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More than half a century of skeletal muscle research is continuing at Padua University (Italy) under the auspices of the Interdepartmental Research Centre of Myology (CIR-Myo), the European Journal of Translational Myology (EJTM) and recently also with the support of the A&CM-C Foundation for Translational Myology, Padova, Italy. The Volume 30(1), 2020 of the EJTM opens with the collection of abstracts for the conference “2020 Padua Muscle Days: Mobility Medicine 30 years of Translational Research”. This is an international conference that will be held between March 18-21, 2020 in Euganei Hills and Padova in Italy. The abstracts are excellent examples of translational research and of the multidimensional approaches that are needed to classify and manage (in both the acute and chronic phases) diseases of Mobility that span from neurologic, metabolic and traumatic syndromes to the biological process of aging. One of the typical aim of Physical Medicine and Rehabilitation is indeed to reduce pain and increase mobility enough to enable impaired persons to walk freely, garden, and drive again. The excellent contents of this Collection of Abstracts reflect the high scientific caliber of researchers and clinicians who are eager to present their results at the PaduaMuscleDays. A series of EJTM Communications will also add to this preliminary evidence., Multiple biological pathways contribute to aging. Some prominent pathways and causes are genome instability, immunosenescence, inflammation, the NAD+ salvage pathways, proteostasis, and mitochondrial dysfunction. Altered gene regulation influenced by epigenetics, circadian rhythms, diet and physical activity levels also influences the rate of aging. Our Institute on Aging and collaborators are involved with multiple clinical trials with the goal to target these pathways and to improve physical and cognitive function or slow the rate of decline typically seen during aging. The ultimate goal is to develop interventions that can maintain health and independence of older adults. Trials ongoing and/or completed have utilized compounds shown to extend health-span and/or life span in invertebrates (c. elegans) or vertebrates (mice/rats). The compounds used range from nutritional (nicotinamide riboside, epicatechin, resveratrol, omega 3, fermented papaya) to pharmacological (metformin, aspirin, rapamycin, telmisartin/losartan, testosterone), lifestyle-physiological interventions (calorie restriction, exercise, fasting regimes). These biological compounds and lifestyle interventions target single or multiple pathways to potentially improve mitochondrial function, mitobiogenesis, autophagy, angiogenesis, nitric oxide production, and levels of NAD+ and/or reduce pathways or biomarkers of inflammation or senescence. In most of these trials we collect skeletal muscle, fat, white blood cells and plasma to determine if the compound alters the proposed biological pathway. We will briefly discuss some clinical trials and highlight findings on a recently completed clinical trial using epicatechins a flavonoid, which improved endothelial and mitochondrial function as well as walking speed. We will also highlight the challenges and limitations in conducting clinical trials, such as dose, duration of study, targeting single vs. multiple pathways, combining an intervention with exercise and/or intermittent fasting.1-5, Age-related cognitive decline has become a major public health concern. Even in the absence of frank neurodegenerative disorders, both cognitive and brain function decline as a function of advancing age. At present, there are few effective intervention options for remediating age-related cognitive and neural decline. Fermented Papaya Product has previously shown promise in human and animal models as a potential nutraceutical compound for impacting mitochondrial function, inflammation, and other age-related processes that contribute to cognitive and neural decline.1-3 The current study presents data from a pilot crossover clinical trials in 28 healthy older adults undergoing 8 weeks of treatment on either 9mg/day FPP vs. 9mg/day placebo (table sugar) with a 6-week washout period. Participants underwent multimodal magnetic resonance imaging and spectroscopy before and after each arm, as well as comprehensive neurocognitive assessment. Results demonstrated promising effect sizes on default mode resting state connectivity (Cohen’s d = .44), measures of verbal fluency (d = 1.16) and attention (d = .61), as well as two out of three novel markers of mitochondrial function from 31P magnetic resonance spectroscopy and neuroinflammation from free-water quantification in diffusion weighted imaging (d = .38). While results failed to survive significance after multiple comparison correction, the pattern of effect size improvement in FPP vs. placebo demonstrate significant promise for further investigation in larger Phase II randomized clinical trials. Potential implications for both healthy older adults and adults with neurodegenerative disease will be discussed., Prolonged oxidative stress may play a key role in tumor development. Antioxidants molecules are contained in many foods and seem to have potential role in future anti-tumor strategies. Among the natural antioxidants, the beneficial effect of Fermented Papaya (FPP®) is known. The aim of this study was to investigate the effects of orally administered FPP® in either prevention or treatment of a murine model of melanoma. The tumor growth was analyzed together with the blood levels of both oxidants (ROS) and anti-oxidants (SOD-1 and GSH). The results showed that FPP® controlled tumor growth, reducing the tumor mass of about 3 to 7 times vs untreated mice. The most significant effect was obtained with sublingual administration of FPP® close to the inoculation of melanoma. At the time of the sacrifice, none of mice treated with FPP® had metastases, the subcutaneous tumors were significantly smaller, and amelanotic compared to untreated mice. Moreover, the FPP® anti-tumor effect was consistent with the decrease of total ROS levels and the increase in the blood levels of GSH and SOD-1. This study shows that a potent anti-oxidant treatment through FPP® may contribute to both preventing and inhibiting tumors growth. The results of the above study suggested that FPP® while showing a clear anti-tumor effect it occurred through the in vivo induction of a potent anti-oxidant reaction. In a new set of experiments, we wanted to verify whether FPP had a clear and scientifically solid in vivo anti-aging effect together with the induction of the anti-oxidant reaction. To this purpose we used a mouse model suitable for aging studies (C576J) treating daily each mouse from 4 weeks of life to 10 months with the same dose of IMMUNEAGE dissolved into the daily water as compared to mice receiving only tap water. At the end of the treatment period (10 months) we measured some biological parameters related to aging cell processes: i) the total anti-oxydant capacity in the plasma of mice treated or untreated with FPP®; ii) the telomerase activity in the plasma of mice treated or untreated with FPP®; iii) the telomeres length in the bone marrow and ovaries of mice treated or untreated with FPP®. The results showed that the blood of treated mice, at the end of the treatment period (10 months) had 2-3 folds more anti-oxidant power and telomerase activities than the untreated mice. In the same mice at the sacrifice, we collected both the bone marrow (from the tibias) and the ovaries. We measured the telomere lengths in both cellular preparations. The results showed that daily FPP® assumption induced 3 folds increase in telomeres length in bone marrow and ovary of treated mice as compared to the untreated mice. This suggests that FPP® induce a clear improvement of the aging biomarkers and that the treated mice were for those variables younger than the untreated ones., Increased intake of a diet high in phosphorus (P) or phosphate (Pi) is associated with an increased risk of mortality in patients with chronic kidney disease or cardiovascular disease. Muscle fibers with ectopic calcification are observed in young patients with severe muscle degeneration. Ectopic calcification was also observed in the skeletal and cardiac muscles of mdx mice. In this study, we determined the effects of dietary phosphorus intake on ectopic calcification in skeletal muscle, muscle performance (specific maximal forces and daily running activity), and pathological features during exercise-induced stress in mdx mice. Increased level of dietary P or Pi intake also increased ectopic calcification and decreased muscle performance in mdx mice. On the other hand, decreased level of dietary P or Pi intake decreased ectopic calcification and muscle degeneration and improved muscle performance. When we added calcium absorber in high phosphorus diet, the ectopic calcification decreased significantly. In C2C12 muscle cell cultures, the increased concentration of Pi in culture medium inhibited myogenesis in a dose-dependent manner. Therefore, we propose that phosphorus (P) or phosphate (Pi) is one of the modifiers in regulating muscle degradation/inflammation in vivo and in vitro. In addition, we also found that the long-lasting Vitamin C, ascorbic acid-2 phosphate added into culture medium supported myogenesis even under high Pi- or low temperature-culture conditions. These results suggest that vitamin C may have therapeutic effect on muscle degeneration caused by increased level of Pi or lowered culture temperature., The neuromuscular junctions (NMJ) are specialized synapses between motor neuron and muscle fibers. They are targets for a variety of neuromuscular diseases. Maintenance and remodeling of NMJ are critical to treat these diseases. Different therapeutic strategies for neuromuscular diseases have been developed ranging from gene therapy to antisense-mediated exon skipping and nonsense mutation suppression by small molecules. We are attempting to search for therapeutic potential molecules from natural sources such as several microorganisms and chemical libraries. Among others, natural bioactive molecules, glucocorticoids and vitamin D have been proposed as potential treatments to delay progression in neuromuscular diseases and aging. However, the direct mechanisms for skeletal muscle have not been fully characterized. Recently, using cultured C2C12 myotubes, we showed that glucocorticoids and vitamin D enhance agrin-induced acetylcholine receptors (AChR) clustering compared to agrin alone. However, sex hormones such as estradiol and testosterone are less effective. To elucidate the physiological role of these compounds in agrin-induced AChR clustering, we investigated and will discuss their effects on expression of key molecules involved in AChR cluster formation and maintenance. This work was supported in part by Japan Society for the Promotion of Science, JSPS Grants-in-Aid for Scientific Research, Grant number:17K10089., Japan is facing a “super-aging” society. Seniors over 65 is 28.4% of the population and the life expectancy is the highest in the world, however, the nation’s annual medical cost is now over 42.6 trillion yen. If this trend continues, the Japanese government will not be able to sustain the social services. Therefore, Osato Research Institute has been conducting researches, aimed at reducing medical costs through Preventive Medicine. Our three core activities for achieving this goal are Education, Researches on Fermented Foods, and Improving QOL of senior generation. Firstly, we think the best preventive medicine is education. For example, AIDS is a typical disease that can be prevented by education. We have been working on young people’s education for AIDS prevention with Prof. Montagnier, president of World Foundation AIDS Research and Prevention at UNESCO. Secondly, we have been focusing our attention on “Fermented Food” since traditional fermented foods are considered to be a key to the world's highest longevity of Japanese people. Among our numerous studies, I will present 5 researches on Fermented Papaya Preparation,1-5 which may be a desired candidate for preventive medicine. Finally, we would like to introduce our activities of “Project ORI wine”. We established vineyards to make a place for social activities especially for local retired people. It would give them fun and challenge to live better and keep them productive and healthy. In this way, we hope we could have happy world not only for young but also for senior people., Mitochondria play a crucial role in health and disease, imposing a growing demand for evaluation of mitochondrial function in inherited and preventable degenerative diseases. These are associated with lack of physical exercise and obesity, causing decline of mitochondrial fitness and early aging. High-resolution respirometry (HRR) is based on the state-of-the-art instrument (Oroboros O2k, Innsbruck, Austria) for mitochondrial and cell respiration.1 The O2k combines real-time respirometry with simultaneous measurement of H2O2 production, mt-membrane potential, ADP-ATP exchange, pH, Ca2+, or nitric oxide. HRR can be applied with a wide spectrum of sample preparations (e.g. isolated mitochondria, tissue homogenate, permeabilized fibers, permeabilized cells, and living cells). Optimized substrate-uncoupler-inhibitor titration (SUIT) protocols offer a powerful tool for comprehensive mitochondrial physiology analysis by HRR.2 Peripheral blood mononuclear cells, platelets and permeabilized fibers are relevant models for diagnostic analysis of oxidative phosphorylation (OXPHOS) in clinical applications (e.g. aging, sarcopenia, obesity). Science and particularly mitochondrial respiratory research face a reproducibility crisis.3 Consequently, Oroboros is strongly committed towards reproducibility by promoting: (1) O2k quality control (i.e. calibrations, instrumental background, DatLab software); (2) proficiency tests with reference samples for inter-laboratory harmonization4,5; and (3) training by scientific experts. Supported by the COST Action MitoEAGLE network, we generate reference values for an Open-Access mitochondrial respirometry database, which requires harmonization of nomenclature.6 With a large O2k-Network, the Oroboros Ecosystem operates in the frame of collaborative Open-Innovation (NextGen-O2k with Q-redox sensor, NAD(P)H autofluorescence and PhotoBiology module) to promote quality and reproducibility in mitochondrial physiology. Support. Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE. Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770., Skeletal muscle plays a significant role in the regulation of whole-body metabolism, and mitochondria are essential organelles for ATP production, thus the studies focused on skeletal muscle mitochondrial activity are necessary to understand the molecular mechanisms controlling the skeletal muscle homeostasis.1 One of the key regulators of mitochondrial metabolism is the second messenger Ca2+. Upon physiological stimuli, skeletal muscle mitochondria rapidly and efficiently accumulate Ca2+ into their matrix via an electrogenic pathway, which relies on the driving force of a steep electrochemical gradient. A large [Ca2+]mt peak occurs dynamically in parallel to agonist-induced [Ca2+]cyt increases, thanks to the activity of the Mitochondrial Calcium Uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ accumulation.2 Mitochondrial Ca2+ stimulates aerobic metabolism, tightly regulating three dehydrogenases of the TCA cycle, thus the alteration of mitochondrial Ca2+ homeostasis leads to a dysregulation of metabolism.3 In this talk, we will show an overview of techniques to monitor mitochondrial activity in skeletal muscle, starting with the ex vivo measure of Ca2+ signaling, in terms of cytosolic Ca2+ concentration and mitochondrial Ca2+ uptake, in single isolated fibers thanks to the use of genetically encoded Ca2+ probes.4 Then, to investigate skeletal muscle mitochondrial metabolism we take advantage of genetically encoded indicators for ATP and NADH (Perceval and Peredox probes), and O2 consumption rate measurements.5 Finally, mitophagy, the removal of damaged mitochondria, is a necessary cellular process to maintain healthy muscle homeostasis. Mt-Keima probe is an easy tool to monitor this phenomenon in muscle fibers. Together, all these techniques allow studying many processes that regulate mitochondrial metabolism, a key aspect in the pathophysiology of skeletal muscle., Mitochondria are implicated in atrophy of aging skeletal muscle, yet the link between specific changes in mitochondrial function and aging muscle atrophy has not been established. Amongst many changes occurring with aging, there is a sensitization to mitochondrial permeability transition (MPT) in aged skeletal muscle.1 Building on this observation, we took advantage of heterogeneity in atrophy with aging between hindlimb muscles of the rat, in combination with a muscle cell culture system, to evaluate the hypothesis that MPT is a mechanism of aging muscle atrophy. Our results showed that, exclusive to muscles that atrophy with aging, mitochondria are sensitized to MPT and there is a marked increase in nuclei positive for the mitochondrial-derived protein, apoptosis inducing factor (AIF). Within a given muscle, aged muscle fibers harboring AIF-positive nuclei are severely atrophied relative to normal muscle fibers. In a muscle cell culture system, induction of MPT using doxorubicin is coincident with AIF translocation to myotube nuclei and causes atrophy that can be blocked using inhibitors of MPT (cyclosporin A, bonkrekic acid), inhibiting mitochondrial reactive oxygen species (ROS; using MitoTempo), or knocking down caspase 3 (using shRNA). Together, these results show that: (1) MPT is linked to aging muscle atrophy from the whole muscle to single fiber level; and (2) MPT causes muscle cell atrophy in cell culture in a manner that depends upon both mitochondrial ROS and caspase 3. Our findings thus support the hypothesis that MPT is a mechanism of aging muscle atrophy, FBXL4-related encephalomyopathic mitochondrial DNA depletion syndrome is an autosomal recessive severe, multi-systemic mitochondrial disease with 48 known pathogenic variants. To better understand FBXL4 function and therapies, we characterized novel C. elegans and D. rerio models of FBXL4 disease. This study particularly focuses on analyzing the neuromuscular function of these genetic models using manual and automated assays. We investigated fbxl-1 worms’ neuromuscular function analyzing pharyngeal pump rate, body bends rate and overall motor activity using manual and automated assays. A novel automated video-tracking approach was used to evaluate overall worms’ activity in liquid media. In the latter case, we created a protocol based on the use of the Zebrabox (Viewpoint), automated software created and used for analyzing zebrafish larvae activity and only in this study, applied to worms. Worms’ neuromuscular function was impaired with significantly increased and uncoordinated pharyngeal pumping rate in young adult and decreased rate during aging. Motility was reduced by ~70% compared to N2 control worms. Automated Video-tracking analysis was also used to analyze swim activity in fbxl4sa12470 zebrafish larvae carrying a missense mutation in the FBXL4 allele. Data shows decreased (~50 %) swimming activity in basic condition and after applying stimuli of light on/off cycles. Overall, these data demonstrate that FBXL4 deficiency can be effectively modeled in simple translational animals; new protocols were created for using automated software applications offering robust translational platforms in which therapies can now be modeled to reverse organ-level and behavioral dysfunction directly relevant to the human disease., The neuromuscular junction (NMJ) plays an essential role in enabling the cross-talk between the motoneuron (MN) and skeletal muscle. With progressive age, oxidative damage due to mitochondrial dysfunction, seems to cause a ‘dying-back’ phenomenon, where damage at the axon terminal, causes degeneration of the entire cell.1 The ensuing death of MNs leads to muscle fibre denervation and loss of the entire motor units, a main cause of sarcopenia.2 NMJ degeneration may also arise from changes at the muscle level. In ageing muscle, several phenomena could also cause NMJ damage: mitochondrial dysfunction, increased ROS production, decreased PGC-1alpha expression, known to trigger NMJ changes with ageing.3 Inactivity also promotes muscle fibre denervation as increased neural cell adhesion molecule (N-CAM), marker of denervation/reinnervation, has been found after short periods of bed rest (10-14 days) in young and older men.4,5 In this study, we tested the hypothesis that regular physical activity, such as recreational dancing, would protect against NMJ degeneration while improving functional performance of older individuals. Thirty-one participants aged 60>years (70.4 ± 5.1 years) were recruited for this study after Ethical approval and participants’ informed consent. Among these, 15 (9 female, 6 male) were sedentary (S), while 16 (10 female, 6 male) practiced regular recreational dancing (D) at least 1.5 h/week for a minimum of 1 year. NMJ degeneration was assessed from serum c-terminal agrin fragment (CAF) levels using a commercially available Elisa kit assay. Gait performance was tested with the timed 10m walk test (10WT), dynamic balance (WD) was tested with a stabilometric tablet measuring ankle oscillations, while timed get-up-and go (TUG) was tested as the time taken to get up from a chair, walk 3m and sit back. Muscle size and architecture of the knee extensors were assessed using ultrasonography. Significance was set at p, Stimulation of myogenic cells with vasopressin (AVP), oxytocin (OT), their analogs and antagonists revealed a previously unknown role of the neurohypophyseal hormones (NH) in modulating myogenic differentiation and trophism1 through the activation of complex intracellular signaling, the expression of the MRFs and the stimulation of the hypertrophy pathways2,3. Other studies demonstrated the expression of functional OT receptors (OTR) in human and murine satellite cells (reviewed in2). Extending these studies to in vivo models, we could show a highly stimulated expression of the V1a AVP receptor (V1aR) in post-injury muscle regeneration and assess that the overexpression of V1aR suffices, without exogenous hormone administration, to sustain muscle regeneration2. Furthermore, the administration of AVP rescued the inhibitory effect of TNF in a mouse model of cancer cachexia (reviewed in2). Works conducted in bovine and ovine livestock showed that animals treated with anabolizing steroids displayed dramatically increased OT expression in skeletal muscle, leading to a significant increase of plasma OT level and that the hypertrophying effect of anabolic steroids is mediated by increased OTR signaling (reviewed in 4). Furthermore, in a mouse model of aging, it was shown that reduced circulating OT levels accompany sarcopenia, and that satellite cells obtained from aged mice express reduced levels of OTR5. Efficient regeneration could be restored in aged mice by administering exogenous OT. Taken together, these studies not only confirm that NH target skeletal muscle increasing differentiation and trophism, but also point to muscle as a physiologic source of OT., Non-coding RNAs are emerging as important players in the regulation of several aspects of cellular biology. The expression and therefore the activity of non-coding RNAs is more tissue and cell specific than that of coding RNAs. Therefore, for a better comprehension of their function, it is fundamental to determine their tissue or cell specificity and to identify their subcellular localization. Myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism. We compiled a comprehensive catalog of ncRNAs expressed in skeletal muscle, associating the fiber-type specificity and subcellular location to each of them. We demonstrated that many ncRNAs can be involved in the biological processes de-regulated during muscle atrophy. Focusing our attention on a specific long non-coding RNA (Pvt1), activated early during muscle atrophy, we revealed that it impacts on mitochondrial respiration and morphology and affects mito/autophagy, apoptosis and myofiber size in vivo. This work corroborates the importance of long non-coding RNAs in the regulation of metabolism and neuromuscular pathologies and offers a valuable resource to study the metabolism in single cells characterized by pronounced plasticity., One of the consequences of aging is the reduction of the growth hormone/insulin-like growth factor I (GH/IGF-I) axis. For skeletal muscle, this leads to a loss of anabolic and regenerative capacity, which, in part, underlies the onset of sarcopenia. Maintenance of activity in aging can help offset functional deficits, and these benefits are both systemic and specific to skeletal muscle. It is an open question as to the contribution of muscle IGF-I activity to these beneficial adaptations. To address this, we imposed an endurance exercise regimen on a recently developed mouse model with muscle specific inducible deletion of IGF-I (MID mouse), and strain matched controls (CON), comparing their performance to sedentary mice of the same genotypes. After 4 weeks of daily endurance training at 15 m/min for 60 minutes, both MID and CON mice displayed similar and significant improvements in a run-to-exhaustion test. Body composition also displayed a reduction in proportional fat with training. However, in the subsequent 4 weeks of daily endurance training at 18 m/min for 60 minutes, only the CON mice continued to improve in the run-to-exhaustion test, even though there were no differences in the body composition changes in the exercised groups. Over the course of 8 weeks, the sedentary mice progressively gained fat, and by the end of the study, the sedentary MID mice ran for less time in the run-to-exhaustion test compared to sedentary CON mice. Taken together, this suggests that benefits to moderate intensity training can occur in the absence of muscle IGF-I. However, the capacity of muscle to adapt to higher intensity regimens may be limited when actions of local IGF-I signaling are impaired., Skeletal muscle atrophy induced by immobilization is one of the greatest issues in athletes as well in general population. The mechanisms underlying skeletal muscle atrophy, are reasonable known, however, there is no evidence of works that was able to avoid it. A strategy that could preclude the large recovery needed after immobilization would be of great importance. Our group made a research protocol that aims to study the effect of Branched-Chain Amino Acids (BCAA’s) administration to avoid skeletal muscle atrophy. In order to achieve our goals, we use an animal model divided in several groups that includes control, cast-immobilization, exercise animals and animals which have taken BCAA’s. We observed the structure of skeletal muscle, fibre type changes and the satellite cells turnover. To evaluate satellite cells, we used antibodies against Pax-7, Myf-5 and c-met, identification by fluorescence which allows to count the immunoreactive cells and study its evolution and differentiation. As expected, cast immobilization caused atrophy and muscle damage. The satellite cells were activated to regenerate the skeletal muscle, as seen by the increase in Pax-7 and Myf-5 expression with a decrease of c-met, suggesting mobilization of those cells. Visible cell niches in the muscle reinforced these data. The supplement had a partial protective effect when it was taken during immobilization, but it seems to hamper the muscle regeneration, when exercise is performed after immobilization. Thus, our results suggested that BCAA prevent skeletal muscle atrophy by acting over its satellite cells. Partially Supported: FCT, Portugal (Strategic Project UID/NEU/04539/2013), FEDER-COMPETE., According to Bartholomaei Eustachii, Aristotle was the first to describe the complicate interlacement of canaliculi which are in the petrosal bone, identifyng the “coclea”. Aristotle, to explain his idea from an anatomical point of view, compared the inner ear to the strovmboi, i.e. spiral shell (cochlea). Regarding the term labirinto, probably Falloppio was the first to use the term labyrinthus – of egyptian and not greek origin - to describe the inner ear: quum haec cavitas tot habeat meatus et cuniculos, merito labyrinthus dicetur, in quem prospicit fenestra ovalis, clausa a stapede, ecc.». The Anatomic school of Padova gave during the XVI century an enormous contribution to the knowledge of the ear: • Andreas Vesalius 1514-1564 • Andrea Falloppio 1523-1562 • Jeronimus Fabricius ab Acquapendente 1537-1619 • Bartolomeus Eustachi 1514-1574 • Matteo Colombo 1515-1559 • Giovanni Filippo Ingrassia 1510-1580 • Iulius Casserius 1552-1616 One of the major contributions of Vesalius was his suggestion that the organ of hearing should be removed from the skull for investigation. We remember here 3 important contributions: Bartolomeus Eustachi in his work entitled “Epistola de auditus organis” (Eustachi, 1564); Girolamo Fabrici ab Acquapendente De Visione Voce Auditu (1600) and Iulius Casserius De Vocis Auditusque Organis Historia 1601. In 1740, Antonio Valsalva published his anatomical observations on the human auditory system in which he pointed out the importance of the ossicular chain and the oval window for hearing and also observed that the innervation target for the auditory nerve was not the osseous spiral lamina, but was instead the membranous portions of the cochlea and that these areas of sensory epithelium represented, in the opinion of Valsalva, the true receptors of sound. Later, Antonio Scarpa, pupil of Giovanni Battista Morgagni and Marco Antonio Caldani, reported a liquid present within the cochlea's inner membranous compartment, that is, scala media. The Count Alessandro Volta is generally qualified as the first to stimulate the ear with the electricity. Volta, carried out on himself in the late 1790s the first experiment on electrical stimulation of the auditory nerve. Because of the unpleasant sensation experienced by the scientist, any other experiment was carried out over the next half century to study this effect. From the first half of the 18th century onwards, tremendous curiosity about electrical phenomena spread throughout Europe. Machines producing electrostatic electricity were produced, and lectures on electricity attracted members of academia as well as the ruling elite. A field known at the time as “medical electricity” emerged following the electrical researches and the discovery of the effects of electricity on the human body. In 1950, Lundberg performed one of the first recorded attempts to stimulate the auditory nerve with a sinusoidal current during a neurosurgical operation. His patient could only hear noise. In 1957 by Djourno and Eyries provided the first detailed description of the effects of directly stimulating the auditory nerve in deafness. They placed a wire on the auditory nerves that were exposed during an operation for cholesteatoma. When the current was applied to the wire, the patient described generally high-frequency sounds that resembled a “roulette wheel”. The signal generator provided up to 1,000 Hz and the patient gradually developed limited recognition of common words and improved lip-reading capabilities. At this point the clinical application of a cochlear implant started. A cochlear implant is a surgically implanted electronic device that provides a sense of sound to a person who is profoundly deaf or hard of hearing; at now approximately 500,000 people worldwide had received cochlear implants. Padova Audio/Otological group is strongly engaged in the diagnosis and treatment of deafness particularly in childhood and in basic research (Padua Bioacustic lab). Recently an international center was established in Venice: I APPROVE, the International Auditory Processing PROject in Venice, Maximal oxygen consumption (V̇O2max) denotes the reproducible upper limit of oxygen (energy) flux through the respiratory system into skeletal muscle mitochondria that can be reached during intense exercise with a large muscle mass. A high V̇O2max is a key requisite for success in all endurance sports such as cycling, cross-country skiing or running over longer distances. However, V̇O2max has also been strongly and negatively associated with cardiovascular diseases and all-cause mortality. V̇O2max can vary by more than twofold between untrained, sedentary subjects with a heritability value greater than 50%.1 Trainability for an individual's V̇ O2max also varies massively between subjects. Trainability is independent of sedentary V̇O2max with a similarly high heritability as sedentary V̇O2max.2 The high heritability of sedentary V̇O2max and trainability and its importance for athletic performance as well as health has prompted a massive search for its genetic underpinning. Candidate-gene studies, gene-expression studies and genome-wide-association studies (GWAS) have failed to identify a genetic signature of the high V̇ O2max phenotype.3 This may be due to the fact that there are vast multigenetic regulatory networks in skeletal muscle and in other organs that are responsible both for the set-point and the malleability of V̇O2max. Multigenetic phenotypes such as V̇O2max appear to be emergent properties of multiple underlying transcriptomic networks modified by epistasis, the epigenome and the epitranscriptome. It is unclear currently whether an artificial intelligence approach on sufficiently large datasets can make reliable predictions on multigenetic phenotypes such as V̇O2max., In the last decades sparse evidence of the therapeutic potentialities of surface electrical stimulation for the treatment of facial palsy has been published.1,2,3 Our study represents one of the few systematic evaluations of this approach within a homogeneous population suffering from complete unilateral facial paralysis. For this study the stimulation was delivered on the region closest to the upper mouth corner and denervation recovery was monitored for the entire period. Five patients were recruited, undergoing surface electrostimulation for a maximum of 1y, twice a day for 15min (5min pause every 5min stimulation). The parameters set during the first visit were confirmed/adapted every month thereafter. At each visit the patients underwent 3D photos, electromyography, ultrasound,4 magnetic resonance controls,5 and Sunnybrook evaluation, answered to the FaCE and FDI questionnaire. For all the patients effective parameters leading to an utterly Zygomaticus m.-specific stimulation could be found below the discomfort threshold. The required stimulation pulse width decreased with time without requiring a significant amplitude increase to remain effective and specific. Sunnybrook results significantly (p=0.04) improved after 6m stimulation. FaCE (total score) showed a significant (p=0.04) improvement already after 2m, while the social component of the FDI significantly (p=0.04) improved after 7m. Short- (hours) and mid-term (weeks) improvements of muscle tone could also be shown by 3D imaging. Our results showed that surface electrostimulation can be used to deliver a specific response of the Zygomaticus m. without adverse events and without non-specific activation of other facial muscles. Patients were extremely compliant with the treatment protocol and received a significant improvement in their quality of life., Age-related cognitive decline has become a major public health concern. There is currently a paucity of effective interventions to prevent or treat cognitive decline and prevent dementia in the elderly. Decline in working memory is a central facet of the cognitive aging process. Prior research has attempted to use transcranial direct current stimulation (tDCS) to enhance working memory performance in older adults with and without neurodegenerative disease, often in the context of cognitive training paradigms(1, 2). Alone, these methods have shown a degree of promise in remediating age-related working memory decline. In combination, these interventions may prove more effective by pairing two interventions targeting neuroplasticity. However, the neural mechanisms and efficacy of combined cognitive training and tDCS improvements in working memory is not well understood. This talk will discuss data from 3 studies investigating the benefits neural mechanisms of cognitive training paired with tDCS to remediate age-related cognitive decline.1-3 Study 1 was a mechanistic investigation of the impact of tDCS with CT during BOLD fMRI in 14 healthy older adults. Study 2 was a pilot randomized clinical trial of 28 older adults randomized to active tDCS or sham tDCS with cognitive training. Study 3 is an ongoing Phase III RCT investigating the benefits of tDCS with cognitive training in 360 older adults. Neural mechanisms of working memory improvement, efficacy of near transfer from tDCS vs. sham with cognitive training in working memory measures, and future directions of investigation will be discussed., We present preliminary results from the ongoing study entitled “Icelandic AVH TMS” which aim is to study the effectiveness of repetitive transcranial magnetic stimulation (rTMS) treatment for patients with schizophrenia and with persistent auditory verbal hallucinations (AVH) using high-density EEG system (256 channels). The main objectives of this work were to describe P50 and P300 cortical topography pre and post treatment, and to define a robust methodology of signal quantification using high density EEG.1-4 Our results show differences in sensory gating and a stronger response to rare audio stimulus 1 week post treatments. Moreover we show the value of assessing brain electrical activity from high-density EEG (256 channels) analyzing the results in different regions of interest., Reduction in muscle mass and muscle strength together with alterations in the metabolic and cellular machinery of the skeletal muscle are common in type 1 diabetes (T1D)1 Collectively these negative changes have been termed diabetic myopathy.2 Moreover, whole-body fatigue is a frequent complaint in individuals living with T1D but it is unclear if this would depend on muscle alterations per se or on a concomitant impairment in motor units (MU) recruitment capacity. MU recruitment can be now investigated non-invasively using high-density surface electromyography (HD-EMGs) using multi-channels electrodes.3 Sixteen participants, 8 T1D (4M; 33.7 ± 5.2 yrs.; HbA1c 8.1 ± 3.1 %) and 8 healthy (4M; 31.5 ± 5.6 yrs.) volunteered for the study. In one 2-hour visit to the lab participants were tested for maximal isometric strength (MVIC) of knee extensors (KE), sustained KE isometric contractions at 10, 20, 40 and 60 (%MVIC), plus a sustained contraction at 40% MVIC until task failure. During these measures we recorded HD-EMGs from vastus lateralis (VL) muscle with a grid of 64 electrodes. At baseline no differences, between the 2 groups, were observed in MVIC, rate of torque development, and HD-EMGs parameters. In addition, and contrary to our expectations, no differences were observed concerning the fatiguing test either in terms of time to fatigue or of changes in muscle fiber conduction velocity (a marker of MU recruitment). In conclusion, no major differences were detected between the two groups, which could justify the presence of diabetic myopathy (whether neurogenic or myogenic) in our T1D participants., Skeletal muscle is the driver of whole-body aerobic capacity measured by spiroergometry as VO2max/M [mL O2∙min-1∙kg-1]. Obesity is defined as accumulation of excess fat tissue mass, MFE=MF-MF° [kg/x]. MF° is the fat mass [kg] per individual [x] in the healthy reference population at a given height and total body mass M° without overweight.1 Body fat excess, BFE=MFE/M°, is directly related to total body mass excess, BME=ME/M°, where ME=M-M°. In model 1, BFE does not reduce VO2max [mL O2∙min-1∙x-1], but BFE lowers VO2max/M by increasing M. Experimentally, however, VO2max/M declines with BME much steeper than predicted by model 1. The more pronounced loss of ergometric fitness is due to the decline of mitochondrial respiratory capacity per muscle mass, mM,2,3 as a function of BME. Yet this model 2 predicts an even lower VO2max/M at overweight. Finally, model 3 includes the well-known ‘weight-lifting’ effect of obesity on increasing muscle mass with low mitochondrial density, providing a quantitatively complete link between low mitochondrial and whole body aerobic fitness in obesity before onset of sarcopenia. The decline of muscular mitochondrial fitness in overweight states is a biomarker of the systemic mitObesity syndrome: Compromised mitochondrial fitness across metabolically active organs provides the mechanistic link between obesity and comorbidities such as diabetes, cardiovascular and neurodegenerative diseases and various types of cancer bound to redox imbalance, inflammation, oxidative stress and insulin resistance. Today mitObesity is the world-wide leading cause of deaths and early aging, which can be prevented by an active lifestyle and improvement of the quality of life by exercise and caloric balance. Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770. Contribution to COST Action CA15203 MitoEAGLE funded by the Horizon 2020 Framework Programme of the European Union., By combining the digital holography with the tomography, Nanolive technology is an example of emerging intelligent nanoscopy techniques1. The 3D Cell Explorer microscope guarantees a high spatial-temporal resolution imaging of transparent unlabeled specimens, with the advantage over fluorescence techniques of not requiring sample labeling, thus reduce potential damages to living cells2. This intuitive and affordable technology enables scientists to investigate macro cellular dynamics like cell health, proliferation, movement and function as well as micro organelle dynamics and interactions in a e.g. mitochondrial network label-free characterization.3, Intracellular aggregates of RNA binding proteins and perturbed RNA metabolism are typical features of many human neurological and neuromuscular diseases, including oculopharyngeal muscular dystrophy (OPMD), a rare genetic repeat expansion disease (1). In OPMD, a short abnormal polyalanine expansion in PABPN1 (poly(A)-binding protein nuclear 1) leads to the accumulation of intranuclear tubulofilament aggregates in muscles of patients and ultimately to muscle dysfunction. Symptoms usually appear in the fifth decade of life and we have shown a good genotype phenotype correlation (2). The disease is characterized by weakness of the eyelid and pharyngeal muscles, leading to eyelid drooping and dysphagia. A strong collaboration with clinicians gave us access to a large collection of muscle biopsies from OPMD patients. We characterized PABPN1 nuclear aggregates to evaluate if age and genotype influence their features (submitted). In addition, using mammalian models of OPMD, we assessed the efficacy of the anti-aggregates molecule guanabenz (GA). We showed that aging and genotype of OPMD patients influence the amount, the size, the percentage and the composition of nuclear aggregates. Treatment of cellular and mouse models of OPMD with GA allowed a reduction in the percentage and the size of nuclear aggregates as well as an improvement of the mice muscle phenotype. GA acts through the unfolded protein response to endoplasmic reticulum stress (ER) showing for the first time that ER stress is activated in OPMD. (3) This study suggests that the use of pharmacological molecules modulating ER stress is a promising strategy to treat OPMD., Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare autosomal recessive lipid storage myopathy, often associated with ETFDH gene mutations.1 This gene encodes electron transfer flavoprotein dehydrogenase, a mitochondrial protein involved in the electron-transfer system.2 In this study we investigated the correlation between ETFDH mutations, expression profile of serum muscle‐specific miRNAs (myomiRs)3 and response to treatment in 2 Italian MADD patients. Patient 1 (53 years) presented late onset of MADD, at the age of 38 years. She carried two different ETFDH missense mutations.4 In patient 2 (54 years) the symptoms appeared at 2 years of age and molecular analysis of ETFDH showed a missense and a splice site variation.5 Muscle biopsies of both patients displayed type I fibers vacuolated and muscle atrophy. Moreover, MRI of patient 2 revealed slight alteration of posterior thigh muscles. Bioinformatic analysis demonstrated that the missense mutations can determine conformational modification of protein structure, decreasing enzyme stability and activity. Serum myomiRs analysis displayed an up-regulation of miR-1, miR-133a, miR-133b and miR-206 expression in the two patients. Treatment led to an improvement of clinical condition in both patients, in particular of muscle vacuolization. In this preliminary study the results seem to indicate that the presence of two ETFDH missense mutations correlate not only with a minor severity of MADD phenotype but also with a partial dysregulation of circulating myomiRs, mainly miR-1 and miR-206. In these cases, various treatments can improve ETFDH stability, resulting in skeletal muscle recovery., Store-operated Ca2+ entry (SOCE) is a ubiquitous cellular Ca2+ influx mechanism, first described in non-excitable cells, that is triggered by depletion of intracellular Ca2+ stores (endoplasmic reticulum, ER). A major breakthrough in the field was the identification of the two essential molecular players in SOCE: STIM1, the Ca2+ sensor in the ER, and Orai1, a Ca2+ permeable channel in the plasma membrane.1,2 SOCE is also well-documented in skeletal muscle4 where it limits muscle fatigue during repetitive stimulation.3 Also in muscle SOCE is mediated by interactions between STIM1 in the SR and Orai1 channels in the PM.4 However, the precise subcellular location of STIM1-Orai1 SOCE complexes in skeletal muscle is still debated. We discovered that exercise in mice drives formation of new junctions between stacks of sarcoplasmic reticulum (SR) cisternae and transverse-tubules (TTs) containing STIM1 and Orai1, two proteins that mediate store-operated Ca2+ entry (SOCE). We proposed that these previously unidentified SR-TT junctions function as Ca2+ Entry Units (CEUs), providing a preferential pathway for rapid reuptake of Ca2+ into the SR during repetitive muscle activity.5 Using electron microscopy and two different functional assays we also studied muscles from mice subjected to an incremental treadmill running and sacrificed within 1hr or after 6-24hrs.6 Data collected indicates that: a) while the number of SR-stacks increased up to at least 6hrs to return to control values only after 24hrs of recovery, the extension of TTs (significantly increased at 1 hr), returned to control values already after 6hrs; b) fatigue resistance of EDL muscles during high-frequency stimulation and Mn2+ quench of Fura-2 fluorescence in FDB fibers were both increased after 1hr, but were not different from control after 6 and 24hrs of recovery. Our work represents a pioneer study that identified exercise-driven dynamic formation of new intracellular structures, a mechanism potentially quite important for the delay of muscle fatigue. As altered SOCE activity contributes to muscle dysfunction in ageing and various myopathies, our findings may also have implications for the understanding of mechanisms involved in muscular dysfunction., The HERG potassium channel is detected as a heteromultimer of 2 alternative splice variants (1A and 1B) in heart and has been shown to be partially responsible for repolarization of the cardiac action potential.1 Both alternative splice variants have been reported in certain cancer cells, but their role in these cells is not clear.2 The HERG1A variant has been detected at low abundance in normal skeletal muscle, but is up-regulated in atrophying skeletal muscle, where it has been shown to increase protein degradation by modulation of both intracellular calcium levels and ubiquitin proteasome proteolysis (UPP).3,4,5 The pathways by which this modulation occurs is not clear. Therefore, we virally transduced C2C12 myotubes with either an adenovirus encoding HERG or an appropriate control virus (n=6). After 48 hours, we extracted total RNA from these cells and reverse transcribed them into cDNA, selecting for coding sequences (i.e., mRNA) by using poly(T) oligomers; the cDNA libraries were sequenced on Illumina’s NovaSeq platform. Sequence quality was assessed using FastQC (v 0.11.7; https://www.bioinformatics.babraham.ac.uk/projects/fastqc) for all samples and quality trimming was done using FASTX-Toolkit (v 0.0.14; http://hannonlab.cshl.edu. fastx_toolkit/) to remove bases with Phred33 score of less than 30. Resulting reads of at least 50 bases were mapped against the reference genome using STAR.6 STAR derived mapping results and annotation file for reference genome were used as input for HTSeq7 (v 0.7.0) to obtain read counts. Counts from all replicates were merged together to produce a read count matrix for all samples and this count matrix was used for downstream differential gene expression analysis (DGEA). DGEA between treatment and control was carried out using ‘R’ (v 3.5.1; http://www.r-project.org/). The results show that HERG does result in numerous changes in gene expression. Limiting results to those with a p, MRI is a powerful non-invasive imaging technique that exploits different contrast mechanisms to provide an unprecedented view into both structure and function. We have used velocity encoded phase contrast technique to map muscle motion in-vivo1 and applied to study age and disuse atrophy related changes. A limitation of this dynamic technique is that it requires of the order of 70 consistent contractions cycles in order to image muscle motion. Thus, it is not easy to extend the MR dynamic studies to senior subjects and to higher % Maximum Voluntary Contractions (%MVC). We have implemented a fairly recent innovation called ‘compressed sensing (CS)’ to dynamic MRI for mapping muscle kinematics during isometric contraction. The implementation of this ‘fast’ technique allowed us to study muscle kinematics in 3D and at three %MVCs in the calf muscle. Eight young and eight senior subjects were imaged using the CS-VEPC sequence and the 3x3 strain and strain rate tensors were calculated in the principle axis and in the muscle fiber basis at 35, 45 and 60 %MVC at much reduced number of contractions cycles. Region of interest measurements are reported for the medial gastrocnemius and in the soleus: principle strains and strain rates were significantly different for 30 and 60% MVC (MG and soleus) and shear strain and shear strain rates were significantly different with age. Surprisingly, the strain and strain rates in the fiber basis were much smaller than in the principal basis and no significant differences were found between %MVCs or in age. The implications of these findings with respect to muscle force loss with age will be discussed., Soft tissue contrast afforded by MRI makes it an ideal candidate for imaging the musculoskeletal system. We will discuss our results using a combination of advanced MR pulse sequences to explore age related changes in tissue composition and tissue microstructure. Tissue composition includes estimation of fat (adipose) fraction, connective tissue/macromolecular fraction and water fraction in different muscles and compartments1. A combination of MRI sequences, IDEAL, UTEs and Magnetization transfer2 contrast sequences were applied to cohorts of ten young and ten senior subjects to determine the intramuscular and intermuscular tissue composition in calf muscle. Initial results show that fat and connective tissue fraction increased while macromolecular fraction (MTsat) decreased with age. Tissue microstructure was extracted from diffusion tensor imaging3,4 using a STEAM-DTI sequence and Random Permeable Barrier Modeling (RPBM)5. DTI studies were performed after IRB approval on a GE 3T scanner on seven young and six senior subjects. Time dependent diffusion was measured at ten values of the mixing time, TM (20 ms to 600 ms). The RPBM fits were made to the time dependence of the average of the secondary and tertiary diffusion eigenvalues λ2 and λ3 (Dᅩ ); the values of Dᅩ were the average over the medial gastrocnemius (MG) muscle segmented from all three slices. Muscle fiber diameters from the fit showed a small increase with age which is surprising as fiber atrophies with age. Fiber membrane permeability increased with age potentially indicating compromised sarcolemma integrity. Initial results of validation with biopsy analysis will also be presented., Chronic inactivity is a main cause of muscle wasting and weakness.1 However, muscle atrophy may not be solely due to a reduction in mechanical loading since muscle denervation and neuromuscular junction damage seem also triggered by inactivity.2,3,4 The identification of biomarkers of muscle atrophy and of neuromuscular degeneration is therefore needed for an early detection of the alterations of the neuromuscular system induced by inactivity. Hence, the aim of the present study was to investigate the onset of muscle atrophy and of neuromuscular alterations during a short-term inactivity period. Ten healthy male volunteers (aged 23±5 years) were recruited for this horizontal 10-day bed rest (BR) study, after Ethical approval and participants’ informed consent. Blood samples were collected every two days for the assessment of NMJ damage, based on serum levels of c-terminal agrin fragment (CAF) measured with a commercially available Elisa kit. Muscle fibre denervation was determined from the expression of neural cell adhesive molecule (N-CAM) in myofibres obtained from vastus lateralis (VL) muscle biopsies collected at baseline, and at 5 and 10 days of BR. Myofiber atrophy was determined from mean fibre cross-sectional area (CSA) measurements on histological sections. Whole muscle atrophy was assessed from changes in muscle architecture (pennation angle and muscle thickness) measured every two days of BR using ultrasonography. Significance was set at p, Over the last 25 years imaging techniques have been extensively used to study the in-vivo muscle and tendon morphology at tissue scale. Whereas Dual-energy X-ray absorptiometry (DXA), computed tomography (CT) and MRI have been widely adopted in health, ageing and clinical populations to track changes in body composition and muscle mass, ultrasound (US) can provide further information about the structure, length, thickness and area/volume of muscles and tendons. Despite these characteristics could also be obtained by MRI, US has the advantage that it can provide the same quantity of information, but during passive joint rotations, isometric or dynamic contractions. Recent advancements in musculoskeletal imaging have also led to the use of US also for the investigation of strain in soft tissues in response to compression (Shear Wave Elastography - SWE). However, some limitations of conventional B-mode US lay on: a) the small field of view used (especially during SWE or the study of in-vivo dynamic changes in muscle and tendon structures) and b) the impossibility of US to provide any in-vivo insights into adaptations at the sarcomeric level. Recent developments in the extended field-of-view US (EFOV) and 3D US techniques may offer further advantages in providing information at a whole-tissue level, providing software-integrated panoramic images and 3D volume reconstructions, allowing to gain insights into regional changes along the muscle-tendon unit. Furthermore, in-vivo microendoscopy has been recently implemented in human skeletal muscle, laying the foundation of new investigations into the remodeling of skeletal muscle “from the micro to the macro”. The purpose of this talk is to present and discuss these advances in imaging techniques for the study of human skeletal muscle in-vivo., Real time Ultrasound (US) imaging is being increasingly utilized by Physical and Rehabilitation Medicine (PRM) specialists to assess abdominal muscle in healthy subjects and in patients with low back pain (LBP).1 The current study set out to evaluate the variability and the intra- and inter-rater reliability of US measurements not only of the thickness of the abdominal muscles but also of the fasciae.2 Three specialists with different levels of training in US measurement techniques followed a standard protocol based on four reference anatomic landmarks to perform US examinations of the abdominal muscles and fasciae of a healthy volunteer in resting and dynamic condition. Each of the specialists measured 17 anatomical structures six times during two sessions (three per session). Their intra-rater reliability was assessed by evaluating the range of relative error and the coefficient of variation (CV). The inter-rater reliability was evaluated using the Kruskal-Wallis test at probability levels of 0.05 and 0.01. There were no significant differences between the measurements that the three raters registered (inter-rater reliability) with the exception of those referring to the anterior fascia of the external oblique muscle (p-value < 0.01), the fascia between the external and internal oblique muscles (p-value< 0.05) and the fascia between the internal oblique and the transversus abdominis muscles (p-value < 0.05).3 Knowledge about the fascial anatomy of the abdominal wall is essential to carrying out accurate US examinations. These findings confirm that US imaging is a reliable, non-invasive, cost-effective instrument for evaluating the abdominal muscles/fasciae.3, Spinal cord injury produces muscle wasting, which is especially severe after complete and permanent damage of lower motor neurons as occurs in complete Cauda Equina Syndrome.1 Even in this worst-case scenario, we have shown that permanently denervated Quadriceps muscle can be rescued by surface electrical stimulation, and a purpose-designed home-based rehabilitation regime, i.e., Home based Functional Electrical Stimulation (hbFES) for human muscles that are completely denervated and degenerating.2,3 Here the aim is to show that the effects are extended to the antagonist muscles and skin of the thighs. Before and after two years of electrical stimulation, the mass and structure of Quadriceps and Hamstrings muscles were quantitated by force measurements.1 Muscle gross cross-sections were evaluated using color computed tomography.4 Biopsies of skeletal muscles,1-3 and of stimulated skin,5 were analyzed by quantitative histology and immuno-histochemistry. The treatment produced: 1) an increase in the cross-sectional area of stimulated muscles; 2) an increase in muscle fiber mean diameter; 3) improvements in ultrastructural organization; and 4) increased force output during electrical stimulation.1-4 The recovery of Quadriceps muscle force was sufficient to allow 25% of the compliant subjects to perform stand-up and step-in place trainings.1-3 Improvements were extended to antagonist hamstrings,5 and skin.6 In conclusion, the cushioning effect provided by recovered tissues is a major clinical benefit. The hope is that new trials may start soon in Europe and beyond to provide stronger evidence of the positive effects of h-bFES for permanently denervated muscles. This will help to extend the results world-wide, and to all persons who are in need of the help they deserve., Patient oriented research in rehabilitation revolves around maintaining, improving or restoring muscle function in conjunction with pain treatment. Based on our findings in treating patients with long-term denervated, degenerated muscle (DDM) after spinal cord injury with electrical stimulation, (Kern et al. 2010)1 our activities in the past were aimed at transferring the knowledge to rehabilitation of elderly people (Kern et al. 2014).2 The current work comprises investigating age-related sarcopenia, a multifactorial disorder which underlying mechanisms are still not fully known. It could be shown, that alterations of mitochondrial Ca2+ homeostasis regulated by mitochondrial calcium uniporter (MCU) affect muscle function. Therefore, improved muscle function and structure are linked to increased protein levels of MCU (Zampieri et al. 2016).3 Future translational research will investigate cellular and molecular mechanisms of aging and physical activity, especially the effect of electrical stimulation training and physical therapy on these mechanisms, because only little research on this topic exists and therefore the evidence for efficacy of these therapy approaches is still missing. Focus of the future clinical research program is the in- and outpatient therapy of orthopedic patients, the remobilization and the rehabilitation of patients after knee and hip replacement surgery. For this reason, standardized assessments, analyzes and documentation will be developed and the results are collected across locations in a data base for in- and outpatient rehabilitation. Overall aim of the program is the evaluation of existing and new approaches in musculoskeletal rehabilitation also concerning the economic aspects with constant or improved quality of treatment. In the next years the following topics will be in focus: i) Underlying cellular and molecular mechanisms of Physical Medicine and Rehabilitation procedures; ii) In- and outpatient rehabilitation after knee and hip replacement surgery; iii) Proof of efficacy and cost-effectivity., Physical inactivity is a global pandemic that not only causes morbidity and mortality, but also represents a major economic burden worldwide. As a longer-term goal, we must strive to integrate physical activity into our everyday lives.1 Increased physical activity, among other things, has an influence on chronic pain of the locomotor system, minimizes the risk of cardiovascular disease, increases self-esteem and cognitive abilities, maintains mobility and autonomy, and thus has direct effects on healthy life years and direct and indirect health expenditure. The Centre of Active Ageing (CAA) project, funded by the EU cross border cooperation program INTERREG, is intended to be in line with the Austrian health targets (R-GZ) for Austria,2 the National Health Program of Slovakia,3 the recommendations of the Lancet Physical Activity Series Executive Committee, and of the WHO.5 Encouraging policymakers to take physical activity seriously, to motivate people, and to create the opportunities to do so on a regular basis. Over the entire project period of three years, more than 1000 people aged 60 years and over will benefit from the offers of the CAAs in the program area. Within the scope of the project, they should carry out a standardized training (10-12 weeks) followed by home training for 12 months. In 2019 the first groups started their “SENIOR aktiv” activity program in Austria. It combines education sessions of training theory and nutrition before and after a 10-week physical activity program for two times a week with alternating strength and gymnastics sessions. The first results show improvements in almost all measured parameters and the subjective feedback of the subjects is consistently positive, When resistance training for the improvement of the neuromuscular function is considered, older adults should base training on submaximal loads (85-80% of their one repetition maximum (1RM); tested or estimated from nRM) and advance to speed-power training regimens (50-75% of 1RM executed explosively). Based on the recent research, the latter is an important supplement facilitating physical ability and functional capacity of older individuals. Recent studies have shown that resistance training stressing performance of repetitions with maximal velocity results in higher performance gains as compared to strength training alone.1 It therefore seems reasonable to include speed-power training into physical conditioning of older adults. As a result, we aim to address this topic in the framework of the current Interreg project Slovakia-Austria, by exposing 60+ subjects to 10-week flywheel speed-power training. This training modality offers innovative solutions for individualised training progression (load, tempo, postural stability), which we will share during with the audience during our conference presentation. Additionally, our plan is to get a better insight into the potential of this kind of resistance training for changing force-velocity-power profile in elderly. This would complement our previously used testing approaches we used in past interventional studies in elderly.2 Namely, improving velocity dominance in the force-velocity profile seems to have significant functional relevance (balance control, gait initiation, change of direction; and prevention of falls as a result).3 Preliminary results on initial methodological considerations and protocols (training and testing) development will be presented, Proper muscle function is controlled by intracellular Ca2+ concentration, which in turn depends on: a) release of Ca2+ from intracellular stores during excitation contraction (EC) coupling, which activates muscle contraction; b) entry of Ca2+ from the extracellular space via store-operated Ca2+ entry (SOCE), a mechanism important to limit muscle fatigue; c) uptake of Ca2+ into the mitochondrial matrix, which stimulates aerobic ATP production; and finally d) sequestration/removal by sarcoplasmic reticulum (SR) and plasma membrane (PM) pumps, which relaxes muscle fibers. Abnormalities in Ca2+ handling underlies many physio-pathological conditions. For instance, reduced SR Ca2+ release has been linked to fatigue and dysfunction in ageing, while excess of SR Ca2+ leak may even underlie life-threatening conditions such as malignant hyperthermia susceptibility (MHS). In the last 15 years we have collected compelling evidence that the proper architecture and function of all those membrane systems involved in Ca2+ handling and aerobic ATP production depends on muscle activity: i) denervation causes disarray of units deputed to EC coupling (calcium release units, CRUs) and mitochondrial apparatuses, while functional electrical stimulation (FES) promotes some rescue of this structural disarray;1 ii) sedentary ageing in mice and humans causes misplacement of mitochondria and partial disarray of CRUs, while long-term training does prevent effectively those changes;2-4 iii) acute exercise promotes functional assembly of SOCE-sites (calcium entry units, CRUs), while post-exercise recovery determines their disassemble;5 iv) sedentary ageing causes accumulation of dysfunctional proteins in tubular aggregates (TAs), while exercise prevents TAs formation (manuscript in preparation). Ca2+ handling is crucial for muscle function and is controlled by diverse membrane systems and intracellular organelles. Our experience collected in different, but complementary, projects: i) indicates the structure and function of intracellular organelles is preserved or rescued by exercise or training; and ii) underlines the importance of physical exercise during ageing to preserve muscle function., The dominant role in muscle regeneration is played by the muscle stem cells known as satellite cells, which reside between the basal lamina and sarcolemma of myofibers. Along with satellite cells, other precursor cells, whose activity is strictly dependent by environmental signals, participate to muscle regeneration.1,2 A tightly regulated interplay between stem cells and other resident cell types, as well as the intimate connection with structural components of the tissue niche, can be responsible for the maintenance of the stem cell pool under steady-state conditions and to guide stem cells activation and differentiation when regenerative signals are provided.1 Because niche factors and components normally control and sustain a physiological stem cell activity and maintenance, the loss of homeostatic input from the niche, as observed under pathological conditions, can deregulate stem cell physiology, critically affecting the ability of muscle tissue to efficiently regenerate and to regain the functional integrity after damage.1-3). Thus, the stem cell niche is not only an anatomical compartment but a complex, integrated network of both cellular and acellular components that provide signals influencing stem cells and muscle homeostasis. The basis of muscle regeneration and the impact of cytokines and growth factors on the physiopathology of skeletal muscle will be discussed.1,4-6, Neuromuscular diseases, ageing, and cancer cachexia are chronic conditions characterized by skeletal muscle wasting (1, 2) resulting from altered muscle metabolism, and catabolic processes. Following muscle pathophysiological changes, proteins are secreted or passively released into systemic circulation. The identification of muscle-derived serum biomarkers can be used to develop a kind of “liquid biopsy” for diagnostic/prognostic approaches and to identify altered muscle physiology. With this aim, we performed combined morphological and biochemical analyses of skeletal muscle biopsies and blood samples obtained from cancer patients, both collected the day of surgery. Samples from non-oncologic patients were also collected as controls. Morphometrical analyses showed a significant reduction of myofiber size in oncologic patients in comparison to controls, and the analyses of myofiber diameter distribution showed a higher prevalence of severely atrophic fibers in oncologic patients, most of them having angulated and flat shaped morphology, typical features of denervation (3). The percentage of in situ Neural cell adhesion molecule (N-CAM) expressing myofibers and serum levels of N-CAM (sN-CAM) and Agrin were also tested, as markers of neuromuscular junction plasticity and remodelling (4, 5). Consistently with morphological observations, in cancer patients with respect to controls, the percentage of N-CAM positive myofibers was significantly higher and circulating levels of sN-CAM and Agrin were also significantly increased. These results correlate morphological changes of skeletal muscle trophism and innervation with serum alterations in NMJ-derived proteins. Moreover, they show that sN-CAM and Agrin are promising and reliable serological biomarkers for neuromuscular junction plasticity and remodeling in conditions characterized by skeletal muscle wasting, Functional electrical stimulation (FES) is used to induce skeletal muscle contractions in persons who are unable or reluctant to move usually by surface neuromuscular electrical stimulation (NMES), also known as transcutaneous electrical nerve stimulation (TENS) in out-patients or hospital. If properly instructed, persons may continue at home with FES-induced training with a daily frequency and for the long duration of time needed to achieve clinically-relevant outcomes. Standing on the results of the EU Project RISE,1 and on those in aging persons,2,3 we are confident that FES may contribute to a better life for an expanding population of aged persons and of early aged patients suffering with mobility impairments due to neuromuscular and metabolic disorders as well as systemic diseases. We will describe in detail results of FES for denervated degenerated muscles (DDM) delivered by large anatomically shaped surface electrodes at the level of the stimulated skin. Indeed h-bFES for DDM is able to recover skin from SCI-induced atrophy and flattening.4,5 Further, we will discuss the value of surface morphometry of neck skin, and of non-invasive blood analyses of circulating myokines (using sweat and mouth fluids6,7 as biological markers of aging progression., Male hypogonadism is a clinical syndrome manifesting with low testosterone (TT) including symptoms like decline in lean mass, muscle strength, increased adiposity, visceral obesity and incidence of insulin resistance and metabolic syndrome. 1 There are only few studies dealing with effect of physical activity on hypogonadal males.2 The aim of the study was to examine the effect of 12-week strength training program on body composition, selected biochemical parameters and physical performance in hypogonadal men. The study compared the effect of resistance training (RT) on hypogonadal patients without hormonal therapy (HP, n=6, 48.41±6.38 yrs, TT= 7.9±1.75 nmol/L) and control group of eugonadal males (EM, n=8, 49.31±5.84 yrs, TT= 15.81±3.99 nmol/L). The subjects performed RT twice a week, the training program consisted of 6 exercises at an intensity from 60-80% of 1RM. Body composition was measured by DXA, muscle strength was measured by predicted dynamic leg press 1RM from multiple repetition maximum, handgrip strength using hand dynamometer. Fasting morning venous blood samples were collected. The parameters analyzed from serum were glucose, total cholesterol, LDL cholesterol, HDL cholesterol, SHBG, insulin, total testosterone, and cortisol. Subjects from both the HP and EM groups significantly decreased relative fat mass by 6.2 % and 4.91% (p>0.05 and p, Central Core Disease (CCD) is a congenital myopathy characterized by presence of amorphous central areas (or cores) lacking glycolytic/oxidative enzymes and mitochondria in skeletal muscle fibers.1 Most CCD families are linked to mutations in ryanodine receptor type-1 (RYR1), the gene encoding for the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle.2 As no treatments are available for CCD, currently management of patients is essentially based on a physiotherapic approaches. Functional electrical stimulation (FES) is a technique used to deliver low energy electrical impulses to artificially stimulate selected skeletal muscle groups.3-5 Here we tested the efficacy of FES in counteracting muscle loss and improve function in the lower extremities of a 55-year-old female patient which was diagnosed with CCD at the age of 44. Genetic screening of the RyR1 gene identified a missense mutation (c.7354C>T) in exon 46 resulting in an amino acid substitution (p.R2452W) and a duplication (c.12853_12864dup12) in exon 91. The patient was treated with FES for 26 months and subjected before, during, and after training to a series of functional and structural assessments: measurement of maximum isometric force of leg extensor muscles, magnetic resonance imaging, a complete set of functional tests to assess mobility in activities of daily living, and analysis of muscle biopsies by histology electron microscopy. All results point to an improvement of muscle structure and function induced by FES suggesting that this approach could be considered as an additional supportive measure to maintain/improve muscle function and reduce muscle loss in CCD patients., Over the last decade, transcutaneous auricular nerve stimulation (tANS) become established method of VNS and has been verified in various disorders including neurological and psychological trauma, addiction of drugs, inflammation and tinnitus. Several groups examined also effects of tANS on central and peripheral nervous system, effects on behaviour in neuropsychiatric populations and effects on subjets with disorder of cognitive and social functioning. tANS of particular areas at the cymba conchae (CC) require electrodes with high spatial selectivity, low impedance and safe reversible charge delivery to specific populations of receptors through the electrode-tissue interface. Cortisol is a naturally occurring steroid hormone that has many important functions in the body and is released into the bloodstream at times of stress. Cortisol is blamed for anxiety, high blood pressure and stroke. It helps to control blood sugar levels, regulate metabolism, help reduce inflammation and assist with memory formulation. All treatments to correct eventual cortisol imbalance involve medication. The present study was aimed at demonstrating that selective tANS can be potentially used as a method for the induction of Cortisol hormone secretion. tANS was accomplished in a 62-year-old subject with angina pectoris, coronary artery disease, and moderate insomnia. Sites at the CC were selectively stimulated using a silicone plug with four platinum cathodes. For the tANS, current regulated stimulating pulses with an intensity of ic=20 mA, a pulse width of 200 μs, and a frequency of f=25, were used. Results show that in six out of eight samples of the mouth fluids, the cortisol level was significantly lower after tANS that before tANS., The environment is critical when, in order of intrinsic morphological or geographical characteristics, or extrinsic features (caused by human presence or human behavior), it induces strenuous changes (independent of emergency accidents) in the physiological conditions of people who require emergency aid.1 Moreover, in an extrinsic critical environment the possibility to have a not homogenous population to be rescued is greater, consequently we can act only on the variable physical capacities and competence of emergency team, to have more success. This is the purpose of this work that has a background of more than 1200 physical evaluations over 10 years, on Italian Alpine Soldiers.2 Every subject had three evaluations of fitness profile during the year (time 0, after three months of programmed training and after six months). Evaluation and training methods are studied in order to be applied to a large number of subjects. The fitness profile includes: anthropometric assessment, aerobic fitness, muscle endurance, heart rate, diastolic and systolic pressure, respiratory rate, hydration, postural assessment by assessing the spine with spinal mouse system.3 The obtained results have shown that the physical preparation carried out, has improved and made homogeneous the physical capabilities of both the team and individual subjects that make it up. In addition to making the members of the emergency team more conscious, the increase in the level of coordinated physical preparation has objectively improved the safety (for team members) and effectiveness of intervention in the various crisis situations., In daily life situations, if we are exposed to unusual, challenging situations, our body reacts in a special way, called acute stress. This mechanism helps to cope with these situations efficiently. Contrary, exposing the body to stress over a longer time period, called chronic stress, like in working environments, can cause various serious health problems.1 The assessment of electrodermal activity (EDA, skin conductance) might be an easy approach to evaluate individual stress conditions over a longer period of time in an objective way. With the help of cvxEDA,2 the monitored skin conductance,3 can be split into the slow skin conductance level (SCL) and the fast skin conductance response (SCR). 20 volunteers were exposed to a relaxing-challenging-relaxing situation to mimic different stress conditions. The SCR rate proofed to be a reliable measure to evaluate sympathetic arousal. During the relaxation phases, the SCR rate was around 6 min-1and during mathematical challenges, and it raised to 9 min-1(p, The nonlinear trimodal regression analysis (NTRA) method,1,2 based on radiodensitometric computed tomography (CT) distributions,3,4 is here used for building predictive models of cardiovascular health parameters, through tree-based Machine Learning (ML) algorithms. This study reports the use of NTRA parameters for classifying elderly subjects from the AGES-Reykjavik database;5 with coronary heart disease (CHD), cardiovascular disease (CVD), and chronic heart failure (CHF). ML models employing the random forests algorithm yielded the highest classification performance for all analyses, and overall classification scores for all three conditions were excellent: CHD (AUCROC: 0.936); CVD (AUCROC: 0.914); CHF (AUCROC: 0.994). The present work introduces a substantial step forward in the construction of non-invasive, standardized tools for associating adipose, loose connective, and lean tissue changes with cardiovascular health outcomes in elderly individuals., Postural Control is the complex feedback system that allows humans to keep balance and maintain the naturally unstable upright stance. This is the result of an articulate interplay between sensorimotor systems (vision, proprioception, somatosensory, etc.) and the Central Nervous System (CNS)1,2. Lesions to the CNS, pathologies like unilateral vestibular loss (UVL) and disorders like Motion Sickness disrupt the postural feedback system, severely impairing balance. In these adverse postural circumstance, prompt adaptive and habituating processes are activated in the CNS to ensure upright posture and gait. By integrating the traditional dynamic posturography with HD-EEG analysis on a cohort of thirtythree healthy subjects3, we investigate cortical involvement in the phases of adaptation and habituation to a postural control challenge, where the balance disruption is induced through a randomized sequence of vibratory stimuli applied to the gastrocnemius muscles of the participant’s calves Keywords: Balance, cerebral cortex, HD-EEG, kinematics, postural control, power spectral density., During skeletal muscle contraction Ca2+ release units (CRUs), the sites of excitation-contraction coupling (EC) provide Ca2+,1 while mitochondria, the organelles deputed to cellular respiration, produce ATP.2 In fast-twitch fibers from adult mice CRUs and mitochondria are structurally linked by small strands or tethers,3 and interact functionally, as the uptake of Ca2+ into the mitochondrial matrix stimulates the respiratory chain.4 Aging causes separation of mitochondria from CRUs and a cross-talk impairment between the two organelles.5 However, whether this age-related uncoupling is the result of aging per-se or the consequence of reduced muscle activity remains still unclear. Here we tested if muscle activity maintains the correct association of mitochondria to CRUs in a) extensor digitorum longus (EDL) muscles from 2 year old mice, either sedentary or trained for 1 year in wheel cages; and b) EDL muscles from denervated adult mice and rats. We analyzed muscle samples using a combination of structural, biochemical, and functional experimental procedures. The results collected in structural studies indicate that: a) ageing and denervation result in partial uncoupling between CRUs and mitochondria; b) exercise and re-innervation either maintains (in old mice) or restores (in transiently denervated rats) the association between the two organelles. Functional studies support the hypothesis that CRU-mitochondria cross-talk is important for mitochondrial Ca2+ uptake, optimal force generation, and muscle performance. Taken together, our results show that muscle activity maintain/improve proper Association between CRUs and mitochondria, providing a potential tool to counteract muscle function decline in elderly and sedentary persons., Wartenberg introduced the pendulum test in the 1950s as a method to assess spasticity in the clinical setting.1 It has proven to be sensitive to the presence and severity of spasticity.2,3 The pendulum test is based on letting the lower leg swing freely under the influence of gravity while recording joint kinematics. In the presented work, the reflex period, extracted from the pendulum test, will be proposed as a new parameter to measure spasticity. The reflex period is defined as the time period from 50% of the maximum velocity of the leg to the first EMG signal indicating muscle contraction. Data from two separate studies, one on spinal cord injury patients (Halla Kristín Guðfinnsdóttir)4 and the other on stroke patients (Belinda Chenery)5 were analyzed. In both studies a pendulum test with goniometers on the knee joint was performed and simultaneously recording electromyography (EMG) of the quadricpes m. Both studies consisted of 3 trials and data from 4 subjects of each study were analyzed. The EMG data was processed with Matlab R2014b (The MathWorks, Inc.) using the open-source toolbox EEGLab. A 4th order Butterworth high-pass filter with cutoff frequency at 10 Hz and a low-pass filter at 500 Hz was applied along with notch filters at 50 Hz, 100 Hz, 150 Hz and so forth. The data was smoothed with a Gaussian filter prior to the analysis. The mean reflex period of SCI patients was 370 ms with a standard deviation of 72 ms. The mean reflex period of stroke patients was 215 ms with a standard deviation of 54 ms. This suggests a difference between the reflex period of stroke and SCI patients which might be explained by the location of their lesion., The demonstrated expression of endocannabinoids receptors in myofascial tissue suggested the role of fascia as source and modulator of pain.1 It is known that the fibroblasts can modulate the production of the various components of the extracellular matrix according to different stimuli: physical, mechanical, hormonal and pharmacological.2 In this work we isolated the fascial fibroblasts from small samples of human fascia lata of the thigh collected from 3 volunteers patients during orthopedic surgery, 2 males and 1 female. We demonstrated for the first time that the agonist of cannabinoid receptor 2, HU-308, permits the in vitro production of hyaluronan-rich vesicles in only 3-4 hours after the treatment, quickly released in the extracellular environment. The cells treated with the synthetic cannabinoid showed a large number of vesicles near the Golgi apparatus of the cells, and also in the cytoplasmic extensions. We demonstrated by Alcian Blue and Toluidin Blue stainings, immunocytochemistry and Transmission Electron Microscopy that the content of these vesicles was rich in hyaluronan. The hyaluronan is a critical element for the extracellular matrix composition and remodeling,3 and in the deep fascia, it affects the movement of hyaluronan-containing fluid layers within and underlying the deep fascia, facilitating the smooth gliding between these structures during movement.4 So its production induced by the cannabinoids can be able to increase the ability of the collagen bundles inside the fasciae to glide one respect to the other, and consequently to improve the tissue adaptability. Furthermore, the stimulation of the endocannabinoid system could be able to provide an anti-fibrotic activity by suppression of pro-inflammatory cytokines and a relief of the myofascial pain.5 These results can help to understand how the cells of fascia can answer to the endocannabinoid system regulating and remodeling the extracellular matrix formation. This is a first step in the comprehension of how the therapeutic applications of cannabinoids for the treatment of pain can have also a peripheral effect, altering the biosynthesis of extracellular matrix in fasciae and consequently remodeling the tissue and its properties., Muscles generate active forces in each half-sarcomere, the smallest contractile Unit, through the coordinated action of myosin motors, assembled in the thick filament, with actin monomers, assembled in the thin filament. Filaments are then arranged in parallel, forming a main axis in the half-sarcomere along which active forces are directed. However, in the musculoskeletal system these active forces must follow non-linear paths from tendon to tendon. This task is achieved thanks to the ability to transmit active forces between half-sarcomeres, along fibre direction (longitudinal axis) through intra-sarcomeric proteins, and between different fibres, in perpendicular directions, through extra-sarcomeric proteins, generally referred as extra-cellular matrix (ECM).1 This ability is affected by age.2 In this work, we analyzed the passive tension generated in elongated fibres alone and when they are arranged in small bundles young and aged healthy humans.3,4 The mechanical properties of the extracellular passive components in a bundle of fibres were deduced by the subtraction of the passive tension observed in single fibres from the passive tension observed in the bundle itself. ECM-related components of passive force are non-negligible in both cases and of the same order of magnitude of intra-sarcomeric components. However, in young humans the increase in passive tension observed in bundles respect to fibres is smaller than in aged humans. Based on our data, we propose to quantitatively characterize the constitutive parameters of a Hill-type three-elements model incorporated in a finite element (FE) mesh representing a fiber bundle. The characterization can be used in future FE models of whole human muscles., Neutral lipid storage disease with myopathy (NLSDM) is a rare autosomal recessive disorder, associated with mutations of patatin like phospholipase domain containing 2 (PNPLA2) gene.1 PNPLA2 encodes adipose triglyceride lipase, that plays a key role in triacylglycerol breakdown1. NLSDM patients mostly present progressive skeletal myopathy, with both proximal and distal involvement.2 Cardiomyopathy, hepatomegaly and diabetes are often observed. NLSDM clinical severity appears to be highly variable and it is difficult to establish the effects of different PNPLA2 mutations on disease phenotype. Recently, alteration in the expression profile of specific microRNAs involved in skeletal muscle development (myomiRs) and lipid metabolism have been observed in neuromuscular disorders.3,4. In this study, we perform the evaluation by qRT-PCR of some circulating microRNAs levels in serum samples obtained from three NLSDM siblings (two brothers and one sister), carrying two PNPLA2 missense mutations.5 Progressive skeletal myopathy was detected in the two brothers and muscle imaging showed fibro-fatty replacement. The sister presented severe hepatosteatosis and diabetes. The analysis of serum microRNAs revealed that NLSDM patients exhibited an increased amount of myomiRs. In particular, a significant correlation between muscle mass and the level of miR-206 and miR-133a was found. Moreover, an elevation of some microRNAs, mainly expressed in liver, was observed, when there was a hepatic involvement. These results show that the changes of serum muscle and lipid metabolism-specific microRNAs might represent biomarkers of skeletal and hepatic involvement. Moreover, the dysregulation of these small molecules might provide a tool to monitor the progression of NLSDM., Spinal muscular atrophy (SMA) is a common autosomal recessive disorder caused by mutations in the gene for the survival motor neuron 1 (SMN 1), and it leads to progressive muscle weakness. A major goal of disease-modifying therapies is to increase the expression of the SMN protein. Although a number of therapies are under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing the efficacy of therapeutic interventions, particularly those targeting the upregulation of the SMN protein levels. Sensitive methods for quantifying SMN protein in peripheral blood are needed. Accordingly, we developed an imaging flow cytometry (IFC) method for evaluation of SMN protein using peripheral blood and fibroblasts. First, we demonstrated that IFC successfully identified different expression patterns and subcellular localization patterns of SMN protein in SMA patient-derived fibroblasts1. Second, we tested the sensitivity and utility of IFC in identifying the differences in the expression of SMN protein between SMA patients and normal subjects using cultured Epstein–Barr virus-transformed B cells2. Subsequently, we developed an IFC method for evaluating the functional SMN protein using < 1.5 mL of peripheral blood. IFC is advantageous for the analysis of peripheral blood because of its capacity to analyze heterogeneous cell populations3. IFC analysis can be implemented in future studies to optimize its application as a tool for assessment of the effectiveness of spinal muscular atrophy treatment., Prolonged oxidative stress may play a key role in tumor development. Antioxidants molecules are contained in many foods and seem to have a potential role in future anti-tumor strategies. Among the natural antioxidants the beneficial effect of Fermented Papaya (FPP®) is known. The aim of this study was to investigate the effects of orally administered FPP® in either prevention or treatment of a murine model of melanoma. The tumor growth was analyzed together with the blood levels of both oxidants (ROS) and anti-oxidants (SOD-1 and GSH). The results showed that FPP® controlled tumor growth, reducing the tumor mass of about 3 to 7 times vs untreated mice. The most significant effect was obtained with sublingual administration of FPP® close to the inoculation of melanoma. At the time of the sacrifice none of mice treated with FPP® had metastases and the subcutaneous tumors were significantly smaller and amelanotic, compared to untreated mice. Moreover, the FPP® anti-tumor effect was consistent with the decrease of total ROS levels and the increase in the blood levels of GSH and SOD-1. This study shows that a potent anti-oxidant treatment through FPP® may contribute to both preventing and inhibiting tumors growth. The results of the above study suggested that FPP® while showing a clear anti-tumor effect it occurred though the in vivo induction of a potent anti-oxidant reaction. In a new set of experiments we wanted to verify whether FPP had a clear and scientifically solid in vivo anti-aging effect together with the induction of the anti-oxidant reaction. To this purpose we used a mouse model suitable for aging studies (C576J) treating daily each mouse from 4 weeks of life to 10 months with the same dose of IMMUNEAGE dissolved into the daily water as compared to mice receiving only tap water. At the end of the treatment period (10 months) we measured some biological parameters related to the aging processes of the cells: i) the total anti-oxydant capacity in the plasma of mice treated or untreated with FPP®; ii) the telomerase activity in the plasma of mice treated or untreated with FPP®; iii) the telomeres length in the bone marrow and ovaries of mice treated or untreated with FPP® .The results showed that the blood of treated mice, at the end of the treatment period (10 months) had 2-3 folds more anti-oxidant power and telomerase activities than the untreated mice. In the same mice we obtained both the bone marrow (from the tibias) and the ovaries at the scarify, and from the cellular preparations we measured the telomere lengths in both. The results showed that daily FPP® assumption induced 3 folds increase in telomeres length in bone marrow and ovary of treated mice as compared to the untreated mice. This suggests that FPP® induces a clear improvement of the aging scientific parameters and that the treated mice were younger than the untreated controls., The muscle spindle (MS) plays an important role in proprioception and coordination,1 which is surrounded by a strong capsule of connective tissue.2 A gelatinous fluid rich in glycosaminoglycans fills the space of capsule, probably mainly hyaluronan (HA).3 Age-related proprioceptive and coordinative deficits are unclear, although some hypothesises have been proposed to explain those mechanisms both on the peripheral and CNS changes. Age-related changes of MS were compared in unmature (1month, M); young adults (4M); and old (27 M) C57BL/6J male mice. Hematoxylin Eosin (HE), Sirius-red,4 Van Gieson staining were used to evaluate age-related MS morphology changes The collagen type I antibody, biotin labeled HA binding protein immunostainings,5 were used to monitor age-related changes in collagen type I and HA of MS. The Purple-Jelley HA assay was used to measure age-related changes HA contents. MS is surrounded by the capsule in continuity with the perimysium and epimysium and HA filled the capsule. The capsule of the MS, perimysium, epimysium underwent thicking with aging, primarily consisting of collagen type I. Van Gieson showed that the presence of elastin in the capsule. The amount of HA in Triceps Surae is 27.74g/g in 4M. The presence of the collagen fiber, elastin and HA in the capsule of MS and its continuous with fascia suggested that they may effect the MS function. If their proportion changed, they will influence the function of MS, which properly explain part of the peripheral Neuro-physiological mechanisms of Age-related proprioceptive and coordinative deficits., Muscle structure,1 and performance2 differ between female and males when compared across the same age groups. Masters athletes compete in age groups of five-year divisions, ranging from 35 to more than 100 years of age. Here, our aim is to investigate the gender differences in the rate of age-related decay using the complete series of female and male normalized Masters’ World records. The World record series are lists of up to 16 data points that can be interpolated with polynomial trend-lines with a very high R2, after normalization to allow comparisons of the values among the different sports events.3 Here, gender comparisons were performed for 19 Track and Field specialties using weighted regression analyses. As expected, the aging decline began at 35 years for both women and men. Despite higher values of the male athletes in all the 19 Track and Field Masters world records,4 the rates of aging performance decay were very similar, if not identical.5 This lack of difference is a unique exception to the general rule of gender differences in sports activities, suggesting that neuro-hormonal mechanisms poorly influence the rate of aging muscle power decay. We discuss implications and limitations of our hypothesis that, at least in humans, the rates of age-induced decline are related to fundamental cellular mechanisms, perhaps those that control energy metabolism., The exponential increase of scientific publications in the mitochondrial field shows the growing interest in mitochondria. However, the lack of methodological consistency in many published projects on mitochondrial respiratory function complicates a quantitative inter/intra-laboratory comparison of datasets. This deficiency manifests the need to improve the quality in science.1 In this context, the MitoEAGLE COST Action is a powerful framework committed to evaluate and enhance the reproducibility in mitochondrial physiology as a basis to establish a novel mitochondrial database related to Evolution, Age, Gender, Lifestyle and Environment. Permeabilized muscle fibers are widely used to evaluate mitochondrial function in health and disease.2 Therefore, our main goals are to: 1) compare protocols used in different research laboratories, 2) analyze factors which contribute to experimental variability, 3) define optimal experimental conditions in muscle studies, 4) elaborate guidelines for evaluating mitochondrial function in muscle tissue, 5) establish reference values on mitochondrial respiration, particularly as a test of the skills in preparing high-quality permeabilized muscle fibers, and 6) generate a database. To achieve our aims, two unique studies are currently in progress: 1) 17 international research groups performing independently experiments on respiration in permeabilized fibers of mouse soleus muscle, following the same experimental procedure;3 2) a blinded international study measuring simultaneously in the same laboratory respiration of permeabilized human skeletal fibers by high-resolution respirometry and assessing the effect of different experimental conditions.4 Our results contribute to face the reproducibility crisis and provide the basis for establishing the first database on mitochondrial respiratory parameters in muscle tissues. Support. MitoEAGLE Task Group WG2. Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE., In the search for systemic changes of biomarkers1 in physical activity, within aging and rehabilitation studies, a major goal is to identify methods less invasive than blood sampling. Thus, the clinical use of mouth fluids is increasing.2 The consensus is that the presence of blood in mouth fluid compromises its diagnostic value. However, we have been looking at its’ contamination as a major opportunity for non-invasive serological analyses of systemic biomarkers. A major preliminary result has been obtained by evaluating the presence of serum in mouth fluids of healthy seniors and the eventual change after a modest trauma, i.e., tooth brushing. Seven healthy persons, aged older than 65, provided the fluids for the analyses by drooling saliva into a test tube. After low speed centrifugation, small aliquots of supernatants were frozen in liquid nitrogen and stored at -80°C until use. Aliquots were thawed and used to quantify by the total protein content using the Lowry method. Serum albumin, fibrinogen and lysozyme were determined by colorimetric ELISA (enzyme-linked immunosorbent assay), and hemoglobin content was quantified by use of spectrophotometry. After a preliminary test using colorimetric ELISA, saliva dilutions were adjusted to better determine the content of analytes. The control reference was a pool of sera from age-matched healthy seniors, to judge the quantity of serum in the fluid obtained from mouths of the elderly. Saliva collected from the seven healthy elderly persons before and after tooth-and-gum brushing presented measurable amount of the analytes, including fibrinogen, which is a minor component of the pooled sera. Tooth brushing did not induce a statistically significant difference in analytes’ contents, suggesting that a measurable blood contamination is a frequent event in elderly persons. In conclusion, fibrinogen is a promising reference to quantify serological biomarkers through a non-invasive procedure. Its determination will increase acceptability and frequency of analyses during follow-up in aging and rehabilitation., Older and oldest persons, i.e., early and late octogenarians, spend small amounts of time performing daily physical activity, which can aggravate their limited independence. This may force them to stay in bed, and/or to incur more frequent hospitalizations. All progressive muscle contractile impairments, including those related to advancing age, need permanent management.1,2 Inspired by the proven capability to recover skeletal muscle by home-based functional electrical stimulation (hbFES) and guided by common sense, we suggest to these elderly persons to perform a 10 to 20 min daily routine of 12 easy and safe physical exercises, that will train almost all the main skeletal muscles of the body. Elderly persons can do many of these exercises in bed (full-body in-bed gym), so that if hospitalized they may continue light training. The routine is an extension of the well-established cardiovascular and ventilatory rehabilitation trainings, usually performed under a guidance and supervision of a physiotherapist.5 If properly instructed in an outpatient clinic or during hospitalization, elderly persons may continue these exercises at home, and eventually integrate into their permanent lifestyle.3,4 Monitored arterial blood pressure before and after the daily routine described demonstrates that peripheral resistance decreases in a few minutes by functional hyperemia of the trained body muscles. The exercise intensity ought to be slightly challenging, up to the manifestation of visible sweating. In the long term, this will add systemic resistance to fatiguing arising from strength training against the own weight of an arm, leg and other body parts.2 Continued regularly, home-based full-body in-bed gym can help to maintain independence of the most frail elderly persons (old and oldest), reducing risks of accidental falls, and thus of serious clinical consequences., Scientific literature suggests that fitness characteristics in patients with Multiple Sclerosis (MS) may be compromised. Because nanotechnological devices have shown improvements in different healthcare application,1,2 the purpose of this study was to investigate the use of a nanotechnological device integrated with a postural exercise program (PEP) on cervical range of motion and handgrip strength in patients affected by MS. Seventeen participants with MS were recruited and randomly assigned to the Experimental Group (EG) and the Control Group (CG) including 9 and 8 subjects respectively. All participants carried out a cervical range of motion evaluation using an inertial motion sensor (Moover®;Sensor Medica®) and a handgrip test using an isometric mechanical dynamometer (KernMap model 80K1;Kern®) before and after a PEP (twenty-session/1 hour each, twice/week). A nanotechnological device (Taopatch®;Tao Technologies srls) was applied to the skin of the participants of the EG at the level of the C7 vertebra throughout the period of the PEP. No differences (p>0.05) were found in the EG on the cervical range of motion, while the CG showed a significantly increase on left rotation (p=0.01). Regards the handgrip test, a significantly improvement was found on the non-dominant hand in the CG (p=0.001) and on the non-dominant hand (p=0.04) and on the dominant hand (p=0.01) in the EG. Our preliminary results suggest that the use of a nanotechnological device could influence muscle strength. The results of this pilot study suggest that we should continue testing larger sample of subjects, diversifying also type and stage of the disease. The authors thank the patients and the Italian Association for Multiple Sclerosis (AISM) of Trapani, Italy for collaboration., Athletes have to pay particular attention on the eating patterns and to dietary intake in the hours before exercise, because pre-exercise nutritional strategies can influence not only exercise performance but also other biological responses to exercise and training including bone remodeling. In fact, inadequate nutrition leads to bone injuries. Twenty-eight preadolescent female gymnasts, playing artistic gymnastics at a pre-competitive level, were examined to investigate the effects of two different pre-exercise meals on athletic performance and bone resorption post exercise. Exercise trial were preceded ninety minutes by an isocaloric meal with carbohydrates or proteins. Urine was sampled at four different time points: pre-meal, ninety minutes post-meal/pre-exercise, ninety and hundred and fifty minutes post-exercise. In urine was analyzed the biomarker of bone resorption CTX by using an established enzyme-linked immunosorbent assay technique. Energy Self-Perception Questionnaire (ESPQ) was used to evaluate energy status of the athletes after the performance. Pre-workout supplementation with a carbohydrate-rich meal reduced significantly post-exercise bone resorption, compared with the protein-rich meal as evidenced by the reduction in CTX levels sixty minutes after the end of physical activity. Moreover, the consumption of both pre-workout meal improves performance indifferently. In conclusion the study shows that a carbohydrate rich pre-exercise meal is able to reduce bone resorption after exercise in pre pubertal age gymnasts and improve the athletic performance., The Main objective of the study was to evaluate the effects of treatment with Cannabis-based drugs combined or not with a proprioceptive training, on patients affected by Multiple Sclerosis (MS), a chronic autoimmune demyelinating disease which can have different symptoms; we estimated the possible synergic interaction between therapy and exercise particularly to reduce the collateral adverse effects of the drugs, over all the spasticity, one of the effect of the disease. We enrolled in our study 10 patients randomly assigned in two groups; both was treated with a cannabinoids drug (group 2) but only one group performed a physical activity for 12 weeks two times per week in session of 60 minutes each (group 1). Blood assessment (serum levels of BDNF, proBDNF, interleukine10 and interleukine17) was performed and the results highlighted a changes. As to concern BDNF levels were 111 vs 33 group 1 compare with 82 vs 22 in group 1; whilst proBDNF levels were 190 vs 153 group 1 compare with 121 vs 138 in group 1. About IL’s, we highlighted this results: IL10 (anti-inflammatory) 61,84 vs 75,52 in group 1 compare with 50,64 vs 54,65 in group 2; IL 17 (pro-inflammatory) 31,12 vs 16,87 in group 1 compare with 24,53 vs 9,68 in group 2. The interpretation of the results is still in progress but the changes has occurred; further investigations are needed improving the sample size and the time of the intervention.

The 14th Meeting of the Interuniversity Institute of Myology (IIM), October 12-15, 2017 - Assisi, Italy gathered together researchers from Italy, European and North-American countries to discuss recent results on muscle research. The program showcased keynote lectures from world-renowned international speakers presenting advances in muscle physiology, bioengineering, metabolism and therapeutics. Based on selection from submitted abstracts, participants presented their novel, unpublished results in seven oral communication and two poster sessions. Particular emphasis was devoted to young trainees. For example, trainees where directly involved in organizing a scientific session and three round tables tailored to the interests of their peers. The meeting attracted a broad audience from Italy, various European countries and from North America. It offered a unique opportunity to all researchers involved in the field of muscle biology to exchange ideas and foster scientific collaborations to better understand the causative mechanisms of muscular diseases and to improve the design of more efficient therapeutic strategies. The friendly and inclusive atmosphere promoted the active participation of junior scientists to exciting discussions, which allowed to identify emerging areas of myology research and encouraged scientific cross-fertilization to facilitate exchanges between different laboratories in different countries. The meeting was a success and this community will continue to deliver major contributions to our understanding of muscle development and function, the pathogenesis of muscle diseases and the development of novel therapeutic approaches. Here, we report abstracts of the meeting discussing recent results of basic, translational and early clinical studies confirming that the field of Myology is strong and articulated, maturing toward clinical development for the treatment of muscle diseases., Force and shortening in striated (skeletal and cardiac) muscles are generated at the level of the sarcomere, the structural unit, by the two bipolar arrays of the motor protein myosin II emerging from the thick filament, during cyclical interactions with the interdigitating thin, actin-containing, filaments. In the classical model of striated muscle regulation the OFF (resting) and ON (actively contracting) states of muscle correspond to the OFF and ON state of the thin filament and the switch between states is controlled by calcium. However, in the resting muscle most of the myosin motors lie on the surface of the thick filament folded back against the myosin tails unavailable for binding to the actin (Woodhead et al. Nature 436:1195, 2005; Zoghbi et al. PNAS 105:2386, 2008) and splitting ATP (Stewart et al. PNAS 107:430, 2011) (OFF state of the thick filament). This implies the presence of a second thick-filament-based switch for contraction: muscle is ON when both the thin and thick filaments are ON. To investigate this point, we used sarcomere-level mechanics and X-ray diffraction interference from striated muscle cells at ID02 beamline of the European Synchrotron (Grenoble, France). In single intact fibres from frog skeletal muscle we showed that, for contraction against high loads, the recruitment of myosin motors from the OFF state is based on a stress-sensing mechanism in the thick filament and that during unloaded shortening a large fraction of motors recovers progressively the OFF state (Linari et al. Nature, 528:276, 2015). The role of thick filament stress-sensing in heartbeat regulation has been investigated by exploiting our recent development of sarcomere-level mechanics in intact trabeculae isolated from the ventricle of rat heart (Caremani et al. PNAS 113:3675, 2016) and the possibility at ID02 beamline to record both the nanometer-scale signals from the contractile proteins along the thick filament (M1-M6, camera length 1.6 m) and the micrometer-scale changes of the length of the sarcomere (camera length 30 m). It was found that, starting from the same resting sarcomere length, at the peak of the twitch force the intensity profile of the M3 reflection due to axial repeat of myosin motors change with the loading conditions, whether sarcomere shortening against the end compliance during force development is allowed (fixed end conditions) or is prevented (length clamp conditions) (Reconditi et al. PNAS 114:3240, 2017). The difference in M3 intensity profiles indicates that, during a cardiac twitch, only a fraction of the motors leaves the OFF state and this fraction depends on the level of the force independently of the diastolic sarcomere length. The different loading conditions of the twitch reproduce the conditions, at the organ level, of the left ventricle beating against a high or a low aortic pressure. We conclude that a stress-sensing mechanism in the heart tunes the energetic cost of contraction to the mechanical task., Skeletal muscle adapts to exercise training through the concerted actions of several transcriptional regulators, among which PGC-1alpha coactivators play a central role. When activated by aerobic exercise, PGC-1alpha1 induces genes relevant to mitochondrial biogenesis, adaptive thermogenesis, lipid and glucose homeostasis, fiber-type switching, among other processes. In addition, exercised skeletal muscle promotes peripheral kynurenine detoxification, which protects from stress-induced depression. Metabolites of the kynurenine pathway of tryptophan degradation play important roles in the regulation of neuroinflammation and mental health. This process is mediated by PGC-1alpha1, which enhances kynurenine aminotransferase (KAT) gene expression in skeletal muscle. Elevated KAT levels convert accumulating kynurenine into kynurenic acid, an end metabolite of the pathway that cannot cross the blood-brain barrier and therefore remains in the periphery. We will discuss some of the consequences of activating muscle kynurenine metabolism for peripheral tissue inflammation and energy expenditure., Human aging is associated with an increase in the number of senescent cells in a variety of tissues. Cellular senescence has been described in vitro as the “Hayflick phenomenon”; whereby, cells fail to replicate. Cellular senescence is associated with other canonical features of aging such as telomere shortening, an increase in free radicals/reactive oxygen species, mitochondrial dysfunction and an increase in inflammatory markers. The senescence associated secretory pattern (SASP) refers to the propensity for senescence cells to release pro-inflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-13), proteases (PAI-1, MMP-3, MMP-1, TIMP-1), growth factors (hepatocyte growth factor, basic fibroblast growth factor, VEGF-a, HGF, EGF) and chemokines (IL-8, CCl2, CXCL, eotaxin, eotaxin-3, MCP2, IL-8, MIP1A). We and others have found that the basal concentration of many of the constituents of the SASP are higher in older humans and mice and are higher in progeroid aging (polymerase gamma-1 mutator mouse model). Our recent data shows that fibroblasts from older adults have far fewer replicate cycles than those derived from younger adults and older athletes are similar to the young sedentary adults. Furthermore, fibroblasts taken from sedentary older adults have greater number of passages until cellular senescence following three months of endurance exercise training. We and others have shown improvements in mitochondrial function and lower oxidative stress in both cross sectional and longitudinal studies in humans and mice. Acute endurance exercise often leads to a “pulse” of higher SASP components; however, long term endurance training leads to a lower basal level of SASP. Given that the consistent observation of increased mitochondrial function and lower oxidative stress in endurance trained mice and humans and the importance of mitochondria to senescence pathways (telomere length, oxidative stress, inflammasome activation) I posit that mitochondrial function is the main factor influenced by exercise training that leads to a reduction in SASP and fewer senescence cells., Neuromuscular junctions (NMJs) are synapses formed between motor neurons and skeletal muscle fibers. Abnormalities in NMJ development lead to various neuromuscular disorders, which are often fatal. Despite their crucial role, the mechanisms that orchestrate NMJ development are still poorly understood. The Dystrophin-associated Glycoprotein Complex (DGC) is a major laminin receptor in the muscle required for proper development of the postsynaptic machinery by linking its components to the extracellular matrix and the actin cytoskeleton. One of the cytoplasmic DGC-associated proteins, α-dystrobrevin-1 (αDB1), was shown to play an important role in the organization of the NMJ postsynaptic machinery. For it’s proper functioning αDB1 needs to be phosphorylated on its C-terminal fragment. To gain insight into the molecular mechanism of αDB1 function we have recently performed a biochemical screen for phospho-specific interacting proteins and identified SH3BP2 as a binding partner. SH3BP2 is a scaffold protein with unknown localization and function in skeletal muscles. We demonstrate that SH3BP2 is concentrated at the NMJ postsynaptic machinery and also at the muscle contractile machinery. Cultured myotubes depleted of SH3BP2 had the impaired ability to cluster AChRs. A similar phenotype was observed at the NMJ upon muscles-specific deletion of SH3BP2. Protein complex purification experiments combined with mass spectrometry analysis revealed that SH3BP2 interacts with several postsynaptic proteins including Lrp4, AChR, and CK2, proteins of the muscle contraction machinery as well as several components of the DGC. Our results suggest that SH3BP2 acts as a scaffold protein involved in the organization of the NMJ postsynaptic specialization. Supported by the National Science Centre grants 2012/05/E/NZ3/00487, 2013/09/B/NZ3/03524, 2015/19/N/ NZ5/02268 and 2016/21/B/NZ3/03638., Muscle atrophy results from transcriptional adaptations occurring in catabolic conditions. The Forkhead Box (Fox) transcription factors FoxO1, FoxO3 and FoxO4 are critical mediators of the catabolic response in skeletal muscle. An exacerbated activation of FoxO family members leads to increased protein breakdown and muscle wasting. Microarray analysis during fasting showed that FoxOs are required for the induction of several atrophy-related genes (atrogenes). However, the activation of already identified atrogenes cannot sustain all the protein breakdown during atrophy. Indeed, the discovery of new unknown players involved in muscle protein degradation is now of potential interest. We identified several new FoxOs-dependent genes, called Rikens, whose functions are still unrevealed. We showed that some Rikens are up-regulated in catabolic condition such as fasting, disuse and cancer cachexia. Interestingly, knocking-down one of the Rikens (Riken1) protected from atrophy during fasting. Moreover, colocalization experiments showed a possible Riken1-LC3/Riken1-Lamp1 interaction, suggesting a potential role in the autophagy-lysosome pathway. Our findings will contribute to the identification of new mediators of muscle mass loss in order to develop new therapeutic approaches against muscle wasting., Muscle activity leads to major swings in mitochondrial [Ca2+], which control aerobic metabolism, survival pathways and cell death. Recently, we showed that mitochondrial Ca2+ uptake positively modulates skeletal muscle trophism by impinging on two major pathways, PGC-1alpha4 and IGF1-AKT/PKB thanks to the use of AAV vectors. Here, we aimed to discern the metabolic route regulated by mitochondrial Ca2+ uptake that is responsible for muscle trophism. For this purpose, we generated a skeletal muscle specific Mcu knockout mouse (mlc1f-Cre-Mcu-/-), by crossing a Mcufl/fl mouse with a line expressing the Cre recombinase under the control of the myosin light chain 1f (mlc1f) promoter. Our preliminary data confirm that PGC-1alpha4 and IGF1-AKT/PKB signaling pathways are negatively regulated in skeletal muscle specific Mcu knockout animals. In addition, we also observed a slight decrease of fibre size in mlc1f-Cre-Mcu-/- skeletal muscles. Most importantly, when these mice were exercised on a treadmill using different training protocols, an impaired running capacity became evident, indicating that mitochondrial Ca2+ accumulation is required to guarantee skeletal muscle performance. Finally, a clear metabolic alteration is present in mlc1f-Cre-Mcu-/- animals. Specifically, mlc1f-Cre-Mcu-/- mice show decreased glucose, increased lactate, free fatty acids and ketone bodies, suggesting an impaired crosstalk between skeletal muscle and liver. Taken together, these data indicate that mitochondrial Ca2+ uptake plays a pivotal role in the control of skeletal muscle trophism. Further investigations of MCU-dependent effects on skeletal muscle homeostasis will represent an important task for the future. Indeed, this research will provide new possible targets for clinical intervention in all diseases characterized by muscle loss, such as dystrophies, cancer cachexia and aging., In skeletal muscle mitochondrial fusion and fission define the mitochondrial network morphology regulating myofiber differentiation, muscle contraction and response to stress conditions. Mitochondrial fission is mainly mediated by dynamin-related protein 1 (Drp1) which represents a critical player in myogenesis and its inhibition suppresses myotube formation. We studied a transgenic mouse overexpressing Drp1 specifically in skeletal muscle (Drp/MC). We have already showed growth defects of Drp/MC mice starting from P7 mainly due to an impairment of glycolytic muscles development; they display an overall 20% reduction of body weight at P100 and a drop of locomotor performance without any increasing in catabolic event. Drp/MC mice exhibit low mitochondrial DNA levels which trigger mitochondrial stress and upregulate the unfolding proteins response (mtUPR) together with an impairment of Growth Hormone anabolic pathway. Interestingly we observe a strong remodeling of mitochondria distribution with a depletion of inter-myofibrillar mitochondria and an enrichment of the sub-sarcolemma pool. In parallel, we observe a perturbation of citoskeleton framework characterized by the disruption of Desmin network (the main skeletal muscle intermediate filament connecting mitochondria to citoskeleton) with the presence of Desmin aggregates inside myofibers and accumulation beneath the sarcolemma. Our study aims at identify the involvement of different molecular effectors, such as the kinesin Kif5b and myotubularin, in dysregulated muscular and mitochondrial phenotype of Drp/MC mice., Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterised by compromised function of the neuromuscular junction (NMJ) manifesting with fatigable muscle weakness. We identified mutations in MYO9A as causative for CMS but the precise pathomechanism remained to be characterised. We hypothesised that defects in MYO9A affect the neuronal cytoskeleton, thus leading to impaired vesicular transport. MYO9A-depleted NSC-34 cells (mouse motor neuron-derived cells) were used to assess the effect on the cytoskeleton using immunofluorescent and immunoblotting techniques. Vesicular transport was analysed using different assays including a secretomic study to identify factors released from the nerve to act on the muscle fibre for NMJ development and function. In addition, an unbiased approach utilising proteomic profiling of control and MYO9A-depleted NSC-34 cells was performed to identify key players of the pathophysiology. Disruption of the cytoskeleton has been identified in MYO9A-depleted cells, with corresponding defects in receptor recycling and regular transport of proteins to the cell surface also observed. Proteomic data support a role for defective vesicular transport and identified affected proteins which are also involved in the manifestation of other neuromuscular disorders. Furthermore, a therapeutic target was identified and treatment of our MYO9A zebrafish model was able to ameliorate movement defects, increase muscle mass and improve muscle innervation. Our combined data allow new insights into the pathophysiology of CMS and show that loss of MYO9A affects the neuronal cytoskeleton, leading to impaired transport and vesicular recycling of proteins. We also identified a protein of potential therapeutic importance for patients., Myotonic Dystrophy is the most common adult onset muscular dystrophy and 2 major forms have been identified based on distinct genetic mutations. Myotonic Dystrophy Type 1 (DM1) is caused by (CTG)n trinucleotide repeat expansion in the 3’ untranslated region of the DMPK gene and displays an autosomal dominant mode of inheritance. Patients present with myotonia, muscular weakness, cardiac arrhythmia, and visual disturbances. The repeat expansion causes the disease through a toxic RNA product clustering RNA processing factors. Mis-splicing of diverse transcripts has been observed in DM1 resulting in presence of aberrant proteins and/or perturbed protein abundances such as for the SR resident SERCA. Affection of the latter protein leading to perturbed Ca2+ SR homeostasis is in accordance with the few descriptions of perturbed ER/SR-homeostasis. As the ER/SR is connected to the Golgi, an affection of these protein processing compartments is very likely and presumably affects folding and glycosylation of further proteins. However, as there remains a lack in the understanding of organelle pathology in DM1, we here systematically addressed this topic: utilizing patient and animal model-derived material we studied (i) organelle integrity, (ii) cellular fitness, and (iii) protein abundances (proteomics and immunoblot) including (iv) the study of glycoproteins. Moreover, (v) RNA-Seq and (vi) measurements of protein clearance have been performed. Our combined data reveal profound overall affection of the ER-Golgi machinery along with affection of the protein clearance machinery and thus strengthen the current understanding of the etiology of DM1 and hereby open new avenues for therapeutic intervention concepts., Muscle development and skeletal muscle regeneration are processes which required specific and synchronized controlled steps. In the laboratory, it has been shown that the transcription factor Nfix is necessary for both processes. During muscle development, Nfix is expressed by fetal myoblasts and regulates the switch from embryonic to fetal myogenesis by activating or repressing specific myogenic genes. In post-natal life, Nfix is expressed by satellite cells (SCs) regulating the proper timing of muscle regeneration upon injury. Interestingly, not only SCs but also macrophages (MPs) express Nfix. Nfix is expressed by MPs at the later stages of muscle regeneration. Specifically, Ly6C+ pro-inflammatory MPs exhibit the same level of Nfix while the percentage of Nfix+ Ly6C- anti-inflammatory MPs always increases over the time during regeneration. Since SCs also express Nfix, we generated LysMCre:Nfixfl/fl mice which selectively lack of Nfix in MPs. After cardiotoxin injection, skeletal muscles of LysMCre:Nfixfl/fl mice exhibit a delay of regeneration characterized by a persistence of necrotic myofibers and a later appearance of newly-formed myofibers. In vitro, Nfix silencing leads to a defect of anti-inflammatory phenotype acquisition and it maintains a pro-inflammatory phenotype upon M2 polarization. Functional experiments on myoblasts shown that LysMCre:Nfixfl/fl M2 MPs adopt WT M1 features. Moreover, we observed that MPs failed to switch from pro- to anti-inflammatory phenotype in vivo. Dystrophies are genetic muscle diseases characterized by continuous cycles of regeneration-degeneration, leading to exhaustion of SCs pool and inflammation. The high amount of MPs infiltration is linked to fibrosis establishment. Interestingly, we observed in two models of dystrophic mice (Sgca null and mdx) that the number of MPs positive for Nfix increases with the progression of the disease. With these studies, we are identifying Nfix as a necessary new interplayer for MPs identity and function during acute muscle regenerative process, while Nfix seems deleterious in chronic dystrophic context., The transition from embryonic to fetal myogenesis is a crucial switch, required for the complete maturation of skeletal muscle. The transcription factor Nfix, specifically expressed during fetal myogenesis, is the master regulator of this transition. Here, we show that the temporal progression of prenatal muscle development is timed by the RhoA/ROCK axis, which maintains the embryonic myogenesis, suppressing Nfix expression. RhoA and ROCK elicit their effects repressing ERK kinase activity, which instead promotes the fetal genetic program. Thus, RhoA/ROCK/ERK axis constitutes one of the major pathways that regulate the temporal progression of prenatal muscle development through the control of Nfix expression. RhoA/ROCK and ERK signaling pathways were also active in the Satellite cells, the postnatal population of myogenic stem cells, which expresses high levels of Nfix. The modulation of RhoA and ROCK axis shows that their role, as Nfix inhibitors, is not conserved in Satellite cells. Conversely, ERK kinases activity is necessary for the induction of Nfix expression not only in prenatal fetal myoblasts but also in Satellite cells. In the light of the fact that dystrophic muscles lacking Nfix have a robust recovery of symptoms and muscle morphology, our results build the basis of a future therapeutic approach for muscular dystrophies by using ERK inhibitors, which are drugs currently used in clinic for the treatment of melanoma., Heat stress is defined as perceived discomfort and physiological strain associated with exposure to a hot environment especially during physical work. In 2009, Dainese and colleagues demonstrated that mice lacking calsequestrin 1 knockout (CASQ1-null) suffer lethal episodes when exposed to high environmental heat (i.e. heat strokes) (Dainese et al. 2009). More recently, a link between hyperthermic episodes and excessive oxidative stress has been established (Michelucci et al. 2015), Finally, we discovered that crisis can be prevented by aerobic training, and that oxidative stress is decreased in muscles of mice that survive from heat exposure after training (Guarnier et al. IIM 2016). However, the mechanisms underlying the protective effect of aerobic training deserve additional investigation. Here, C57Bl/6 and CASQ1-null male mice had their individual maximal exercise capacity evaluated at 2-2.5 months of age before being subjected to aerobic training for 2 months (60% of maximal speed, 5x/week). In addition, groups of C57Bl/6 and CASQ1-null with the same age were sacrificed and muscles collected to evaluate mitochondrial damage and proteolysis, and cytochrome c and SERCA activities. At 4-4.5 months of age, controls, untrained and trained mice were first re-evaluated: CASQ1-null displayed a raised aerobic capacity and, when submitted to the heat stress protocol (41°C/1h), the mortality rate of trained CASQ1-null was greatly reduced (16.6%) when compared to untrained mice (85.6%). Muscles from CASQ1-null mice that survived from heat-stress protocol revealed that aerobic training was effective in: a) decreasing mitochondrial damage (13% vs 7 % in trained mice); b) improving energy generation, by increasing cytochrome c activity, while decreasing SERCA’s; and finally c) decreasing calcium-dependent proteolytic activity. Taken together, these results indicates that the protective effect of aerobic training against heat strokes is mediated by reduction of oxidative stress which, in turn, reduces mitochondrial damage and proteolysis and improves energy expenditure. Supported by: a) Brazilian Ciências Sem Fronteiras: CNPq 233892/2014-1 to FAG; b) Italian Telethon ONLUS Foundation Rome, Italy: GGP13213 to FP; c) Italian Ministry of Health: GR-2011-02352681 to SB., Tubular aggregates (TAs), ordered arrays of sarcoplasmic reticulum (SR) tubes, are frequently found in ageing fast-twitch fibers of male C57Bl6 mice. TAs are also found in biopsies from patients affected by Tubular Aggregate Myopathy (TAM), a muscle disorder linked to mutations in STIM1 and Orai1, STIM1 and Orai1 are the two main players in store-operated Ca2+ entry (SOCE), a mechanism that allows recovery of extracellular Ca2+ during repetitive activity and fatigue. First, we investigated presence of TAs, expression and subcellular localization of STIM1 and Orai1, and contractile force during repetitive stimulation in EDL muscle from male adult (4 months) and aged (24 months) mice. While TAs are never found in 4 month old muscle, 50 % of fibers contains TAs in aged EDLs. Furthermore, we have found that: i) ageing causes STIM1 and Orai1, to accumulate in TAs; and ii) EDL muscles from aged mice exhibit a decreased capability to maintain contractile force compared to young animals (relative force after 10 tetani: 55.0±1.9% vs. 65.6±3.5%). Secondly, we analyzed EDL muscles from 3 male mice that were exercised in wheel cages for voluntary running for 15 months, from 9 up to 24 months of age (each mouse ran on the average a total of ~396±29 km) and discovered that: a) wheel cage running significantly reduced formation of TAs, with a great reduction in the percentage of fibers containing TAs (50% vs. 7%, respectively in untrained vs. trained animals); b) EDL muscles from aged mice trained in wheel cages exhibit an increased capability to maintain contractile force compared to untrained aged match animals (relative force after 10 tetani: 63.1±4.9% vs. 55.0±1.9%). Taken together these findings point to: a) TAs as structures that lead to dysfunctional accumulation of STIM1 and Orai1 in inactive mice; and b) voluntary exercise as an effective measure to prevent formation of TAs and maintain a functional SOCE in muscle., Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor-neuron injury and skeletal muscle atrophy. Transgenic animals carrying mutations in the SOD-1 gene develop similar symptoms than those observed in clinic. In this animal model skeletal muscle has been demonstrated to be primarily involved in SOD-1-mediated toxicity (Dobrowolny et al. Cell Metabolism. 8, 425–36, 2008). In this context, sarcolemma ion channels play a crucial role for muscle function. Resting chloride conductance (gCl), sustained by the ClC-1 channel, controls sarcolemma excitability, indeed a large reduction of gCl produces myotonic-like symptoms (Pierno et al. Brain 125, 1510–21, 2002). At the moment, there are no data describing the involvement of skeletal muscle ion channels functions in this pathology, thus, we measured the resting gCl and the potassium conductance (gK), as well as muscle excitability in extensor digitorum longus muscle of 4-months old transgenic SOD-1 mice, at the onset of the symptoms, by using the two-intracellular microelectrodes technique. We found that resting gCl was strongly reduced in SOD-1 mice as compared to wild-type (WT), being it 1593±100 μS/cm2 (19 fibers) and 2410±79 μS/cm2 (22 fibers), respectively. Resting gK was increased in SOD-1 animals by 67±27%. Preliminary patch clamp studies showed different activity of the KATP channels and an altered sensitivity to ATP in accord with the increase of gK. Also sarcolemma excitability, evaluated as the maximum number of action potentials, was accordingly increased from 7.1±0.8 (16 fibers) in WT to 12.6±1.5 (10 fibers) in SOD-1 muscle fibers. Resting intracellular calcium level was increased in these animals and an altered response to caffeine was found. In order to evaluate the muscular involvement in the pathology we also examined an animal model in which the mutated SOD1 G93A gene is selectively overexpressed in skeletal muscle under the control of the MLC promoter. Similar modifications were found in skeletal muscle of these animals, since resting gCl was reduced to 1694±146 μS/cm2 (19 fibers). We observed that the ClC-1 mRNA expression was significantly decreased in muscles of SOD-1 mice respect to WT but do not change in muscles of MLC/SOD1G93A mice. In this animal model we tested the in vitro effect of acetazolamide, previously found to beneficially improve ClC-1 function in Myotonia Congenita through voltage-dependence regulation (Desaphy et al. Exp Neurol. 248: 530-40, 2013). Interestingly, acetazolamide application, increased resting gCl toward the control value, being 2097±119 μS/cm2 (19 fibers). Accordingly, sarcolemma hyperexcitability was improved. In conclusion, chloride channel function is modified in skeletal muscle of the SOD-1 transgenic animals suggesting their contribution to ALS and opens the possibility to investigate on acetazolamide as a promising therapy., The octopus arm is composed by uninucleated myofibers packed in a complex matrix of connective tissue. Two main muscle types compose the arm bulk: the longitudinal (L), located at the outer layer, and the transverse (T), surrounding the arm core. Here we investigated the mechanical properties of these muscles using a Dual-Mode Lever System on in-vitro preparations. We show that L and T muscles differ in their biophysics although single myofibers share the same physiological properties. To explore the bases of these different mechanical features, we studied the arm muscle-connective interaction and the possible role of gap junctions in the coordination of muscle ensembles. We performed confocal microscopy of arm sections and employed a waviness index (W.I.) analysis to measure the coiling of elastic fibers. W.I. was found to be higher in L muscles than in T, where elastic fibers are sparsely organized. This architecture contributes to accommodate strain during movements and to coordinate contraction of antagonistic muscles. Moreover, muscle fiber synchrony is achieved despite monosynaptic innervation and might be based on gap junction intercellular connections. Invertebrates gap junctions are formed by innexin, a family of protein analog to vertebrate connexin. As the presence of innexins might be an indicator for gap junction, we sequenced octopus unc-9 innexin and found a good identity and a comparable topology with invertebrate C. elegans unc-9 innexin. The presence of gap junction might suggest that octopus arm muscles evolved together with a fine-tuned coordination control mechanism., Skeletal muscle exhibits a high capacity to regenerate, mainly due to the ability of satellite cells to replicate and differentiate in response to stimuli. Epigenetic control is effective at multiple steps of this process, regulating the transition from quiescence to activation and differentiation of muscle stem cells1. The chromatin-remodelling factor, HDAC4, has been shown to regulate satellite cell proliferation and commitment2; however, the underlying molecular mechanisms are still uncovered. To study HDAC4 in satellite cells, we generated a conditional inducible KO mouse line, in which HDAC4 is deleted in Pax7 positive cells, upon tamoxifen administration. Despite having similar amounts of satellite cells, HDAC4 KO mice show compromised satellite cell proliferation and differentiation in vitro. To identify the molecular targets regulated by HDAC4 in satellite cells, we performed a ChIP-seq analysis by immuno-precipitating the endogenous HDAC4 in proliferative muscle cells and we performed a RNA-seq analysis by comparing control and HDAC4 KO satellite cells. By intersecting the results of the ChIP-seq with the list of the genes significantly up-regulated in HDAC4 KO satellite cells from the RNA-seq, we identified two candidates, P21 and Sharp1, as direct targets of HDAC4 in proliferating muscle cells. By interfering with their expression, we demonstrated that HDAC4-mediated repression of the cell cycle inhibitor P21, promotes satellite cell amplification; while HDAC4-mediated repression of Sharp1, allows satellite cell differentiation and fusion. These data identify HDAC4-mediated regulation of genes that control two sequential stages of satellite cell activity during myogenesis, such as proliferation and differentiation into new fibers., Skeletal muscle inflammation is an important feature of different myopathies and supportive evidence for the relevance of inflammation in promoting dystrophin-related diseases come from the beneficial effect of corticosteroids in the treatment of Duchenne muscular dystrophy (DMD). Although many studies have been focused on the role of bioactive sphingolipids in inflammatory-associated disorders, the involvement of hydrolase acid sphingomyelinase (A-SMase) in DMD-related inflammation is currently unknown. Our results showed that A-SMase is overexpressed in skeletal muscles of mdx mouse model of DMD, both at 4 and 12 weeks of age. The initial inflammatory response during chronic muscle injury is similar to the response to acute damage. To study a possible role of A-SMase in muscle regeneration we injected cardiotoxin in Tibialis Anteriors of wild-type (wt) and A-SMase knock-out (KO) mice thus inducing acute muscle damage. Of interest, the expression of Myogenin, a marker of muscle differentiation, significantly increased in A-SMase-KO mice when compared to wt after cardiotoxin administration. In addition, A-SMase-KO mice muscles showed enhanced levels of the anti-inflammatory cytokines and Arginase, an enzyme which is positively related to muscle regeneration. These data, although preliminary, provide the first evidence that in the absence of A-SMase muscle regeneration is accelerated and suggest a role of A-SMase in the pathophysiology of DMD., Skeletal muscle tissue engineering represents a revolutionary approach for the treatment of musculoskeletal tissue pathologies1. However, there are some limitations related to the complex muscle architectural organization and to the difficulty to find a reliable and appropriate source of myogenic progenitors able to sustain swift vascularization. Recent studies have led to the identification of a new muscle progenitor cell population namely pericytes2. These are perivascular muscle progenitors able to undergo a robust myogenic differentiation beside a preserved angiogenic ability3,4. Human derived pericytes (hPeri) were isolated from human muscle biopsies by enzymatic digestion and selected for low confluence plastic adhesion and alkaline phosphatase (AP) expression5, showing colony forming capability and a remarkable spontaneous myogenic activity. hPeri were characterized in vitro for mesodermic differentiation and in vivo by intramuscular injection, revealing their astonishing myogenic capability. Moreover, for skeletal muscle tissue engineering purpose, it was evaluated the combination of hPeri and PEG-Fibrinogen (PF) based biomimetic scaffold6 for building a human derived artificial muscle in vivo upon subcutaneous implantation. The results obtained showed that PF based matrix encapsulating hPery promoted the generation of an engineered human derived muscle, moreover presenting an important vascularization upon hPeri angiogenic action. Hence the perivascular compartment with hPeri can be considered a remarkable reliable source for myogenic stem/progenitor cells., Sarcopenia is defined as the age-related loss of muscle mass, strength and function, associated to regenerative difficulties by satellite cells (SCs), adult muscle stem cells. The oxidative stress and mitochondrial dysfunction are closely linked. The aim of this study was to investigate the metabolic profile of young and aged mitochondria in SCs isolated from human Vastus Lateralis skeletal muscles of young (20-35 y) and elderly (65-80 y) subjects, correlating with ROS production. The mitochondrial superoxide anion (O2•-) production on myoblasts and myotubes was assessed with Mitosox Red Probe. It was performed in the presence of anti-oxidant N-acetylcysteine (NAC) or glucose and sodium pyruvate (burst). Young myoblasts with NAC and burst presented a significant reduction of O2•- levels. In elderly myoblasts, both treatments did not give effects while elderly myotubes increased O2•- levels. The Seahorse Analyzer measures the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR). The basal and the maximal OCR in elderly myoblasts were lower respect to the young ones independently from NAC. In presence of NAC elderly myotubes were affected. The spare respiratory capacity and the ATP-linked OCR were impaired only in elderly myoblasts independently from NAC. The glycolytic parameters were higher in young myoblasts compared to elderly ones while an opposite situation observed in myotubes. In conclusion we demonstrated that elderly muscle cells are unable to handle high glucose concentrations that is associated to impaired mitochondrial function resulting in high ROS levels., A major challenge in the muscle field is to understand how growth arrest is coordinated in satellite cells (i.e. muscle stem cells) during muscle homeostasis/maintenance and repair. Skeletal muscle shows a remarkable capacity to regenerate after severe injuries, which is attributed to its satellite cell population. Once muscle growth is completed at early postnatal life, this stem cell population enters into a non-cycling, quiescent state. However, in response to specific needs, such as injury, it is rapidly activated to provide differentiated progeny for muscle repair as well as to self-renew the quiescent pool. We have designed a protocol to isolate the satellite cells following direct fixation and defined molecularly the early activation following exit from quiescence. In addition, muscle differentiation is a coordinated process of tissue-specific gene expression and irreversible cell cycle exit. We have analysed the mechanism of growth arrest during terminal differentiation, including the role of Cyclin Dependent Kinase Inhibitors p21 and p57. We will present data regarding p57 and p21 expression and function ex vivo and in vivo, in adult myofiber culture and regeneration models., Skeletal muscle is of high clinical interest since many congenital or acquired conditions can affect its function, leading to irreversible loss of tissue – volumetric muscle loss, VML. Tissue engineering aims to mimic neo-organogenesis for producing tissues to be applied in regenerative medicine, and holds great potential for the treatment of incurable skeletal muscle pathologies. Recent translational technologies are based on the use of natural or synthetic scaffolds that ideally can be implanted in patients and aid functional maturation of skeletal muscle in vivo. In order to investigate the real application of such scaffolds in muscle regenerative medicine, VML mouse models were generated for extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. Decellularised rat EDL were used to rescue the VML of 90% EDL ablation, while a synthetic photo-crosslinkable biomaterial was used for repairing 30% TA ablation. Scaffolds materials were able to be repopulated by recipient cells and supported myogenesis and functional muscle formation. The preservation of native ECM components and 3D organization, strongly improved the ability of the scaffold to support myogenesis., Muscle regeneration is a complex, multi-staged process in which macrophages play a fundamental role not only as scavengers of damage tissue but also as modulators of both myogenic and non-myogenic precursors. To study the effects of macrophage-released factors in this context we used the murine macrophage cell line J774 to obtain highly active conditioned medium (mMCM) upon exposure to LPS. We have previously shown that mMCM can enhance the proliferation and differentiation of rat primary myoblasts as well as of normal and dystrophic human myoblasts. We now show that mMCM exert different effects on murine myogenic cells, depending on their physiological state. Specifically, macrophagic factors turned out to have an anti-apoptotic action on freshly isolated satellite cells and a pro-proliferative effect on established cultures of satellite-derived myoblasts. When used to challenge muscle-derived murine fibro-adipogenic precursors, mMCM did not appear to modify their proliferation rate but it showed a potent anti-adipogenic effect, which did not appear to involve the triggering of apoptosis. Interestingly, the effect on FAP differentiation seems to be at least in part related to the exosomes found in mMCM. Lastly, we tested the effects of mMCM on human, muscle-derived pericytes, a population of multi potent precursor cells known to exhibit myogenic potential. Similarly to what we found in FAPs, mMCM did not affect the proliferation rate of cultured pericytes, but, somewhat surprisingly, it seemed to actually decrease their capability to differentiate towards skeletal muscle cells and form myotubes., Skeletal muscle regeneration requires coordinated remodeling of the extracellular matrix (ECM), and matrix metalloproteinases (MMP)s play a critical role. MMP-13, a collagenase, was shown to have high expression during regeneration and regulate C2C12 myoblast migration, yet MMP-13’s function in skeletal muscle in vivo has not been studied. Under baseline conditions the Mmp13-/- mouse does not exhibit a muscle phenotype; including muscle mechanics, fiber size, capillary density, or ECM area. However, in muscle injured with cardiotoxin, regeneration is delayed at 2 weeks post injury. To determine if these effects were mediated by satellite cell deficiencies when lacking MMP-13, live cell imaging of single fiber cultures were conducted to directly measure migration velocity. Mmp13-/- satellite cells had 33% lower migration velocity than controls. In addition, 3D invasion assays through basement membrane extract (BME) revealed that Mmp13-/- cells had a 53% reduced ability to migrate through BME substrate.. These data extend evidence of MMP-13 being a critical component of myoblast migration in primary cells in 2D and 3D. To further investigate the contribution that MMP-13 mediated migration had on the repair process, we generated a mouse with inducible and satellite cell specific deletion of Mmp13. These mice mirrored many of the phenotypes of the global knockout, including a reduction in satellite cell migration, and delayed resolution of damage by cardiotoxin injection. In sum, MMP-13 mediated satellite cell migration is an important factor for efficient muscle regeneration. Supported by NIH R01 AR057363., Histone Lysine Methyltransferases (KMTs) are epigenetic modifiers that dynamically control gene expression during stem cell differentiation. Among the different KMTs, EHMT2/G9a and EHMT1/GLP, responsible of mono- and di-methylation of Lysine 9 of histone H3 (H3K9), are of particular relevance in the context of myogenesis since they have been shown to control the repression of muscle-specific genes in myogenic precursors and to prevent their premature differentiation. Modulation of their activity might therefore be exploited to promote the expression of muscle-specific genes and to enhance muscle differentiation in tissues whose myogenic capacity is compromised due to a pathological condition, as in the case of muscles affected by Duchenne Muscular Dystrophy (DMD). DMD is a severe X-linked neuromuscular degenerative disorder that leads to progressive muscle weakness associated with loss of muscle tissue and replacement with adipose and connective infiltrates, in coincidence with the final stages of disease. Despite recent progresses in genome editing approaches, the cure for DMD is still a big challenge and pharmacological therapies aimed to counteract the fibro-adipogenic degeneration and to promote the compensatory regeneration, typical of the early stages of disease, hold great promise to slow-down DMD progression. Here we provide evidence of the pro-regenerative effect of G9a/GLP specific inhibitors in vivo. Our results show that in vivo inhibition of G9a/GLP-mediated H3K9me2 improves skeletal muscle regeneration. This is caused by an accelerated myogenic capacity of muscle stem cells (MuSCs) and by an impaired adipogenic differentiation of fibro-adipogenic progenitors (FAPs), which rather unmasks a previously silent myogenic capacity. Our preliminary results provide proof of concept of the use of H3K9 KMTs specific inhibitors as potential pharmacological strategy to promote the regenerative response of diseased, dystrophic, muscles, while concomitantly blocking their fibro-adipogenic degeneration., Several therapies to treat Duchenne Muscular Dystrophy (DMD) are under development; unfortunately many lack efficacy in the heart. Despite the primary genetic defect being identical in skeletal and cardiac muscle, the symptoms and severity differ suggesting the involvement of secondary organ-specific pathways that are yet to be fully understood. We have developed an in vitro model of cardiomyocyte hypertrophy using cardiomyocytes isolated from mdx (mouse model of DMD) hearts. Additionally, a transcriptomic approach has been applied to this model, to help elucidate the secondary organ-specific pathways involved. Proteomic analysis of 25 week-old mdx hearts was carried out to identify changes in the early stage of the disease. Using this model, we have been able to utilise various therapeutic approaches to lessen the severity of the phenotype. This model could be a fast, efficient way to screen new and existing approaches for therapeutic efficacy. Restoration of truncated dystrophin protein to these cells via a viral vector rescues the hypertrophic phenotype in addition to the application of pharmaceutical compounds. RNA-Seq data from the hypertrophic cardiomyocytes implicated the involvement of pathways such as angiogenesis, fibrosis and calcium handling. Using data generated from transcriptomics, differentially regulated transcripts were targeted for investigation on a protein level. Data from proteomic analysis of mdx hearts revealed perturbations in the immune response and actin regulation. Identification of genes, proteins and pathways differentially regulated in the mdx heart provides novel insights into the cellular pathophysiology of this organ in DMD and allows the identification of potential therapeutic targets., In the adult muscle the process of regeneration is necessary to replace and rebuild mature fibers. Following a muscle injury, inflammatory cells and tissue resident fibroblasts are recruited to the site of injury where, among other functions, they contribute to the remodeling of the extra cellular matrix (ECM), a process known as reparative fibrosis. FAPs (fibro-adipogenic progenitors) are thought to be the main source of matrix producing cells in skeletal muscle. In conditions of chronic or unresolved muscle damage, aging, or muscular dystrophies, FAPs persist and cause excessive scar tissue and/or ectopic fat infiltration, ultimately responsible for the progressive decline of muscle function. Here we identify a novel mechanism by which the Polycomb Ezh2 chromatin modifier modulates the number and proliferation of FAPs within skeletal muscle through the repression of several pro-fibrotic cytokines secreted into the extracellular space. We found that specific deletion of Ezh2 in satellite cells results in the accumulation and hyperproliferation of FAPs in adult and aged muscles. Transcriptome studies showed that component of the TGFb, Wnt, TNF-alpha and PDGF signaling are expressed at higher level in Ezh2 knock out satellite cells, escaping Ezh2 mediated repression and are also found up-regulated in aged satellite cells. Inhibition of these signaling is sufficient to block FAPs proliferation. Importantly treatment of aged FAPs with supernatant isolated from young wild type satellite cells rescue the aged FAPs phenotype, providign further evidence that satellite cells modulate the production of fibrogenic cells in a paracrine fashion. Altogether these results show that epigenetic changes driven by Ezh2 in satellite cells result in functional changes in the muscle niche and point to a role for Ezh2 in regulating persistent fibrosis during aging. Modulation of satellite cells epigenetics and specifically Ezh2 acitivity may therefore offer an additional therapeutic route for the treatment of chronic fibrosis., Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, caused by a (CTG)n expansion within the 3’ untranslated region of the DMPK gene and characterized by progressive myopathy, myotonia and multiorgan involvement. Expression of the mutated gene results in production of toxic transcripts that aggregate as nuclear foci where RNA-binding proteins are sequestered, resulting in mis-splicing of several transcripts, defective translation and microRNA dysregulation. No effective therapy is yet available for treatment of the disease. In this study we exploited the CRISPR/Cas9 gene editing strategy to remove the pathogenetic repeat expansions for future therapeutic use. To this aim we generated DM1 myogenic cell models from DM1 patient-derived fibroblasts, exhibiting typical disease-associated alterations. We will show that by using the CRISPR/Cas9 and NHEJ gene-editing system CTG expansions can be removed permanently from DMPK gene, resulting in phenotypic reversion of edited cells., Loss of muscle innervation occurs in several pathological conditions and mainly depends on the imbalance between protein synthesis and degradation (1,2,3). The ubiquitin proteasome system (UPS) and autophagy are the two main proteolytic pathways in skeletal muscle, which are activated upon denervation and contribute to the maintenance of muscle homeostasis (4,5). Histone deacetylase 4 (HDAC4) is an epigenetic factor that mediates skeletal muscle response following denervation. Under this condition, HDAC4 mediates skeletal muscle atrophy (6,7), suggesting the use of HDAC4 inhibitors as pharmaceutical treatment for neurogenic muscle atrophy. Accordingly, mice lacking HDAC4 in skeletal muscle are resistant to neurogenic muscle atrophy. However, the effects of HDAC4 inhibition in a condition of long-term denervation have not been clarified yet. To investigate the role of HDAC4 in skeletal muscle following long-term denervation with a genetic approach, we analyzed mutant mice with a tissue-specific deletion of HDAC4 (HDAC4mKO mice). Strikingly, while HDAC4mKO muscles were resistant to neurogenic muscle atrophy two weeks following denervation, muscles degenerated following 4 weeks of denervation. We demonstrate that, upon denervation, HDAC4 mediates both the UPS and the autophagic flux, which resulted crucial for maintaining muscle homeostasis. Indeed, by triggering either one of these two catabolic pathways in HDAC4mKO mice, muscle degeneration was significantly reduced 4 weeks after denervation. These findings reveale that HDAC4 mediates the activation of the catabolic pathways in skeletal muscle and that inhibition of HDAC4, in a condition of long-term denervation, leads to skeletal muscle degeneration., Facioscapulohumeral muscular dystrophy (FSHD) is caused by a contraction of the D4Z4 macrosatellite repeat on the subtelomere of chromosome 4. This genetic lesion results in epigenetic derepression and aberrant transcription of the pathogenic Double Homeobox Domain-Containing Protein 4 (DUX4) gene. Understanding the pathogenesis of FSHD and development of a DUX4-silencing therapy will require characterization of the molecular players that direct transcriptional activation of the D4Z4 locus. We developed one of the largest banks of disease-affected human embryonic stem cells (hESCs), including FSHD-affected cell lines. In addition, we developed and published a fast, robust and highly efficient differentiation method for skeletal muscle that does not rely on the overexpression of myogenic transcription factors or cell sorting to enrich myogenic populations (Caron et al. 2016). Together this technology provides a valuable platform to study development and disease states in FSHD with relevant human pathologies that cannot be recapitulated in animal models. It also provides an unlimited, uniform resource to develop cell-based primary and secondary phenotypic assays for drug discovery. Here, we discuss our recent findings on DUX4 expression in stem cell-derived myotubes, its epigenetic regulation and secondary FSHD phenotypes observed in these cells in the context of our overall aim to develop a small molecule drug to treat FSHD patients., Sarcoglycanopathy is the collective name of four rare genetic diseases caused by defects in the genes coding for alpha-, beta-, gamma- and delta-sarcoglycan (SG), which form a key structural complex that assures sarcolemma stability during muscle contraction. Most of the reported cases of sarcoglycanopathy are due to missense mutations. The resulting folding-defective SG is degraded by the quality control of the cell, leading to the secondary deficiency of the wild type partners. Many missense mutants retain their function as the entire SG-complex can be properly rescued by skipping the degradation of the defective protein. Presently, major effort is devoted to the development of novel therapeutic approaches based on the use of small-molecules either inhibiting the degradation or helping the folding process of SG mutants. Indeed, the application of this strategy allowed the recovery of the SG-complex in both cell models and, notably, primary myotubes from a patient with sarcoglycanopathy. To confirm in vivo efficacy and tolerability of this successful strategy, we are now generating novel sarcoglycanopathy models in zebrafish, which will carry a mutated SG, recoverable by drug treatment. We are focused on zebrafish because it is an excellent vertebrate model for muscular disorders, drug screening, and it is relatively easy to introduce any desired mutation by genome-editing technologies. Here we report data showing knock-down of delta-SG in zebrafish, that leads to severe muscular abnormalities, well mimicking the human disease, and first results concerning the generation of beta-SG and delta-SG knock-in and knock-out zebrafish lines by the CRISPR/Cas9 technique., Muscular dystrophies (MD) are genetic diseases, all characterized by a progressive muscle wasting in time, leading patients to wheelchair and premature death caused by respiratory and cardiac failure. Nowadays there is still no definitive cure for these pathologies. Only chronic treatment of corticosteroids is widely used to slow down the symptoms, but with several sides effects for the patients. It is also known the myofibers affected by MD are more susceptible to oxidative stress than healthy fibers due to a lacking of antioxidant signal. In this study we investigate on the effects of a diet enriched with a natural antioxidant (Cyanidin, red diet RD) on the dystrophic mouse model Sgca null. We observed a delay on the typical markers of the MD onset, as more homogeneous CSA distribution, reduction of fibrosis and also a rescue in the muscle performance. We also addressed the molecular pathways triggered by RD, identifying an exclusion of the transcription factor NF-kB from the myonuclei as an anti-inflammatory pathway. On the other side we found that RD promotes Nrf-2 translocation into the nuclei to trigger an antioxidant response through AMPK activity., Gene transcription is strongly influenced by various posttranslational modifications of the N-terminal tails of histones. The aim of our study is to understand which role histone modifications play during exercise-induced chromatin remodelling in skeletal muscle. In our analyses, we found a rapid and significant increase in phosphorylation of Histone 3 on Serine 10 after eccentric contractions. Moreover, we have found that this histone phosphorylation is followed by an increase in the acetylation of the nearby lysine 14, suggestive of an activation of gene transcription. We identified p38-MSK1, whose activity is strongly up regulated during exercise in muscles, as the kinase involved in this phosphorylation. We are now investigating the upstream signals responsible for p38-MKS1 activation and the histone modifications observed. In addition to our results in mice, we confirmed our results in human biopsies taken immediately after high intensity exercise where we have observed similar changes in signaling and histone phosphorylation. In our model of exercise, the activation of the early genes FOS, JUN and EGR1 have been confirmed by CHIP analyses on the phosphorylated histone. We are now exploring the expression of immediate early genes in MSK1/2 ko mice to understand the importance of histone phosphorylation in the early gene response to exercise., Fibro adipogenic progenitors (FAPs) play a leading role in muscle regeneration by positively regulating satellite cells differentiation. However, in pathological conditions, they are responsible for fibrosis and fat infiltration. Despite the established importance of FAPs in both regeneration and degeneration, the signals that regulate these opposing roles are not fully characterized. Our results support a model whereby Notch plays an important role in the regulation of FAP differentiation. In addition, when co-cultured ex vivo, myotubes inhibits the adipogenic differentiation of FAPs in a Notch dependent way. Interestingly, this control mechanism is impaired in FAPs isolated from young dystrophin-deficient (mdx) mice or in FAPs from ageing wild type mice. To further investigate these phenotypic differences, we performed deep proteomics profiling and multiparametric analyses of mdx and wild type FAPs, by exploiting single cell mass cytometry and mass spectrometry based proteomics. Both analyses reveal clear differences in the phenotypes of the mdx and wild type cell populations and in their proteome profiles. Significantly we observed a striking perturbation of the concentration level of metabolic enzymes where a significant decrease of enzymes of the TCA cycle and fatty acid oxidation pathway was balanced by an increase of the enzymes of the glycolytic pathway. In addition an increase in proteins involved in exosome formation or in response to activation of tol-like receptors was also observed., Mutations in the LMNA gene encoding the nuclear lamins A/C cause a variety of diseases, including lamin-related congenital muscle dystrophy (L-CMD). The underlying mechanisms by which LMNA mutations cause tissue specific disease remain elusive. Given the apparent contribution of lamins A/C to cellular mechanotransduction, we hypothesized that defective mechanical properties of LMNA mutated muscle contribute to the dystrophic phenotype in L-CMD.To assess muscle plasticity, plantaris muscle (PLN) of WT and LMNA+/ΔK32 mice were mechanically overloaded by synergy ablation of the gastrocnemius and soleus muscles. PLN muscles were analyzed 1 or 4 weeks after mechanical overload. In addition, human derived myoblasts carrying the same mutation in the lamin AC gene were differentiated into myotubes and subjected to cyclic stretch. In response to 1 and 4 weeks of mechanical overload, WT PLN mass/body mass increased 1.9 and 2.3 fold respectively compared to control WT, but LMNA+/ΔK32 mice failed to produce a comparable hypertrophic response (1.1 and 1.6 fold increase respectively compared to WT controls). Paradoxically, anabolic signals (Akt/P70S6K) were similarly activated between mouse strains, however the LMNA+/ΔK32 mice displayed a significant up regulation of both total and de-phosphorylated FoxO3a and its downstream gene targets (Murf1, MAFbx, Musa1, Fbxo31, SMART). The average number of Pax7+ cells per cross-section and Pax7 protein expression were significantly lower in LMNA+/ΔK32 PLN compared to WT. In addition, we observed a high rate of fragmentation of the neuromuscular junction (NMJ) following overload whereas WT PLN were characterized by NMJ hypertrophy. In vitro data showed that myotube morphology was markedly different between cell types with increased myotube size, nuclei per myotube and cytoskeletal disorganization observed in LMNA+/ΔK32 vs WT myotubes. Acute mechanoresponsiveness of LMNA+/ΔK32 myotubes was aberrant in vitro as determined by abnormal localization and activity following stretch of the mechanosensitive proteins YAP and MKL1 accompanied by a lack of cytoskeleton reorganization in LMNA+/ΔK32 myotubes. In conclusion, our results show that the muscle ability to sense and respond to mechanical overload is severely disturbed in L-CMD. These data highlight the importance of the nuclear envelope in mechanosensing, shedding light on the intrinsic force sensing mechanisms in skeletal muscle., Met Activating Genetically Improved Chimeric Factor 1 (Magic-F1) is a human recombinant protein, derived from dimerization of the receptor-binding domain of hepatocyte growth factor (HGF). Previous experiments demonstrate that in hemizigous transgenic mice, the skeletal muscle specific expression of Magic-F1 can induce a constitutive muscular hypertrophy, improving running performance and accelerating muscle regeneration after injury [1]. Furthermore, the microarray analysis of Magic-F1+/+ satellite cells evidenced transcriptomic changes in genes involved in the control of muscle growth, development and vascularisation [2]. In this study we demonstrate that Magic-F1 mice show an alteration of the heart morphology. Data obtained by morphometric analysis and three-dimensional reconstruction of the hearth revealed that circulating Magic-F1 proteins are able to induce a dilatation of the left ventricle chamber of transgenic mice. Interestingly, we found in Magic-F1 hearts an alteration of Phd2 and HIF1 protein levels. These two oxygen sensors are found dysregulated in cardiac ischaemic conditions, where generalised hypoxia causes functional impairments in cardiomyocytes and structural tissue damage [3-4]. These preliminary results support the involvement of oxygen sensors in Magic-F1-induced cardiac hypertrophy. In addition, Magic-F1+/+ mice can be used as non-pressure overload model to further investigate the role of oxygen-sensors in ischaemic heart disease., Skeletal muscle is a dynamic organ, characterized by an incredible ability to rapidly increase its rate of energy consumption to sustain activity. Muscle mitochondria provide most of the ATP required for contraction via oxidative phosphorylation. Here, we found that skeletal muscle mitochondria express a unique MCU complex containing an alternative splice isoform of MICU1, MICU1.1, characterized by the addition of a micro-exon that is sufficient to greatly modify the properties of the MCU. Indeed, MICU1.1 binds Ca2+ one order magnitude more efficiently than MICU1 and, when heterodimerized with MICU2, activates MCU current at lower Ca2+ concentrations than MICU1-MICU2 heterodimers. In skeletal muscle in vivo, MICU1.1 is required for sustained mitochondrial Ca2+ uptake and ATP production. These results highlight a novel mechanism of the molecular plasticity of the MCU Ca2+ uptake machinery that allows skeletal muscle mitochondria to be highly responsive to sarcoplasmic [Ca2+] responses., Pluripotent stem cells (PSCs) have a high proliferative capacity and can give rise to all three embryonic germ layers. Due to these characteristics, PSCs have been extensively researched in the field of muscular dystrophies as a large number of cells is required to restore the entire muscle pool. Recently, our group has successfully isolated induced PSC–derived mesodermal progenitors (MiPs) that can regenerate both cardiac and skeletal muscle1. Nevertheless, improvements need to be made to work towards a clinical relevant protocol that has an efficacy high enough to allow functional improvements. First of all, MiPs are obtained through the formation of embryoid bodies using serum-containing media thereby limiting their clinical translatability. Secondly, although MiPs showed to contribute to both cardiac and skeletal muscle, functional improvement was only moderate. For this study, a serum-free monolayer approach, adapted from Shelton et al. (2014), was used to derive mesodermal progenitors from induced PSCs in a clinical relevant manner2. These progenitors were tested in vitro for their potential to contribute to the myogenic lineages. Next, in order to further enhance their myogenic capacity and later engraftment, stimulation of the Dll1/Notch1 signaling pathway is being explored as this has been shown to promote the myogenic commitment of adult stem cells3. With the purpose of cell therapy in mind, two small molecules, namely valproic acid and resveratrol, are being investigated for their potential to improve the myogenic capacity of the obtained mesodermal progenitors and whether this effect can be attributed to the stimulation of the Dll1/Notch1 pathway., Sarcoglycanopathy is caused by mutations in sarcoglycans (SG), four glycoproteins that form an essential complex for the integrity of muscle cells. Most sarcoglycan mutations are missense mutations generating a folding defective protein, which is prematurely degraded by the cell’s quality control. We recently unveiled the pathway responsible for the disposal of defective alpha-SG and demonstrated that many missense mutants retain their function. Indeed, the complex can be properly rescued by targeting the degradative pathway. Thanks to these knowledges, we design a novel therapeutic approach for sarcoglycanopathy based on the use of small molecules able to improve the folding process of defective sarcoglycans. Once structurally stabilized, they can skip disposal and traffic at the proper site of action. To this intent, we tested compounds of the library of the CFTR correctors and several of them were effective in rescuing SG mutants in cell models and, notably, in myotubes from a patient with alpha-sarcoglycanopathy. Although we need to clarify the mechanism by which CFTR correctors exert their activity in sarcoglycanopathy, these data represent the proof of principle of a novel pharmacological strategy for this, still incurable, disease. Now we are focused on the development of unconventional animal models for in vivo studies, essential to confirm efficacy as well as safety and tolerability of these compounds. At present, we are characterizing at the histological and molecular level, mouse models obtained by the AAV-mediated gene delivery of mutated versions of the human sequence in the null alpha-SG background., Dental pulp pluripotent-like stem cells (DPPSC) isolated from human third molar pulp express embryonic stem cell markers 1 and show pluripotent-like behavior,2 making DPPSC an appealing tool for tissue repair or maintenance. In this study, DPPSC obtained from young patients were characterized and their secretome was analyzed. We then evaluated DPPSC differentiation potential towards endothelial, smooth and skeletal muscle lineages. The in vivo contribution of DPPSC was tested in a wound-healing mouse model and in two immuno-deficient mice resembling Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2E (Scid/mdx and Sgcb-null Rag2-null γc-null [3], respectively). Our results showed that DPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and that DPPSC treatment improved vascularization in all mouse models. In dystrophic mice, DPPSC integrated in muscular fibres and vessels, and induced larger cross-sectional area of type II fast-glycolytic fibres. In addition, DPPSC treatments resulted in reduced fibrosis and collagen content and changes in macrophage polarization. This is likely due to the observed cytokine profile modification, with higher levels of interleukin-10 in DPPSC-injected muscles compared to controls. Overall, DPPSC represent a source of stem cells with the potential to enhance wound healing and slow down dystrophic muscle degeneration., Disruption of the development and maintenance of the neuromuscular junction (NMJ) through mutations in the genes encoding its components lead to congenital myasthenic syndromes (CMS). These disorders result in impaired neurotransmission and muscle weakness; current treatments for these conditions are limited. Development and maintenance of the NMJ depends on the neuronal factor agrin and its downstream pathway. Neurotune AG has developed a modified form of agrin (NT-1654) which has been show to stimulate the NMJ development pathway. The aim of this project is to test NT-1654 in mouse models of human CMS, and provide evidence for its use as a therapeutic compound in humans. We have conducted dose finding studies to identify safe, non-toxic concentrations of the drug in three animal models of CMS: DOK-7(c.1124_1127dupTGCC), COLQ(-/-), and Agrin (nmf380), and one of hereditary neuropathy: GARS(C201R). Daily subcutaneous injections of either 1mg/kg, 5mg/kg, 10mg/kg, or PBS, were administered and animals monitored for signs of adverse reactions and toxic effects. So far all animals have all tolerated the drug well, with no adverse changes being noticed. Full studies administering NT1654 have now been initiated in two of the CMS models with further models expected to begin soon. Preliminary data regarding body weight, grip strength, swallowing ability, and survival will be presented. It is hoped that these investigations will provide the rationale for beginning human trials of NT1654 in patients., α-Dystroglycanopathies are a group of muscular dystrophies characterized by α-DG hypoglycosylation and reduced extracellular ligand-binding affinity. Among other genes involved in the α-DG glycosylation process, fukutin related protein (FKRP) gene mutations generate a wide range of pathologies from mild limb girdle muscular dystrophy 2I (LGMD2I), severe congenital muscular dystrophy 1C (MDC1C), to Walker-Warburg Syndrome and Muscle-Eye-Brain disease. FKRP gene encodes for a glycosyltransferase that in vivo transfers a ribitol phosphate group from a CDP –ribitol present in muscles to α-DG, while in vitro it can be secreted as monomer of 60kDa. Consistently, new evidences reported glycosyltransferases in the blood, freely circulating or wrapped within vesicles. Although the physiological function of blood stream glycosyltransferases remains unclear, they are likely released from blood borne or distant cells. Thus, we hypothesized that freely or wrapped FKRP might circulate as an extracellular glycosyltransferase, able to exert a “glycan remodelling” process, even at distal compartments. Interestingly, we firstly demonstrated a successful transduction of MDC1C blood-derived CD133+ cells and FKRP L276IKI mouse derived satellite cells by a lentiviral vector expressing the wild-type of human FKRP gene. Moreover, we showed that LV-FKRP cells were driven to release exosomes carrying FKRP. Similarly, we observed the presence of FKRP positive exosomes in the plasma of FKRP L276IKI mice intramuscularly injected with engineered satellite cells. The distribution of FKRP protein boosted by exosomes determined its restoration within muscle tissues, an overall recovery of α-DG glycosylation and improved muscle strength, suggesting a systemic supply of FKRP protein acting as glycosyltransferase., Ferroptosis is a recently discovered form of cell death causally linked to the ability of iron to induce oxidative damage by peroxidation of polyunsaturated fatty acids (PUFAs). Misregulated ferroptosis has been implicated in a number of pathological processes and there is a growing interest in the pre-clinical use of ferroptosis inducers against tumors. Cells to prevent ferroptosis mostly engage in the activity of glutathione peroxidase 4 (GPx4), a selenoenzyme that uses glutathione for neutralizing lipid hydroperoxides. Two major ferroptosis inducers mediating GPx4 inhibition have been identified, namely Erastin (eradicator of RAS and ST-expressing cells) and RSL3 (RAS selective Lethal 3). In this work we have investigated their effect on mouse skeletal C2C12 myoblasts and cell lines of rhabdomyosarcoma (RMS), the most frequent soft-tissue tumor affecting children and adolescents. As evaluated by using specific fluorescent probes, treatment with Erastin or RSL3 agents resulted in a marked production of both cytoplasmic/mitochondrial ROS and lipid ROS, which correlated in a dose-dependent manner with a decreased cell viability, as evaluated by means of Neutral Red assays after 48 hours. In Erastin-treated cell lines ferroptosis was enhanced in the presence of iron supplementation (through ferric ammonium citrate), while it was prevented by pre-treatment with agents sequestering iron (bathophenanthrolinedisulfonic acid), antioxidant scavengers (glutathione and N-acetylcysteine) and lipid ROS scavengers (ferrostatin-1). We observed Erastin to be more effective to promote ferroptosis in the cell lines showing a higher proliferation rate. Indeed, inhibition of ERK signaling, as observed during differentiation or upon pharmacological treatment with PD090859 agent, prevented ferroptosis in Erastin-treated human RMS embryonal RD and C2C12 cell lines. Furthermore, we found Erastin and RSL3 to be more effective in inducing ferroptosis in RD subclones characterized by higher ERK1/2 phosphorylation and proliferation rate. Taken together, our data suggest that iron metabolism could play a key role in the cell fate of muscle cells; in addition, the use of ferroptotic inducers could offer a novel alternative to improve the efficacy of conventional antineoplastic cocktails utilized against RMS., Muscle wasting occurs during various chronic diseases and precedes death in humans as in mice. Assessing the degree of muscle atrophy in diseased mouse models is often overlooked since it requires the sacrifice of the animals for muscle examination or expensive instrumentation and highly qualified personnel, such as Magnetic Resonance Imaging (MRI). We developed a non-invasive procedure based on micro-computed tomography (micro-CT) without contrast agents to monitor hind limb muscle wasting in mouse models of neuromuscular disease and cancer cachexia: the transgenic SOD1G93A mouse and the colon adenocarcinoma C26-bearing mouse, respectively. We established the scanning procedure and the parameters to consider in the reconstructed images to calculate the Index of Muscle Mass (IMM). We performed longitudinally micro-CT scan of hind limbs in SOD1G93A mice at presymptomatic and symptomatic stages of the disease and calculated the IMM. We found that IMM in SOD1G93A mice was lower than age-matched controls even before symptom onset. We also detected a further decrease in IMM as disease progresses, most markedly just before disease onset. We performed the same analyses in the C26-based mouse model, losing progressively body and muscle mass because of cachexia. Interestingly, we found a strong correlation between IMM and Tibialis Anterior and Gastrocnemius muscle weights in both disease models. We developed a fast, easy-to-conduct and cost-effective imaging procedure to monitor hind limb muscle mass useful in preclinical therapeutic trials but also in proof-of-principle studies to identify the onset of muscle wasting. This procedure could be widely applied to other disease models characterized by muscle wasting, to assist drug development and search for early biomarkers of muscle atrophy. #Equally contributing authors., Physical activity extends life span of patients affected by certain types of cancer, also by contrasting the associated muscle wasting (i.e. cachexia). The most effective type of physical activity against muscle wasting during cancer seems to be aerobic exercise. So, we asked whether it promotes secretion of proteins by muscles (i.e. myokines) that may contrast cancer cachexia. To mimic aerobic exercise, we infected C2C12 myotubes with PGC1α-expressing adenoviruses, because PGC1α is the main transcriptional coactivator involved in muscle adaptation during aerobic exercise. Our microarray analysis showed musclin as a PGC1α-induced myokine. We further immunoprecipitated it only from supernatants of PGC1α expressing-myotubes. By Q-PCR, we found musclin expression unchanged in myotubes hypertrophying because of activated AKT (to mimic anaerobic exercise). Among other PGC1α-induced myokines, we found only musclin strongly downregulated in cachectic muscles and plasma of C26-bearing mice even at times when their body weights were not lost yet. Thus, we electroporated Tibialis Anterior (TA) of C26-bearing mice with musclin-encoding plasmids and found musclin to preserve fiber area. Dexamethazone-treated myotubes or FoxO3-expressing myotubes undergo atrophy as measured by increased rates of proteolysis and MuRF1 induction. Unlike GFP, musclin was able to contrast the dexamethazone induced-MuRF1 expression in Luciferase assays. Notably, musclin-containing supernatants of PGC1α expressing-myotubes restrained the FoxO3-induced rates of long-lived protein degradation. Musclin is a myokine induced specifically by PGC1α, typically increased upon aerobic exercise and musclin overexpression is beneficial against muscle wasting during C26 growth or in atrophying myotubes. Overall, musclin would be a good drug option for cancer patients that cannot exercise and are at risk of developing cachexia., Muscle wasting is responsible for severe debilitating weakness and it is well known as sarcopenia when it occurs in aged people causing loss of muscle mass, strength and function [1]. Recent literature revealed that human satellite cells are impaired in elderly subjects [2], however, how interstitial muscle cells affect tissue regeneration and fatty deposition upon ageing has not yet been elucidated. Firstly, we isolated the non-satellite cell fraction from human muscle biopsies of young and elderly subjects, referred here as CD56- interstitial cells. The elderly CD56- interstitial cells showed a larger number of CD15+ and PDGFRα+ cells in comparison with young interstitial cells. Moreover, CD56-/ALP+ cells were the most abundant plastic cell population able to differentiate into smooth muscle cells and adipocytes, and the unique interstitial population with myogenic differentiation potential. They expressed pericyte markers including NG2, αSMA and PDGFR-β and were referred as mesoangioblasts (MABs), vessel-associated stem cells. Interestingly, elderly MABs displayed a dramatic impairment in the myogenic differentiation ability in vitro and after transplantation in immunodeficient dystrophic mice (Sgcb-null Rag2-null γc-null mice). In addition, elderly MABs proliferated less, but yet retained other multilineage capabilities. Taken together, our results suggest that the CD56- cell fraction is modulated in skeletal muscle during ageing, it is possibly involved in the fibroadipogenic deposition and could represent a feasible target for future treatments aimed to reduce muscle loss and wasting., The dystrophin-associated glycoprotein complex (DGC) serves as a mechanical link between the cytoskeleton and the extracellular matrix of muscle cells. Disassembly of this protein complex renders the sarcolemma vulnerable to contraction-induced injury, leading to progressive fiber damage, membrane leakage and cell death. DGC interacts with Filamentous-actin (F-Actin) fine regulated by Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing disaggregation and preventing repolymerization. MICAL2 has been shown to transduce semaphorin/plexin external signaling into cytoskeletal modifications [1]. Interestingly, in a genome profiling study, MICAL2 has been found among a set of ten functionally linked genes involved in muscle degenerations of mdx mice [2]. In this study, we focus on the role of MICAL2 in skeletal, cardiac and smooth muscle differentiation. In particular, we found that MICAL2 increases during myogenic differentiation of C2C12 cells and primary satellite cells. Intriguingly, perturbation studies of MICAL2 levels impair myogenic differentiation, both in C2C12 and satellite cells. Murine mesoangioblasts, vessel associated stem cells, also express high levels of MICAL2 when differentiated into smooth muscle cells. Finally, murine hearts express MICAL2 protein both in embryonal and adult conditions, as well as human induced pluripotent stem cells during differentiation into cardiomyocytes. Taken together, these data demonstrate that modulations of MICAL2 have an impact on muscle differentiations. Further experiments are necessary to understand whether the absence of MICAL2 affects smooth and cardiac muscle differentiation. Moreover, gain-of-function experiments might shed light on the role of MICAL2 in myogenic commitments., Cachexia is a common complication of cancer characterized by several metabolic alterations and a massive skeletal mass loss occurring in up to 60% of cancer patients. In addition to increasing morbidity and mortality, aggravating the side effects of chemotherapy, and reducing quality of life, cachexia is considered the direct cause of death of a large proportion of cancer patients. However, no efficacious treatment exists today. 25(OH) vitamin D3 (25OHD) blood levels have been correlated with the incidence and evolution of some cancers, suggesting that vitamin D (VD) can play a role in improving patients’ prognosis. VD has been also shown to be important in the maintenance of muscle homeostasis and functionality. This led to the hypothesis of using VD as anti-cachectic treatment, but the direct effect of VD supplementation on muscle and its mechanism are still not clear. Here we show that 25OHD protects C2C12 myotubes from cytokine-induced atrophy by activating Akt-FOXO3 signaling, and that the intracellular conversion of 25OHD to 1,25(OH)2 vitamin D3 is crucial for its anti-atrophic function. Moreover, 25OHD treatment promotes hypertrophy. Altogether, our data indicate that 25OHD has a protective effect on skeletal muscle cells in vitro. Thus, 25OHD could be a possible candidate for further studies in cancer-cachexia treatment., Cachexia is a metabolic syndrome characterized by an involuntary loss of skeletal muscle and adipose tissue that leads to progressive functional impairment. This syndrome affects about 50-80% of cancer patients and there are no standard therapy regimens for its management. Acylated and unacylated ghrelin (AG and UnAG) are circulating peptide hormones generated mainly in the stomach due to fasting or caloric restriction. AG, through binding to GHSR-1a, induces strong GH release, stimulates food intake, adiposity, and positive energy balance. Acylation of ghrelin is essential for its binding to GHSR-1a, since UnAG does not activate this receptor. In both patients and animal models, AG ameliorates cachexia induced by several pathological conditions in a GHSR-dependent manner. However, we have shown that both AG and UnAG directly protect skeletal muscle from experimentally-induced atrophy, independently of GHSR-1a, thus providing the evidence for the existence of an alternative ghrelin receptor. Anamorelin (ANAM) is a selective agonist of GHSR-1a with appetite-enhancing and anabolic effects. Different clinical studies have highlighted its ability to improve the cachectic state in cancer patients by increasing total body mass. Nevertheless, the effects of ANAM on muscle tissues have not yet been completely elucidated. The data herein presented show that ANAM not only binds to GHSR-1a, but also recognizes the alternative receptor responsible for the anti-atrophic activity shown by ghrelin. Indeed, ANAM protects C2C12 myotubes from experimentally-induced atrophy and activates protein synthesis. These effects are GHSR-1a independent, since they are also visible in culture of primary myotubes obtained from GHSR-1a knock-out mice., Insulin-like growth factor-1 (IGF-1) is a growth factor with multiple roles in various aspects of normal and pathological growth and differentiation. The translation of the IGF-1 gene gives rise to an immature IGF-1 peptide, which has a signal peptide at the 5’ end of the gene, a core region and an Ea-peptide at the 3’ end. The signal peptide is removed after facilitating the passage of the polypeptide into the endoplasmic reticulum and give rise to the IGF-1 prohormone (proIGF-1Ea), retaining C-terminal Ea peptide. Recent studies demonstrated that intracellular IGF-1 is mainly expressed as prohormone, not mature IGF-1. Moreover, we recently demonstrated that the Ea peptide is an intrinsically disordered region (IDR) enriched in regulatory elements including a highly conserved N-glycoylation site. In this study we investigate the role of Ea peptide glycosylation on proIGF-1Ea stability and secretion. After transient transfection of Hek293 cells with IGF-1Ea transgene two IGF-1 prohormones were produced intracellularly: glycosylated (~17kDa) and non-glycosylated forms (~11-12kDa). Subsequently, we wondered whether glucose withdrawal or direct inhibition of N-glycosylation by tunicamycin (Tun) might interfere with IGF-1Ea production. To achieve this aim, we overexpressed proIGF-1Ea in Hek293 cells cultured in glucose depleted medium or treated with Tun. Notably, the band corresponding to glycosylated proIGF-1Ea completely disappeared in the absence of glucose or after treatment with Tun. Moreover, the analysis of culture media of IGF-1Ea-transfected Hek293 cells showed that the inhibition of glycosylation by glucose deprivation or Tun completely abrogated the glycosylated proIGF-1Ea secretion and markedly reduced the mature IGF-1 secretion. After that, using the protein synthesis inhibitor cycloheximide, we demonstrated that the turnover rate for non-glycosylated IGF-1Ea was faster than glycosylated IGF-1Ea. To test the involvement of 26S proteasome machinery, we subsequently treated IGF-1Ea-transfected Hek293 cells with proteasome inhibitor MG132 and we found an increase of non-glycosylated IGF-1Ea, while IGF-1Ea glycosylated was marginally affected by proteasome inhibitor. In conclusion, these results show that glycosylation of Ea peptide enhances the export efficiency of proIGF-1Ea and it is necessary for IGF-1 secretion. We hypothesize that proIGF-1Ea glycosylation ensures proper prohormone folding and secretion preventing its entry into the ER-associated degradation (ERAD) pathway., Duchenne and Becker muscular dystrophies (D/BMD) are caused by a variety of mutations in the dystrophin gene and collectively comprise the most prevalent congenital skeletal muscle disorders. Although a recent therapeutic treatment has been approved by the FDA, long term therapeutic benefits are pending and subpopulations of D/BMD patients do not benefit from it. Drug discovery in D/BMD, as well as other orphan disorders, is typically hampered by the lack of adequate disease models, clinically irrelevant assays or a combination of both. Herein, we present a human pluripotent stem cell (hPSC) derived two- and three-dimensional (2D and 3D) D/BMD disease modeling platform, amenable to use with hPSCs carrying any type of dystrophin mutations. Skeletal muscle differentiation is achieved using Genea Biocells' simple three media process as previously described by Caron et al. (Stem Cell Trans Med, 2016). For 2D modeling, monolayer hPSCs are plated onto collagen I-coated dishes and differentiated through three myogenic developmental stages (e. g. satellite-like cells, myoblasts and myotubes), generating cultures suitable for morphological and metabolic analyses and cell-based drug screening assays. For 3D modeling, similar to previously published work using primary human myoblasts (Madden et al., eLife, 2015), hPSC-derived myoblasts generated in the 2D system were encapsulated in hydrogel based myobundles to promote differentiation into myotubes using a 3D culture environment. The 3D myobundles are suitable for histology, as well as contractile force measurements, both critical clinical outcome measures. Collectively, these platforms provide a patient-specific disease-in-a-dish model with clinically-relevant assay endpoints for drug discovery programs. Moreover, this approach can be extended to model a variety of other skeletal muscle disorders., Sarcopenia is the progressive aging-related loss of skeletal muscle mass and function occurring during aging. It is the result of multiple factors, including changes in the metabolic state or in the neuromuscular system, inflammatory pathway activation, altered turnover of contractile proteins and organelles as well as altered production and tissue responsiveness of trophic factors. In particular changes in hormonal level, including decrease of insulin-like growth factor (IGF-1), contribute to sarcopenic condition. We demonstrated that the overexpression of IGF-1 in skeletal muscle was able to counter the decrease in muscle mass, CSA and strength, in 24 months age old mice. Moreover, overexpression of IGF-1 improved insulin sensitivity and glucose tolerance.., 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. Along this line, transgenic MCK-PGC-1α, which overexpress this transcription factor specifically in the skeletal muscle, are characterized by enhanced exercise performance in comparison with wild-type animals1; 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 ones2, we found a high number of fibers with centrally located nuclei, which is indicative of muscle regeneration. Moreover, myogenic stem cells are more abundant in transgenic mice compared to wild-type animals, and when isolated and cultured in differentiating medium, they form larger myotubes. Starting from this point, the aim of the study was to investigate if stem cells from MCK-PGC-1α mice can improve myogenesis. Muscles from male wild-type and MCK-PGC-1α mice were subjected to mild digestion and mononuclear cells were isolated by filtration. These cells were then transplanted into the tibialis anterior muscle of female wild-type mice, either injured (BaCl2 i.m. injection 8 hours before cell transplantation) or not. The animals were euthanized 12 days after BaCl2 injection. Hematoxylin/eosin staining of muscles transplanted with WT-derived cells shows improved regeneration. On the contrary, all the muscles injected with MCK-PGC-1α-derived cells show an increase of centrally located nuclei, altered myofiber cross-sectional area distribution and marked SDH staining. The expression of molecular markers of regeneration is consistent with the histological pattern. The results obtained in the present study suggest that cells isolated from MCK-PGC-1α donor mice are able to fuse with recipient muscle myofibers partially inducing a shift towards oxidative metabolism and affecting regeneration., Fast-twitch fibers are more affected during the muscle waste induced by cancer cachexia. The current literature correlates the proteolysis-related atrophy (which usually occurs in later stages of cachexia syndrome) to oxidative damage of proteins. In the present study, we investigated oxidative modifications, ultrastructural modifications in mitochondria and sarcoplasmic reticulum (SR), and the correlation with muscle loss in EDL and Soleus muscles (respectively fast- and slow-twitch muscles) in an early stage of cachexia (pre-cachexia). Male Wistar rats were subcutaneously inoculated with a suspension of Walker-256 tumor cells, and divided in 2 groups: tumor bearing rats (T), and tumor bearing rats treated with N-acetylcisteine (T-NAC; NAC 1% ad libitum in drinking water), a drug known to promote increased antioxidant capacity in tissues. A control group (C) without tumor implantation or NAC treatment was also added. After 5 days, pre-cachexia was characterized in T animals according to the criteria pre-established by Fearon et al. (2011; Lancet Oncol). Tumor implantation caused decrease in general body weight (-2.77%), followed by a significant weight loss in EDL muscle (-15.33%), contrasting with the small mass loss of soleus (-5.14%). We measured both protein and membrane oxidation and found that only membrane oxidation was significantly increased in EDL from tumor bearing mice. As a result, we found that the volume of intracellular membrane organelles such as SR and mitochondria was significantly decreased (-18.89% and -22.41%, respectively) compared to group C. Interestingly, all differences were significantly rescued in T-NAC group, suggesting a central role of oxidative stress in the modifications of membrane compartments. These data suggests that in pre-cachectic stages: 1) glycolytic muscles are already more prone to mass waste, but independently of protein oxidation; and 2) loss of membrane organelles could play an important role early in these initial stages. Supported by: a) Brazilian Ciências Sem Fronteiras: CNPq 233892/2014-1 and Brazilian Continuous Flow Program to support special projects (973/2013) to FAG; b) MIUR Future in Research: RBFR13A20K to SB., Caveolin-3 (Cav-3), muscle-restricted coiled coil MURC (also referred to as Cavin-4) and the recently identified large tripartite motif Trim72 are proteins controlling a number of important processes in skeletal muscle, ranging from myogenesis and insulin signaling up to sarcolemma repair. In this study we investigated their expression in rhabdomyosarcoma (RMS), a soft tissue tumor showing morphological and biochemical traits of the skeletal muscle lineage. Immunohistochemical analysis showed a similar pattern of expression of the three proteins in different human RMS samples. Immunoblotting analysis carried out using different RMS cell lines showed an increased expression of these proteins during myogenic differentiation as compared to proliferating cells. In addition, confocal microscopy analysis revealed that Cav-3, MURC and Trim72 all co-localize at the plasmalemma of differentiated embryonal RD cells. Ectopic expression of a point mutated Cav-3 P104L form in RD cells was sufficient to mislocalize both MURC and Trim72 into perinuclear Golgi-like compartments, leading to a cell phenotype characterized by increased ERK phosphorylation and severe impairment of myogenic differentiation. Overall, these data suggest that a concurrent expression of Cav-3, MURC and Trim72 configures as a specific signature predicting and regulating cell differentiation in RMS., It has been shown that mitochondrial dysfunction modulates intracellular pathways involved in skeletal muscle mass control and plays a major role in disuse atrophy. As aerobic exercise can promote an oxidative metabolic program, we hypothesized that exercise training before hindlimb suspension could prevent mitochondrial dysfunction and skeletal muscle atrophy. To test the latter hypothesis the effects of aerobic physical preconditioning on mechanisms involved in muscle mass maintenance and muscle mass were studied. Mice were divided into 4 groups: 7 days aerobic training before suspension (EX+HU); 7 days exercise only (EX); 3 days of hindlimb unloading (HU); control (CTRL). Mice were trained daily on treadmill for 7 days. After 3 days HU, gastrocnemius showed atrophy (CSA: CTRL 1557±26 vs HU 1300±134), ubiquitine proteasome system (Murf1 and atrogin1 increase), autophagy activation (LC3II/LC3I increase) and no change of synthetic pathway (mTOR) compared to control mice. Gastrocnemius of EX+HU mice did not show muscle mass loss (CSA: HU 1300±134 vs 7EX+HU 1479±5). Such prevention of muscle atrophy in EX+HU was associated with: (i) a significant enhancement of TFAM and NRF-1 mRNA levels, two transcription factors regulating mitochondrial biogenesis; (ii) a significant increase of mRNA levels of key factors involved in mitochondrial dynamic, namely profusion proteins Mfn2, OPA1 and profission proteins DRP1 and Fis1; (iii) an increased phosphorylation of mTOR compared to control and HU mice; (iv) a significant persistent activation of catabolic pathways. The data suggest that exercise training can prevent skeletal muscle atrophy due to acute periods of unloading by modulating mitochondrial dynamics., The muscle-tendon interface, called myotendinous junction (MTJ), is the key anatomical area through which the contractile strength can be transmitted between tissues. The MTJ is a dynamic interface and different physiological and pathological conditions may induce morphofunctional changes. Mechanical loading seems to have a key role in the MTJ plasticity. In fact, MTJ may reduce or increase its complexity and the contact surface between tissues, in relationship to muscle atrophy and exercise protocols, respectively. The molecular mechanisms and the different stages of these morphofunctional adaptations need to be further deepened. An interesting high presence of tenocytes near the MTJ has been revealed by means of transmission electron microscopy. These cells showed an increased amount of rough endoplasmic reticulum in exercised rats, compared to sedentary ones. In literature, the exercise ability to increase tenocyte rough endoplasmic reticulum amount has already been demonstrated. This increase has been associated with enhanced production of collagen fibers and extracellular matrix components. Morphological observations of tenocyte behaviour near MTJ reveal their activation following exercise protocol, suggesting a key role of these cells in the modulation of MTJ morphology., Regular exercise has emerged as an effective strategy to prevent and treat metabolic diseases. The beneficial effects of exercise can be easily observed in muscle tissue but also on a variety of distant organs, such as brain, heart, lungs, adipose tissue and liver, thus suggesting that muscle has endocrine activity. A growing body of evidence shows that contracting muscle releases cytokines with autocrine, paracrine and endocrine functions; hence, they are now recognized as myokines. In the last years a new mechanism for intercellular communication mediated by extracellular vesicles (EVs) has emerged. EVs are spherical structures bound by a lipid bilayer, which is similar in composition to the cell membrane from which the vesicle originated. Among these, exosomes are well-defined vesicles (ranging in size from 40 to 100 nm) that originate from multivesicular bodies. In order to understand EV release in working skeletal muscles, we took advantage of electrical pulse stimulated (EPS) C2C12 myotube model to investigate the characteristics of secreted EVs in response to EPS-evoked contractile activity. Five to six days after differentiation, C2C12 myotubes were placed in a chamber for electrical stimulation. EPS was carried out as follows: 6 hours stimulation with continuous 10-ms duration pulse of 40V/60mm voltage and 1 Hz frequency. Conditioned medium was first cleared and then exosomes were collected using standard ultracentrifugation protocol. EPS applied for 6 hours resulted in contraction of the myotubes and no other dramatic morphological changes were detected. Although EPS promoted the contraction of C2C12 myotubes, it did not appear to induce cell death, as assessed by LDH release, since cytotoxicity did not differ significantly between cell cultures with vs. without EPS treatment. The immunoblotting results showed that phosphorylation of AMP-activated protein kinase (AMPK; Thr172), and ERK1/2 (Thr202/Tyr204) was slightly but significantly increased by 6-h EPS. Then we examined whether EPS-evoked contraction results in cytokine and miRNAs modulation and, as expected, IL-6 mRNA and mir-146a expression were remarkably upregulated in contractile myotubes. Nanoparticle tracking assay showed that EPS-induced contraction determined an increased release of EVs in the medium compared with control. These data were further corroborated by western blot analysis of purified EVs using antibodies against well-defined exosomes markers (Tsg101 and Alix). Taken together, these findings indicate that myotube contraction may induce the active release of membrane vesicles, in particular exosomes potentially involved in mediating communication between muscle and other organs. Supported by: Italian Ministry of Health: GR-2011-02350264 to MG., Cancer is a recognized cause of the prominently reduced muscle mass known as cachexia. Inflammatory cytokines such as TNF-α, IL-1β, IL-6 and IFN-γ are main atrophy-inducing factors in cachexia causing excess catabolism of myofibrillary proteins.1 RAGE (Receptor for Advanced Glycation End-products) and its physiological ligands, S100B and HMGB1, are involved in muscle regeneration, inflammation, and tumor growth, which represent key processes in cancer cachexia.2,3 We found that: i) RAGE signaling has a trophic effect in myotubes in physiological conditions; ii) excess RAGE ligands leads to myotube atrophy; iii) high amounts of S100B and HMGB1 are found in cachectic muscles, and elevated levels of S100B are present in the serum of cachectic mice, likely released by tumor cells; iv) atrophying muscles re-express RAGE; v) RAGE, S100B and HMGB1 are involved in the mechanism through which TNFα±IFNγ induces atrophy in myotubes in vitro and in muscles in vivo; vi) Lewis lung carcinoma (LLC)-bearing RAGE-null (Ager‒/‒) mice show reduced loss of muscle mass and reduced atrogenes expression, and a dramatic increase in survival rate compared with LLC-bearing WT mice, likely due to reduced systemic inflammation, maintenance of spleen morphology and a different tumor-derived cytokine profile. Thus, increased expression/activity of RAGE and its ligands, S100B and HMGB1 at both systemic and muscle levels appears to concur to muscle wasting in cancer conditions., Flywheel-based isoinertial exercise was originally designed to maintain muscle health of astronauts during spaceflight. It employs isoinertial technology rather than gravity dependent weights, which allows for maximal concentric and eccentric muscle actions, with brief episodes of eccentric overload. Isoinertial exercise produces greater muscle hypertrophy and peripheral neural adaptations than weight-loaded resistance exercise in healthy subjects probably due to the eccentric overload. The purpose of this study was to analyse the modulation of circulating and local molecular markers of muscle damage and growth/repair over 48h after an isoinertial exercise. Eight male collegiate students (with at least 2 years of squatting experience) performed a session of isoinertial squat exercise (5 sets of 10 maximal reps; 3’ rest in-between) on the D11 flywheel device (Desmotec, Italy). Circulating markers analyzed were: muscle creatine kinase (CKM), insulin-like growths factor-1 (IGF-1) and interleukin-6 (IL-6); measured before and 2, 24, 48h post-exercise. In addition, miRNAs (miR-1, -133b, -206, -146a, -126 and -423) encapsulated in circulating exosomes were quantified before and 2h after exercise. Local markers investigated were: mRNA levels of genes involved in myogenesis and cell cycle control such as IGF-1 isoforms (IGF-1Ea, IGF-1Eb, and IGF-1Ec), myogenin, myogenic regulator factor-4 (MRF-4) and cyclin D1; determined in vastus lateralis muscle using fine needle aspiration coupled with real-time PCR before and 2h post-exercise. Exercise-induced inflammatory response was also analyzed by quantification of mRNA encoding cytokines and chemokines (IκB-α, MCP-1, TNFα, IL-6 and IL-6R) in muscle and in peripheral blood mononuclear cells (PBMC). Circulating CKM increased significantly after 2h post-exercise, the maximum peak occurred within 24h restoring to baseline level within 48h. Isoinertial exercise significantly increased total serum IGF-1 24h post-exercise and IL-6 at 2, 24 and 48h post-exercise. Nanoparticle tracking assay revealed a 2-fold increase in circulating exosomes in response to acute exercise, which was paralleled by higher levels of the mir-146a ad mir-126. Exercise increased muscle MCP-1, TNF-α and IL-6 and peripheral PBMC IkB-α and MCP-1 mRNA levels 2h post-exercise. On the contrary, muscle IGF-1Ea, IGF-1Eb, IGF-1Ec myogenin and cyclin D1 mRNA content was down-regulated 2h after the exercise bout. Muscle IkB-α, IL-6R and MRF-4 and PBMC TNF-α, IL-6 andIL-6R mRNA levels were unaffected. In conclusion, a single isoinertial exercise session increased serum markers of muscle damage (CKM and IL-6) and pro-inflammatory gene expression in muscle and PBMC of healthy, recreationally resistance-trained men. On the contrary, the mRNA level of genes related to muscle growth and repair decreased 2h post-exercise, suggesting that these processes had not yet been activated while those responding to muscular injury were prevalent. Accordingly, the plasma levels of IGF-1 increased only after 24 h post-exercise. These results indicate early molecular adaptations of skeletal muscle to loading, supporting the hypothesis that eccentric-overload inferred by isoinertial machinery offers a potent stimulus to critically optimize the benefits of resistance exercise., Nemaline myopathy (NM) is among the most common non-dystrophic congenital myopathies. Recently, a novel implicated gene was discovered - KBTBD13. NM patients with mutations in KBTBD13 (NEM6) exhibit muscle weakness and a typical muscle slowness. Here, we aim to gain insight in the pathophysiology of NEM6 myopathy muscle slowness. In vivo muscle relaxation was assessed using Transcranial Magnetic Stimulation (TMS) in NEM6 patients (n=10) and controls (CTRL) (N=24). Calcium-handling protein levels were determined in skeletal muscle biopsies by Western blot. Contractile parameters were measured in isolated single fibers and in myofibrils that were isolated from skeletal muscle biopsies. Next, the nanoscale structure and acto-myosin interactions in these muscle fibers were studied by X-ray diffraction. In vivo TMS revealed slower muscle relaxation in NEM6 patients. The phosphorylated phospholamban/phospholamban ratio was lower in NEM6 muscle biopsies, which might contribute to slower calcium-uptake. Relaxation kinetics of both single muscle fibers as well as individual myofibrils were slower in NEM6 compared to CTRL. X-ray diffraction studies show that the peak position of the actin layer line 6 was reduced in NEM6 compared to CTRL, suggesting a compressed, stiffer thin filament. Modelling of sarcomere kinetics revealed that a stiffer thin filament slows muscle relaxation. Here, we studied the pathophysiology of muscle slowness in NEM6 patients. We used a top-to-bottom approach: from the patient in vivo level to the nanoscale acto-myosin in vitro level. The data suggest that changes in the skeletal muscle thin filament level contribute to the clinical phenotype of NEM6., Myogenic helix-loop-helix proteins such as MyoD, Myf5, myogenin and MRF-4 regulate the skeletal muscle development and differentiation. In addition, a few zinc finger proteins have been described as helix-loop-helix proteins and are also regulators of muscle development and muscle gene expression. Among those proteins, Zinc finger E-box- binding homeobox 2 (Zeb2, also known as SMAD interacting protein 1) is poorly characterized although showing similar molecular characteristic of skeletal muscle regulators (1). In this study we investigate whether Zeb2 could play a role in regulating myogenic differentiation using genetic tools and transgenic mouse embryonic stem (mES) cells. We report that mES cells genetically modified for the absence or the overexpression of Zeb2 maintain pluripotency and are capable to differentiate in skeletal muscle cells. However, Zeb2-null mES cells display impaired myogenic differentiation and more interestingly the overexpression of Zeb2 in both mES cells and adult myogenic cells impacts positively the myogenic commitment in vitro and in vivo. To deeper investigate the role of Zeb2 in myogenic differentiation and in the context of cellular microenvironment we employed the single cell sequencing (RNA-Seq) in transgenic mES cells. This technology examines the sequence information from individual cells with optimized next generation sequencing technologies (2). The single cell RNA-Seq analysis shows that myogenic regulatory factors including Id family and SMAD/TGF-β pathway genes are affected by the absence or the overexpression of Zeb2. These findings reveal a critical role of Zeb2 in skeletal muscle differentiation, thus providing a novel possible target to improve skeletal muscle regeneration in muscle diseases., Mitochondrial calcium uptake plays a key role in modulating cell metabolism, cell survival and other cell specific functions. Calcium accumulates into the mitochondrial matrix through the mitochondrial calcium uniporter (MCU). Few years ago a MCU homolog has been discovered, which has been called MCUb. MCU and MCUb shares 50% sequence and structure similarity although some conserved differences in the primary sequence prevent MCUb from forming a Ca2+-permeable channel, thus acting as a dominant-negative subunit. RT-PCR experiments demonstrated that MCUb expression levels dramatically increase during skeletal muscle regeneration after cardiotoxin-induced injury. In addition, high MCUb expression levels have been detected in anti-inflammatory macrophages (M2). The latter are one of the most important effectors of the later stages of tissue repair. Preliminary results demonstrated that MCUb has a key role in macrophages skewing from M1 to M2 phenotype. In order to confirm our hypothesis, we performed skeletal muscle regeneration experiments on a MCUb KO mouse model. Our preliminary results demonstrate that the lack of MCUb causes a delay in skeletal muscle regeneration process that occurs in parallel with a reduction of the expression level of known markers of M2. We hypothesized that this altered muscular phenotype might be due to an impairment in macrophages skewing from an M1 to M2 phenotype. Indeed, macrophages from MCUb KO animals have lower phagocytic capacity compared to wild type animals and this affects skeletal muscle regeneration. These results are in line with published data demonstrating that phagocytic activity is fundamental for M2 polarization., Duchenne muscular dystrophy (DMD) is a pathological condition caused by the absence of a functional dystrophin protein. To date, there is no effective therapy for DMD; however, alternative therapeutic approaches have been attempted. Cell-based therapies are promising methods for treating DMD but stalled by a limited impact of transplanted stem cells on the long-term muscle cell replacement. Gene therapy, including exon-skipping, gained interest because of optimistic results in clinical trials but antisense oligonucleotides (AO) needs to be chronically injected to maintain a clinical efficacy. Our working hypothesis is that the hostile dystrophic microenvironment might interfere with and limit the efficacy of DMD therapies. A potential candidate that contributes to sustain a more hospitable microenvironment in dystrophic muscle is the Insulin-like growth factor-1 (IGF-1). We previously demonstrated that local IGF-1 over-expression plays a critical role in muscle regeneration modulating muscle-specific genes associated with maturation of regenerating fibers. Thus, mIGF-1 over-expression in dystrophic context could be useful for cell-based therapy, sustaining resident and transplanted stem cells survival and could improve the efficacy of the exon skipping-mediated approach trough the stabilization of the differentiated phenotype in mdx muscle. In this work we show how mIGF-1 over-expression in mdx mice is able to modulate pathological mechanisms affecting muscle niche. Moreover, we analysed the impact of the modulation of dystrophic environment by mIGF-1 on both stem cell therapy, based on mesoangioblasts transplantation, and gene therapy performed using PMO-Pip6a, a new generation PMO-based AO conjugated to the Pip6a peptide enhancing the delivery in mdx mice., Histone deacetylase (HDAC) and histone acetyltransferase (HAT) catalyse the reactions that maintain the homeostatic level of lysine side chain acetylation in thousands of cellular proteins. In muscle cells, alteration of this equilibrium is found in a variety of pathological conditions characterized by progressive degeneration and atrophy. Muscle atrophy may occur as a consequence of genetic diseases such as Duchenne muscular dystrophy (DMD). DMD is caused by mutations in the dystrophin gene that lead to the absence of the functional protein, myofiber membrane instability and damage upon contraction. Consequently, muscle cells release cytoplasmic components including the so-called Damage Associated Molecular Patterns (DAMPs). These activate both the resident immune cells and the muscle cells thus generating an inflammatory environment that progressively increases and amplifies the muscle damage. NF-kB activation is one of the downstream effect of TLR activation through DAMPs and proinflammatory cytokines such as TNF-α These pathways are subjected to regulation involving the acetylation of effector molecules. HDAC inhibitors are endowed with pleiotropic activities that influence the biology of muscle cells at different levels. Givinostat is a pan-HDAC inhibitor currently in phase III clinical trial for the treatment of DMD. Immunohistochemistry analysis of patients’ biopsies demonstrated that treatment with givinostat lead to a reduced inflammation, necrosis, fibrosis and increase of the cross sectional area of the myofibers that occupy a larger fraction of muscle tissue. To further characterize the molecular mechanism of action of givinostat on muscle cells induced atrophy we used in vitro differentiated human skeletal myotubes stimulated with the atrophy inducer cytokine TNF-α. We found that givinostat countered the upregulation of some key atrogenes such as TRIM63 and Atrogin-1 induced by TNF-α -α activated the expression of HDAC4 and HDAC6, both involved in muscle atrophy. Furthermore, HDAC6 has been recently described as an atrogene that interact with Atrogin-1 multiprotein complex. As expected, givinostat induced tubulin hyperacetylation, a pharmacodynamic marker of HDAC6 inhibition. Since givinostat inhibits HDAC4 activity, both enzymes are dually inhibited, at transcriptional and functional level. In agreement with these gene modulations, we found that givinostat upregulated the gene expression of myosin heavy chain I and II that are targeted to proteasomal degradation during atrophy. Taken together, these data indicate that givinostat counters the atrophy program induced by TNF-α in normal skeletal muscle cells and suggest that a similar mechanism may take place in DMD muscle cells., With an incidence of 4.5 cases per million adolescents, the rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma. It develops in different tissues, most commonly in the head and neck, in the extremities and in the genitourinary tract. According to its histological and pathological characteristics, RMS can be classified in two major subtypes, embryonal (eRMS) and alveolar (aRMS), which seem to share the same initiating cell type(s), even if this point is still debated. In fact, some evidence supports the notion that skeletal muscle progenitors, such as satellite cells, could give rise to RMS even though alternative theories point to mesenchymal stem cells or even progenitors of the adipocyte lineage, as possible tumor-initiating cells. The clinical differences between the two RMS types result from different molecular genetic mechanisms of origin. To study the eRMS, which is our main focus, we adopted the KrasG12D/+Trp53Fl/Fl conditional mouse model to induce cell transformation, by in vivo or in vitro infecting cells with an Adenovirus vector expressing the CRE recombinase that leads to the constitutive activation of the oncogene KRAS along with the inactivation of the P53 tumor suppressor gene. Since our goal is to identify which cell population(s) can give rise to eRMS, we triggered embryonal RMS formation by infecting purified muscle mononuclear cell populations with the CRE recombinase adenovirus which activates expression of Kras(G12D) and inactivates the p53 gene. Both satellites and FAPS are transformed in vitro by this approach and induce the formation eRMS like tumors when grafted into nude mice. We are in the process of characterizing the changes in the cell populations from the tumor mass, at different stages of development, by flow cytometry techniques., Mammalian neuromuscular junctions (NMJs) undergo a postnatal topological transformation from a simple oval plaque to a complex branch-shaped structure called “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, assemblies formed in vitro undergo similar stages in developmental remodeling from “plaques” to “pretzels” as the NMJ postsynaptic machinery 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 scaffolding protein Amotl2, which is localized at podosomes. We now report that in muscle cells Amotl2 interacts with one of the key synaptic components, Homer1. Mice lacking Homer1 expression exhibit skeletal muscle myopathy characterized by disturbed calcium homeostasis. Additionally, it has been shown that Homer1 expression is affected in mouse models of Amyotrophic Lateral Sclerosis (ALS) and Duchenne’s Muscular Dystrophy (DMD). We show that Homer1, together with Amotl2 are concentrated at postsynaptic areas of NMJs in the indentations between the acetylcholine receptor-rich branches. We also demonstrate that Homer1 is indispensable for AChR clustering in the cultured myotubes and identify Homer1-interacting proteins that are involved in this process. Our results provide novel insight into molecular machinery that is orchestrating postsynaptic machinery development. Supported by the National Science Centre grants 2012/05/E/NZ3/00487, 2014/13/B/NZ3/00909 and 2016/21/D/NZ4/03069., Caveolin-3 (CAV3) is a muscle specific protein localized to the sarcolemma where it interacts with the dystroglycan complex (DGC) and is thus involved in the connection between the extracellular matrix (ECM) and the cytoskeleton. Muscle diseases caused by mutations in the CAV3 gene are called Caveolinopathies. So far, more than 40 dominant pathogenic mutations have been described leading to different phenotypes molecularly associated with a mis-localization of the mutant protein to the Golgi. Hereby, associated Endoplasmic Reticulum (ER)-stress has been demonstrated for the p.P104L mutation. However, the further patho-physiological consequences of mutant CAV3 mis-localization and ER-stress remained elusive. Utilizing a transgenic (p.P104L) mouse model of Caveolinopathy and performing proteomic profiling along with immunoblot and morphological studies (including electron and CARS microscopy) we systematically addressed these consequences. Our morphological studies revealed Golgi and ER proliferations as well as the build-up of protein aggregates. These observations were confirmed via immunological studies and are in accordance with our proteomic data showing altered abundance of 120 proteins in diseased quadriceps muscle fibres. Proteomic findings indicated ECM remodeling and cytoskeletal vulnerability. Moreover, our proteomic findings suggested that further DGC components are affected by the perturbed protein processing machinery leading to the formation of protein aggregates which could be confirmed via CARS microscopy. Hence, our combined data classify (p.P104L) Caveolinopathy as an acquired protein folding disease with sarcolemmal affection and thus expand the pathophysiological knowledge of this disorder, an important aspect in the therapeutic management of CAV3-patients., Skeletal muscle is a highly plastic tissue able to adapt to different stresses, in part due to its remarkable regenerative capacity. Recently, it has been found that myoblasts and myotubes release exosomes and exosome-like vesicles in the extracellular environment during myogenic differentiation. Within the satellite cell niche, muscle stem cells exchange signals with other cell types, and among these, complex interactions between skeletal muscle and the immune system have been reported. The aim of this study was to ask whether EVs (Extracellular Vescicles) released by differentiating myocytes can mediate cell-communication between muscle cells and macrophages, one of the key actors of muscle remodelling. To this end, RAW 264.7 cells (a model of macrophages) have been used as target cells. Myocytes undergoing myogenic differentiation are subjected to deep structural rearrangements, so that a complex mixture of EVs (comprising exosomes and shedding microvesicles) and cellular membrane fragments may be released in the extracellular environment. A serial ultracentrifugation protocol was specifically adjusted to remove cellular debris and isolate shedding microvesicles and exosomes. The EVs collected during myogenic differentiation process were characterized using TEM, western blot, density gradient and real-time PCR analyses and used to treat RAW 264.7 cells. Gene expression analysis performed 24 h after treatment highlighted a significant up-regulation of IL-6 and IL-10, two cytokines involved in muscle differentiation and anti-inflammatory process, respectively, but not of TNF-alpha (a proinflammatory cytokine). IL-6 stimulates the production of the classical anti-inflammatory cytokines IL-1Ra and IL-10, moreover it has been reported that IL-10 plays a central role in regulating the switch of muscle macrophages from a M1 to M2 phenotype in injured muscle in vivo. Collectivelly, these data suggest that EVs could be involved in the regulation of normal growth and regeneration of muscle. Supported by: Italian Ministry of Health: GR-2011-02350264 to MG., Muscle growth and regeneration following injury, is regulated by the myogenic stem cells, called satellite cells. The satellite cells are located beneath myofiber basement membranes and closely associated with capillary endothelial cells. We previously observed that 90% of capillaries were associated with pericytes in adult mouse and human muscle. We also have shown during post-natal growth that, by promoting post-natal myogenesis through Insulin like-growth factor 1 and stem cell quiescence through Angiopoietin-1, pericytes play a key role in the microvascular niche of satellite cells. Consistently, here we show that in a mouse model of muscle pericytes depletion, the loss of the perivascular cells induces a spontaneous necrosis of the muscle. We also observed after induced chemical injury of the muscle in a mouse model of depletion of microvascular Angiopoietin-1, a delayed regeneration process associated with Type 2 myofibers hypotrophy along with an elevated number of remaining cycling Pax7+ cells. In conclusion, pericytes associated with endothelial cells are essential to maintain adult muscle homeostasis and exert paracrine effects on adjacent myogenic cells during muscle repair in adulthood., Muscular Dystrophies are severe disorders due to mutations in structural genes that cause skeletal muscle wasting compromising patient mobility and respiratory functions. Although previous works suggested enhancing regeneration and muscle mass as therapeutic strategies, these led to no long-term benefits in humans. Here we propose a conceptually new idea based on making a dystrophic muscle slower in regeneration and more oxidative, by silencing of the transcription factor Nfix. In different forms of Muscular Dystrophy, lack of Nfix rescues histopathological and functional hallmarks of dystrophic muscle. More importantly, silencing Nfix in post-natal dystrophic mice, when the first signs of the disease already occurred, rescues the pathology. On the contrary, Nfix overexpression in dystrophic muscles pushes regeneration and markedly exacerbates the pathology. We therefore provide evidence that current strategies are based on a misconception and offer a proof of principle for a novel therapeutic approach., Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder caused by mutations in the dystrophin gene. Oxidative stress and chronic inflammation have been proposed as important mechanisms involved in DMD. Under pathological conditions, elevated levels of ROS in skeletal muscle can overwhelm cellular antioxidant defenses, leading to oxidant-related damage. Moreover, an alteration in the redox signaling might stimulate inflammatory response collaborating to produce muscle fiber necrosis. To date, inflammation is considered the principal determinant of degenerative processes in dystrophic muscle and treatments are limited to glucocorticoids. However, a central role of oxidative stress in DMD pathology has been evidenced by clinical and pre-clinical studies. A potential candidate linking inflammation and ROS production is Interleukine 6 (IL6), a critical factor involved in the switch between acute and chronic inflammation. We generated a severe animal model of DMD, the mdx/IL6 mouse, that better recapitulates disease progression in human pathology and approximates the antioxidant expression profile observed in DMD patients. In particular, increased circulating levels of IL6 alter the redox signaling cascade in dystrophic muscle., Caveolin-1 (Cav-1) is a plasma membrane scaffolding protein that was shown to control the ERK pathway in muscle satellite cells. Oncogenic transformation of satellite cells is responsible of the generation of rhabdomyosarcoma (RMS), a soft tissue tumor affecting childhood and adolescence. We previously reported that Cav-1 is a marker of proliferating RMS cell lines and that its overexpression promotes increased malignancy of RMS cells in vitro and in vivo. Here we show that tail vein injection of the human embryonal RD cells with Cav-1 overexpression (RD Cav-1) into NOD/SCID mice resulted in formation of lung metastasis in about 9 weeks as compared to control cells that did not form metastasis. After performing ex vivo transplantation of lung metastases we isolated one cell population, termed lung metastatic RD1, which injected in mice again gave rise to lung metastases in 5 weeks; from these disseminated lungs we were able to isolate the lung metastatic RD2 cell population. All the distinct cell populations, including RD Cav-1 and lung metastatic RD1 and RD2 clones, retained high Cav-1 expression and showed high phosphorylation levels of ERK1/2, which completely prevented their ability to undergo myogenic differentiation. In addition, lung metastatic RD1 and RD2 clones exhibited an increased migration, adhesion and production of angiogenic stimuli in comparison to non-metastatic control RD and RD Cav-1 lines. Taken together, these data suggest a key role of Cav-1 in promoting both local tumor growth and metastasis of RMS through cooperation of the ERK signaling pathway., Oxidative stress is involved in the pathogenesis of Duchenne muscular dystrophy (DMD), an X-linked genetic disorder caused by mutations in the dystrophin gene and characterized by progressive, lethal muscle degeneration and chronic inflammation. In this study, we explored the expression and signaling pathway of a master player of the anti-oxidant and anti-inflammatory response, namely NF-E2-related Factor 2, in muscle biopsies of DMD patients. We classified DMD patients in two age groups (Class I, 0–2 years and Class II, 2–9 years), in order to evaluate the antioxidant pathway expression during the disease progression. We observed that altered enzymatic antioxidant responses, increased levels of oxidized glutathione and oxidative damage are differently modulated in the two age classes of patients and well correlate with the severity of pathology. Interestingly, we also observed a modulation of relevant markers of the inflammatory response, such as heme oxygenase 1 and Inteleukin-6 (IL-6), suggesting a link between oxidative stress and chronic inflammatory response. Of note, using a transgenic mouse model, we demonstrated that IL-6 overexpression parallels the antioxidant expression profile and the severity of dystrophic muscle observed in DMD patients. This study advances our understanding of the pathogenic mechanisms underlying DMD and defines the critical role of oxidative stress on muscle wasting with clear implications for disease pathogenesis and therapy in human., Sarcopenia is a multifactorial syndrome defined as the irreversible loss of skeletal muscle mass and functionality occurring during aging. Muscle atrophy and impaired regeneration are the main features of this syndrome but the underlying mechanisms and etiology remain poorly defined. Acylated and unacylated ghrelin (AG and UnAG, respectively) are circulating peptide hormones mainly produced in the stomach. AG, through GHSR-1a, induces a strong release of GH and orexigenic effects. Although UnAG does not activate this receptor, shares common activities with AG on skeletal muscle counteracting atrophy and promoting myoblast differentiation. Moreover, UnAG enhances muscle regeneration stimulating satellite cell functions. AG/UnAG plasmatic levels change during aging and this may contribute to sarcopenia establishment. We are evaluating the role of AG/UnAG in sarcopenia prevention by means of Myh6/Ghrl transgenic (Tg) mice (characterized by high levels of circulating UnAG) and of Ghrl-/- (KO) mice. Although no difference in muscle functionality were observed among Tg, KO and WT at 6 and 12 months, fat accumulation and glucose clearance rate were significantly different in the different genotypes. Moreover, muscle regeneration in KO mice was impaired, suggesting a potential role of AG/UnAG system in the sarcopenia onset. Supported by Fondazione Cariplo, Circular RNAs represent a recently re-discovered class of covalently closed RNAs, derived from a non-canonical splicing event (back-splicing), ubiquitously expressed among Eukaryotes and conserved among different species. We identified several circular RNAs expressed during myogenesis. Thanks to a knockdown-based phenotypic screening, we identified a circRNA, named circZNF609, involved in the regulation of human myoblast proliferation. Upon its depletion, the percentage of proliferating cells is highly reduced with respect to the control sample. CircZNF609 also has an open reading frame generated upon circularization and it can encode a peptide. Here we focus on circZNF609 role in regulating cell cycle progression. An RNAseq experiment performed on human myoblasts revealed that the expression of 300 genes is altered upon circZNF609 specific depletion, of which 60% are down-regulated, and specifically enriched for cell cycle related genes. To deepen our knowledge about circZNF609 role in proliferation, we studied its expression and function in rhabdomyosarcoma (RMS), a pediatric muscle malignancy. We found that circZNF609 is strongly up-regulated in biopsies from the two major RMS subtypes, the embryonal and the alveolar, and we discovered that its knockdown blocks proliferation of an RMS-derived cell line, promoting an accumulation of cells in G1 cell cycle phase, with a reduction of cells in S phase. These results suggest that circZNF609 could be a good target for therapeutic approaches against RMS. We are now testing the effect of its depletion on tumor growth in xenograft mice, injected with a stable RMS cell line expressing an siRNA against circZNF609 upon doxycycline induction., Sertoli cells (SeC), which are crucial for germinal cell development, have demonstrated trophic and immunomodulatory effects in numerous experimental setting.1 A single injection of microencapsulated SeC into the peritoneal cavity of mdx mice, an experimental model of Duchenne muscular dystrophy (DMD), results into recovery of muscle architecture and performance in the absence of any pharmacological immunosuppression, opening to new perspectives for DMD treatment.2 Besides restraining muscle inflammation, treatment of mdx mice with microencapsulated SeC induced at the sarcolemma of myofibers the expression of the dystrophin paralogue, utrophin, thus conferring an additional advantage to dystrophic muscles.2 However, the direct effects of SeC on myoblasts/myotubes themselves and on myotubes of higher mammals have not been investigated yet. C2C12 myotubes treated with TNFα/IFNγ or cultured in PBS (phosphate buffered saline) to mimic atrophying conditions show reduced levels of myosin heavy chain (MyHC), and we found that SeC protect against loss of MyHC in both conditions in a dose-dependent manner, high numbers of SeC being the most efficacious. Moreover, we demonstrated that, as for mdx myotubes/myofibers,2 SeC are able to induce utrophin expression in myotubes from GRMD (golden retriever muscular dystrophy) dogs and dystrophic humans. Our data further support the use of microencapsulated SeC for treatment of DMD patients., Creatine (Cr) is a nutritional supplement promoting a number of health benefits, whose use is spreading in the prevention of muscle aging and treatment of neuromuscular maladies (Wallimann, 2007). Indeed Cr has been shown to be beneficial in disease-induced muscle atrophy, improve rehabilitation and afford mild antioxidant activity (Tarnopolsky, 2011). The beneficial effects of its supplementation are likely to derive from pleiotropic interactions. In accord with this notion, we previously demonstrated that multiple, pleiotropic effects account for the capacity of Cr to prevent the differentiation arrest caused by oxidative stress in C2C12 myoblasts, namely: increased expression of muscle regulatory factors mRNA, antioxidant activity, amelioration of energy status and preservation of mitochondrial damage (Sestili, 2015). Given the importance of mitochondria in supporting the myogenic process, here we further explored morphologically, cytofluorimetrically and biochemically the protective effects of Cr on the structure, function and networking of these organelles in C2C12 cells differentiating under oxidative stressing conditions (acute exposure to 0.3 mM H2O2); the effects on the energy sensor AMPK, on PGC-1α, which is involved in mitochondrial biogenesis and its downstream effector Tfam were also investigated. Our results indicate that damage to mitochondria is crucial in the differentation imbalance caused by oxidative stress and that the Cr-prevention of these injuries is invariably associated with the recovery of the normal myogenic capacity. We also found that Cr- activates AMPK and induces an up-regulation of PGC-1α expression, two events which are likely to contribute to the protection of mitochondrial quality and function., Duchenne Muscular Dystrophy (DMD) is a lethal X-linked disease caused by mutations in the dystrophin gene that progressively lead to severe respiratory and cardiac failure that cause premature death. DMD pathogenesis is characterized by continuous cycles of muscle contraction/degeneration. At early stages of disease, muscle degeneration is counterbalanced by a compensatory repair driven by muscular stem cells (MuSCs). As the disease progresses, the regeneration potential is exhausted and muscles are replaced by fibrotic scars and fat infiltration (Dalkilic & Kunkel, 2003). The identity of the cellular source of fibrosis and fat deposition has been recently assigned to fibro-adipogenic progenitors (FAPs; Joe et al., 2010; Uezumi et al., 2010). Although there is currently no available cure for DMD, several therapeutic strategies are undergoing investigation. In this context, it is of particular interest the pharmacological approach based on the histone deacetylase inhibitors (HDACi), which represent the first generation of epigenetic drugs used to counteract the DMD progression. Recently, our lab has identified a regulatory network targeted by HDACi to repress FAPs differentiation into pro-fibrotic and adipogenic cells, while enhancing their ability to support MuSCs-mediated compensatory regeneration (Saccone et al., 2014). Interestingly HDACi pharmacological effect is restricted to the early stage of DMD (Mozzetta et al. 2013). The mechanism during the aging of DMD FAPs that confers resistance to HDACi treatment is still unknown. The aim of this project is thus to investigate the epigenetic and transcriptomic changes that leads FAPs stage dependent response to HDACi. By ChipSeq bioinformatic analysis of MDX FAPs histone acetylation we uncovered an indiscriminate histone hyperacetylation at late stages of DMD that is paradoxically reduced by HDACi treatment. This data could represent the first evidence of an HDACi counter-active effect at late stages of DMD. Integrating various histone modification and the transcriptomic profile of FAPs could unveil a possible mechanism behind DMD aging and HDACi efficacy at late stage of DMD. References: Dalkilic I, Kunkel LM (2003) Muscular dystrophies: genes to pathogenesis. Curr Opin Genet Dev 13: 231–238. Mozzetta C, Consalvi S, Saccone et al.,Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice., Iron is an essential metal for a plethora of biological reactions in light of an elevated reactivity, which is also the reason of its elevated toxicity when it is not properly compartmentalized. While iron overload is known to cause muscle dysfunction, we still have a limited knowledge of the role played by iron deprivation in the pathogenesis of skeletal muscle atrophy. Since iron is commonly limited in several systemic diseases characterized by muscle atrophy, we speculated that iron availability might directly regulate skeletal muscle mass homeostasis. Consistently, iron deprivation in vitro is sufficient to promote myotubes atrophy as seen as myotubes thickness reduction and atrogenes expression. Datasets analysis indicated transferrin receptor 1 (the receptor involved in transferrin uptake) as one of the primary targets to be downregulated during muscle atrophy upon various stimuli, while treatment with conditioned media derived from cancer cells or dexamethasone is sufficient to impair transferrin recycling in myotubes, as well as total transferrin receptor protein levels. These data further suggest that iron metabolism might be directly involved in the pathogenesis of muscle wasting. Implications of the effects of iron dysbiosis on mitochondrial metabolism and altered gene expression will be discussed., Alterations of mitochondrial Ca2+ homeostasis regulated by MCU has been recently shown to affect muscle trophism in vivo. Mice lacking MCU exhibit functional abnormalities in conditions that require a rapid increase in the skeletal muscle work load. During aging skeletal muscle undergoes a progressive loss of muscle mass, with decline in specific force and functional impairment. The etiology of this phenomenon is complex and involves the interplay of numerous factors whose underlying mechanisms are currently not fully understood. Physical exercise is known to have beneficial effects on muscle trophism and force production modulating signaling pathways also via intracellular Ca2+ and specific mitochondrial adaptations. To understand the relevance of MCU-dependent mitochondrial Ca2+ uptake in aging and to investigate the effect of physical exercise on MCU expression and mitochondria dynamics, we analyzed skeletal muscle biopsies from 70yrs old seniors, either sedentary, 9 weeks and lifelong trained in comparison to young subjects. We demonstrate that improved muscle function and structure induced by physical exercise are linked to increased protein levels of MCU. Ultrastructural analyses by Electron Microscopy showed remodeling of mitochondrial apparatus in trained muscles that is consistent with an adaptation to physical exercise, a response likely mediated by an increased expression of mitochondrial fusion protein OPA1. Altogether these results indicate that the physiological effects of exercise on skeletal muscle size and force are associated with changes in mitochondrial-related proteins involved in Ca2+ homeostasis and mitochondrial shape. These findings observed for the first time in aging human skeletal muscle confirm the data obtained in mice and propose MCU and mitochondria related proteins as potential pharmacological targets to counteract age-related muscle loss, promoting healthy aging. Sandra Zampieri was supported by A&CM Carraro Foundation for Translational Myology, Padua, Italy to attend the 2017 IIM Meeting., Mammalian target of rapamycin (mTOR) plays a central role in cell growth. mTOR assembles into two distinct multiprotein complexes, namely the rapamycin-sensitive complex mTORC1 and the rapamycin-insensitive complex mTORC2. One of the key members of the mTORC1 complex is a. 150kDa protein called Raptor, which has been shown to be able to recruit mTOR substrates S6K1 and 4EBP1 on mTORC1. Mice lacking Raptor only in skeletal muscle from birth show a pronounced myopathy leading to a premature death. However, treating adult mice with the specific mTORC1 inhibitor rapamycin does not lead to a myopathic phenotype, and even improves muscle physiology in aged mice. Here we want to examine the role of Raptor and mTORC1 using a new CreER-inducible transgenic mouse in which we can delete Raptor in muscles of adult mice (Raptor k.o.). Activation of Cre by treatment with tamoxifen leads to a rapid loss of Raptor transcript and protein levels. Also the phosphorylation levels of ribosomal protein S6, a known mTORC1 target, are strongly reduced in Raptor ko mice. One month after Raptor deletion, muscle weight and basic histology are unchanged. A longer deletion of Raptor, however, leads to a myopathic phenotype with central-core structures and a high number of small and large muscle fibers., The causes of skeletal muscle (SkM) loss in aging and diseases are complex and largely unknown. Intensive research in simple model organisms, such as the nematode Caenorhabditis elegans, which offers the prowess of sophisticated genetic approaches and recapitulates aspects of the human pathologies, may contribute in understanding the molecular mechanisms of SkM cell degeneration. C.elegans has distinct striated and non-striated muscle systems, the sarcomere contains the same or homolog morphological features described in mammals and functional age-related changes of C.elegans SkM, including the decline of locomotor behavior, are similar to the age-related or disease-induced modifications observed in mammals. Sphingolipids (SLs), essential components of eukaryotic cell membranes and bioactive factors involved in the regulation of a variety of different cellular processes, such as cell growth and differentiation, are also emerging as key regulators of SkM cell biology. In particular, sphingosine 1-phosphate attenuates the muscle damage, protect the muscle fiber from apoptosis and preserve satellite cell viability and renewal (Sassoli et al., 2011; Meacci et al., 2008). Since the principal components of SL biochemical pathways are also described in the nematode, here, we provide evidence of S1P signaling involvement in the maintaining of SkM phenotype. Grants from MIUR and Fondazione Cassa di Risparmio di Pistoia e Pescia.

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.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-08-04T18:29:32Z No. of bitstreams: 1 2017 - Roberta Benic? Sartori.pdf: 2251565 bytes, checksum: 2cc548260ea3d0f5856e36bbb514bf7f (MD5) Made available in DSpace on 2017-08-04T18:29:32Z (GMT). No. of bitstreams: 1 2017 - Roberta Benic? Sartori.pdf: 2251565 bytes, checksum: 2cc548260ea3d0f5856e36bbb514bf7f (MD5) Previous issue date: 2017-02-22 Banana is a tropical fruit cultivated in warm places and harvested during the whole year, as well as its consumption is intense. It is known that banana productivity, however, is threatened due to the vulnerability of the cultivars to some pests and diseases. Because of this, some agrochemicals are recommended to avoid the attack of these hindrances such as: azoxystrobin, chlorothalonil, diphenoconazole, imazalil, bifenthrin, and chlorpyriphos. All of them can cause serious health problems in humans such as cancer and neurological diseases and adverse reproductive effects are associated with the consumption and/or exposure to these hazardous substances. However, even when applied in conformity with Good Agricultural Practices, pesticides can leave residues, that can still be present in the soils as well as in the fruits. Due to these considerations, this study has as aim the technical evaluation of supercritical fluid in the extraction of pesticides present in banana flour (Musa spp). Therefore, the whole fruits (8 g), previously submitted to lyophilization and milling were fortified, during a stable period of overnight, with a solution containing the agrochemical standards mentioned above, diluted in methanol. The experimental unit consisted basically of a 42 mL extractor, a high pressure pump and a micrometering valve to sample removal. Different operational conditions were investigated (60 ?C ? 200 bar; 46 ?C ? 244 bar; 74 ?C ? 244 bar; 40 ?C ? 350 bar; 60 ?C ? 350 bar; 80 ?C ? 350 bar; 46 ?C ? 456 bar; 74 ?C ? 456 bar and 60 ?C ? 500 bar) based on an experimental design. The analysis of the extracted pesticides was performed using an ultra performance liquid chromatography coupled to a mass spectrometer (UPLC-MS). It was observed that the yield of the extract increased with the temperature increasing. According to the results, the extraction at 80 ?C and 350 bar was able to remove 1.4194% of the extracts in the banana. The yield of pesticides increased with the pressure increasing and the better condition was at 500 bar and 60 ?C. The experimental data were well modelled by the models of Esqu?vel et al. (1999), Reverchon & Osseo (1994), Zekovi? et al. (2003) and Sovov? (1999). The Chrastil (1982) model could represent the solubility behavior of the pesticides with the variation of temperature and pressure. A banana ? uma fruta tropical cultivada em locais quentes e colhida durante todo o ano, assim como seu consumo ? intenso durante todo o ano. Sabe-se que a produtividade da banana, no entanto, est? amea?ada devido ? vulnerabilidade de tais cultivares a algumas pragas e doen?as. Devido a isso, alguns agroqu?micos s?o recomendados para evitar o ataque destes entraves ? produtividade da banana, tais como: azoxistrobina, clorotalonil, difenoconazol, imazalil, bifentrina e clorpirifos. Todos esses compostos podem causar s?rios problemas de sa?de em seres humanos, como o c?ncer, assim como doen?as neurol?gicas e efeitos reprodutivos adversos est?o associados ? ingest?o e/ou exposi??o a estas subst?ncias perigosas. No entanto, mesmo quando aplicados em conformidade com as Boas Pr?ticas Agr?colas, os pesticidas podem deixar res?duos, que ainda podem estar presentes tanto nos solos como nos frutos. Devido a estas considera??es, este estudo tem como objetivo a avalia??o t?cnica do fluido supercr?tico na extra??o de agrot?xicos presentes na farinha de banana (Musa spp). Assim, os frutos inteiros (8 g), previamente submetidos ? liofiliza??o e moagem foram fortificados, durante um per?odo est?vel de um dia para o outro, com uma solu??o contendo os padr?es dos pesticidas mencionados acima, dilu?dos em metanol. A unidade experimental consiste basicamente em um extrator de 42 mL, uma bomba de alta press?o e uma v?lvula microm?trica para remo??o da amostra. Foram investigadas diferentes condi??es operacionais (60 ?C a 200 bar, 46 ?C a 244 bar, 74 ?C a 244 bar, 40 ?C a 350 bar, 60 ?C a 350 bar, 80 ?C a 350 bar, 46 ?C a 456 bar e 74 ?C - 456 bar e 60 ?C - 500 bar) com base em um planejamento experimental. A an?lise dos pesticidas extra?dos foi realizada utilizando cromatografia l?quida de ultra desempenho acoplada ao espectr?metro de massa (UPLC-MS). Observou-se que o rendimento do extrato aumentou com o aumento da temperatura. De acordo com os resultados, a extra??o a 80 ?C e 350 bar foi capaz de remover 1,4194% dos extratos na banana. Para o rendimento de pesticidas, observou-se um aumento com o aumento da press?o e a melhor condi??o foi a 500 bar e 60 ?C. Os dados experimentais foram modelados pelos modelos de Esqu?vel et al. (1999), Reverchon & Osseo (1994), Zekovi? et al. (2003) e Sovov? (1999), apresentando boa correla??o. O modelo de Chrastil (1982) conseguiu representar o comportamento da solubilidade dos agrot?xicos com a varia??o de temperatura e press?o.

Atrav?s de avan?os na concep??o de ci?ncia, valendo-se de Kuhn (1962), Poeppel (2004) e Giere (2006), Costa (2007b) lan?a a Metateoria das Interfaces e sugere um novo tratamento para a Lingu?stica e para a significa??o. A Lingu?stica deixa de ser vista como uma disciplina pr?-existente e passa a ser tratada como Ci?ncias da Linguagem e, portanto, como o resultado de constru??es de interfaces internas e externas. Da mesma forma, o significado em linguagem natural, para fins de investiga??o, deixou de ser um objeto observ?vel, no e do mundo, para ser compreendido como um objeto complexo constru?do atrav?s dessas interfaces. A partir dessa concep??o de interfaces e objetos complexos, respaldada pela Metateoria das Interfaces, a fim de estudar o significado por um vi?s inferencial, propomos a elabora??o de um construto que tem na articula??o de teorias de car?ter inferencialista a capacidade de promover uma investiga??o mais interessante desse objeto tanto do ponto de vista descritivo quanto do ponto de vista explanat?rio. Para tanto, foram reunidas, ? luz do Perspectivismo e da no??o de cross-fertilization, a Teoria das Implicaturas Conversacionais (GRICE, 1975), a Teoria das Implicaturas Conversacionais Modelo Ampliado (COSTA, 1984), a Teoria da Relev?ncia (SPERBER; WILSON, 1986, 1995), a Teoria das Implicaturas Conversacionais Generalizadas (LEVINSON, 2000), a Teoria da Conectividade N?o-Trivial (COSTA, 2002, no prelo, 2005a) e a Teoria do Di?logo (COSTA, 2013). Dessa forma, foi poss?vel, a partir da cria??o de interfaces, em especial da TCNT e da TR, fortalecer os fundamentos que explicam por que usamos a linguagem, mais do que para obter informa??es e melhorar nossa representa??o de mundo da maneira mais eficiente por menos esfor?o, assumimos que o homem usa a linguagem porque est? geneticamente programado para a conex?o, e a linguagem ? uma das formas pelas das quais se obt?m isso, atrav?s, contudo, de uma forma n?o-mec?nica, n?o-redundante, mas criativa e informativa. As interfaces tamb?m permitiram verificar que, via processamento inferencial, devido a elementos da l?gica, podemos n?o apenas monitorar nossos racioc?nios, mas nosso conhecimento de mundo como um todo. Al?m disso, pela problematiza??o das no??es de benef?cio e custo, verificou-se a necessidade de se considerar que aspectos emocionais e est?ticos ? neste caso, em especial, a partir da concep??o de que o meio ? a mensagem (McLUHAN, 1969) ? passassem a compor o que entendemos por significado. Ele, portanto, n?o se constitui apenas de aspectos racionais e informativos ? no??o est? igualmente revisitada. Por fim, foi poss?vel verificar que essas novas interfaces nos permitiram identificar que infer?ncias de diferentes naturezas, l?gico-lingu?sticas, a saber, as multiformes, e elementos de diferentes naturezas, emocionais, est?ticos, etc., interferem e interagem nas trocas conversacionais, das mais diferentes situa??es comunicativas, para a produ??o dos mais ricos e variados efeitos de significa??o, reorientando online, inclusive, as inten??es dos interlocutores. Todos esses aspectos foram investigados a partir dos discursos jornal?stico, pol?tico e jur?dico, cujas exist?ncias sociais concretas os tornam locus perfeito para a gera??o dos fen?menos que nos interessam, e cujas naturezas fazem com que se valham dos tantos recursos aos quais o uso da linguagem natural d? origem. Por fim, tais discursos podem ser vistos reunidos em um evento, a elei??o presidencial norte-americana, em 2000, cujo debate entre democratas e republicanos mostrou-se o local ideal para testar o construto e identificar os fascinantes efeitos de significa??o que inten??es movidas por raz?o e emo??o, de olho nos mais diferentes tipos de benef?cios, mesmo que estes se caracterizem pelo baixo custo, podem produzir atrav?s da perfeita intera??o entre ditos e implicados mediada por infer?ncias l?gico-lingu?stico-cognitivo-comunicacionais. Concerning the breakthroughs science has experienced, using Kuhn?s (1962), Poeppel?s (2004) and Giere?s (2006) work, Costa (2007b) launches the Metatheory of Interfaces and suggests a new approach to Linguistics and meaning. Linguistics thus is no longer seen as a preexisting discipline, but now is considered as Language Sciences, i. e., it is now regarded as a result of external and internal interface construction. All the same approach is given to natural language meaning when it comes to research, for it is no longer an observable object in and from the world, but now it is understood as a complex object built through the establishment of these interfaces. Regarding the complex objects and interfaces conception, backed by the Metatheory of Interfaces, in order to study meaning from an inferential point of view, we suggest the development of a model which has in the articulation of inferential theories the capacity of investigating such a complex object in a more interesting way regarding both the descriptive and explanatory perspectives. To do so, concerning the Perspectivism position and the cross-fertilization notion, the Conversational Implicature Theory (GRICE, 1975), the Conversational Implicature Theory ? Enlarged Model (COSTA, 1984), the Relevance Theory (SPERBER; WILSON, 1986, 1995), the Generalized Implicature Theory (LEVINSON, 2000), the Non-Trivial Connectivity Theory (COSTA, 2002, unpublished, 2005a) and the Dialog Theory (COSTA, 2013) were brought together to form an articulated model. Therefore, it was possible, from the devise of interfaces, specially, using the Non-Trivial Connectivity Theory and the Relevance Theory, to strengthen the theoretical foundations which explain why we use language not only to acquire information and to improve our world representation in a more efficient way for less effort, but by assuming that man uses language because he is genetically programmed to connection, and natural language is one of the various forms through which man can obtain it, in, however, a non-mechanical, non-redundant, but creative and informative way. The interfaces also allowed to verify that, through inferential processing, due to logic traits, we can not only monitor the way we reason, but also our world knowledge as a whole. Besides, because of the questioning of notions such as benefit and cost, it was possible to verify the of considering that aesthetic and emotional aspect ? in this case, especially regarding McLuhan?s (1969) maxim ?the medium is the message? ? started to explain what we understand as meaning. So it is not only based on rational and informative aspects. Finally, it was also possible to verify that these new interfaces allowed us to identify inferences of different natures, such as linguistic and logic, known as multiform inferences, and identify elements of different natures, such as aesthetic, emotional, etc., dabbling and interacting in conversational exchanges regarding the most different communicative situations concerning the production of rich and various types of meaning effects, to the point of online reorienting interlocutors intentions. All these aspects were investigated using journalistic, political and legal discourses, whose concrete social existence makes them a perfect locus for the generating of the phenomena we are interested in and whose natures make them exploit the many resources that the use of the natural language gives rise to. Such discoursed can all be seen together in one event, the 2000 American presidential election, whose debate between Democrats and Republicans also is an ideal place to test the model we are building and to identify the fascinating meaning effects that intentions, moved by reason and emotions, regarding different kinds of benefits, even being the sort of low cost, can produce through the perfect interaction between what is said and what is implicated mediated by communicative-cognitive-linguistic-logic inferences.

Animals are imbued with adaptive mechanisms spanning from the tissue/organ to the cellular scale which insure that processes of homeostasis are preserved in the landscape of size change. However we and others have postulated that the degree of adaptation is limited and that once outside the normal levels of size fluctuations, cells and tissues function in an aberant manner. In this study we examine the function of muscle in the myostatin null mouse which is an excellent model for hypertrophy beyond levels of normal growth and consequeces of acute starvation to restore mass. We show that muscle growth is sustained through protein synthesis driven by Serum/Glucocorticoid Kinase 1 (SGK1) rather than Akt1. Furthermore our metabonomic profiling of hypertrophic muscle shows that carbon from nutrient sources is being channelled for the production of biomass rather than ATP production. However the muscle displays elevated levels of autophagy and decreased levels of muscle tension. We demonstrate the myostatin null muscle is acutely sensitive to changes in diet and activates both the proteolytic and autophagy programmes and shutting down protein synthesis more extensively than is the case for wild-types. Poignantly we show that acute starvation which is detrimental to wild-type animals is beneficial in terms of metabolism and muscle function in the myostatin null mice by normalising tension production.