline re-evaluation at our hospital, have been treated for the last seven months with an additional personalized home-based Electrical Stimulation protocol using triangular currents for denervated muscles. At the end of the follow up we observed clinical, radiological (False-color CT)1 and neurophysiological (needle-EMG)2 improvements. The case report provides the opportunity to discuss a rehabilitative pathway for diagnostics and rehabilitation of patients suffering of peripheral denervation, a condition that is still a challenge for clinicians., The Chronic low back pain (CLBP) is a disabling condition affecting a majority of people of the western countries. It also deeply affects the quality of life as it is often linked to multidimensional disturbances such as poor sleep, mood disorders, chronic fatigue and joint pain. There is no condition with higher social and economic expenses. It has been reported that only a minority of patients with gut inflammation suffers from intestinal symptoms. In a previous paper it was proposed that gastrointestinal disturbances, beyond mechanical issues, could be overlooked in the management of these patients. Dietary changes were successful in the positive resolution of the described clinical case. Here we further test this hypothesis. We measured on 5 subjects specific parameters related to gastrointestinal and digestive physiology that have been associated with metabolic and immuno-related pathological conditions. Inflammation in the gut can lead to altered mucosa permeability. The entrance in the blood stream of abnormal molecules activates the immune system in a cascade of events affecting remote systems and possibly the integrity of structures like the neuromuscular junction or the pathways of energy production. Conditions that are currently managed by orthopaedists, reumatologists and neurologists could benefit from a screening of the gastrointestinal functionality., In the last decades, a growing body of literature focused on the use of mechanomyography (MMG) as a means to study non-invasively skeletal muscle mechanical activity. MMG signal is detectable at the skin surface during the dimensional changes of active muscle fibres that generate pressure waves due to voluntary or evoked contractions.1,2 A novel application is the use of an electromyographic (EMG), MMG, and force (F) signals combined approach as a tool to partition the electrochemical and mechanical events underpinning the electromechanical delay during muscle contraction (EMD) and relaxation (R-EMD).3,4 This approach has been utilized to evaluate the changes in the electrochemical and the mechanical components of EMD and R-EMD under several physiological conditions (local fatigue, muscle temperature manipulation and muscle-tendon unit stretching). Under all these circumstances, the approach presented a high reliability and sensitivity. Myotonic dystrophy type 1, the most frequent form of inherited muscular dystrophy5 involves a broad spectrum of systemic complications. The main features at the skeletal muscle level are muscle weakness and grip and percussion myotonia. Distal muscles are generally more compromised than the proximal ones. In clinical settings, muscle weakness and myotonia are usually determined on patients with DM1 qualitatively or semiquantitatively by the Medical Research Council scale, by dynamometry, and/or by physician’s handgrip evaluation. Hence, a valid, non-invasive, and reliable tool to assess the degree of muscle dysfunction in DM1 could be of great interest for clinical trials involving new therapies. Therefore, the aims of the study were: (i) to assess the reliability and sensitivity of the measurement of the electromechanical delay components during both contraction and relaxation in patients with DM1; and (ii) to evaluate and discuss possible differences in delay components’ duration between patients with DM1 and healthy, age-matched controls (HC). EMD and R-EMD electrochemical and mechanical components duration and reliability of the measurements were investigated during skeletal muscle contraction and relaxation in a group of patients with DM1 (n = 13) and in healthy controls (n = 13). EMG, MMG, and F were recorded from the tibialis anterior (distal muscle) and vastus lateralis (proximal muscle) muscles during maximum voluntary and electrically-evoked isometric contractions. The electrochemical and mechanical components of the electromechanical delay during muscle contraction and relaxation were calculated off-line. Maximum strength was significantly lower in DM1 than in controls under both experimental conditions. All electrochemical and mechanical components were significantly longer in DM1 in both muscles. Measurement reliability was very high in both DM1 and controls. The high reliability of the measurements and the differences between DM1 patients and controls suggest that the EMG, MMG, and force combined approach could be utilized as a valid tool to assess the level of neuromuscular dysfunction in this pathology, and to follow the efficacy of pharmacological or nonpharmacological interventions., The optimum metric for assessing changes in skeletal muscle quality remains debated. Identifying a novel quantitative method for muscle assessment in this regard would allow for the generalizability of such studies to clinical practice and therefore aid in the indication of compensatory targets for clinical intervention.1-3 While there is much extant literature reporting the use of average HU values to investigate muscle quality and its utility as a comorbidity index, no studies have yet to utilize the entire radiodensitometric distribution. The increasing prevalence of sarcopenic and cachexic muscular degeneration necessitates the establishment of a robust quantitative muscle assessment methodology. Herein, we hypothesize that rigorously quantifying entire HU distributions can elicit much more information regarding muscle quality than extant methods that, to date, only utilize average HU attenuation values. This study reports the development and use of this method, wherein we assess upper leg muscle quality utilizing nonlinear trimodal regression analysis with radiodensitometric distributions from computed tomography (CT) scans of a healthy young adult, a healthy elderly subject, and an SCI patient with complete lower motor neuron denervation. Results from this assessment highlight the utility of utilizing entire HU attenuation value distributions and identify novel parameters from these analyses that could provide further insight into how muscular degradation can be optimally quantified., During the last decade we contributed to rehabilitation in aging studying effects of physical exercise induced by Functional Electrical Stimulation (FES) in the special case of Spinal Cord Injury patients affected by complete injury of the Conus Cauda, a syndrome in which the denervated leg muscles are fully disconnected from the nervous system. Denervated human muscles become unexcitable with commercial electrical stimulators and undergo ultra structural disorganization within a few months from SCI, while severe atrophy with nuclear clumping and fibro-fatty degeneration appear within 3 and 6 years, respectively.1-4 To counteract these progressive changes a novel therapy concept for paraplegic patients with complete lower motor neuron denervation of the lower extremity was developed in Vienna: home-based functional electrical stimulation of long-term denervated muscles (h-b FES). New electrodes and a safe stimulator for h-b FES have been designed to reverse severe atrophy by delivering high-intensity (up to 2,4 J) and long- duration impulses (up to 150 ms) able to elicit contractions of denervated skeletal muscle fibers in absence of nerves.5,6 Specific clinical assessments and trainings were developed at the Wilhelminenspital Wien, Austria,7 based on sound evidence from animal experiments.8 Main results of the clinical study on patients which completed the 2-year h-b FES training were: 1. significant increase of muscle mass and of myofiber size, with striking improvements of the ultra- structural organization; 2. recovery of tetanic contractility with significant increase in muscle force output during electrical stimulation; 3. capacity to perform FES-assisted stand-up and stepping-in-place exercise.9-12 The study demonstrated that h-b FES of permanent denervated muscle is an effective home therapy that results in rescue of muscle mass, function and perfusion. Additional benefits are improved leg cosmetic appearance and enhanced cushioning effect for seating., Functional recovery after peripheral nerve injury is reduced when axon growth is misdirected to reinnervate muscles other than their original targets.1-3 Here we review the effects of chronic electrical muscle stimulation (EMS) following peripheral nerve injury in rat, canine, and equine models of peripheral nerve injury. Specifically, we examine whether EMS accelerates reinnervation of muscular targets and if these targets are appropriately reinnervated by their original axons following nerve injury. Methods: In the Sprague Dawley rat, the lateral gastrocnemius nerve was transected and immediately repaired. The soleus muscle was implanted with electrodes and connected to a mini stimulator implanted intraabdominally. Muscles were stimulated daily using a 12-hour day time pattern of a 10 second burst of 20 Hz once per hour followed by a 12-hour night time pattern of 20 Hz (10 seconds on, 20 seconds off). This stimulation pattern was delivered for 2 months. Appropriate reinnervation of the soleus muscle was assessed using retrograde labeling of the soleus nerve. Functional recovery was assessed by measuring isometric soleus muscle forces. In the dog, the recurrent laryngeal nerve was transected bilaterally and immediately repaired. Electrodes were implanted to stimulate the posterior cricoid arytenoid (PCA) muscles bilaterally. Muscles were stimulated continuously using either a 10 or 40 Hz pulse train for ninety days. Appropriate reinnervation was measured using electromyography methods. Functional recovery was assessed using a treadmill exercise test and vocal fold movement during hypercapnia. In the horse, the right recurrent laryngeal nerve was injured using a stainless steel probe pre-chilled in liquid nitrogen and placed on the nerve for two minutes. Electrodes were implanted into the right PCA muscle and connected to an implanted stimulator. Muscles were stimulated for one hour once every 12 hours at 22 Hz (3.5 seconds on, 6.5 seconds off) for twenty weeks. Functional recovery was assessed using a treadmill exercise test and examining arytenoid abduction and tracheal inspiratory pressure endoscopically. Results: Retrograde labeling in the rat, demonstrated that EMS of the soleus muscle had no effect on directing the original soleus neurons back to reinnervate the muscle following nerve injury and repair. Muscle twitch forces were significantly greater, however, tetanic forces were not different whether the muscle was stimulated or not. In the dog, progressive addition of samples to the study showed that exercise tolerance and glottal area following hypercapnia was maximal in dogs that received PCA stimulation at 10 Hz.4 EMG measurements in 3 dogs suggest that PCA muscles stimulated at 10 Hz were preferentially reinnervated by their original motoneurons whereas those muscles stimulated at 40 Hz or were unstimulated had random reinnervation. Horses that had the PCA muscle stimulated had improved function as demonstrated by lower negative tracheal inspiratory pressures. These improvements occurred sooner after injury and at lower exercise intensities than horses that did not have the PCA muscle stimulated. However, functional recovery returned to near baseline in all horses suggesting that the original nerve injury was not severe enough. Conclusions: Despite stimulating the soleus muscle in rats using a pattern resembling natural activity before and during the time of reinnervation, EMS did not encourage the original motoneurons that were connected to the stimulated muscle to return. However, in the dog, 10 Hz stimulation promoted selective reinnervation. One limitation in the dog study is the small sample size which needs to be expanded to provide adequate statistical power. In the horse, EMS enhanced the speed of functional recovery despite a nerve injury that was not as severe as one in both dogs and rats. Nevertheless, in all animal models stimulation did not negatively impact functional recovery with muscle forces in the rat being higher with stimulation and dynamic airway measurements being enhanced in both dogs and horses that had their muscles stimulated following nerve injury., The cellular mechanisms underpinning the maintenance, gain and loss of muscle mass are of great interest at present, given the popularity of bodybuilding, the potential for increased muscle metabolism to reduce the damage caused by diabetes and insulin resistance, and the key role of muscle function associated with the decline in mobility and well-being associated with ageing. There are many model systems in which to make experimental investigations of muscle hypertrophy in rodents.1-9 It is generally considered that increased average force generation (loading) is important, although hypertrophy can be achieved in some muscles by pharmaceutical agonism of the androgen receptor family, or by genetic manipulation of, for example, the response to IGF. Models have included removal1,2,3 or denervation of agonists to generate constant overload, various training modalities such as squats,3 lifting,4,5 or jumping for a food reward, and treadmill or ladder climbing exercise,6 sometimes with added weight7 to increase the muscular effort. We have designed a hypertrophy model using programmed exercise by stimulating agonists and antagonists simultaneously. In the rat hind limb, the dorsiflexor muscles that lift the foot are supplied by the common peroneal nerve whereas the plantarflexor muscles are supplied by the tibial nerve. The plantarflexors are the larger and stronger group so it is possible to generate loaded contractions of the dorsiflexors by activating them fully at the same time as a partial activation of the plantarflexors. We have achieved this with a single channel implant by careful positioning of the electrodes with the cathode under the common peroneal nerve and the anode near to the tibial nerve as it runs on the proximal posterior surface of the gastrocnemius muscle. The key to success in this model is the ability to adjust remotely the stimulating current and to choose a stimulation pattern that generates high force contractions with minimal disturbance to the subject. Using the new miniVStim device developed between Vienna and Liverpool we are able to programme an ‘adaptation’ pattern so that the subject is accustomed to the sensation of muscle activation at a low level before the loaded contractions are made. With stimulation in one session per day of 5 sets of 10 repetitions at 100Hz (2s ON 2s OFF) and 2.5 minutes between sets, we have achieved hypertrophy of the tibialis anterior muscle giving an increase in wet weight of between 11,5 and 13,7% in 5 rats over 4 weeks. We will use this model to investigate further the sensitivity to hypertrophy of the various fibre types and the cellular pathways that are activated in this response., One of the main determinants of the size of neural implantable pulse generators is the size of the battery. The challenge for engineers is to design devices that are small in volume whilst fulfilling their stimulation task as long as possible. Efficient stimulation methods are crucial for their success. Wongsarnpigoon pointed out three different types of efficiency relating to nerve activation.1 A “charge-efficient” stimulation has the positive effect of reducing tissue damage. As the battery size is proportional to the maximal instantaneous power required, “power-efficient” stimulation could reduce battery-size and therefore the overall size of an implant. An “energy- efficient” stimulation on the other hand, increases the battery lifetime. We have compared different waveforms according to their “energy-efficiency”. Six different waveforms have been investigated (rectangular monophasic, rectangular biphasic, rectangular biphasic with interphase gap, gaussian biphasic, exponential biphasic, asymmetric rectangular biphasic with interphase gap) to clarify some of the potentially useful efficiencies noted in other studies.2-7 Another interesting aspect that has been investigated in our latest experiments, is a comparison between monopolar and bipolar stimulation, and some investigation of the transition between the monopolar and the bipolar configuration in terms of stimulation efficiency. For bipolar stimulation, two stainless-steel loop electrodes were placed under the common peroneal nerve of rats under buprenorphine/isofluorane anaesthesia. In the monopolar situation the anodal electrode was a hypodermic needle placed under the skin more than 50mm away from the cathode. The cathode was the same in both cases. The isometric force produced at optimal length by the extensor-digitorum-longus muscle was measured using a load-cell. Our results showed a noticeable difference between monopolar and bipolar stimulation. Monopolar stimulation showed generally higher energy levels than bipolar stimulation to activate the motor neurones of the common peroneal neve. While the introduction of an interphase-gap increased the threshold current, where the force was 10% of the control-force, in the bipolar case, a reduction was observed in the monopolar situation. In previous bipolar experiments we found an increased energy requirement for threshold activation with asymmetric waveforms while the monopolar stimulation again showed a reduction. The results show similar effects as described in literature when using monopolar stimulation. Nevertheless we achieved different results for the bipolar stimulation regime. It is therefore important to bear these differences in mind, when designing electrodes and patterns of stimulation to improve stimulation efficiency., According to PubMed roughly 10% of the annually added publications in the Life Sciences describe findings obtained from animal models. Since half of these studies are done in mice and rats it can be assumed that there is a need for implantable electrical stimulators which are flexible, reliable and small enough (~1 cm3) that implantation is possible in mice. It is important that animals do not have to be isolated during stimulation periods and that they can run freely. MiniVStim 12A is a battery powered implantable electrical stimulator able to deliver constant current monophasic, rectangular pulses up to 2mA and 1ms pulse width (@1kOhm). It is easy to use because the required stimulation pattern is preprogramed during manufacturing. On, off or different stimulation patterns can be cyclically activated with a strong magnet, also through the skin. This implant has an outer diameter of 15 mm and a volume of 1.2 cm3. MiniVStim 12B has the same mechanical dimensions but can be fully programed via a transcutaneous bidirectional data link. Both types of implants are already successfully used in studies.1 The latest generation of implants is represented by the new MiniVStim18B. It is slightly larger (22mm outer diameter, 5.3 cm3) than its predecessors but offers an 8 fold longer battery life. Additionally, it can deliver biphasic constant current pulses and extends the stimulation parameter range up to 8mA at a maximum output voltage of 10V and with a pulse width of 5ms (@1kOhm) for monophasic and 2x5ms for biphasic pulses. Lifetime is strongly dependent on the chosen stimulation pattern. For example, monophasic stimulation with a duty cycle of 20% (20% on, 80% off time) and 2mA, 100Hz, 250µs pulse width, 1kOhm load, leads to a battery lifetime of 300 days and when stimulating with 8mA life time comes to 70 days. Under the same circumstances except choosing stimulation frequency of 10Hz a lifetime of 1000 days and 450 days could be expected. The very low standby current consumption (, Muscle atrophy as part of the ageing process also affects the larynx, where it constitutes the major cause of presbyphonia. Current treatment options are mainly conservative or phonosurgically based and are far from being satisfactory. Electrical stimulation of motor neurons constitutes a promising strategy. Materials and Methods: Using aged sheep as an animal model electrical chronic stimulation of laryngeal muscles was achieved via a mini-electrode that targeted the right recurrent laryngeal nerve (RLN; unilateral stimulation). Functional electrical stimulation (FES) implants were programmed to deliver a pattern able to evoke supramaximal muscle stimulation over a period of 29 days. At the end of the study, vocalis and posterior crico-arytenoid muscles were excised and analyzed molecularly and histologically. To quantify the expression levels of genes related to distinct muscle fiber types, a real-time PCR (RT-qPCR) analysis pipeline was newly established. Results: First results showed a shift towards larger muscle fiber diameters of the stimulated side, compared to the unstimulated control side. Based on this, chronic electrical stimulation of the RLN can induce hypertrophy of the vocalis muscle even after a relatively short stimulation period of 29 days. Upcoming trials will focus on longer stimulation periods as well as more intense stimulation algorithms., Vocal fold paralysis is a pathological motion impairment of one vocal fold, mostly caused by laryngeal nerve damage. If the vocal fold does not reinnervate a flaccid paralysis occurs due to denervation of the vocalis muscle and its atrophy.1 Patients with unilateral vocal cord paralysis suffer from hoarse and weak voice since there is always a remaining glottic gap during phonation. Today’s standard treatment of unilateral paralysis includes surgical medialization through either injection augmentation or laryngoplastic framework surgery.2 We want to investigate whether it is possible to selectively stimulate the denervated muscle fibers of the vocalis without causing pain or excitation of sensory nerve fibers or activation of innervated muscles in the neck region. The goal is 1. a verivication of functionality for screening and 2. a strengthening and increase in total volume of the target muscle on order to improve voice quality in patients with unilateral paralysis. In combination with voice therapy also electrical stimulation of laryngeal muscles has alraedy been used in order to achieve hypertrophy.3 Furthermore research with functional electrical stimulation of patients with long time denervated limb muscles showed very promising results.4 The selective stimulation of denervated muscles has been investigated in rabbits with unilateral paresis of the recurrent laryngeal nerve. It could be shown that with triangular ramping and very long pulses (> 200ms) the afferent nerve fibers where not stimulated but only denervated muscle, with change in muscle fibers confirmed through histology.5,6 It is to be investigated if these findings can be repeated with surface electrodes positioned in the neck area and successful stimulation of the denervated vocalis muscle can be performed without causing pain and excessive contraction of neighboring neck muscles rendering treatment impossible. The optimal stimulation parameters for this application and ideal position of the surface electrodes have yet to be investigated., Facial nerve paralysis as a peripheral nerve injury results in neuromuscular atrophy. The symptoms include significant aesthetic, functional and often life-altering consequences. Several procedures such as Nerve Grafting, Facial Reanimation and Rehabilitation have been developed to treat functional and cosmetic aspects of this disease.[1] Nerve grafting is a sophisticated surgery, which requires experience but offers promising results. Although cable grafting is state of the art, the method suffers the disadvantage of long nerve regrowth time. [2]Facial Pacing systems too show promising results to treat facial paralysis. [3] [4] Former research showed good results stimulating denervated extremity muscles using FES.[5] Nevertheless this field of research is still lacking optimal stimulation settings to selectively recruit denervated atrophic or simply age-related atrophic facial muscles under non painful conditions. Methods: Several Devices are considered to investigate optimal stimulation settings. To encourage noninvasive screening methods for facial pacing, surface electrodes are used to estimate the optimal settings for stimulations. The use of surface electrodes causes the need for optimized electrode positioning, which is also investigated. Results: Martin et al. [6] showed that recruitment of denervated muscles requires exponentially shaped pulses with long phase durations(>200ms). The outcome of our investigation confirms these findings as well, showing best performance when recruiting paralyzed facial human muscles with biphasic long-duration impulses. It is crucial to position the surface electrodes appropriately in order to avoid stimulation of innervated muscles, for instance the masseter muscle. Conclusions: Surface electrodes, combined with the optimal stimulation settings, offer a screening possibility for facial pacing. Since muscles affected by age-related atrophy could be recruited too, further research is necessary to show effectiveness of training using the determined exponential patterns., Artificial stimulation of the vagus nerve, the main nerve of the parasympathetic nervous system, gained importance in the last years. Due to the modulatory interaction with the autonomous nervous system, the stimulation re-establishes the sympathovagal balance, counteracts over-inflammation responses or improves peripheral perfusion.1-3 Thus, the clinical applications have a wide range from depression to acute/chronic pain or cardiovascular dysfunction up to various neurological disorders.3-7 Neuromodulation is mediated via either implanted cervical, transcutaneous cervical/auricular or percutaneous auricular stimulation devices. While implanted stimulation devices are interrelated with high risks/costs and transcutaneous devices lack precision and require strong stimuli during their operation, percutaneous devices seem to avoid these drawbacks.8 Current percutaneous stimulators use needle electrodes in the auricle to stimulate afferent nerve fibers by the use of simple monophasic or biphasic stimulation patterns with the need for an additional reference electrode.5 No adaptation of the stimuli is possible to account for the specific pathology to be treated as well as the current physiological state of the patient.8 Our group has developed a multi-punctual percutaneous stimulator which operates three independent stimulation channels without any additional reference electrode.8 Stimulation patterns, with specific triphasic pulses, seem to reduce adaptation processes and to establish a pathology specific efficient stimulation. The pattern can be advantageously adapted throughout the stimulation duration to account for the current treatment state and physiological state of the patient [9]. Specific and precise positioning of needles - based on electrical and optical approaches10 - close to auricular nerves is performed, which is of high importance for efficient nerve stimulation. Preliminary studies of our group show positive effects of this percutaneous stimulation on heart rate variability, cerebral/peripheral blood perfusion, pain, sleep, diabetic food syndrome and cervical dystonia.1,6,8,9,11,12, Use of electrical stimulation (ES) of skeletal muscle as a tool to restore normal control of movement and ability to perform motor tasks has lately received increasing attention. Its capability to elicit muscle tissue contractions through the delivery of current impulses is commonly exploited in clinical settings,1 when damage to the nervous system, either central or peripheral, produces rapid denervation of muscle, resulting in weakness or paralysis. Possible mechanisms of muscle fibre recruitment have previously been studied using stereotyped electrical pulses delivered at variable pulse frequency, width and current amplitude.2 However, these protocols often exhibit several significant limitations, resulting in an overall decreased efficiency of contraction, ultimately leading to the development of muscular fatigue, as well as the elicitation of unpleasant symptoms. In the present study, the influence that different parameters of ES protocols exert on the efficacy of skeletal muscle cell contractions was investigated in skeletal myotubes in culture. The efficiency of a “noisy” stimulus waveform, derived from human muscle electromyogram (EMG) recordings, used as templates for the delivery of ES, was compared with conventional stereotyped 1 Hz and 40 Hz electrical stimulation delivery.3 EMG traces obtained by recording human gastrocnemius medialis muscle activity during sessions of real overground locomotion, were used to design the “noisy” stimulation pattern (EMGstim). ES protocol efficiency in inducing contractile activity of cultured skeletal muscle cells was compared by measuring intracellular Ca2+ dynamics and patch-clamp electrophysiological recordings. Collected data demonstrated that EMGstim was more efficient in inducing myotube cell action potential firing, [Ca2+]i changes and contractions, when compared with more conventional electrical stimulation using stereotyped rectangular pulses. Furthermore, it was demonstrated that EMGstim strength was also considerably lower than the minimum current amplitude required to induce contractions via canonical stimulation protocols. These results demonstrate the peculiar properties of the “noisy” EMGstim pattern to enhance the efficiency of muscle cell recruitment, minimizing charge transfer and therefore preventing possible tissue damage. We suggest this could represent a promising new approach for the optimization of ES protocols and for future design of electrical devices to stimulate the rehabilitation/recovery of weakened or injured muscles in human patients., Spinal cord injury is a traumatic injury of descending spinal cord tracts that alters the spinal neural circuitry.1-3 Spasticity is a common result of spinal cord injury (SCI) and can restrict daily living activities, cause pain and fatigue and, therefore, decrease the quality of life for SCI individuals.4-5 The aim of this study is to evaluate the effects of transcutaneous spinal cord stimulation (tSCS) on individuals with post-traumatic SCI for the alleviation of lower limb spasticity. Methods: In total, 8 subjects, 5 males and 3 females, aged between 31 – 63 years old (M = 49,9; SD = 11,5) were studied, with complete- and incomplete SCI. The evaluation of the effects of tSCS was done by means of electrophysiological evaluation and evaluation of residual motor control functions. The protocol consisted of four stages: first assessment/evaluation (control data), application of 30-min tSCS, a second assessment immediately after the treatment and a third assessment two hours after stimulation. The assessments consist of the evaluation of the spasticity level through the Ashworth scale, clonus beet quantification, 10-m walking test (if possible), electrophysiological evaluation (Brain Motor Control Assessment, BMCA [1]) and the Wartenberg pendulum test (WPT).6 Results: The index of spasticity R2n, derived from the WPT is the primary variable and the results of the WPT show increase in muscle tone in four subjects while the others presented average index values ≥ 1, indicating non-spastic conditions. During the BMCA, there was a significant difference of the normalized EMG activity of all muscles before the stimulation and immediately after stimulation for all participants, which indicates amelioration of intrinsic phasic and extrinsic spasticity. Enhancement of motor control was also observed. Conclusion: The similarity of the effects of tSCS with those induced by epidural SCS, strongly suggests that both techniques are able to activate similar neural structures. From our results we can see that the application of low-intensity tSCS for 30 minutes leads to the alleviation of lower limb spasticity regardless of the clinical profile of the subjects and enhancement of voluntary motor control in the motor incomplete SCI subjects., Whether via sarcopenia, cachexia, or sequela of trauma, the degeneration of muscle has been consistently identified as an independent risk factor for mortality.1 Many recent investigations have realized the quantitative potential of CT image analysis to describe skeletal muscle volume and quality.2-4 However, the optimum metric for assessing these data remains debated. Identifying a novel quantitative method for muscle assessment in this regard would allow for the generalizability of such studies to clinical practice and therefore aid in the indication of compensatory targets for clinical intervention. While there is much extant literature reporting the use of average HU values to investigate muscle quality and its utility as a comorbidity index, standardized methods for this analysis have yet to be defined, and no existing studies have explored the utility of an entire radiodensitometric distribution. Herein, we hypothesize that rigorously quantifying entire HU distributions can elicit much more information regarding muscle quality than extant methods that, to date, only utilize average HU attenuation values. This study reports the development and use of this method, wherein we assess upper leg muscle quality utilizing nonlinear trimodal regression analysis with radiodensitometric distributions from computed tomography (CT) scans of a healthy young adult, a healthy elderly subject, and a spinal cord injury patient exhibiting complete lower motor neuron denervation. Results from this assessment highlight the utility of entire HU attenuation value distributions and identify novel parameters from these analyses that could provide further insight into how muscle degeneration can be optimally quantified., Physical activity plays an important role in preventing muscle atrophy and chronic diseases in adults and the elderly. Voluntary physical exercise is not always feasible and other therapies should be applied such as electrical stimulation (ES).1 The process of calcium storage, uptake and release (EC-coupling) and, in a broader framework, Ca2+ cycling is essential in activity-induced muscle adaptation.2 De-codification of Ca signals is accomplished by an heterogeneous class of decoders such as transcription factors (i. e NFATc1, PGC1α) kinases (CaMks) and phosphatases (Calcineurin).3,4 We investigated the effects of either passive ES (acute or long-lasting) or voluntary physical exercise (leg press, LP), on expression of Ca2+ handling proteins of the sarcoplasmic reticulum and on the activation of key Ca2+ signal decoders in human vastus lateralis (VL) of elderly sedentary persons. Muscle sections and total homogenates were obtained from biopsies performed before and seven days after nine weeks of ES, before and 30 minutes after one session of ES and before and seven days after LP volitional exercise on a group of volunteers.1,5 Expression of Sarcalumenin, SERCA and p-CaMKII were evaluated by western blot while NFATc1/PGC1α nuclear translocation and muscle remodeling were determined by immunofluoresence. Evidence of kinase and phosphatase activation after both ES and LP were obtained. NFATc1 translocation to nuclei 30 minutes after one ES session training was obtained, after 9 weeks of ES NFATc1 translocation lasted at least 7 days. Moreover, mixed SERCA 2/MHCII fibers and Ca2+ handling proteins Sarcalumenin and SERCA 2 increased after ES. Conclusions. These results show that ES influences expression of muscle components deputed to Ca2+ cycling and promotes fiber remodeling essential to improve muscle performance in old sedentary people. This work identifies a set of molecules which are modifiable by ES, easy to measure and gender and age independent suitable as biomarkers for skeletal muscle response to neurorehabilitation., Most organisms experience changes in regenerative abilities through their lifespan. The principles that underlie the decline in regenerative abilities through lifespan are currently being unraveled. However, it is already clear that both cell-intrinsic (such as cellular senescence) as well as cell-extrinsic (such as alterations in the regenerative environment) factors play significant roles. During aging, numerous tissues exhibit a progressive decline in homeostasis and regeneration that results in tissue malfunction, pathology and degeneration. With age, both stem and progenitor cells undergo a series of alterations including loss of self-renewal capacities, altered proliferative activity, declines in functionality and potency. These changes have been shown to contribute to the dysfunction and degeneration of a number of tissues and systems including most epithelia and endothelia, blood, skeletal and cardiac muscle, bone, cartilage, the peripheral and central nervous system (CNS), and organs such as the pancreas, liver, kidney and lungs. The regenerative capacity in the skeletal muscle system experiences a marked decline with age in many organisms, as reflected by a decrease in the generation of muscle fibres and an increase in fibrotic tissue upon muscle injury. In humans, this is an underlying cause of sarcopenia, the loss of muscle mass that accompanies late aging. The decline in muscle regenerative potential is largely attributed to changes in satellite cells, the muscle stem cells, which undergo age-related declines in proliferative and myogenic capacities. Indeed, satellite cell numbers decline gradually in mammalian muscles with advancing age. Age-specific changes have also been reported for mesenchymal stem cells (MSCs), stromal cells that can differentiate into multiple cell types such as osteoblasts, chondrocytes, and adipocytes. Alterations include a loss in chondrogenic potential leading to impaired chondrocyte formation, which results in decreased cartilage repair in aged mammals. Furthermore, studies in human-derived bone marrow MSCs revealed age-dependent decreases in their capacity to differentiate to osteoblasts, which are related to increases in the level of MSCs senescence and apoptosis upon aging. Together, these alterations contribute to conditions such as osteoporosis and reduced bone repair capacity that are characteristic of human aging. A number of cellular and molecular mechanisms have been associated with the decline in regenerative abilities observed during aging in humans. These include intrinsic factors such as genomic instability (including telomere attrition), mitochondrial dysfunction, epigenetic changes, loss of proteostasis and metabolic alterations, as well as cell-extrinsic factors such as disruption of the regeneration niche and alterations in systemic signals. Though most of these factors can contribute to age-related impairment in regenerative capacity, a consensus on their relative importance in this process is currently lacking. Furthermore, emerging evidence suggests a high degree of interconnectivity between them, stressing the importance of identifying the common denominators. The advances in our understanding of the factors that modulate the decline in regenerative abilities have pinpointed areas of potential clinical relevance. In this view examining the influence of systemic factors on aged progenitor cells from tissues activated during neurorehabilitation training may prove to be clinically relevant. Our activity will be focused on the study of 3 different markers present in blood and tissue biopsies, such as long and small noncoding RNAs,1-3 growth factors,4-6 and transcription factors, such as NFAT.7,8

In 1940 and thanks to L.E. Heilbrunn1 the scientific hypothesis on the possible role of calcium ion in muscle contraction was published. Nevertheless, Ca2+ was not recognized as an indispensable factor of contraction up to the paper of A. Weber that appeared in 1959 and in which it was shown that Ca2+ exerts its effect having the filament proteins as target; just a ionic concentration of 0.2 mM was capable to induce the muscle contraction.2 The goal of the calcium ion on the filaments is a protein, troponin C, identified some years later by Ebashi and Kodama.3 It is important to note that a few years earlier in 1954, at the same time and in the same issue of the Nature, AF Huxley and Niedergerke4 and HE Huxley and Hanson,5 published the hypothesis of “sliding filaments theory” on the mechanism of action of muscle contraction. Starting from the seventies of the last century, the studies on Ca2+ exploded (together with calcium related proteins or Calcium Binding Proteins or CBP) linked to the mechanisms of signal transduction with problems and surprises related to mechanisms of phosphorylation and de-phosphorylation of protein substrates. In particular, in skeletal muscle were identified: the principal proteins (Ryanodine receptor or RYR, Dhyidropiridine receptor or DHPR and Calsequestrin or CSQ) capable of coupling the electrical phenomena occurring in the sarcolemma with the release of Ca2+ from the terminal cisternae of the sarcoplasmic reticulum.6 To assure a precise coordination of Ca2+handling regulation during contraction of skeletal muscle, several different CBP, as well as S100, were proposed.7 In striated muscle, S100 demonstrated highest expression in cardiac muscle, followed by slow twitch and fast twitch skeletal muscle, respectively. S100 binds specifically to a CaM binding site on RyR1 and enhances the Ca2+ release flux resulting from coordinated opening of multiple RyR1s.8 Muscle requires two cofactors to function: load and innervation. If both or one of the them are impaired, muscle becomes atrophic through a mechanism of protein degradation that develops into proteasomes and lysosomes (autophagy).9 However, the muscle is a surprising tissue because even when it seems to have almost disappeared, as a consequence of a long time denervation, it is able to regenerate, if exposed to daily cycles of functional electrical stimulation (FES).10 Ageing of muscle tissue is a complex process (Sarcopenia) that is usually associated with a decrease in mass, strength, and velocity of contraction triggered by reactive oxygen species (ROS) that have accumulated throughout one’s lifetime. Exercise as a method to prevent or at least delay sarcopenia has been discussed in many scientific reports. While on the one hand, it seems clear that exercise is effective in reducing the loss of muscle mass, on the other it appears that physical activity increases both the mechanical damage and the accumulation of free radicals as results of increase in the aerobic metabolism of the involved muscles.11 How to take advantage of physical exercise, limiting the adverse effects, is the main goal for further successful managements of age-related power decline., The marked fall in birth and mortality rates that has taken place over the last century has modified the demographic structure of the Italian population: there are now 13,220,000 persons over 65; they represent more than 21% of the population and make Italy the country with the highest percentage of elderly persons in Europe (it has been estimated that in 2050 persons over 65 will exceed 33% and those over 85 will make up approximately 8% of the population).1 The primary aim of geriatric and gerontology research is that of gaining information to reduce disability and increase the functional autonomy in elderly adults in order to guarantee individuals’ greater wellbeing during their later years. As far as physical disability is concerned, particular interest has been dedicated to sarcopenia which has been defined by the European Working Group on Sarcopenia in Older People as a syndrome characterized by the progressive and generalized loss of skeletal muscle mass and strength. It has been estimated that the prevalence of sarcopenia in the general elderly population in Italy is at 10%; those affected have three times the risk of physical disability and two times the risk of mortality.2, Both strength and power developed by human skeletal muscles decline with increasing age.1,2 The athletic world records of the Master athletes of age ranging from 35 years to 100 years are an excellent proof of such decline in every track and field competition. The world record performances of running, jumping and throwing events can be transformed into dimensionless parameters proportional to the power developed in the trials. Such parameter ranges from 1 for the Senior world record (i.e. the maximum human performance) to lower values for the Master athletes of increasing age down to 0 for a null performance.3,4 With this procedure the declines of the power parameter with increasing age can be analysed and compared: the trend-lines start to decline very close to the age of 30 years and arrive to 0 around the age of 110 years for every athletic discipline (running, jumping and throwing).5 The comparison of the various trend-lines shows significantly different rates of decline. Each declining trend-line reaches a “critical” threshold at different ages for the running, jumping and throwing activities. Such thresholds indicate different age limits for most of everyday tasks: walking, climbing stairs and lifting weights above a table. The decline of the Master world records, transformed into a dimensionless power parameter declining from 1 toward 0 with increasing age is the decline of the power developed by each one of us, starting from 1 in our youthful age and declining toward lower values with increasing age. There are no reason, for each one of us, to decline differently from the world record-men, provided that each of us remains in stable fitness condition without disabling pathologies., The age-related loss of skeletal muscle mass, and strength contributes to disability and mortality associated with advanced aging. The aetiology of this syndrome is multifactorial including: alterations in endocrine function, chronic syndromes, and inflammation, however muscle deconditioning caused by reduced mobility appear to play a pivotal role in this phenomenon. Recently, utilizing the human-model of the oldest-old (~90 years old) and varying levels of limb disuse, our group demonstrated that the progressive fall in skeletal muscle use, plays a significant role in the exacerbation of cellular ageing and the loss of muscle mass.1 Moreover, in a different study we investigates both in vivo and in vitro muscles properties, of locomor- and non-locomotor muscles, which experience differing degrees of disuse during the lifespan.2 The outcomes of this study indicate than in the oldest-old, neither advanced aging nor disuse, per se, impact intrinsic skeletal muscle function, as assessed in vitro and in vivo, but volitional muscle function is attenuated by age and exacerbated by disuse. These observations imply that the limiting factors for the reduction in force generation capacity with advanced aging resides outside the contractile machinery of the skeletal muscle cells. Moreover, the loss of muscle mass is the consequence of a greater loss of muscle fibres, caused by denervation 3, and a reduction in size of the remaining fibres. Interestingly, the assessment of the single fibres utilized for contractile measurements did not reveal this latter effect. Therefore, this dissociation between atrophy and the more pronounced reduction in skeletal muscle voluntary force with advancing age imply that additional physiological mechanisms are responsible for this phenomenon. In this scenario it has been suggested that age- and disuse-related changes in neural control play a significant role in this decline in muscle voluntary force. Specifically with regards to aging, it has been documented that there is a significant age and neural-dependent decrease in dihydropyridine receptor (DHPR) functional expression that is responsible for an uncoupling of the excitation–contraction process, which results in an incomplete activation of the myofibrillar machinery.4 Moreover, increasing evidence points to a decline in neural influence on skeletal muscle at later ages, and this might also lead to changes in muscle structure which together result in excitation–contraction uncoupling. 5 Taken together, these studies support the concept that in vivo force characteristics, are largely dependent upon neural activity, including a possible involvement of DHPR, and consequently influence excitation–contraction coupling., Sedentary persons, but even healthy persons experience a steady decline in their life. Understanding the biologic changes that occur with age and their social implications is essential for a physiatrist to maximize an aging patient’s quality of life and functional independence. Sarcopenia, contributes to the functional consequences of aging because of changes in muscle fibers. By age 80 years, up to 50% of peak skeletal muscle mass can be lost, leading to functional decline. Age-related decreases in androgens and other growth factors may contribute to this process with selective loss of type II muscle fibers.1 Older adults have larger postural sway, slower gait velocity, and slower reflexes. Slow walking speeds have been associated with an increased risk of fractures, hospital admissions, institutionalization, and death. Postural hypotension, impaired proprioception, vision, and other sensory losses affect balance and coordination2,3 and increase the risk of falls. An aggressive multidisciplinary approach for the elderly may help to continue or resume a productive and functional existence. Multimodal approach includes: patient education, exercise, physical, occupational, and kinesiotherapy, the use of the orthotics, the physical modalities, and psychosocial, medical and pharmacologic treatment. Physical Activity and exercise: Regular physical activity reduces the risk of many conditions associated with aging, including cardiovascular disease, diabetes, and stroke and it is recommended as treatment for many of these same conditions. Physical training increases muscle mass, helping to combat age-related sarcopenia.4 Balance training reduce falls and injuries from falls. Physical activity decreases risk of dementia and cognitive decline in older adults, it improves mood and is effective for treatment of depression. With appropriate guidance and a personally tailored exercise program based on patient’s fitness goals and functional abilities, older persons can achieve improvements in performance and general well-being. Exercise programs generally consist of four major components: strength, endurance, balance and proprioception, and flexibility.5 Exercise is therapy: some conditions warrant further diagnosis and treatment before beginning exercise.6 A staged prescriptive approach is better, first targeting the urgent needs of the patient, and then including other modalities. Supplemental Treatments: Modalities are physical agents that are used to produce a therapeutic response in tissues. They are supplemental treatments in a multimodal management program that should always include exercise, physical, occupational, or kinesiotherapy regimens. The major modalities used in common practice are heat, cryotherapy, lasertherapy and electrotherapy. Before selecting a modality, one must understand the physiologic effects each exerts on tissues. The target tissue, the depth and intensity of heat or cooling desired, and patient characteristics are all factors to consider when writing the prescription: especially in the elderly, the use of modalities is not without limitations. It is of fundamental interest of aging Europe the healthcare and research for the elderly. By employing a multimodal approach the physiatrist may restore, preserve, and, hopefully, even enhance cognitive and motor functions of older population, improving their quality of life.7,8, All permanent or progressive muscle contractility impairments (including aging-related muscle power decline and ventilation insufficiency) need permanent managements. Beside eventual pharmacology therapy a home-based physical exercise approach is helpful. Awaiting development of implantable devices for muscle stimulation, i.e., of electroceuticals, as effective as pace-makers for cardiac arrhythmias or cochlear implants for hearing loss, education of hospitalized patients to take-home physical exercise managements is an effective low cost alternative. Frail elderly due to advanced age or associated diseases are often hospitalized for long periods of time. There, their already modest amount of daily physical activity is reduced, contributing to limit their independence up to force them to the bed. Inspired by the proven capability to recover skeletal muscle contractility and strength by home-based Functional Electrical Stimulation (h-bFES) and functional Magnetic Stimulation (fMaS) even in the worse cases of neuromuscular traumatic injuries,1-3 but, mostly, by common sense, we suggest a short (15-20 minutes) daily sequence of ten simple physical exercises that may be performed in bed (Bed-Gym). In a few days in hospital Bed-Gym may increase muscle strength, fatigue resistance and independence in daily life activities. Bed-Gym helps also to mitigate the bad mood that accompanies mobility limitations, strengthening confidence in recovering partial or total independence.4 Continued regularly, Bed-Gym may help to maintain the independence of frail older people and to reduce the risk of the possible serious consequences of accidental falls. Bed-Gym may also mitigate eventual high blood pressure, a major risk factor in elderly.5

Injury to peripheral nerves is not uncommon, and spinal injury with fracture dislocation of the vertebrae can damage roots as well as the cord. Both types of trauma leave the corresponding muscles denervated, resulting in a flaccid paralysis and catastrophic loss of muscle mass. When this occurs in the lower limbs, a loss of cushioning over bony prominences, combined with a deterioration in skin condition, greatly increases the risk of developing pressure sores. Furthermore, bones become osteoporotic and the wasted appearance of the affected limbs can be a source of great distress to patients. Although there has been a longstanding interest in the potential therapeutic value of electrical stimulation of denervated muscles in humans it was always regarded as impractical. In the absence of the nerve or intramuscular nerve branches the muscles must be excited directly. The charge delivery needed for this is so high that the approach has been frustrated by regulatory restrictions and a lack of suitable equipment. More recently, however, these problems were addressed, and the value of stimulation was clearly demonstrated, in a remarkable research programme pursued with the support of the EU Commission Shared Cost Project “RISE”. It is hard to explore the benefits and limitations of the technique in patient groups, which are small and inhomogeneous in age, nature and duration of injury, and compliance. Moreover, the intense surface stimulation elicits co-contraction of antagonistic muscle groups, which interferes with the measurement of force or torque. In Project “RISE”, the important clinical work1 was therefore complemented by Laboratory studies. The usual model, total sciatic section in the rat, is unsatisfactory on two counts. First, denervated rat muscles show evidence of extensive degeneration in a few months,2 differing in this respect from the muscles of other species. Denervated human muscles, in particular, do not undergo significant necrosis for at least a year post-injury (U. Carraro, personal communication), and we could confirm that this was also true of the rabbit.3 Second, most published studies of stimulation in the denervated rat start at, or soon after, the moment of lesion, which does not correspond to the clinical situation. The “RISE” experimental studies to be discussed were conducted by the Muscle Research Group, University of Liverpool, UK, in a long-term model of established selective denervation in the rabbit. The Department for Biomedical Engineering and Physics, University of Vienna, designed the implantable stimulator,4 ultrastructural studies were performed at the Interuniversity Institute of Myology, Chieti, and valuable input was provided by clinical colleagues at the Ludwig Boltzmann Institute of Electrostimulation and Physical Rehabilitation, Wilhelminenspital, Vienna, Austria. Through this joint programme we were able to assemble comprehensive physiological, histological, biochemical, and ultrastructural data on muscles subjected to selective denervation alone for up to 1 year, and muscles subjected to denervation and stimulation for up to 3 months.3,5,6 Although this data settled several issues, and may serve to take some of the guesswork out of the design of stimulation protocols for clinical use, it raised some tantalizing questions. These will be worth addressing in future studies., This work initially began within a study called AGES-Reykjavik Study, that was initiated to examine genetic susceptibility and gene/environment interaction as these contribute to phenotypes common in old age, a collaborative study between the National Institute on Aging, NIH and the Icelandic Heart Association.1 In this frame we are assessing over 3,200 patients from ages 66-93 for changes in muscular and fat content within thigh cross-sections as a function of various measured conditions and pathologies. This wealth of data is historically unique in both its size and variety of explored elderly patient conditions, and discerning how best to analyze both the CT images and patient database could greatly impact modern geriatric medicine and the scientific understanding of aging. We have begun to develop a novel computational method for analyzing each patient’s CT contrast histogram by employing analytical methods that allow to create subject specific muscle profiles.2,3 These profiles are used to assess and correlate muscle quality with subject co-morbidities. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration., Resistance training (RT) is still nowadays considered as the main counter actor of skeletal muscle loss (sarcopenia) and weakness in ageing scenarios.1-3 Traditionally, Conventional RT consists in lifting and lowering a constant external load: however, a physiological de-recruitment of motor units (MUs) naturally occurs during the load-lowering, eccentric phase.4 A substantial number of works reported considerable strength gains after Conventional RT in older adults,5 but only few investigated the muscular adaptations in response to pure concentric vs. pure eccentric RT. As widely known, according to the F-V curve, pure eccentric actions can develop greater forces compared to concentric ones,6 and therefore greater training loads could be used. Thus, the general consensus up to date was that eccentric exercise could potentially be preferable over concentric in terms of muscle growth promotion. Lastayo et al (2003),7 using backward cycling to maximise eccentric loading (avoiding de-recruitment of MUs), showed the superiority of pure eccentric RT to traditional RT in enhancing muscle mass and strength in older individuals. This apparent superiority of eccentric RT in combating sarcopenia and weakness in old age, prompted a series of investigations by our Lab to explore the mechanical, structural, molecular and metabolic mechanisms of such adaptations. When comparing 14 wk 3 times/week of pure eccentric RT (ECC) vs Conventional RT (CONV RT) in older males aged 65-77 years, both regimes resulted in a similar increase in vastus lateralis (VL) muscle size (12%) but with distinctly different VL architectural changes.4 The ECC RT promoted a greater increase in VL fascicle length (Lf) (20%) than CON RT (8%), while CON RT promoted a greater increase in pennation angle (PA) (+35%) than ECC RT (5%). When investigating the underlying molecular mechanisms of these findings in a following study in young people,8 we found morphological adaptations similar to those observed in the older individuals (increase in VL vol of +6% with ECC RT and +8% with CON RT (pure concentric RT), greater Lf increase with ECC (12%) than CON (5%) and greater increase in PA with CON (30%) than with ECC RT (5%). Interestingly, MAPK activation (p38MAPK, ERK1/2, p90RSK) was specific just to ECC RT, while neither mode affected AKT-mTOR or inflammatory signalling 30 min after exercise. Hence the present findings do not support the belief that ECC loading leads to greater hypertrophy and strength gains than CON loading, neither in young nor in older individuals. However, muscle hypertrophy is obtained through distinctly different architectural adaptations: while ECC RT appears to stimulate preferential addition of sarcomere in series, CON training seems to promote preferential addition of sarcomere in parallel. The main difference in the hypertrophic responses to CON and ECC RT may actually be not in the amount but then in the location where sarcomere addition occurs (Franchi et al. unpublished)., We have used micro-CT imaging with iodine potassium iodide contrast to provide high resolution images of cardiac muscle1,2 and skeletal muscle.3 The technique is based on differential attenuation between muscle cells and surrounding connective tissues, which in turn is based on the differential uptake of aqueous iodine into the various tissue types. Cardiac myocytes are on average only about 20 microns wide, and so are just at the limit of resolution of this technique, using high resolution scans of sample preparations we are beginning to reveal the much debated micro-anatomy of the complex cardiac mesh. There is considerable interest in the internal 3D structure of cardiac muscle, because the orientation and interweaving of myocyte aggregations determines the functional movement of the walls that provides the remarkable pumping function of the heart. The myocytes do not produce a force against a unique external structure but rather populations work both together and antagonistically to pressurise and to eject the momentarily contained blood volume. The disposition of the myocyte aggregates also influences the temporal sequence of their own rhythmic depolarisation, because conduction is faster in the long axis of the myocytes than in the short axis. In skeletal muscle in which individual fibres are unbranched and cylindrical, it is possible to distinguish individual fibres using micro-CT3 In skeletal muscle, the mechanical situation is often thought of as simpler in that fibres act relatively independently to generate a force at their myotendinous junction which acts to draw insertion points towards the fibre origins. However we know that fibre orientation can also be complex in skeletal muscles, particularly in the myotendinous regions. There is also evidence of transverse distribution of force such as illustrated by PA Huijing.4 Physical therapists are particularly interested in painful trigger points in muscle that can be identified by ultrasound examination as hypoechoic and with increased resistance to blood flow.5 They are often interpreted as areas of fascial tissue that have sub-normal motility, or areas of adhesion, restricting the smooth movement of one muscle relative to another, or one part of a muscle relative to another. Physical therapy in this case aims to improve internal movement. Micro CT of fixed skeletal muscle, along with ultrasound of working muscle will help us to understand the internal as well as the external transmission of force., Great technologic and clinical progress have been made in the last decades in identifying genetic defects of several neuromuscular diseases. However, the diagnosis is usually challenging, due to great variability in genetic abnormalities and clinical phenotypes, the complexity of the molecular genetic approaches and the poor specificity of complementary analyses. Muscle biopsy represents the gold standard for the diagnosis of genetic neuromuscular diseases, clinical imaging of muscle tissue is an important diagnostic tool to identify and quantifies muscle changes. Radiologic imaging is, indeed, increasingly used as a diagnostic tool to describe patterns and extent of muscle involvement.1-5 Computer tomography (CT) and ultrasound (US) due to some of their shortcomings have given way in the diagnosis of neuromuscular diseases to Magnetic resonance imaging (MRI), which is a technique that doesn’t use ionizing radiation, has a higher contrast resolution and allows early recognition of the initial phases of inflammation and dystrophies fatty degeneration of some early myopathies. In conclusion, radiologic imaging is increasingly playing a relevant role in neuromuscular disorders, in particular in those of genetic etiology that are difficult to be characterized., Muscle fibers are multinucleated single cells originating from myoblasts fusion which, under the control of developmental cues, develop distinct molecular composition and physiological properties. The four basic fiber types (slow type1, and fast type 2A, 2X and 2B) vary greatly in their contractile and metabolic properties and are identified based on the molecular properties of the corresponding myosin heavy chain isoforms.1 Interestingly, primary muscle diseases and metabolic disorders often affect, or spare, specific fiber types. Despite recent advances in mass spectrometry (MS)-based proteomics,2,3 single cells have been beyond reach so far, preventing fiber type-resolved studies in skeletal muscle. Although large compared to average mononuclear cells, muscle fibers contain relatively limited protein amounts. They have a highly unfavorable dynamic range dominated by highly abundant sarcomeric proteins, which has also limited proteomic studies at the whole muscle level. Furthermore, a muscle has a heterogeneous composition, including not only muscle fibers but also connective and adipose tissue, blood vessels and nerves, with possible variations of their relative proportions in pathophysiological conditions. With these challenges in mind, we set out to develop a high-sensitivity proteomics workflow that has allowed us to obtain the proteome of single mouse muscle fibers. We could also analyse and compare the proteomes of different segments of the same fiber. Our strategy allowed unbiased fiber-type assignment, leading to the discovery of novel type-specific features. Our results show fiber type-specific patterns of mitochondrial specialization, revealing alternative utilization of metabolic intermediates that fine-tune fiber types to their tasks.4 As the single cell proteomics analysis performed here is rapid and robust, it can be applied to a wide variety of physiological and pathological conditions., Functional electrical stimulation (FES) has been used for over 15 years in equine rehabilitation and hundreds of case studies show the positive clinical outcomes of FES to reduce equine muscle spasms.1 FES is the most applicable electrotherapy for equine rehabilitation due to the ability of the device to stimulate deep enough to reach the skeletal support muscles of the horse while obtaining a high compliance.2 FES is also used for early mobilization after injury or surgery to obtain controlled, precise movement while the horse is confined.3 In addition FES has been used as a treatment for recurrent laryngeal neuropathy in horses.4 The goals of FES treatments for use in equine rehabilitation is to: 1. Reduce muscle spasms therefore reduce pain 2. Produce symmetrical muscle movement to improve skeletal alignment 3. Stimulate deep skeletal support muscles 4. Reeducate muscle memory to improve movement patterns 5. Strengthen muscle 6. Reduce muscle atrophy and 7. Recruit fast and slow twitch muscle fibers in all stages of rehabilitation. The fundamental purpose of the FES treatments in horses is to improve functional movement so that the horses are more comfortable and balanced, and therefore can perform better and have fewer chances of injury due to improved mechanics. The FES system used on horses has specific characteristics that may or may not be found in human FES systems. The signal must be produced by a microcontroller so that the clarity and control of the signal is precise. The stimulus must be able to reach comfortably 20cm deep to activate the deep core muscles of the horse. The system must be portable and must be able to be run off a battery so that no external source of electricity is needed. The FES system for horses weighs about 2 pounds and attaches to a surcingle, which is strapped around the thorax of the horse. The waveform is rectangular, with a zero net charge and the pulse duration is 250 microseconds positive/negative. Carbon electrodes are placed in a pad about 55cm long, which is used for treatments to the axial skeleton. Self-stick electrodes are placed on the neck and are also used for other site-specific applications. The typical voltage to produce strong muscle contractions, resulting in functional movement, along the top line of the horse and on the neck is approximately 7-9 volts. Treatment time varies from 20-35 minutes. Typically, 2 adjacent sites are treated on the same day, for example, the neck and thorax. A clinical change in the reduction of the severity of muscle spasms usually occurs after 2-4 FES treatments. Positive results utilizing this FES system and protocols have been obtained by a variety of veterinarians and equine physiotherapists. Functional electrical stimulation has produced encouraging rehabilitation outcomes in horses, however it is not extensively utilized in veterinary medicine. Equine case studies have shown examples of the use of FES to reverse muscle atrophy and decrease muscle spasticity. Exploring the application and outcomes of the use of FES for rehabilitation in horses may provide some interesting information for the utilization of FES in human rehabilitation., Functional Electrical Stimulation (FES) has been used extensively over several decades to reverse muscle atrophy during rehabilitation for spinal cord injury patients.1-3 The benefits of the technology are being expanded into other areas, and FES has been recently utilized for injury rehabilitation and performance enhancement in horses.4 FES can obtain precise, controlled functional movement and therefore can be used to initiate conservative movement early in the rehabilitation plan, as well as obtain more aggressive movement during the later stages of healing. Six retired horses, that had been previously used mainly for dressage riding, were selected for this study. The horses ranged in age from 10 to 17 yr and had all been clinically evaluated by veterinarians for axial musculoskeletal skeletal pathologies and none had been noted. Clinical evaluation found epaxial muscle spasms in all horses with minimal to no pelvic extension when manually palpated. FES treatments were performed on the sacral/lumbar region 3 times per week for a period of 8 weeks. The Modified Ashworth Scale for grading muscle spasms found a one grade improvement after approximately 4 FES treatments, indicating improved functional movement of the sacral/lumbar region, supporting the evidence by clinical palpations that a reduction in epaxial muscle spasms occurred. Skeletal muscle biopsies Pre and Post FES treatments were obtained from the longissimus lumborum muscle at a depth of 3 cm on the same side of each horse. Cryosections were stained with a Hemotoxylin-Eosin (H-E), and nicotinamide adenine dinucleotide tetrazolium reductase reaction (NADH-TR). The eventual size change of the muscle fibers due to FES or co-morbidities were evaluated by morphometry in H-E and NADH-TR stained cryosections, while in the NADH-TR slides the density and distribution of mitochondria were also determined.5 Main results of the morphometric analyses were: 1) As expected for the type of FES treatment used in this study, only a couple of horses showed significant increases in mean muscle fiber size when Pre- vs Post-FES biopsies were compared; 2) In the older horses, there were sparse (or several in one horse) severely atrophic and angulated muscle fibers in both Pre- and Post-FES samples, whose distribution suggests they were denervated due to a distal neuropathy; 3) The hypothesis of generalized FES-induced muscle fiber damage during epaxial muscle training is not supported by our data since: 3.1) Denervated muscle fibers were present in the Pre-FES biopsies and 3.2) Only one horse (age 15 yr) presented with high numbers of long-term denervated muscles fibers Post-FES; 4) Preliminary data indicate that the increased density and distribution of mitochondria in Post-FES biopsies suggests that the clinical improvements in the treated horses may be related to the increased muscle contractions, therefore improving muscle perfusion which is induced by FES training. In conclusion, FES in horses is a safe treatment that provides clinical improvements in equine epaxial muscle spasms. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration., FES in neuromuscular dystrophy diseases has been a controversial topic through the past decades, generally associated with the concern, that voluntary or stimulated activation of nerves and muscles can lead to metabolic overuse and acceleration of degenerative processes.1 More recent literature on clinical treatments with FES with vastly different parameters has found strong hints for beneficial influence and no real indications for adverse effects. In the particular case of ALS with progressive denervation of motor units, rapid loss of muscular functions and inevitable development towards respiratory insufficiency threshold and dependence on artificial respiration, usually in intensive care environment. A recent development of a stimulator with percutaneous electrodes, which can activate still innervated motor units of the diaphragm and train size, contraction force and endurance of their muscle fibers to compensate part of the denervation induced capacity loss.2 It has been shown in studies, that respirator dependence can be delayed from in average 40 month after disease onset, by about 16 months. After the European project RISE has shown that denervated muscle fibers can be maintained, trained and functionally activated by non-invasive application of long-duration stimuli,3-6 and earlier studies have demonstrated, that similar results can be achieved with implantable electrodes, there is a high probability, that in case of ALS already denervated muscle fibers can be activated and maintained with similar approaches, which could lead to further delays in respirator dependence., Respiratory failure is the most common cause of death in ALS. Early symptoms of respiratory insufficiency are diverse and most commonly occur during sleep. They may severely impede the patients’ quality of life. It is important that respiratory dysfunction is recognized and discussed with patients early enough. Several methods are in use to alleviate such symptoms or even prolong life of these patients. Non-invasive intermittent ventilation (NIV) is primarily aimed at lessening symptoms severity rather than prolonging life. It improves the quality of sleep and of cognitive function, and relieves morning headaches. The alternative is invasive ventilation through tracheostomy which prolongs life but does not affect disease progression. It also precludes the patients’ ability of oral communication. They greatly increase care needs and, consequently, deteriorate quality of life, at least that of the carers. The search for new approaches to treatment of respiratory failure has led to the development of diaphragm pacing, that is currently an approved method only in the USA. However, there are important unanswered questions regarding its benefit and impact. It may only improve patients’ quality of life, with better sleep and daytime functioning, better breathing, and less fatigue but it does not prolong life. A small number of patients decide not to use any of the above methods while others, mostly those with severe bulbar involvement, do not tolerate NIV. In them, the feeling of shortness of breath can be reduced by the administration of morphine. There are substantial differences in the use of the above mentioned methods in different countries especially in the use of mechanical ventilation via tracheostomy.1-5 We recently analysed data of 271 ALS patients treated at the Institute of Clinical Neurophysiology in the 10-year period between 2003 and 2012. Their mean age at symptoms onset was 62.7 ± 11.4 years, and mean survival from the time of enrolment 16.4 ± 15.1 months. One hundred seventy nine (66.1%) patients had spinal onset and 71 (26.2%) bulbar onset of the disease. In total 34.7% of all patients consented to the non-invasive assisted ventilation at some point. The proportion of those using non-invasive respiratory support was rising through the analysed years and has reached over 50%, reported also by other tertiary care Centres. Survival after institution of non-invasive ventilation was 7.8 ± 7.3 months. Six patients used invasive ventilation, two of them started using it before the diagnosis was established. This is less than in some other countries. Four patients had a tracheostomy done solely for the airway hygiene. Diaphragm pacing is currently not in use by the Ljubljana ALS team., In managements of ALS, a syndrome that is very often fatal due to a ventilation crisis, palliative therapies are only possible, at least for the predictable future.1 On the other hand, we are confident we may extend function of respiratory muscles, and thus postponing the need of air pumping that damages even more the diaphragm muscle,2 if we will be able to show in experimental models that some additional muscle contractile function is achievable by combining proven approaches to maintain/recover contractility of “denervated” muscle fibers of the diaphragm in animal models of ALS.3 The left hemi-diaphragm of 3-and 30-month old rats (adult and oldest old, respectively) was denervated under general anesthesia by section of the left phrenic nerve, reached through an intercostal approach. The left sciatic nerve was also severed. Rats were sacrificed after 7 days. Isometric mechanical measurements were performed in vitro in a vertical muscle apparatus (300B, Aurora Scientific Inc, Canada) containing a Ringer solution of the following composition: 120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 3.15 mM MgCl2, 1.3 mM NaH2PO4, 25 mM NaHCO3, 11 mM glucose, 30 µM d-tubocurarine, pH 7.2–7.4, 30°C, bubbled with 95% O2–5% CO2. Two strips from each hemi-diaphragm were examined. Each strip was stretched to the optimal length (i.e. the length that allowed maximal tension development in response to a single pulse) and electrically stimulated, by two parallel electrodes, with supramaximal pulses (0.5 ms duration) delivered by a Grass S44 electronic stimulator through a stimulus isolation unit (Grass SIU5). Muscle response was recorded through an isometric force transducer (Harvard) connected to an AT-MIO 16AD acquisition card (National Instruments) and data were analyzed by a specific module of the National Instruments Labview software. Force-frequency curve was determined by stimulating the muscle at 1, 15, 30, 60, 80, 120 and 150 Hz. All tensions were normalized to the muscle wet weight (specific tension, N g-1). Contraction and relaxation times, and twitch and tetanic tensions were similar in adult and old innervated diaphragm. Denervation caused similar slowing of contractile properties in adult and old muscles. Interestingly, denervated diaphragm produced a significant higher specific tension with respect to the contralateral innervated up to 15 Hz stimulation in adult but not in old muscles. Instead, a similar drop of tension was evident at higher frequency both in adult and oldest old denervated diaphragm, compared to innervated one. In conculsion contractile properties of diaphragm were not weakened by ageing. On the other hand, mitochondrial density and distribution determined by electron microscopy and histochemistry were substantially changed by age and even more by 7 day denervation, confirming that the muscles were denervated seven days after neurectomy. We are confident, that this animal model, i.e., muscle denervation in oldest-old animals, will be useful in testing cellular (injecting myogenic stem cells derived from adipose mesenchymal cells and physical (FES) approaches. The first step of a long way will be to test if mitochondrial density and distribution may recover to young rat levels by neuromodulation of the hemidenervated diaphragm in oldest-old rats. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration., Muscle dysfunction and degeneration of skeletal muscle lead to a perturbation of the regenerative process, causing the premature exhaustion of satellite cell reservoir due to continuous cycles of degeneration/regeneration. The in vivo approach in animal models can be helpful to study the mechanism underneath the operating principle of the stem cell reservoir, namely the niche, which holds great potential to understand the onset of muscle pathologies. To this end, the success of skeletal muscle reconstruction depends on finding the most effective, clinically suitable strategy to repair the damaged tissue. We1 designed and developed the delivery of either SCs or muscle progenitor cells (MPCs) via an in situ photo-cross-linkable hyaluronan-based hydrogel, hyaluronic acid–photoinitiator (HA-PI) complex in a mouse model of muscle mass depletion and more recently,2 we systemically injected amniotic fluid stem cells in a mouse model of Spinal Muscle Atrophy. Through these two approaches muscle reconstruction and rejuvenation was associated with the formation of neural and vascular networks in the first model and the reconstitution of a functional SC niche in the second., Despite the the severely destructive effects of long term denervation there is structural and ultrastructural evidence for survival of muscle fibers in mammals, some surviving at least ten months in rodents and 3-6 years in humans.1 Further, in rodents there is evidence that muscle fibers may regenerate even after repeated damage to aneurally regenerated muscle,2 this potential being maintained for several months after aneural regeneration.3 While permanently denervated human muscle sooner or later loses the ability to contract, even many months after denervation the muscles may almost maintain or recover their size and ability to function if electrically stimulated by long impulses.4 During the past decade, we have studied muscle biopsies from the quadriceps muscle of Spinal Cord Injury (SCI) patients suffering with Conus and Cauda Equina syndrome, a condition that fully and irreversibly disconnects skeletal muscle fibers from their damaged innervating motor neurons. We have indeed demonstrated that human denervated muscle fibers survive surprisingly years of denervation5,6 and can be rescued from severe atrophy by home-based Functional Electrical Stimulation (h-bFES).5-8 After permanently denervated human muscles reach the minimal 10% residual volume/weight, we discovered that many severely atrophic muscle fibers still persist, but they present peculiar clusters of centrally located myonuclei.6 This features are seemingly the result of complete loss of contractile structures in both fast and slow types muscle fibers and of the redistribution of myonuclei from their spiral subsarcolemmal distribution to groups of central nuclei separated by long stretches of amyofibrillar sarcoplasm.5 These peculiarly severe atrophic muscle fibers are present in rodent muscles from seven-months after neurectomy and in human muscles between 30 and 70 months after a spinal cord injury that results in a permanent complete Conus and Cauda Equina Syndrome.5,7,8 These severe atrophic muscle fibers are therefore structurally distinct from early myotubes, one of the early stages of myogenesis during both development and regeneration of adult muscle fibers after necrosis. Whether in humans this is a result of persistent de novo formation of muscle fibers is an open issue we explored using immunohistochemistry in both non-stimulated and h-b FES stimulated human muscles.1,4,5,7 We have indeed observed the persistent presence of muscle fibers which are positive to labeling by an antibody which specifically recognizes the embryonic myosin heavy chain (MHCemb). Relative to the total number of fibers present, only a small percentage of these MHCemb positive fibers are detected, suggesting that they are regenerating muscle fibers and not pre-existing myofibers re-expressing embryonic isoforms).1,4,5,7 Although embryonic isoforms of acetylcholine receptors are known to be re-expressed and to spread from the end-plate to the sarcolemma of all muscle fibers in early phases of muscle denervation, we suggest that the MHCemb positive muscle fibers resulted from the activation, proliferation and fusion of satellite cells, the myogenic precursors present under the basal lamina of the muscle fibers.9 Beyond reviewing evidence from rodent and human studies, we add some ultrastructural evidence of muscle fiber regeneration in long-term denervated human muscles (i.e., fusing myoblasts, myotubes and the presence of muscle fibers with a double layer of basal lamina) and discuss the options to substantially increase the regenerative potential of long term denervated human muscles not having been treated in time with h-b FES.1,10 Some of the mandatory procedures, are indeed ready to be translated from animal experiments to clinical studies to meet the needs of persons with long-term irreversible muscle denervation.10 An European Project, the trial Rise4EU (Rise for You, a personalized treatment for recovery of function of denervated muscle in long-term stable SCI) will hopefully follow. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration., Duchenne Muscular Dystrophy (DMD) is the most common childhood onset dystrophy affecting 1:3,500-6000 live male births. It is a rapidly progressive muscle wasting condition which, without treatment, causes death in teenage years due to cardiac and respiratory failure. Since the 1990s survival has improved due to the introduction of non-invasive nighttime home ventilation and early and aggressive treatment of cardiomyopathy using with ACE inhibitors and beta blockers. 1,2 More recently, the routine use of corticosteroid treatment as the ‘gold standard of care’ has resulted in prolonged ambulation and preservation of cardiac and respiratory function.3 The future for newly diagnosed boys with DMD looks even more promising since a new generation of drugs targeted towards RNA processing have been developed and include drugs designed to either readthrough nonsense mutations or skip exons with deletions or duplications (antisense oligonucleotides).4-6 Some of these agents are currently in phase 2 and phase 3 clinical trials, the first of these (translarna otherwise known as ataluren) has recently been given provisional regulatory approval in Europe.7, Skeletal muscle channelopathies are rare inherited disorders that are characterised by disabling episodes of muscle paralysis or myotonia. The periodic paralyses include hypokalaemic periodic paralysis, hyperkalaemic periodic paralysis, Andersen-Tawil syndrome and thyrotoxic periodic paralysis. The non-dystrophic myotonias include paramyotonia congenita, sodium channel myotonia and myotonia congenita. Accurate diagnosis forms the foundation of appropriate treatment. Symptoms are frequently triggered by specific provoking factors including certain foods, temperature or activity. Treatment can begin with education of these factors and how to avoid them. Exercise is often limited but careful technique and modification can allow many forms of activity to continue. Pharmacological therapies for the periodic paralyses include acetazolamide and diuretics (either potassium sparing or potassium losing). Emergency treatment of an attack of hypokalaemic periodic paralysis with intravenous potassium can sometimes be needed if there are ECG changes or severe muscle weakness causing respiratory compromise. Caution is needed however with very strict ECG and serum potassium level monitoring as a rebound hyperkalaemia can occur. Thyrotoxic periodic paralysis is most common in Asian populations. It may present with an attack of hypokalaemic paralysis without obvious systemic features of thyrotoxicosis but is an important differential to consider as treating the thyroid dysfunction abolishes attacks of periodic paralysis. Andersen-Tawil syndrome is the only skeletal muscle channelopathy to also affect the heart and cardiology input is needed for any conduction abnormalities. Usually there is a triad of periodic paralysis, dysmorphic features and cardiac disease but the dysmorphic features can be very subtle and many patients are asymptomatic from a cardiac perspective prompting a high degree of suspicion of this diagnosis. Sodium channel blockers form the mainstay of treatment for the myotonic disorders. Pain is probably an under-recognised symptom and as a result under-treated. Simple analgesics are usually ineffective and although sodium channel blockers may provide some relief, this is often incomplete. A severe neonatal phenotype with respiratory compromise often requiring intensive care support has recently been recognised. Symptoms do seem to respond to sodium channel blockers in the majority but less than 20 children with this phenotype are so far described and two fatalities have been reported suggesting optimal management is yet to be determined. Although the most prominent symptoms in the channelopathies occur in an episodic manner a fixed proximal weakness can also occur. Its relationship to the frequency or severity of these episodic symptoms is unclear. This poses difficulties in knowing if treating the episodic attacks will prevent the development of myopathy, the onset of which seems to be at least in part unrelated to age with severe weakness sometimes described in children. An MRI can be helpful in distinguishing between the presence of oedema which suggests ongoing attacks that are amenable to treatment and fatty infiltration suggesting an irreversible myopathy. If irreversible myopathy is established consideration needs to be given to functional and home modifications., Sarcoglycanopathy is the name of rare autosomal recessive disorders affecting mainly the proximal musculature, hence belonging to type 2 Limb Girdle Muscular Dystrophy family (LGMD2). Age of onset and disease severity is variable, ranging from severe forms (with onset in the first decade and rapid evolution) to milder forms (with late onset and slow progression)1 Four different forms (LGMD2C, LGMD2D, LGMD2E, LGMD2F) are caused by defects in the genes coding for γ-, α-, β- and δ-sarcoglycans (SG), respectively. It is important to note that about 75% of α-SG, 59% of β-SG, 40% of γ-SG and 57% of δ-SG genetic defects are missense mutations.1 Until now, 52, 29, 20 and 8 different missense mutations have been reported for α-, β-, γ- and δ-SG, respectively. Sarcoglycans form a tetramer, the SG-complex, member of the dystrophin associated protein complex and key element to assure membrane stability during muscle contraction. The presence of a missense mutation generates a folding-defective, although potentially functional sarcoglycan, that is rapidly recognized by the cell’s quality control and targeted to proteasomal degradation. The loss or strong reduction of the mutated sarcoglycan leads to a secondary variable deficiency of the other sarcoglycans, thus compromising sarcolemma stability. Disease severity is strictly related to the residual level of sarcoglycans in the sarcolemma, with the most severe forms characterized by the almost complete loss of the proteins. Interestingly, the entire SG-complex can be rescued at the cell membrane by blocking different steps of the degradative pathway of the sarcoglycan mutants,2,3 a result that opens a new perspective for the therapy of these neglected diseases. In particular, we have designed two therapeutic approaches named “protein rescue” and “protein repair” strategy. The former aims “to save” the mutant from degradation, whereas the second intends “to assist” folding-defective sarcoglycans to reach the native or a native-like conformation and thus overcome the quality control check point. In both cases the recovered protein is expected to assemble with the SG partners and traffic toward the plasma membrane. The pharmacological inhibition of the E3 ligase HRD1, key element of the sarcoglycan degradative route, allowed the quantitative and functional rescue of an α-SG mutant both in a cell model and in primary myotubes derived from a patient suffering from LGMD2D,4 validating the “protein rescue” strategy. For the “protein repair” strategy, we are testing a panel of 12 small molecules known as protein folding correctors screened for the treatment of cystic fibrosis. Although the way of action on sarcoglycans of these compounds is still unknown, we demonstrate in a heterologous cell model and in myotubes derived from an LGMD2D patient the accumulation of different α-SG mutants, that are competent to assemble with the wild type partners and traffic to the cell membrane.5 These data represent the proof of principle of the two conceived pharmacological strategies which validation in vivo is currently undergoing. We believe our work is moving toward the development of a cure for the most frequently reported cases of sarcoglycanopathyr, Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy [1]. Over the years, we have tested disease models as well as patients for their response to cyclophilin inhibitors [1-4]; in parallel, we have developed novel PTP inhibitors through high-throughput screening methods. I will report on our progress in the development and treatment of muscular dystrophies with PTP inhibitors., McArdle’s disease, the most common muscle glycogenosis, is characterized by exercise intolerance and recurrent exercise induced myoglobinuria. The condition is associated with severe limitation not only in the capacity to sustain even brief acute strenuous efforts, but also in the significant reduction of skeletal muscle aerobic metabolism. The exercise limitation is typically eased after 6-7 minutes of warm-up, giving rise to the so called “second wind” phenomenon, likely linked to the mobilization and muscle uptake of blood borne glucose as fuel to sustain muscle contraction. 20-30% of patients develop fixed weakness and muscle atrophy, especially in the upper limbs and report difficulties even for routine mild activities like walking.1. No causal treatment is available for this myopathy. The supplement of oral sugar prior to acute effort and the aerobic training have been showed to provide relief and improve functioning. Diet manipulation with preference to carbohydrates has been also proposed as management strategy.2 Prescriptions in this direction are regular part of the medical management of the disease and form the core of the indications given to patients.In spite of the available evidences however, when interviewed at follow-up visit, patients often admit not being able to consistently follow the instructions in terms of regular exercise, being either overwhelmed by the fear of suffering exercise induced pain and acute muscle damage, or just not being able to regulate the intensity and frequency of the exercise sessions. Given these premises we designed a one week patient-tailored program of intensive functional evaluations, including direct assessment of peak VO2 and customized oversighted training, followed by detailed indications for home-work. The session was followed by active weekly telephone monitoring. We tested our novel strategy on a first group of 8 molecularly defined McArdle’s patients. The intensive session was well tolerated by all but one patient who complained of growing pain and was later found to be suffering for an herniated lumbar disc. No rise in CK was observed after 45 min exercise sessions repeated twice a day for 4 days. The functional evaluations confirmed the significant reduction in aerobic power but also the inappropriate response to even mild exercise (e.g. 12min walking test). The first post training evaluation at 3 months was available for 3 subjects, and provides evidence for mild but consistent improvement in most functional measures. Most importantly, the program was successful in modifying patient behavior and improve attitude towards regular exercising and more appropriate dietary habits. An appropriately designed and customized program of training coupled with active monitoring appears as a promising and efficient strategy to address functional limitations and reduce disability in McArdle’s patients., Functional electrical stimulation (FES) is a widely accepted rehabilitative treatment. Its effects have been extensively investigated in patients with stroke1 or spinal cord injury2 and FES is also currently used in clinical and rehabilitative management of patients presenting these diseases. Nevertheless, only few studies3,4 show that this kind of electrical stimulation can be useful also in neuromuscular diseases. Through the present report we will present a case series that shows our initial experience and the obtained results by the treatment with FES of patients affected by two different neuromuscular diseases: Myotonic Dystrophy (Type 1) and Hereditary Spastic Paraparesis. The aim of the study was to assess the feasibility and the usefulness of FES treatment in neuromuscular diseases, and also verify the presence of any adverse effects. We selected 4 patients affected by Myotonic Dystrophy and 4 patients affected by Spastic paraparesis and all subjects were treated as inpatients in IRCCS San Camillo Hospital, for 4 consecutive weeks. The FES devices used in IRCCS San Camillo Hospital are: Cycling-FES for lower limbs (Hasomed RehaStim2 MOTOmed® viva2) and peroneal neuroprosthesis FES for foot drop (NESS® L300TM). Functional tests and clinical scales [i.e. Medical Research Council (MRC) Scale for Muscle Strength; Active Range of Motion (ankle; knee); Timed Up & Go; 6 Minutes Walking Test] were performed, before and after treatment, in order to assess the effects of therapy. Our preliminary results show that there are no adverse effects of FES treatment in patients affected by this kind of neuromuscular disorders. FES treatment also seems to be useful in improving functional performances during deambulation, but further research with a larger number of treated patients is required to confirm this data., Myotonic Dystrophy type 1 is a dominantly inherited disease comprehending multiple features such as myotonia, muscle weakness and multisystemic involvement. Fatigue and exhaustion during exercise are two common symptoms presented by dystrophic patients, thus representing the most significant factors that could negatively influence their quality of life and compliance to rehabilitation programs.1 Mitochondrial abnormalities and a significant increase in oxidative markers had been previously reported, suggesting the hypothesis of a mitochondrial functional impairment in the pathogenesis of the disease.2 The study aims at evaluating oxidative metabolism efficiency in Myotonic Dystrophy patients, through analysis of lactate levels at rest and after an incremental exercise test, in order to propose a safe and valid method to guide specific aerobic training in rehabilitation program of these subjects. We analyzed, in 18 Myotonic Dystrophy patients, resting and exercise-related blood lactate values, as an indirect marker of oxidative metabolism, comparing results to 15 healthy subjects forming the control group. The exercise protocol consisted of a submaximal incremental exercise on an electronically calibrated treadmill: patients performed 11 consecutive steps consisting of walking for two minutes at a constant speed of 3 Km/h, while the inclination degree ranged from 0° at baseline to 2.5% for each step. This way the physical exercise was maintained in predominantly aerobic condition. Lactate levels were assessed through venous blood samples, collected at rest and at 5, 10 and 30 minutes after the end of the exercise. Analysis of the differences in mean values in the two groups were performed using the Z test.3,4 The results showed early exercise-related fatigue in Myotonic Dystrophy patients, as they performed a mean number of 9 steps, while controls completed the whole exercise. Moreover, while resting values of lactate were comparable between the patients and the control group (p=0,69), after the exercise protocol, dystrophic subjects reached higher values of lactate, at any recovery time (p, In heart, the functional ERG1 (ether-a-gogo related gene) K+ channel is composed of both ERG1a and 1b splice variant proteins and is known to be partially responsible for late phase repolarization of the cardiac action potential. 1,2] We have shown that the mouse ERG1a (MERG1a) splice variant protein is increased in skeletal muscle of mice experiencing atrophy as a result of hindlimb suspension (disuse). Additionally, we have shown that ectopic expression of Merg1a in mouse skeletal muscle (a result of the electroporation of expression plasmid into gastrocnemius muscle) increases activity of the ubiquitin proteasome pathway (UPP),3] a proteolytic pathway responsible for the majority of protein degradation that contributes to muscle loss in atrophic skeletal muscle.4 Further, we have shown that this ectopic expression increases protein levels of the UPP component E3 ligase MURF1, although not that of ATROGIN1. Because the calcium activated enzyme calpain is also known to contribute to proteolysis in skeletal muscle atrophy, we wanted to determine if Merg1a expression would also affect intracellular calcium levels and calpain activity. Thus, we infected C2C12 myotubes with an adenovirus containing the human ERG1a (HERG1a) construct and determined that, as in mouse muscle, ERG1a expression increased levels of MURF1 protein, but not that of Atrogen. Interestingly, it also increased levels of intracellular calcium in C2C12 myotubes by 86% in response to depolarization. Further, Merg1a expression also increased calpain activity in mouse gastrocnemius muscle by 58.8% (p, The power developed by the skeletal muscle decreases with aging: no question. The decline rate can be deduced from the decline of the world records of Masters athletes in various track and field discipliness.1,2 Studies on this subject are numerous and the results of our, in line with those of others, indicate trend-lines for the power decline starting at the age of 30 and pointing to zero at the age of 110 years.3,4 The normalized decline rate (from 1 to 0) of the skeletal muscle power with aging of any ordinary people, if always healthy, is the same of the normalized decline rate of the world records of the Masters athletes. The most important everyday functional tasks can be related to the main disciplines of the track and field competitions. A short walk can be associated to the short runs; a long walk to the endurance runs; the stairs climb1 to the jumping events (all imply mainly the legs power to raise the body centre of gravity) and finally the raise of a weighted supermarket bag on to the kitchen table can be associated to the throwing events (they require mainly the power of the upper limbs).4,5 The decline rate of the Masters world records for selected events (short runs, long runs, simple jumps and selected throwing events) have been analysed and plotted vs age. The declining trend lines of the Masters word records can indicate the upper level of the age limits of the key everyday tasks for everyone (short walk; long walk, stairs climb and bag raise). Accepting some simple assumptions for the performance of the main functional tasks (limiting power from 20%, to 5% of the youthful power), then the limit age ranges from 85 to over 105 years. An earlier occurrence is, thus, the product of a disease, severe muscle disuse included. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration., The prolongation of skeletal muscle strength in neuromuscular disease has been the objective of numerous studies employing a variety of approaches. Stem cell therapy represents a promising tool to cure genetic diseases. However, this approach is not definitive yet and several hurdles limit the immediate translation of this strategy into clinic. One of the crucial parameters of tissue regeneration is the microenvironment in which the stem cell populations should operate.1 Stem cell microenvironment, or niche, provides essential cues that regulates stem cell proliferation and that directs cell fate decisions and survival. It is therefore plausible that loss of control over these cell fate decisions might lead to a pathological transdifferentiation and contribute to the exacerbation of a pathologic condition, such as muscular dystrophy. Among critical parameters, the activation and persistence of inflammatory and fibrotic pathways may render the dystrophic muscle incapable to sustain and complete an efficient muscle regeneration,2 leading to a progressive loss of muscle tissue due to chronic degeneration of muscle and to the exhaustion of satellite cells that replace damaged fibers.3 Indeed, the progressive loss of tissue function and integrity observed in dystrophic muscles are the eventual consequences of a history of continuous rounds of degeneration and regeneration. Specific factors are required to trigger stem cells toward a specific lineage, to improve their survival, and to render them effective in contributing to tissue repair. Studies on stem cell niche leaded to the identification of critical players and physiological conditions that improve tissue regeneration and repair. Preliminary evidences demonstrated that the local form of Insulin-like Growth Factor–1 (mIGF-1)4,5 sustains muscle hypertrophy and regeneration in senescent skeletal muscle, enhances the recruitment of circulating stem cells in injured muscle and counteracts muscle wasting in mdx dystrophic mice, reducing the inflammatory response and improving muscle mass and strength and elevating pathways associated with muscle survival and regeneration. Among the factors modulated by mIGF-1, we observed a specific down-regulation of the inflammatory cytokines IL-6, which has been associated with the switch from acute to a chronic inflammatory response that therefore can exacerbate the dystrophic phenotype. We will discuss the role of mIGF-1 and IL-6 in the modulation of muscle regeneration under physiological and pathologic conditions., Ageing is associated with detrimental changes in function, mass and structure of skeletal muscle that are also predisposing factors to disability and increased the risk of falling in elderlies. Lifestyle interventions including increased physical activity are one of the primary approach to prevent age related muscle decline. Our recent results from a peculiar group of lifespan trained seniors, suggest that long term physical exercise have beneficial effects on age related decay not only affecting muscle trophism and phenotype, but also counteracting denervation atrophy by promoting reinnervation.1-3 By this rationale, we trained 70 years sedentary seniors either with leg press (LP) or electrical stimulation (ES) for 9 weeks. We investigated the effects of training on mobility and muscle trophism by functional tests and morphological, biochemical and molecular analyses of Vastus Lateralis muscle biopsies, before and after the training. The effects of increased muscle activity with respect to sedentary lifestyle were also analysed focusing on the effect on mitochondrial proteins associated with Ca2+ uptake and respiratory chain function. The results show that either LP or ES training induce similar force and functional improvements without damaging skeletal muscle fibers. Indeed, ES more efficiently in comparison to LP, attenuate muscle mass decline maintaining the overall size of muscle fibers and increasing the number and the size of the fast type fibers, also activating satellite cells. Both trainings, without significantly affecting the overall number of mitochondria and respiratory chain enzymes (SDH and COXIV), induced an increased expression of MCU regulating the mitochondria Ca2+ uptake in post training biopsies, which seems to be associated to the increase of fast fibers diameter in ES trained subjects.4-6 Altogether these findings show that training sedentary elderlies with ES is a safe and effective intervention to counteract muscle fiber atrophy and to improve the performances of aging muscles. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration., Cancer cachexia is a degenerating syndrome characterized by a severe body weight loss occurring in the advanced stage of tumor progression. Muscles wasting, together with a remarkable loss of fat tissue, are known hallmarks of cancer cachexia. Clinically, cancer-related cachexia accounts for almost 2 million of deaths per year and until 80% of tumors can develop cachexia. So far, many studies identified a pivotal role of several circulating tumor factors (i.e. proteolysis-inducing factor, PIF) in mediating muscle degeneration observed in skeletal muscle tissue. In particular, pro-inflammatory cytokines (i.e. IL-1, IL-6 and TNF-α) from tumor origin are the main players in triggering muscle wasting.1 They sustain the inflammatory network between tumor and skeletal muscle tissue accountable for the main catabolic pathways activated into the skeletal muscle fibers such as the ubiquitin-proteasome pathway (UPP), proteases-mediated degradation and autophagocytosis.2 Since the inflammatory burden is considered the most important mediator of muscle degenerative processes, many efforts pointed to the treatment of cancer-related cachexia with anti-inflammatory drugs. Nevertheless, soluble antibodies against TNF-α and their receptors still showed poor beneficial effects in the clinics.We investigated cancer-related cachexia in Gastrointestinal Stromal Tumor (GIST) xenograft-mouse model. GIST is the most common mesenchymal tumor of the gastrointestinal tract, characterized by high expression of KIT, PDGFR3 and DOG1, a calcium activated chloride channel.4 By transplanting of human GIST fragments in nude mice, we found that 30% of tumor bearing mice developed severe muscle wasting characterized by a strong reduction muscle fibers area associated with increase of Atrogin1 expression, a known marker of muscle atrophy.5 Expression profile analysis of inflammatory markers showed that muscle wasting in cachectic mice was not sustained by the increasing of pro-inflammatory cytokines from tumor, nor by changes in their expression in skeletal muscles. Furthermore, histological analysis revealed the presence of tumor cells within the connective tissue surrounding the fibers. In vivo analyses showed that both tumor cell migration and colonization of cachectic muscles are processes closely associated with the expression of DOG1. Loss of function experiments further supports our hypothesis. Finally, histological analysis performed on skeletal muscle tissues of colon carcinoma C26-bearing mice shows the colonization of tumor cells in muscle microenvironment. Thus, tumor cells can also colonize skeletal muscle tissues in immunocompetent murine model of cancer cachexia., Human aging is characterized by the progressive loss of muscle mass and strength, phenomenon known as sarcopenia. The etiology of sarcopenia is still not clear and involves several factors. Among these, sedentary lifestyle acts synergistically with age to determine the decrease of muscle mass and strength.1 Aging and inactivity play a role also in the accumulation of inter-muscular adipose tissue, another factor involved in the development of sarcopenia.2 In skeletal muscle, fat accumulates also as intra-muscular triglycerides (IMTGs). Little is known about the possible role of IMTGs in muscle aging and atrophy. IMTGs accumulate in form of lipid droplets (LDs) characterized by the presence of Perilipins (Plins). In skeletal muscle the most abundant are Plin2 and Plin5. The exact role of these Plins is currently unknown, however it is well established that these proteins correlate positively with the levels of IMTG and are up-regulated by lipid accumulation under both physiological and pathological conditions.3 Recently, we found that the expression level of Plin2 increases with age and inactivity, and it is inversely associated with muscle mass and strength.4 On the basis of these findings, we compared the expression of Plin2 and Plin5 during muscle aging and inactivity, and analysed the possible consequences of such expression on muscle mass and strength. To this purpose, we investigated the levels of these two proteins in Vastus lateralis muscle biopsies from subjects of different age: healthy donors and patients with limb mobility impairment, that gave us the possibility to analyse the expression level of Plins in condition of chronic physical inactivity. Our data indicate that of Plin2, but not Plin5, increases with age and inactivity. Moreover Plin2, but not Plin5, is associated not only with active form of p53, but also with muscle atrophy-related genes, such as MuRF-1 and Atrogin, suggesting a role only for Plin2 in muscle aging and atrophy. To further reinforce our observation on this association, we took advantage of an experimental model of muscle atrophy induced by cutting the sciatic nerve. In this model, an increase in Plin2 and a concomitant decrease of Plin5 was observed, adding further evidence that these proteins have different role in skeletal muscle and notably during atrophy., Interest, development and market offer of sensor based systems have grown tremendously over the last few years. Today technology allows us to monitor all segments of our lives, from health aspects to living environment and others. Current multi-sensor systems usually operate in a way that the measured data are stored and analysed only later (post hoc) by the user. Ergo-Cell is a novel multi-sensor device that offers the ability to transfer data from multiple sensors to the cloud in real time. Coach, doctor or any other end user can log into a web based application from anywhere in the World and check and/or compare the measured parameters from one or more remote systems simultaneously. Ergo-Cell is an embedded modular system composed of three main parts: i) the base board, ii) the battery module and iii) wireless sensors. The biggest challenge while developing the Ergo-Cell was the ability to use multiple sensors without affecting the measuring performance. A real time operating system is therefore used to ensure that all operations are executed on every cycle at a given time, without any delay. The beating heart of the new device is the ARM Cortex-M3 microcontroller; FreeRTOS is used for the operating system.1 The program runs on multiple threads, one task reads data of board sensors (temperature, humidity, accelerometer and gyroscope), the second reads data from the GPS (Global Positioning System), while the third task takes care for the wireless communication etc. ANT+ is the main wireless technology that allows devices to talk to each other. The main feature is the interoperability which means that ANT+ products can be used from multiple brands2 The first version of EC incorporates ANT module for reading heartbeat data, later on we plan to extend the list of supported sensors to cadence, speed, blood pressure and other. We also plan to take ANT+ advantages and produce custom wireless sensors for our needs. The last important part of the new multi-sensor system is the GPRS (General Packet Radio Service) modem, used for sending data to the cloud. EC is connected to the server in a "transparent" mode so bidirectional communication is possible in real time. The dimensions of the first Ergo-Cell version are 10x10x5 cm, weight is about 500 g, battery life 20+ hours. We believe that Ergo-Cell offers new interesting opportunities for studies in the field of general mobility, ergonomics and specific physical activity in elderly., The study will examine acute response of bone turnover in pre- and postmenopausal women to resistance exercise.1 A randomized repeated measures crossover design will be used to find out the effect of two single sessions of resistance exercise on serum bone metabolism markers: bone alkaline phosphatase (bALP), amino-terminal propeptide of type 1 procollagen (P1NP), beta-crosslaps (bCTx) and sclerostin. All the subjects will perform one session with a constant resistance (75 % of 1RM) and one isokinetic session with repeated (10 Hz) counter-movements in concentric and accelerations in eccentric phase of the movement on different days. Each session will consist of 6 sets of 6 repetitions with a 2-minute rest and will be conducted between 8 and 9 AM. Blood samples will be obtained by venipuncture of antecubital vein before (after a 10-12 hour over-nigh fast), 15 minutes, 24 and 48 hours after the exercise., Cachexia is a metabolic syndrome characterized by skeletal muscle wasting and weight loss, associated with an underlying disease and chronic inflammation. Cancer cachexia affects the majority of cancer patients, leading to weakness, decreased mobility and quality of life and negatively interfering with anticancer therapies. Furthermore, no effective therapy of cachexia exists as far.1,2 One of the reasons of this failure is the lack of a deep knowledge of the basic mechanisms underlying muscle wasting in this condition3 The goal of this study is to characterize muscle deficits in cancer cachexia, and to develop novel approaches to counteract cachexia. The muscular function of 15 oncologic patients (13 males, 2 females, 47.07 ± 17.58 years of age) and 16 controls (13 males, 3 females, 49.13 ± 17.55 years of age) was analyzed in this study, by means of: (a) the analysis of the oxidative metabolism, indirectly evaluated trough dosage of blood lactate levels before and after a submaximal incremental exercise on a treadmill; (b) the analysis of strength and resistance, in both proximal and distal muscles, by means of an isokinetic dynamometer, expressed as the average of strength applied during 3 maximal voluntary contractions (MVC) lasting 3 seconds each and the decay of the strength exerted, expressed as a percentage of maximum voluntary contraction (%MVC) during an isometric contraction sustained for 60 seconds. Results were then analyzed and compared with obtained by a group of healthy subjects. Mann-Whitney U test was used for statistical analysis. Patients were affected by solid tumors of different severity located in different sites (neck, lung, testicle and neuroendocrine tumors) and in different stages of disease. During the study patients were being subjected to chemotherapic treatments. Analysis of oxidative metabolism during the incremental exercise on the treadmill showed that patients performed a reduced number of steps respect to the controls. Lactate levels were significantly (p, The maintenance of a functional mitochondrial network is particularly important in highly structured and metabolically active tissues like muscle, but whether and how changes in mitochondrial fusion and fission cause extramitochondrial responses at the cellular and organismic level is unknown. Here we show that age related muscle loss and weakness in humans (sarcopenia) correlates with the decreased expression of the inner mitochondrial membrane fusion protein Optic Atrophy 1 (Opa1). Mechanistically, the endoplasmic reticulum (ER) senses conditional and inducible skeletal muscle ablation of Opa1 and signals via the Unfolded Protein Response (UPR) a nuclear program that triggers muscle and adipose tissue loss. Conditional as well as inducible Opa1 deletion in muscle altered mitochondrial morphology and function but not DNA content, ultimately reducing growth and lifespan. Mechanistically, UPR activation mediated muscle stem cells loss, protein synthesis inhibition, ubiquitin-proteasome activation and FGF21 expression leading to a fasting-like condition. Indeed, pharmacological inhibition of ER stress blunted the atrophy program and restored a normal muscle mass. Thus, the communication between mitochondria and ER controls muscle and systemic response to mitochondrial malfunction., Dysfunctional mitochondria and insufficient autophagy are common in aged muscle and neuronal tissues. The mitochondrial theory of aging is based on the premise that cumulative damage caused by the production of oxidants can alter nuclear and mtDNA (e.g., point mutations and deletions). It is well established that defects in mtDNA lead to a decline in mtDNA abundance (content) and a reduced number of genes encoding mitochondrial proteins that are essential for the proper assembly and maintenance of mitochondria. Additionally, lower mitochondrial protein synthesis rates, disturbances in mitochondrial enzyme activities, altered import protein machinery, and lower oxidative capacity and ATP synthesis have been documented to occur in aged tissues. Besides being less bioenergetically efficient, damaged mitochondria also produce increased amounts of reactive oxygen species, specifically under stressed conditions (ischemia, reperfusion, environmental toxins). The age-related accumulation of dysfunctional mitochondrial likely results from the combination of impaired clearance of damaged organelles by autophagy and inadequate replenishment of the cellular mitochondrial pool by mitochondriogenesis. Muscle has been studied extensively, but less attention has been paid to the declining function of peripheral nerves with age. We recently showed in animals a drastic decline in the expression of glial and neuronal proteins in myelinated peripheral nerves with age, which is significantly ameliorated by lifelong calorie restriction. We also noted an improvement in nerve architecture with caloric restriction due to a sustained expression of protein chaperones, markers of the autophagy–lysosomal pathway and marked reduction in oxidative stress and inflammation. In muscle of humans, we recently examined whether mitochondrial regulation differed in muscle from elderly subjects classified as high- or low functioning, when compared to young subjects. Mitochondrial respiration rates, PGC-1α, a mitochondrial regulator, Sirt3, a mitochondrial deacetylase, the mitochondrial fusion protein Opa1, were all markedly suppressed in both high and low functioning subjects compared to healthy controls. In addition, we will present data related to a human study in which we show that a combination of diet and exercise has a beneficial effects on protein quality control, autophagy and mitochondrial biogenesis in muscle. This study was to assess the effects of a 6-month weight loss program combined with moderate-intensity exercise on the cellular quality control mechanisms of autophagy and ubiquitin-proteasome, as well as mitochondrial function in the skeletal muscle of older obese women. Taken together, the age-related decline in functional molecules benefitting mitochondrial function (and impaired mitochondrial regulatory pathways) and autophagy (including alterations in other cellular protein homeostatic mechanisms), play a major role to cellular dysfunction with age and provide us with biological targets for intervention studies. Future studies should consider combining exercise with other metabolic and/or anabolic factors (i.e., testosterone, resveratrol, or other bioactive compounds or drugs) to enhance response on major parameters of biological and physical health compared to either regimen alone. Our goal is to provide additional validation to findings that exercise combined with natural and/or pharmaceutical compounds have the most powerful hormetic response to achieve optimal health., Muscle atrophy contributes to the poor prognosis of many pathophysiological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca2+] which control aerobic metabolism, cell death and survival pathways. We have investigated in vivo the effects of mitochondrial Ca2+ homeostasis in skeletal muscle function and trophism, by overexpressing or silencing the Mitochondrial Calcium Uniporter (MCU). The results demonstrate that both in developing and in adult muscles MCU-dependent mitochondrial Ca2+ uptake has a marked trophic effect that does not depend on aerobic control, but impinges on two major hypertrophic pathways of skeletal muscle, PGC-1α4 and IGF1-AKT/PKB. In addition, MCU overexpression protects from denervation-induced atrophy. These data reveal a novel Ca2+-dependent organelle-to-nucleus signaling route, which links mitochondrial function to the control of muscle mass and may represent a possible pharmacological target in conditions of muscle loss., Mitochondrial ultrastructural and morphological changes have been implied in the control of several physiological and pathological changes, including the progression of apoptosis.1 However, the precise role of mitochondrial dynamics in the response to reversible and irreversible cellular damage is not completely understood. Today we will present our recent data obtained in genetic models of ablation and up-regulation of the key mitochondrial shaping proteins Optic atrophy 1 (Opa1) and mitofusin in D. melanogaster and in the mouse. The in vivo experiments of tissue damage by inducing atrophy, apoptosis or ischemia/reperfusion indicate that the master cristae biogenetic regulator Opa1 can prevent multiple forms of tissue damage by controlling mitochondrial cytochrome c release and metabolic efficiency. On the other hand, the interplay between mitochondria and the endoplasmic reticulum is highlighted by the ablation of the multifunctional mitofusin of the fruitfly, that results in the development of ER stress that contributes to the lethality of the phenotype. Our data indicate that the shape of mitochondria dictates function of the organelle and therefore complex tissue responses, opening the possibility for treatment of pathological conditions where mitochondrial dysfunction plays a crucial role., Background. EC coupling in muscle links the transverse (T)-tubule depolarization to release of Ca2+ from the sarcoplasmic reticulum (SR).1,2 These membranes communicate in specialized intracellular junctions, i.e. calcium release units (CRUs), thanks to a cross-talk between voltage-dependent C2+ channels CaV1.1 (or dihydropyridine receptors, DHPRs) in the T-tubule and Ca2+ release channels, or ryanodine receptors type-1 (RYR1), in the SR. Mutations in the gene encoding for RYR1), the SR Ca2+ release channel, underlie debilitating, life-threatening muscle disorders such as central core disease (CCD) and malignant hyperthermia (MH).3,4 To date, MH is only seen as a clinical syndrome in which genetically predisposed individuals respond to volatile anesthetics in the operating room with potentially lethal episodes characterized by elevations in body temperature and rhabdomyolysis of skeletal muscle fibers. However, virtually identical over-heating episodes have been reported in individuals also after exposure to environmental heat, physical exertion, or even during febrile illness.5 The life-threatening nature of MH and environmental heat stroke (EHS) underscore the critical need for a deeper mechanistic understanding of these syndromes and for the development of new and effective treatments. Specific Gaps of Knowledge. A) Mutations in RYR1 have been found in many, but not all, MH cases suggesting the potential involvement of additional genes in the pathogenesis of this syndrome. B) The relationship between classic MH and over-heating episodes triggered by different stressors (heat, exertion, fever, etc.) is not yet widely recognized. C) The cascade of molecular mechanisms that from SR Ca2+ leak leads to rhabdomyolysis of muscle fibers are still unclear and needs to be fully elucidated. Recent breakthroughs. In the last years, thanks to the support of Telethon (GGP08153 and GGP13213), we have moved significant steps forward demonstrating in animal models that: A) MH episodes can result not only from mutations in RYR1, but also from mutations in proteins that interact with RYR1 (such as Calsequestrin-1, CASQ1); B) the mechanisms underlying hyperthermic episodes triggered by anesthetics and by heat/exertion are virtually identical, suggesting that these syndromes could be possibly treated/prevented using similar treatments; C) during lethal MH/EHS crises Ca2+ leak from intracellular stores results in a feed-forward mechanism mediated by excessive production of oxidative species of oxygen and nitrogen (ROS and RNS), which eventually will lead to depletion of the SR and to massive activation of Store Operated Ca2+ Entry (SOCE)., Age related changes of the muscle and its adjacent structures also affect the larynx. This is not only important for speech production, but plays a significant role for swallowing function too. Chronic electrical stimulation of the afferent nerve (recurrent laryngeal nerve) is a completely new therapeutic option that has not been tested before. 18 male Wistar rats were implanted with a unilateral nerve stimulator. Stimulation period was 8 weeks in all animals, twice daily. Changes were observed on the muscular level (cross section area, fiber size) as well as (immuno)-histochemically (e.g. myosin-heavy chain distribution). Compared to control group, chronical stimulation lead to changes in the parameters mentioned above. In conclusion, chronic electrical stimulation can be a new treatment option for age related changes of the larynx. The findings need to be proven in larger animals before going into human studies., The access to different structures in the larynx - especially to the intrinsic muscles in vivo - is limited. Additionally the volumetric quantification is problematic due to their covering with mucosa. Nevertheless it is necessary to generate accurate models of these structures for the purpose of answering muscle-specific issues. Nowadays this is possible with modern imaging procedures such as micro-CT scanning. This technology has advantages over MRI in terms of better resolution and the samples are not destroyed during the imaging process as in histologic sampling. To differentiate the muscles from the soft tissue, the samples are fixed and preserved in neutral buffered formalin (NBF) and stained with iodine to enhance contrast in the CT-scan. The purpose of this study is to generate a 3D-model of the skeleton and the intrinsic laryngeal muscles with the segmentation and finite-element generation and 3D-analysis-software Amira®. This modeling technique will be used in further experiments in the field of muscle stimulation for analyzing of the results, especially muscle volume and structure., Bilateral vocal fold paralysis (BVFP) is a potentially life-threatening medical condition, which causes persistent dyspnoeic symptoms and has a significant impact on the patient’s quality of life. Endoscopic enlargement techniques have been the standard treatment for BVFP for decades.1,2 Laryngeal pacing is a potential treatment based on the electrostimulation of the posterior cricoarytenoid (PCA) muscle. The data herein present the results of the long term performance of a new Laryngeal Pacemaker (LP) System implanted for 24-months in one patient. In a prospective multicentre study, 9 patients were implanted unilaterally with the LP System. 2 left the study prematurely. Of the 7 patients who completed the study, 1 has been implanted for 24-months. Respiration quality; swallowing capacity; quality of life (SF-36 and GBI); six minute walk test (6MWT); jitter; maximum phonation time (MPT); voice range profile (VRP); Dysphonia Severity Index (DSI); Voice Handicap Index-12 (VHI-12); roughness, breathiness and hoarseness (RBH) were evaluated pre-operatively, 1-, 6-, 12-, and 24-months after implantation. Results: Respiratory, voice and life quality generally improved 6-months after implantation and remained stable between 6- and 24-months post-implantation. The results of VHI-12, MPT, DSI, Peak Expiratory Flow (PEF) and Peak Inspiratory Flow (PIF) further improved between 6- and 24-months after implantation. Swallowing quality remained unchanged. These preliminary results showed that the LP System is effective in reducing the symptoms of the BVFP in both the middle- and long-term. In particular, the implantation of the LP System correlated with improved respiratory, voice and life quality. Improvements were stable or increased further between 6- and 24-months after implantation. Evaluation after 24-months of follow-up of all the other patients of the study and larger cohort studies are planned to confirm these initial findings., A unit's "optimal pulse sequence" does not depend on the number of pulses in the train. This property ensures that a motor unit will always develop maximum tension-time area per pulse without a priori knowledge of how long its discharge burst will be, as long as its spikes are generated in time with a pattern corresponding to its optimal pulse sequence. Optimal pulse sequences always began with one or two short interpulse intervals. To produce the repetitive, submaximal, short duration muscle contractions with a rapid rate of force production that are needed during many of the movement patterns elicited during FES, high frequency, moderate-intensity, short-duration trains could be used. These trains have the disadvantage of producing a rapid rate of fatigue of muscle tension. Lower-frequency trains (e.g., 20 pps) produce much less fatigue, but produce a much slower rate of rise of force than higher-frequency trains. Properties of electrical stimulus pulses, patterns and trains producing maximum force, tension, minimum fatigue, to reduce the level of stimulus pain during high levels of muscle stimulation or reducing electrical energy for powering the stimulation will be presented., Much has been learned about the response of muscle to changes in activity by means of implantable neuromodulators.1-3 The pattern of daily activity of a particular muscle group can be reliably changed over periods of weeks, and in many cases, an internal control (for example an unstimulated contralateral limb) is available. One limitation of such studies is that the load on the stimulated muscle is more difficult to control than the activation and thus experiments on resistance exercise in small animals have relied on methods of weight lifting that require specific training or repetitive anaesthesia.4 We have investigated in experiments under anaesthesia the possibility of loading the dorsiflexors of the foot by co-activation of the plantarflexors. We have made preliminary experiments to determine the difference in force produced in the tibialis anterior tendon with: 1) unloaded contractions (common peroneal stimulation, foot free to move), 2) isometric contractions (common peroneal stimulation, foot held externally) and 3) co-contractions (common peroneal stimulation with spill-over stimulation of the tibial nerve to recruit plantarflexion). We have used a new design of in-line load cell to report the force experienced by the tibialis anterior tendon. We can begin to give numerical values to the difference in force experienced by the musculoskeletal system when movement is restricted. This will allow us to include not only the activity pattern but also the loading pattern in our analysis of changes in muscle due to increased activity., Facial nerve pacing may restore eye blink, and to a lesser degree facial expression, using electrical stimulatory techniques. The feasibility of facial pacing via functional electrical stimulation (FES) has already been assessed in animal models (e.g. rabbit and dog) and healthy human volunteers. We performed a feasibility study of whether eye blink can be elicited by transcutaneous electrical nerve stimulation in patients with acute facial palsy, and obtained real-time sensation feedback from participants to determine whether stimulation would be tolerable for daily eye blink restoration. Methods: A cohort of 9 individuals (4 males and 5 females) experiencing paralysis of orbicularis oculi muscle were enrolled at 6-42 days from onset. Unilateral stimulation of zygomatic facial nerve branches to elicit eye blink via orbicularis oculi contraction was achieved with brief bipolar constant-current pulse trains, delivered transcutaneously by epicutaneous electrode placement. Stimulation trains patterns fell in the range of 0.2-1.2 ms pulse duration, 150-250 Hz pulse frequency, and 1-15 mA pulse amplitude. The relationship between stimulation parameters and cutaneous sensation was obtained using the the Wong-Baker Faces Pain Rating Scale in a continuous manner throughout stimulation trials. By studying an initial 9 individuals, we established descriptive statistics regarding the average stimulation thresholds for initial twitch, complete closure, and the relationship between those thresholds and the corresponding level of stimulation discomfort (Wong-Baker Pain Rating Scale scores). In conclusion, facial nerve pacing can potentially restore eye blinks in individuals with acute facial paralysis. Despite variability in reported levels of stimulation-related discomfort, effective pulse trains could be delivered at tolerable current levels. These patients would benefit from a biomimetic device to facilitate eye closure during waking hours in the weeks between paralysis onset and the return of normal blink., Neuromuscular basic research is mainly done in small Laboratory animals, particularly in mice and rats. Long term electrical activation of investigated muscles can only be reliably done when implantable pulse generators are used. A major criterion for such implants is a small volume. The applied daily stimulation patterns depend strongly on the research goal and can be rather different. Consequently, highly adjustable pattern generation and easy handling are essential. The presented implantable neuromuscular stimulator, called MiniStim fulfills these requirements. The MiniStim is powered by a single Lithium primary cell and generates monophasic constant current pulses followed by a charge balancing exponential reverse current. The amplitude can be set with a resolution of 8bit up to 2mA. The minimum pulse duration is 130us and can be increased in steps of 130us. All pulse parameters and stimulation sequence parameters are stored and controlled by a microcontroller. Two different stimulator types have been developed and tested up till now.1 MiniStim type A is pre-programmed during the manufacturing process. Different stimulation patterns can be selected by the use of a permanent magnet. Each touch close to the pulse generator is detected by the internal reed switch and advances to the next in the pre-programmed palette of patterns. Ministim A is intended for experimental studies, which do not require adjustment of stimulation parameters during a period of programmed activity. MiniStim type B can be programmed freely by a bidirectional RF link between a programming tablet and the implanted device. Stimulation data are designed or modified on an Android based tablet computer, which is linked by Bluetooth to the programmer device. Amplitude shift keying is used for data transfer from programmer to the pulse generator and pulse shift keying for transmission in the opposite direction, all operating on a carrier frequency of 400kHz. The transmission link works reliably within an axial and radial displacement of up to 40mm. Epoxy resin or silicone rubber are used for implant encapsulation. Both implant types have been tested successfully in rats for a period of up to 2 months. The implemented stimulation patterns differ markedly and range from 24 bursts daily up to 1452 bursts. There is therefore the possibility to test the effect of complex patterns of stimulation such as those made up of intensive bursts of activity separated by variable rest periods., The Vienna/Liverpool implantable stimulator is the result of many years of development taking advantage of the steady progress in miniaturisation and gate density of integrated circuits. Functions such as digital to analogue conversion and amplification that formerly required the use of additional chips are now packaged into very small plastic packages that are suitable for experimental implants. We have demonstrated that there is sufficient processing power now for a device that is remotely programmable via a short range radio frequency link that allows complete specification of amplitude and pattern a stimulation regime at any stage of an experiment., The use of chronic electrical muscle stimulation for treating partially or completely denervated muscle is controversial. Recently, we used a daily electrical muscle stimulation paradigm over a two week period after nerve injury and immediate repair. We showed that muscle stimulation significantly increases the number of motor nerves reinnervating muscles and axon outgrowth within the distal nerve stump.1 Activity-dependent intramuscular trophic factor release acts on regenerating axons, which may explain the increased early regeneration in stimulated muscle. However, chronic electrical muscle stimulation applied throughout the entire reinnervation period has not been previously assessed. In the present study we hypothesized that stimulation would enhance functional recovery over three months and that stimulation enhances early intramuscular trophic factor release. Six groups of Thy1 -GFP transgenic male rats underwent tibial nerve transection and immediate repair using two epineurial sutures. One group of rats underwent daily electrical muscle stimulation of the gastrocnemius with a paradigm producing 600 equally separated contractions throughout one hour, delivered 5 days per week. Rat gastrocnemius muscles were electrically stimulated for 1, 2, or 3 months and then we assessed muscle force, contractile properties, motor unit numbers, and wet muscle weight. Rats in the 3 month group were serially evaluated using a tapered beam test to evaluate skilled locomotion. Muscles underwent immunohistological examination of motor end plate reinnervation. Two additional groups of rats were subjected to the same nerve injury and were used to investigate early intramuscular trophic factor release following two weeks of electrical muscle stimulation or no treatment. The number of motor units was significantly increased after daily muscle stimulation for all three time points (1, 2, and 3 months). Mean motor unit sizes were significantly smaller in stimulated muscles, suggesting that muscle stimulation may inhibit terminal sprouting as reported by others. This may allow for a more natural course of reinnervation resulting in improved functional recovery. Indeed, skilled locomotion tests showed that stimulated muscles enhanced and maintained recovery at levels no different than normal functioning rats, whereas non-stimulated controls became progressively worse and did not recover to baseline. After two weeks of stimulation, BDNF was significantly upregulated in stimulated muscle compared to non-stimulated muscle. In conclusions, treatment of denervated muscle using electrical stimulation significantly enhanced muscle reinnervation, and upregulation of BDNF may explain this enhancement. As the muscle continues to reinnervate, tailoring the stimulation paradigm to improve muscle force and fatigability may further enhance muscle recovery., The method commonly used in FES is in general a non-selective nerve stimulation, which in turn causes frequent occurrence of undesirable side effects. To alleviate such problems, various models and electrode systems that selectively stimulate certain features have been developed. In this regard, we review the functional performance of quasitrapezoidal current biphasic stimulating pulse for fibertype selective nerve stimulation. The stimulus should predominantly stimulate myelinated Aβ-fibres, minimize the stimulation of the myelinated Aα-fibers and Aδ-fibres, and bypass the stimulation of the non-myelinated C-fibres. The study including nerve conduction velocity and compound action potential (CAP) measurements is performed on an isolated segment of a rat sciatic nerve. A stimulus is applied to the nerve using a pair of hook platinum wire electrodes while CAP is measured at the two sites along the nerve using two couples of identical recording hook platinum electrodes. Positive recording electrodes of the two couples, are situated at the distance 9.6 for the first couple and 19.2 mm for the seconfd couple from the stimulating cathode. Results of the study show, that ascending parameters of the stimulus, namely, ic - intensity of the cathodic phase, tc - width of the cathodic phase, texp - width of the cathodic exponential decay and τexp – time constant of the cathodic exponential decay, influences the peak values and all the temporal parameters of the CAP1 and CAP2. Results also show that a peak value of the CAP1max - CAP1 maximum and CAP2max - CAP2 maximum can be obtained when parameters of the stimulus are set within the following range: ic=2.5 to 3.35 mA, τexp=330-440 µs, tc=325-430 µs and texp=330-440 µs, respectively. The most important finding of the present study however, is that when certain parameters of the stimulus waveform are selected, the contribution of the myelinated Aα-fibers and Aδ-fibres can be minimized, the contribution of the non-myelinated C-fibres bypassed and the contribution of the myelinated Aβ-fibres actualized. The confirmation of this statement can be found in the measured CAP1 and CAP2, where in spite of the three aforementioned types of nerve fibers are present within the nerve, only one peak in both the measured CAP1 and CAP2 is observed. It was also shown that only tc slightly shorter and slightly larger than 365 µs and texp slightly shorter and slightly larger than 270 µs are appropriate to be used with the pre-defined stimulus waveform and arrangement of stimulating and recording electrodes within this particular stimulating/recording setup. It can be concluded that the design of stimulating electrodes and stimuli waveform based on the obtained results, could act as a useful tool for nerve stimulating electrodes development that potentially enable fibre-type selective stimulation of nerve fibers., Adaptive changes of muscle fibers can occur in response to variations in the pattern of neural stimulation, loading conditions, availability of substrates, and hormonal signals These signals stimulate pathways which may lead to changes in fiber size and/or fiber type. Fiber size is in a dynamic equilibrium between protein accumulation (hypertrophy) and protein loss (atrophy). The paradigmatic human models for muscle hypertrophy is resistance training (RT). But RT can influences not only muscle mass but also many other metabolic pathways that may affect positively health. Recently our group has demonstrated the different effects of circuit training carried out at low or high intensity on some anthropometric and metabolic variables. One of the major issue regarding the effects of RT on muscle mass is the extreme complexity of such kind of exercise. Resistance training may be carried out via different methods that have been shown to have differing effects on muscle metabolism and signalling pathways. As a matter of fact a resistance training program is a composite of several important variables including: 1) muscle action used, 2) type of resistance used, 3) volume (total number of sets and repetitions), 4) exercises selected and workout structure (e.g., the number of muscle groups trained), 5) the sequence of exercise performance, 6) rest intervals between sets, 7) repetition velocity and 8) training frequency that could be taken into account. For this reason we have investigated the effects of two different kind of RT on muscle signalling. 12 healthy and physically active subjects performed in two different moments and with different legs an high intensity resistance training (HIRT) and traditional resistance training (TRT). HIRT consisted in 2 sets at the leg extension performed with the following technique: 6 repetitions, 20 seconds rest, 2/3 repetitions, 20 secs rest, 2/3 repetitions with 2 min 30 secs rest between the sets. TRT consisted of 4 sets of 15 repetitions with 1 min 15 secs of rests between the sets. Biopsies from the vastus lateralis were taken one week before training (Tb) sessions, immediately after training (T0), 6 hours after (T6) and 24 hours after (T24). Western blot analysis was performed to investigate mTOR, Akt, 4EBP1, S6, AMPK and ACC. Our results showed that different RT execution affects muscle pathways in a specific manner. These results suggest that a specific kind of RT should be studied to counteract atrophy mechanism related to disuse or ageing., The aim of our study was to investigate whether stem cell (SC) therapy with human amniotic fluid stem cells (hAFS, fetal stem cells) and rat adipose tissue stromal vascular fraction cells–GFP positive cells (rSVC-GFP) was able to produce favorable effects on skeletal muscle (SM) remodeling in a well-established rat model of right heart failure (RHF). RHF was induced by monocrotaline (MCT) in Sprague–Dawley rats. Three weeks later, four millions of hAFS or rSVC-GFP cellswere injected via tail vein. SMremodeling was assessed by Soleus muscle fiber cross sectional area (CSA), myocyte apoptosis,myosin heavy chain (MHC) composition, satellite cells pattern, and SC immunohistochemistry. hAFS and rSVC-GFP injection produced significant SC homing in Soleus (0.68±1.0 and 0.67±0.75% respectively), with a 50% differentiation toward smoothmuscle and endothelial cells. Pro-inflammatory cytokines were down regulated to levels similar to those of controls. SC-treated (SCT) rats showed increased CSA(pb0.004 vsMCT) similarly to controlswith a reshift toward the slow MHC1 isoform. Apoptosis was significantly decreased (11.12.±8.8 cells/mm3 hAFS and 13.1+7.6 rSVC-GFP) (pb0.001 vs MCT) and similar to controls (5.38±3.0 cells/mm3). RHF rats showed a dramatic reduction of satellite cells(MCT 0.2±0.06% Pax7 native vs controls 2.60±2.46%, pb0.001), while SCT induced a repopulation of both native and SC derived satellite cells (pb0.005). In conclusion, SC treatment led to SM remodeling with satellite cell repopulation, decreased atrophy and apoptosis. Modulation of the cytokine milieu might play a crucial pathophysiological role with a possible scenario for autologous transplantation of SC in pts with CHF myopathy., The existing healthcare systems built around the traditional paradigm of patients suffering from a single acute illness are largely unprepared to face the increasing demands for health services that can specifically address the medical needs of older, multimorbid people. As a consequence, a large and growing segment of the older European population is currently suffering from medical conditions that cannot be efficiently managed by the available healthcare services.1 In this scenario, the geriatric syndrome of frailty plays a major role. Frailty is defined as a multidimensional condition characterised by decreased reserve and diminished resistance to stressors.2 Such extreme vulnerability exposes the older individual at an increased risk of morbidity, disability, inappropriate healthcare use, institutionalization, poor quality of life, and mortality.2 Detecting and contrasting frailty are thus crucial for impeding the progression of the syndrome, preventing its detrimental clinical consequences, and ensuring the sustainability of healthcare systems. Unfortunately, to date, no healthcare programs or pharmacological treatments are available for frail older people, largely because of the lack of a precise, universal definition of the condition. Such a barrier may be overcome by developing and validating a robust conceptual framework to achieve a practical operationalisation of frailty. The recognition of sarcopenia as a central component of physical frailty may allow overcoming the existing uncertainties in the field, while providing a biological substrate for preventive and therapeutic interventions.3 In this regard, it should be noted that monodimensional interventions may be insufficient at reversing the complex frailty status. Conversely, multi-component interventions have shown to be particularly useful when dealing with age-related syndromic conditions.4 Indeed, the simultaneous targeting of multiple and heterogeneous mechanisms underlying the disabling cascade may enhance the intervention effects.5 At the same time, multi-component interventions resemble what is commonly done in usual clinical practice, in which the intervention is designed around the needs and resources of the individual. Based on the existing evidence, it is expected that the combination of physical exercise and nutrition, with the eventual support of appropriate e-health services, may provide the greatest benefits in the management of physical frailty and sarcopenia., Aging involves the progressive functional decline of tissues, which includes the dysfunction of the mitochondrial respiratory complexes leading to a reduced adenosine triphosphate (ATP) synthesis and to a decreased cell bioenergetics capability. Mitochondrial Transcription Factor A (TFAM) is regarded as a histone-like protein of mitochondrial DNA (mtDNA), performing multiple functions for this genome. Due to the close connection between mtDNA transcription and replication, TFAM has been supposed to participate to the regulation of mtDNA copy number. TFAM is also involved in the constitution of mtDNA nucleoids and might be part of a system responsible for sensing and repair of oxidative damage to mtDNA. Because of the relevant involvement in the regulation of mitochondrial biogenesis and transcription, TFAM expression has been investigated in tissues such as the brain, heart and skeletal muscle that have a high dependence on oxidative metabolism either in metabolically very active tissues, such as the liver. Aging affects mitochondria in a tissue-specific manner and so far only calorie restriction (CR) is able to delay or prevent the onset of several age-related alterations also in mitochondria. Age-related changes in mtDNA content were reported in various tissues. We detected TFAM amount, TFAM-binding to mtDNA and mtDNA content in three aged rat tissues. Samples of the frontal cortex 1 and soleus skeletal muscle 2 from 6- and 26-month-old ad libitum-fed and 26-month-old calorie-restricted rats and of the livers 3 from 18- and 28-month-old ad libitum-fed and 28-month-old calorie-restricted rats were used. We found an age-related increase in TFAM amount in the frontal cortex, not affected by CR, versus an age-related decrease in the soleus and liver, fully prevented by CR. The semi-quantitative analysis of in vivo binding of TFAM to specific mtDNA regions, by mtDNA immunoprecipitation assay and following PCR, showed a marked age-dependent decrease in TFAM-binding activity in the frontal cortex, partially prevented by CR. An age-related increase in TFAM-binding to mtDNA, fully prevented by CR, was found in the soleus and liver. MtDNA content presented a common age-related decrease, completely prevented by CR in the soleus and liver, but not in the frontal cortex. The common age-related loss of mtDNA might be explained by two different tissue-specific mechanisms. The age-related decrease in TFAM-binding in the frontal cortex might imply decreased mtDNA replication and/or increased mtDNA damage, not counteracted by the usual repair mechanisms. On the contrary, the age-related increased TFAM-binding at both origins of replication in the soleus and liver might explain the mtDNA loss through a hindered mtDNA replication. The modulation of TFAM expression, TFAM-binding to mtDNA and mtDNA content with aging and CR showed a trend shared by the skeletal muscle and liver, but not by the frontal cortex counterpart. A fine modulation of TFAM-binding to mtDNA may contribute to several tissue-specific changes involved in the age-related mitochondrial dysfunction as well as in the preventive effects of CR. This could have relevant consequences for future applications of CR or other nutritional regimens to delay the onset of pathologies, such as sarcopenia and neurodegeneration, related to the age-dependent mitochondrial dysfunction., Ageing is associated to a dramatic increase in the incidence of heart failure, even if the existence of a real age-related cardiomyopathy remains controversial.1-3 In this study we performed a morphological study of cardiac cells in hearts from adult and old mice (4 months and ≥ 24 months of age, respectively) using confocal and electron microscopy. Our results indicates that the cross sectional areas (CSA) of cardiomyocytes is on the average increased in old hearts (adults: : 189 ± 93 µm2; aged: 282 ± 155 µm2), with the greater variability in size indicating also the presence of several atrophic cells. The increased average CSA may be the result of an increased presence of amorphic (and apparently empty) cytoplasmic space between myofibrils (adults: 2.2 ± 0.3; aged: 9.7 ± 0.6). As effective contraction and relaxation of cardiomyocytes also depends on Ca2+ supply to myofibrils, handled by calcium release units (CRUs) and sarcoplasmic reticulum (SR) and on efficient ATP production (provided by mitochondria), we have also performed a qualitative and morphometric analysis of these intracellular organelles. The analysis of CRUs indicates that SR/transverse-tubules (TT) couplons becomes shorter with age and that the number of CRUs/50 µm2 is decreased of about 24% (adults: 5.1 ± 3.3; aged: 3.9 ± 2.6). Also mitochondria present structural modifications, with a significant increase in the percentage of organelles presenting severe alterations (3.5% vs. 16.5%, respectively in adult vs. aged). Importantly, both CRUs and mitochondria undergo a spatial re-organization with respect to sarcomeres/myofibrils: CRUs are may be miss-oriented (longitudinal) or miss-placed (found at the A band instead of being correctly placed in proximity of Z-lines), while mitochondria are often grouped in an abnormal fashion. These age-related ultra-structural changes may underlie an inefficient supply of Ca2+ and ATP to contractile elements, providing a possible structural explanation for heart dysfunction., Detection of the pull of earth’s gravity (graviception) is essential to life and shared by all living things on earth, both plants and animals. Simple creatures as jellyfish through invertebrates have a simple form of graviception where biological mass (otoliths) act upon hair cells to detect the pull of gravity. With it's complex shape, sculpted in the petrosal part of the temporal bone, the mammalian inner ear is responsible of a dual function i.e. hearing and balance. The functional nominator is force transduction i.e. changing mechanical forces into electrical signals, called action potentials.1 The basis behind this force transduction property is two folded, i.e. inner ear morphological construction and physiological qualities. The later is based on potassium rich endolymph responsible for the endocochlear potential, hair cells depolarizing properties and recycling of potassium in the organ of Corti and nerve conduction of hair cell signal for central processing. The morphology of the inner ears are of highest interest, i.e. paired (right/left) construction with almost 100% symmetry not only regarding the size, but importantly regarding orientation of the semicircular canals, configuration of utricular and saccular maculae and coiling of the cochlea. This morphology was evolved in terrestrial environment in response to forces associated with sound conduction in air, gravity and fast head and body movements. In fish that live in aquatic environment, graviception is made possible through otolith that act upon hair cells within the utriculus and sacculus, i.e. big calcium crystals forms that allow the fish to detect the pull of earth’s gravity, extremely reduced in the aquatic environment. During sea-to-land evolution the terrestrial environment forced upon the living creature much stronger gravity forces, that resulted in splitting of the otolith into otoconia, small calcium crystals embedded in gelatinous membrane in utriculus and sacculus of terrestrial vertebrates inner ears. Terrestrial environments allow also much quicker and more agile movements that calls upon more sophisticated semicircular canals which sets ground for vestibulo-ocular reflex essential for eye focusing.2 Muscle tone, the force with which a muscle resists being lengthened, depends on three fundamental issues. The intrinsic elasticity or stiffness of the muscles, the stretch reflex feedback loop and “higher” neural contribution. The fundamental of this higher neural contribution are signals from the inner ear i.e. the graviceptal information relayed through vestibulo-spinal pathways described partly by Sherrington some 100 years ago.3 Whales are mammals that have adapted holaquatic life style, where similar terrestrial mechanical forces are severely reduced and due to that the inner ear has changed dramatically again. The driving forces of those evolutional changes are probably due to seasickness., Dizziness, vertigo, impairment of balance and fear of falling are common complaints in elderly. The underlying processes are not fully understood but a significant number of symptoms have been linked to vestibular pathology, and inadequate postural control. Dizziness ranks among the most common complaints in medicine, affecting in some forms approximately 40 % of general population during the course of life.1 In a Swedish cohort study the daily occurrence of balance problems was present in 33 % among elderly at age 70 years and increased to 50 % for elderly at age of 80 years or more.2 In literature the terms dizziness and vertigo have been used to identify the site of pathology, i.e. that dizziness would originate from non-vestibular sites and vertigo would be specific to the vestibular part of the inner ear. Finally the balance symptoms have been addressed less specifically to conflicts in postural control. The AGES Reykjavík study is based on the Reykjavik study, which started in 1967 and comprises health information of more than 20.000 individuals who at present are older than 69 years. The study, started in the year 2001 and is ongoing. The study includes several aspects of human aging and a total sample of more than 5000 subjects have been evaluated for balance and hearing functions, i.e. the ageing of CNS and its postural control have been thoroughly studied.3 Beside cognitive tests and MRI of the CNS, the focus is on following balance research: Questionnaire regarding brain, inner ear and balance problems. Motor functions test as timed up and go and 6 meter walk. Strength in the lower extremities. Posturography (force) platform, where four main tests are performed: a) chair stand b) quiet stands with open and closed eyes c) target hunting d) step test. Hearing evaluation (PTA, impedens audiometry). Main findings are that the reaction time for both men and women decrease as well as the stabilization time in the chair test with increased age. Hearing thresholds in all frequencies tested decreases with increased age in both ears. All data available is now under thorough investigation schedule, which can be coupled with all other fields of data, harvested in the AGES Reykjavík study. The AGES Reykjavík study is one of the largest epidemiology studies in to ageing carried out., FMS is a non-invasive technique to induce eddy currents in living tissue and activate action potentials in neuron structures. The main advantage in comparison to electrical stimulation via skin attached electrodes is the fact that pain sensors in the upper skin layers are not excited by FMS, as due to tissue impedance properties induced currents remain more or less below threshold.1,2 On the other hand, induction of effective electrical field strength requires power electronics capable of driving application coils with high impulse current in the range of kA, which is an engineering challenge and rises problems of heat management and size and weight of equipment. Application of FMS has for long time been limited to delivering single and double stimuli to the human cortex for diagnostic assessments (transcranial magnetic stimulation), with bulky and expensive equipment. Just in the recent years, FMS devices enter the market that can deliver trains of stimuli with up to 100 Hz repetition rate and capable of inducing strong but comfortable muscular contractions. A first very useful application is offered for efficient and painless pelvic floor training for therapy of continence problems, with stimulator electronics and application coil integrated in a comfortable chair. Meanwhile also systems with stationary electronics module and mobile coil for manual positioning near target muscles are available, though at much higher costs than comparable electrical stimulators. Portable solutions are not realistically to be expected due to technical limitations. Even though we can expect, that FMS will become an important alternative treatment option where therapy stations can be provided in appropriate environment - like the mentioned pelvic chair centrally in retirement homes or outpatient clinics. Mobile solutions and affordable devices for home based training will in foreseeable future remain relying on FES technology., EMG signals are a physiologically appropriate control source that can provide the user with direct control over a device and their exploration is an important step in the prosthetic development of an intuitive human-machine interface.1 Due to several technological hindrances, like high pressures and shear forces on the residual limb and thus upon surface electrodes within the socket, as well as traditional surface EMG drawbacks,1 no commercially available lower limb prosthetics yet utilise the surface EMG signal for control. This study explores a surface electrode measurement technique for attaining reliable EMG signals from the residual muscles of a transfemoral amputee, with the future prospect of controlling functions of lower limb prosthesis. Neuroline surface electrodes (Ambu® Denmark)2 were used inside the socket, on the hamstring, quadriceps and adductor muscles, and passed proximally above the socket rim to the ground electrode on the subjects back. Nickel-plated brass triode electrodes(T340 Lifematters US)4 were placed outside of the socket, on the tensor fasciae latae (TFL) and gluteus medius (GM) muscles and were also used as reference electrodes. Placing of electrodes on the TFL and GM muscles followed the SENIAM recommendations.4 Same recommendations could not be applied for muscles whose distal end had been amputated but instead the optimal electrode position was determined by palpation and the area of the strongest signals was defined by iterative placement of the triode electrodes in the area of interest, taking in account the anticipated displacement by the liner and socket (usually proximal direction). An array of 3 to 4 electrode pairs were placed over the region of greatest activity and the EMG signals were measured after the amputee donned his socket to locate the best electrode position. The EMG signals were recorded and analyse using the wireless Kine System (Kine ehf. Reykjavík, Iceland).5 EMG signals were collected while the subject contracted individually the respective muscles, first prior to liner placement and again after liner and socket placement. The same signals were subsequently collected collectively during treadmill walking, stair decent and sitting-to standing. The study shows that EMG signals can with this method be reliably identified and collected using surface electrodes underneath a silicone liner. The signals were more distinguished after liner and socket placement. The technique for placement of an array of electrodes was critical for success as it permits anticipation of the displacement by liner and socket system during weight bearing. This technique does not make the placement of electrodes practical for home use. Still we conclude that the results are promising regarding the prospects of using an array of surface EMG signals for the control of lower limb bionic prosthesis., Low back pain is the most common health problem of musculo-skeletal system of today’s population. It is a big social and economic problem in developed countries and in developing countries. People with low back pain have many changes in neuro-muscular functions of the human trunk.1-3 To assess postural control muscle activation during mechanical perturbation is measured with the use of electromyography (EMG). Processing of EMG signals is usually done manually and the expert must have good knowledge of the used methods.4,5 This makes it very slow, error prone and unsuitable for large set of EMG signals. Our aim in this study was to develop methods that will be suitable for automatic processing of EMG signal with no user input. Processing of EMG signals can be divided into three phases: noise removal, detection of a response onset and offset and assessment of an event (attribute calculation). In noise removal phase we must deal with several types of noises from which the electrocardiogram (ECG) artifact is very common in EMG signals of the trunk and it is very hard to remove.6 Several methods (high-pass filtering, gating, template subtraction, empirical mode decomposition with independent component analysis, adaptive wavelet transform) have been proposed in the literature to remove ECG artifact, however, none of them turned out to be completely successful. We have applied, tested and proposed a new method for ECG artifact removal that is based on dynamic time warping method and it first identifies ECG artifacts in the EMG signal and then adaptively removes the artifacts with subtraction. For onset and offset detection a variety of different approaches has been used (methods based on threshold, optimal estimator, approximated generalized likelihood-ratio detector (AGLR), integrated profile, sample entropy and some others). We developed a new method for this task that is based on AGLR and have more advanced false event detector which helps eliminates false detections. There has been several attributes for an event assessment used in the literature: attributes in time domain, attributes in frequency domain and attributes in time-frequency domain. We add several attributes known from needle EMG and sound processing domain and identified subsets that are the best for complete EMG event assessment. Our proposed method for ECG artifact removal outperformed other methods and proved to be more reliable in ECG detection. AGLR with advanced false detector showed better results compared to previously used methods, especially in false detection prevention. A few subsets for complete EMG event assessment were identified as suitable for this task. Newly introduced attributes from needle EMG and sound processing domain improved the reliability significantly. Newly proposed methods makes EMG processing more reliable, much faster and are suitable to process large datasets of EMG signals from human trunk., The paper is concerned with closed-loop On-Demand control of Deep-Brain Stimulation (DBS) for Parkinson’s disease (PD) patients and it efficacy. On-Demand control implies that DBS is applied in pulse trains of finite pre-computed durations only when necessary, rather than being applied continuously. The paper thus investigates the efficacy of On-Demand DBS control, in terms of maximizing the ratio of durations where no tremor occurs while stimulation is OFF, relative to the duration of stimulation that precedes the period of no-stimulation. It also investigates the efficiency of a neural-network-based algorithm to predict onset of tremor in order that On-Demand control of DBS will avoid any tremor from occurring via efficient prediction of onset of tremor before rather than after it occurs. Data were obtained from 9 PD patients, tested at the University of Illinois Hospital in Chicago, IL, all of whom had been implanted with DBS in at the University of Illinois Hospital, Chicago or at Rush University Medical Center, Chicago over the past 15 years. Non-invasive surface EMG (sEMG) data from patients’ limbs were used to predict onset of tremor stochastic signal processing algorithms, including entropy analysis, were used to process the raw sEMG signals. During testing, DBS stimuli of standard and FDA-approved protocols were applied in packets of pulse-trains of varying durations. We show that there exists, for each patient, an average duration of Stimulation that maximizes the ratio of duration of No-Tremor-No-Stimulation over duration of Stimulation (denoted as R). In 3 out of 9 patients tested above, the ratio R (as above) is between 2 and 10.3, in the vast majority of trials performed in these patients (25 of 30 individual trials), when a stimulation duration that maximizes R was used. In conclusion, we show that: a) On-Demand control of DBS using non-invasive sEMG sensors can reduce total stimulation time by 55% to over 90% (namely [R+1]/R), depending on the patient involved, in a significant number of PD patients with tremor, when adequately selected durations of stimulation pulse-trains are employed; b) An optimal duration of stimulation pulse-trains as in (a) exists and is computable; c.) Prediction of onset of tremor from non-invasive sEMG signals is possible, to allow applying fixed duration DBS pulse-trains before actual tremor starts. Our past work [Basu et al., J Neural Engineering, vol. 10, No. 3, 2013] on predicting onset of tremor following a DBS pulse-train via sEMG yields the average duration (Tp) from end of stimulation to predicted tremor to be more than 0.8To where To is the duration from end of stimulation to onset of actual tremor. Hence, an on-demand predictive controller, using the latter or similar prediction algorithm, makes a non-invasive sEMG-based ON-DEMAND control of DBS feasible and effective for at least a sub-population of PD patients who have tremor. It requires no change in present (uncontrolled) DBS implantation procedures or in DBS pulse-generation protocols already approved by the regulatory authorities; d) Prediction can be made in sufficient time ahead of the onset of tremor, while still keeping the tremor-free time without stimulation reasonably long compared to the duration of stimulation being on; e) It is reasonable to use tremor as a proxy for all Parkinsonian symptoms (i.e. rigidity, bradykinesia, etc.), as tremor is the first symptom to reappear after DBS is switched off and the first symptom to resolve after DBS is turned back on.1, Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron degeneration, muscle atrophy and weakness, eventually leading to muscle paralysis and death. Several factors account for the development of ALS, including accumulation of oxidative stress in skeletal muscle1. A positive correlation between the expression of the histone deacetylase 4 (HDAC4) and the progression of the disease has been recently reported in ALS patients, suggesting the use of HDAC4 inhibitors as a promising therapeutic approach for the treatment of this neurodegenerative disease2. HDAC4 in skeletal muscle plays a crucial role in the regulation of muscle mass and reinnervation following denervation3. However, the molecular pathways controlled by HDAC4 in ALS onset or progression, as well as in response to oxidative stress in skeletal muscle are not delineated yet. We investigated the role of HDAC4 in ALS by deleting HDAC4 in skeletal muscle of SOD1G93A mice, a mouse model of ALS. Lack of HDAC4 in skeletal muscle anticipated body weight loss and induced more pronounced muscle atrophy in late stage SOD1G93A HDAC4 mKO mice, compared with age-matched SOD1G93A mice, indicating a protective role of HDAC4 in ALS. To study the molecular mechanisms underlying HDAC4 function in response to a chronic denervation, such as in ALS, we cut the sciatic nerve of one limb of HDAC4 mKO mice and analyzed muscles over time. HDAC4 mKO mice did not undergo muscle atrophy for two weeks following denervation, but muscles degenerated at later time points. Moreover, contralateral innervated muscle of HDAC4 mKO mice presented ultrastructural defects in myofiber organization and higher levels of ROS, while alteration of sarcomeric architecture and the molecular responses to oxidative stress were blunted following denervation. From our results, we conclude that HDAC4 protects skeletal muscle in ALS and is important to maintain muscle integrity and oxidative stress response following denervation. Further studies are necessary to delineate the role of HDAC4 in skeletal muscle integrity and in response to chronic denervation., Amyotrophic Lateral Sclerosis (ALS) is a major neurodegenerative disorder, which involves the degeneration of upper and lower motor neurons, muscle weakness, atrophy, fasciculations and paralysis. The deambulation is quantitatively and qualitatively reduced. However, pervious evidence in related field suggests usefulness of conventional rehabilitation for the ALS patients. Furthermore, there is no evidence regarding the specific treatment modality. Functional Electrical Stimulation (FES) has been assessed for neurorehabilitation treatment (Spinal Cord Injuries, Stroke, Multiple Sclerosis), but there are shortage scientific reports for the ALS. Relying on neurophysiological mechanisms, we hypothesized that FES could be a beneficial muscle’s treatment in the patients affected by ALS. Therefore the aim of the present study is to investigate the clinical and functional effects of the electrical muscle stimulation in ALS patients. Matherials and Methods: Two patients, admitted to the Department of Neurorehabilitation of the Care & Research Istitute San Camillo in Venice, with confirmed primary ALS were treated according to the experimental Cycling-FES clinical protocol. The protocol was an additional 1 hour a day treatment to the conventional neuromotor rehabilitation. The lower limbs training was performed by the use of Cycling-FES (Hasomed RehaStim2 MOTOmed® viva2). The treatment consisted of 15 sessions, half an hour a day, 5 days per week, for 3 weeks. The therapeutic session was divided as follows: 5 minutes of warm-up (pedal without FES), 20 minutes pedal + FES and 5 minutes of cool-down (pedal without FES). The following muscles (bilateral) were considered for the FES: flexors and extensors of the knee, dorsal and plantar flexor of the ankle. The surface adhesive electrodes were placed according to the operation manual of Rehastim2 (i.e. 9X4 cm for the proximal muscles and 4X4 cm for the distal muscles). The parameters of electrical stimulation were as follows: biphasic rectangular waveform, frequency range 40-45 Hz, pulse duration within 150-200 µsec, and the muscles contraction was obtained through intensity within 30-50 mA, as well. The synchronized cycling was provided by a motorized cycle ergometer (20-30 Revolutions per Minute). Functional tests and clinical scales [i.e. 10 Meters & 6 Minutes Walking Test; Medical Research Council (MRC) Scale for Muscle Strength; Timed Up & Go; Modified Ashworth Scale] were performed, before and after treatment, in order to assess the effects of therapy. All the same, was monitored the spasticity, through an instrumental examitation (H-reflex test). Results: Both subjects improved their motor performance according to the clinical and functional tests. Furthermore, the modified Ashworth Scale and H-reflex test did not show increased spasticity of lower limb muscles. There were no reports of adverse events during the assessment and treatment sessions, as well. Discussion and Conclusions: To our knowledge this is the first study which described the application of Cycling-FES for the ALS treatment. Obtained results are encouraging, but, due to limited subjects, we cannot draw any conclusion, therefore we are keeping on working, because further research is needed. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration., Central Core Disease (CCD; OMIM# 117000), one of the most common human congenital myopathies, is characterized by hypotonia and proximal muscle weakness with slow (or non progressive) clinical course.1 Diagnosis of CCD is confirmed by histological examination of muscle biopsies showing amorphous central areas or cores (typically found in type I muscle fibers), lacking glycolytic/oxidative enzymes and mitochondria. Usually, orthopedic complications limit the ability of CCD adult patient to perform physical exercise. Most CCD families have been associated with C-terminal mutations in the gene encoding for the Ca2+ release channel of skeletal fibers, i.e. ryanodine receptor type-1 (RYR1). We have recently successfully used functional electrical stimulation (FES) to rescue muscle mass and force in spinal cord injury patients and in elderly subjects,2,3 but the use of FES has never been considered to improve muscle function in CCD patients. The purpose of this study is to test the efficacy of FES in counteracting muscle loss and improve function in the lower extremities of a 55-year-old female patient affected by CCD. The patient presented, since the adolescence, fatigue and progressive proximal limb weakness with hyperCKaemia. Her family history was inconsistent for neuromuscular diseases. At 44 years of age she was diagnosed with CCD as a muscle biopsy in the left brachial biceps revealed the presence of “central cores” in most type-1 muscle fibers. Methods. a) Genetic Screening. PCR primers for all RYR1 exons were designed with the Primer-3 software (http://frodo.wi.mit.edu/cgibin/primer3/primer3_ www.cgi). Mutation analysis of the RYR1 gene has been performed as previously described;4 b) FES protocols. Before beginning of FES training protocols, the patient performed stabilometry test 1, maximal isometric voluntary force (MVF) of leg extensor muscle test by dynamometer 2 and a complete set of functional tests to assess mobility and function in activities of daily living (ADL). These tests included: time up and go test (3, TUGT); 10m-walking test with habitual and fastest walking speed 4 and short physical performance battery (5, SPPB). In the first phase of FES training (5 months) from Time-0 to Time-1 (T0 to T1), upper leg muscles of the patient have been stimulated by FES (60Hz), 3 sessions per week, 3x10min of treatment for each muscular group. c) Electron microscopy (EM) analysis. A new muscle biopsy was collected from the right vastus lateralis using a semi-automatic needle (Precisa 13 Gauge; Hospital Service, Rome, Italy) to perform EM analysis. Neurological examination revealed a hyperlordotic posture, bilateral pes cavus, limb-girdle hypostenia (MRC score: 3-4 in the upper and 2-3 in the lower) with waddling gait and difficulty into climbing stairs, steppage. The study is still in progress: EM structural analysis has been only performed at T0, whereas most functional assessments have been so far only performed at T1 = 5 months). Analysis of the RYR1 gene identified a missense mutation (c.7354C>T) resulting in the substitution of arginine in position 2452 with a tryptophan (p.R2452W). In addition, a duplication of 4 amino acid residues (Thr, Ala, Ala, Thr: p.Thr4285-Thr4288dup) was also observed. At the EM ultrastructural analysis, a high percentage of muscle fibers analyzed (80%) revealed the presence of large regions of hyper-contraction devoid of intra-myofibrillar organelles, i.e. calcium release unites (CRUs) and mitochondria. The outcome of the functional tests at T1 was only partially encouraging: whereas improvement in the Stabilometric and MVF tests were encouraging (indication of an improvement of force induced by FES), results in the battery of ADL tests were unfortunately negative. The reason for this partially negative outcome may reside in low back-pain lamented in the last months by the patient, a problem that has temporally caused a significant reduction in her daily walking activities. In conclusion, the next immediate goal is, obviously, to determine the reason causing low back-pain and reduced mobility of the patient (by magnetic resonance imaging). The study will then proceed in the next months to a) stabilize the lower back of patients with the goal of improving her mobility and independence and b) proceed with the next step in FES training (at increasing loads) with the goal of improving ADL functional tests. A new muscle biopsy for EM will be performed only if (and when) functional performance will be significantly improved., Amyolateral Sclerosis (ALS) is a neurodegenerative disease, attacking motor neurons. The degeneration of the motor neurons causes muscle paralysis and death will eventually result, often from a ventilation crisis. Only a small percentage of ALS cases are known to be genetic. The causes of sporadic ALS cases (those without a known genetic origin) are not understood. There is no known cure for ALS and only palliative therapies are available, at least for the predictable future.1 However, we believe that respiratory muscle function may be extended, thus postponing the need for pumping air which further damages the diaphragm muscle. We must first show in animal models that some additional muscle contractile function can be achieved by combining proven approaches to maintain/recover contractility of “denervated” muscle fibers of the diaphragm.2-4 The aim of the Project Save-ALS is to experimentally test a number of procedures on animals experiencing unilateral sciatectomy of leg muscles and unilateral section of the phrenic nerve. Working with mammals, first with rodents (rats and mice, the last both wild type and SOD-/-),6-8 and then with larger mammals (e.g., rabbits, sheep, and pigs) we will test the effects of the following: 1) new molecules, 2) new improved methods of production and injections of gliogenic and myogenic stem cells derived from adipose tissue, and 3) effects of physical stimuli (i.e., direct electrical stimulation of denervated muscle fibers in the leg muscles and the diaphragm, using flexible ring multi-wire electrodes). The development of new protocols and stimulation devices (e.g., electrodes and implantable mini-stimulators) for denervated muscle will complement the effects of existing commercial electro-stimulators used for ventilation support to delay the necessity for use of supported ventilation by pneumatic devices. This is important because controlled mechanical ventilation is known to exacerbate this progressive disease by unloading the diaphragm, thus decreasing the diaphragmatic force generating capacity and potentially causing ventilator-induced diaphragmatic dysfunction (VIDD Syndrome).9-11 In short, we would like to test if the accumulating evidence that brief ES, a useful method to improve functional recovery for delayed repair of peripheral nerve lesions, may be extended to ALS.12-15 It is important to note that devices already were approved for ALS-treatment and are reported to prolong life expectancy based on a large study (http://www.synapsebiomedical.com/). Nevertheless, the reported devices work by activating the remaining innervated muscle fibers and not the denervated ones (http://www.synapse biomedical.com/als/neurx-als.shtml). To test if denervated fibers could be recovered, we will continue preliminary experiments in oldest-old rats (> 30 months of age) which show that, even in these trial animals, short-term phrenicotomy does not significantly change the contractile characteristics of the diaphragm, when tested in vitro. If activation of muscle by ES can improve the setting, differentiation and survival of myogenic and gliogenic stem cells administered to the experimental animals, then it will be an achievable goal to support ventilation in ALS subjects. Thus, our goal will be able to design and implement a multi-faceted strategy based on activity-driven enhancement of the effects of molecular, cellular and physical approaches. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration., Mitochondrial Transcription Factor A (TFAM) is a histone-like protein for mitochondrial DNA (mtDNA), involved in multiple functions for this genome. Aging markedly affects mitochondrial biogenesis and functions in a tissue-specific manner and calorie restriction (CR) diet is, so far, the only intervention able to delay or prevent the onset of several age-related alterations, also in mitochondria, in different organisms. TFAM amount, mtDNA content and TFAM-binding to mtDNA were analyzed in samples of frontal cortex and soleus skeletal muscle from 6- and 26-month-old ad libitum-fed and 26-month-old calorie-restricted rats and of liver from18- and 28-month-old ad libitum-fed and 28-month-old calorie-restricted rats. We found an age-related increase in TFAM amount in the frontal cortex, not affected by CR, whereas an age-related decrease was present in the soleus and liver, fully prevented by CR. The semi-quantitative analysis of in vivo binding of TFAM to specific mtDNA regions, by mtDNA immunoprecipitation assay and following PCR, showed a marked age-dependent decrease in TFAM-binding activity in the frontal cortex, partially prevented by CR. An age-related increase in TFAM-binding to mtDNA, fully prevented by CR, was found in the soleus and liver. A common age-related decrease in mtDNA content, completely prevented by CR, was found in the soleus and liver, but not in the frontal cortex. The modulation of TFAM expression, TFAM-binding to mtDNA and mtDNA content with aging and CR showed a trend shared by the skeletal muscle and liver, but not by the frontal cortex counterpart. Considering the above mentioned findings, aging and CR appear to induce similar mitochondrial molecular mechanisms in the skeletal muscle and liver, different from those elicited in the frontal cortex., It was generally believed that no effective treatment was available for muscle that underwent severe atrophy due to chronic denervation. Under the Gutmann’s1 view of the trophic influence of nerves on muscle, the effect of a mimicking approach, electrical stimulation, played an important role, but over the years the value of electrically stimulating the denervated muscle has been disputed because of the difficulties to obtain strong contraction by electrical stimulation and of its possible unfavorably effects on any remaining potential for reinnervation. In the last 15 years, we studied the possibility to effectively train permanently denervated human muscles by means of Functional Electrical Stimulation (FES). The results of the EU Project RISE2-4 show a new perspective in stimulating muscle fibers in the absence of nerves and after prolonged denervation, enabling: i) restoration of muscle fiber ultrastructure; ii) recovery of conduction velocity of the excitation-contraction apparatus up to a level that allows tetanic contractility; and thus iii) astonishingly recovery of fiber size, muscle mass and FES-induced force. Our training strategy is based on two combined stimulation programs. Within continuous clinical assessments, the stimulation parameters and training protocols should be progressively modified according to the patient’s time span of denervation, the current condition of muscle and function. At the beginning of the treatment, biphasic stimulation impulses of very long-duration (120-150 ms, 60-75 ms per phase) at high intensity should be applied to improve membrane excitability and muscle structure. The next period of the routine daily training consists of combined stimulation patterns one eliciting single twitches (impulse duration of 120 ms) and the other tetanic contractions (2 – 3 s bursts with an impulse duration of 36-50 ms and impulse pause of 10 ms). After tetanic contractility is achieved and the subject is able to provide full extension of the leg during stimulation of the quadriceps muscles, the ankle should be progressively loaded following the training theory for healthy people. Finally, few patients who have achieved a good muscle and functional condition can be able to stand and perform step-in-place and walking exercise with stimulation to train the cardiovascular system, upper body, sense of balance and thigh muscles., Adult and aged population provide the vast majority of potential diseased people in Europe, the Americas and Japan. Their needs of mobility support and rehabilitation may be categorized as minimal (young light subjects during and after minor traumatic events), medium (old subjects with impairments in the normal life activity and sarcopenia), severe (advanced sarcopenia in oldest old subjects, oncology, neuromuscular and skeletal disorders, subjects weaning from long hospitalization for intensive care and/or heavy surgery) and extreme (severe neuromuscular disorders, permanent flaccid paraplegia due to lower motoneuron injury in the spine, cachexia due to severe nutritional, metabolic, oncologic and septic conditions). I will here briefly describe a comprehensive approach to help people with border line mobility and standing impairments, in particular those in surgical units that will need to rise soon after surgical interventions to decrease the risks of thrombo embolisms and falls. Surgical patients have a special need of voluntary mobility to minimize the need of personal to help them in every day toeletting and physiologic intestinal evacuation (catheterization solve the urology needs), but even more to stand-up as soon as possible after surgery. Indeed, day-hospital surgery although substantially increased is NOT possible for any surgical need. Any subject that is hospitalized arriving in a wheel-chair, is a potential subject that needs special care. The burden on the families and the services are increasing the number of aged people that use temporary or permanent wheel-chairs, more because it is easier and safer to be wheel-chaired to stand and walk. A program of rehabilitation in the days that precede hospitalization or after hospitalization the surgery (patients spend some days for mandatory analyses to grant that the patients may stand the surgical act) may rescue borderline seniors from their “immobility” syndrome, and allow them to self-care with self-evident advantages for the individual and the Health System. The majority of these patients may reach independent (or supported, in particular if they are overweight and long-lasting disused) standing and walking by a program of volitional exercise to reactivate and strength enough their muscles to stand-up and walk. In particular cases, functional electrical stimulation (FES) and functional magnetic stimulation (FMS) may shorten the period of pre-standing volitional activity. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration., Meat eating traits, such as tenderness and juiciness, are known to be linked to total fat levels, and to the associations between intra-muscular connective and fat distribution. The visual appearance of the fat could additionally affect the consumers overall acceptability of the product and therefore the choice and the selection of meat products before buying .1,2 Bioimaging, image processing and 3D modeling has showed a fundamental role in assessing denervated muscles during electrical stimulation.3,4 Similar techniques have been recently employed to monitor Extracellular Matrix Mineralization in biological scaffolds using X-ray µCT technology.5 A combination of these two approaches was used to analyse meat samples with the General Electric nanotom x-ray µCT system, this system has 200 nm detail detectability. The aim is to study the feasibility of developing an alternative methodology for meat quality assessment based on image processing that could be in the future correlated with specific sensory analysis. A salted-smoked-fermented meat sample (Tiroler-speck, a typical product of the north part of Italy) of 10×10×1 mm was scanned with step of 3,5 µm using µct technology. The µct data scan data are imported into a special image processing and editing computer program called MIMICS.6 In this software environment we isolate: muscles, intra muscular connective tissue and fat tissues. the discrimination between tissues is possible because of the different linear attenuation coefficient and consequent gray value intensity. False colors are assigned to the muscles, connective and fat tissues within the sample and the amount of each tissue have been quantified.7 Moreover, it was also possible to isolate few single muscle fibers of 40-60 µmm diameters. Correlation of the 3D color analysis of each sample product with expectations of buyers may open large application to the imaging approach. U.C. thanks the Interdepartmental Research Center of Myology at the Department of Biomedical Sciences, University of Padova, Italy for collaboration and hospitality and the Ludwig Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation of Vienna at the Department of Physical Medicine, Wilhelminenspital, Vienna, Austria for support and collaboration.

BACKGROUND: Graded Motor Imagery (GMI) is a new approach that is thought to promote graded cortical brain activation and may promote motor recovery after stroke. AIM: This non-randomised controlled trial investigated the feasibility and clinical effect of GMI in motor recovery after stroke. DESIGN: Non-randomised controlled trial. SETTING: Inpatient subjects of neurorehabilitation hospital. POPULATION: Twenty-eight patients (i.e. 14 experimental and 14 control matched) with first-ever stroke. METHOD: Patients were assessed before and after a 4-week intervention. Assessors were blinded to the protocol. The experimental group underwent 20 sessions (1-hour each) based on GMI principles; the control group received the same amount of conventional rehabilitation. Primary outcomes were Wolf Motor Function Test (WMFT) and the 66-points motor section of the Fugl- Meyer Assessment (FMA) RESULTS: Groups were comparable under demographical and clinical features. Mean duration since stroke was 19 weeks. Patients were satisfied and adhered well to the protocol. Ten patients in the GMI group and four in the control group reached the minimal clinically important difference. Mean (SD) improvement in the GMI group was 0.72 (0.5) for WMFT, and 10.3 (8.9) points for FMA. The control group improved a mean (SD) of 0.21 (0.35) points at WMFT and 2.7 (0.35) points at FMA. Between-group analysis show that GMI provided significantly greater improvements for both motor functions at WMFT (p=0.05) and in the pain section of FMA (p=0.006), respectively. CONCLUSION:GMI is a feasible treatment for stroke patients with better outcomes than conventional therapy. A randomised controlled trial is warranted to minimise risk of selection bias. CLINICAL REHABILITATION IMPACT: Clinicians should implement GMI treatment in their clinical practice, being a feasible, clinically relevant, costless, and easy-to-do treatment. Refereed/Peer-reviewed