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Endothelial inflammatory dysfunction, reflected by reduced NO bioavailability, is recognized as promoting atherosclerosis. NO activates the soluble guanylate cyclase (sGC) and the subsequent generation of cyclic guanosine monophosphate (cGMP), which in turn, serves as a final modulator of vascular relaxation. PDE5A, is a phosphodiesterase that catalyses the hydrolysis of cGMP into GMP, thus curtailing NO signalling and favouring pro-angiogenic effects. The ω-3 fatty acids DHA and eicosapentaenoic acid (EPA) are considered health-promoting nutrients. However, molecular mechanisms underlying their effects remain incompletely understood. Since recent data suggest a role for endothelial phosphodiesterase (PDE)5A in the modulation of endothelial-dependent vasodilation and angiogenesis, we investigated whether inflammatory stimuli known to be involved in the inflammation-mediated endothelial dysfunction and angiogenesis affect the endothelial PDE5A expression, and whether cell exposure to DHA modifies such PDE5A expression.Methods: Human umbilical vein endothelial cells (HUVEC) were treated with inflammatory or pro-angiogenic stimuli interleukin (IL)-1, tumour necrosis factor (TNF), IL-6, vascular endothelial growth factor (VEGF) and phorbol myristate acetate (PMA) for 0-24 hours. After this time, PDE5A expression was assessed by Western analysis, while mRNA expression was investigated by quantitative PCR (qPCR). In a subset of experiments HUVEC were treated with 0-50 μmol/L DHA for 48 hours before stimulation with IL-1, and PDE5A expression was then evaluated by Western analysis and qPCR.Results: PDE5A protein expression increased significantly after stimulation with 10 ng/mL IL-1 and TNF for 12 h (P<0.01 vs control), but not after stimulation with IL-6, PMA and VEGF. At the same concentrations, IL-1 also increased mRNA expression by 40% (P<0.05 vs control). DHA treatment of HUVEC before IL-1 stimulation reduced PDE5A induction at both protein and mRNA levels (P<0.05 vs IL-1).Conclusions: Classical pro-inflammatory - but not specifically pro-angiogenic - stimuli significantly induce PDE5A expression in endothelial cells, thus suggesting the involvement of an inflammatory signaling in the mechanisms leading to PD5A expression. Since PDE5A inhibitors are now approved for use in erectile dysfunction and pulmonary hypertension and have a potential in treating other disease states featuring endothelial dysfunction, downregulating the inflammation-mediated expression of PDE5A by DHA may positively impact the NO/sGC/cGMP axis and reproduce, at least partially, the therapeutic potential of PDE5 inhibitors. [ABSTRACT FROM AUTHOR]

Introduction: Human skeletal muscle displays an epigenetic memory of resistance exercise induced by hypertrophy. It is unknown, however, whether high-intensity interval training (HIIT) also evokes an epigenetic muscle memory. This study employed repeated training intervention interspersed with a detraining period to assess epigenetic memory of HIIT., Methods: Twenty healthy subjects (25±5yrs) completed two HIIT interventions (training and retraining) lasting 2 months, separated by 3 months of detraining. Measurements at baseline, after training, detraining and retraining included maximal oxygen consumption (V̇ O 2max ). Vastus lateralis biopsies were taken for genome-wide DNA methylation and targeted gene expression analyses., Results: V̇ O 2max improved during training and retraining (p<0.001) without differences between interventions (p>0.58). Thousands of differentially methylated positions (DMPs) predominantly demonstrated a hypomethylated state after training, retained even after 3-months exercise cessation and into retraining. Five genes; ADAM19, INPP5a, MTHFD1L, CAPN2, SLC16A3 possessed differentially methylated regions (DMRs) with retained hypomethylated memory profiles and increased gene expression. The retained hypomethylation during detraining was associated with an enhancement in expression of the same genes even after 3 months of detraining. SLC16A3, INPP5a, CAPN2 are involved in lactate transport and calcium signaling., Conclusions: Despite similar physiological adaptations between training and retraining, memory profiles were found at epigenetic and gene expression level, characterized by retained hypomethylation and increased gene expression after training into long-term detraining and retraining. These genes were associated with calcium signaling and lactate transport. Whilst significant memory was not observed in physiological parameters, our novel findings indicate that human skeletal muscle possesses an epigenetic memory of HIIT.

Background: Degeneration of the motoneuron and neuromuscular junction (NMJ) and loss of motor units (MUs) contribute to age-related muscle wasting and weakness associated with sarcopenia. However, these features have not been comprehensively investigated in humans. This study aimed to compare neuromuscular system integrity and function at different stages of sarcopenia, with a particular focus on NMJ stability and MU properties., Methods: We recruited 42 young individuals (Y) (aged 25.98 ± 4.6 years; 57% females) and 88 older individuals (aged 75.9 ± 4.7 years; 55% females). The older group underwent a sarcopenia screening according to the revised guidelines of the European Working Group on Sarcopenia in Older People 2. In all groups, knee extensor muscle force was evaluated by isometric dynamometry, muscle morphology by ultrasound and MU potential properties by intramuscular electromyography (iEMG). MU number estimate (iMUNE) and blood samples were obtained. Muscle biopsies were collected in a subgroup of 16 Y and 52 older participants., Results: Thirty-nine older individuals were non-sarcopenic (NS), 31 pre-sarcopenic (PS) and 18 sarcopenic (S). A gradual decrease in quadriceps force, cross-sectional area and appendicular lean mass was observed across the different stages of sarcopenia (for all P < 0.0001). Handgrip force and the Short Physical Performance Battery score also showed a diminishing trend. iEMG analyses revealed elevated near fibre segment jitter in NS, PS and S compared with Y (Y vs. NS and S: P < 0.0001; Y vs. PS: P = 0.0169), suggestive of age-related impaired NMJ transmission. Increased C-terminal agrin fragment (P < 0.0001) and altered caveolin 3 protein expression were consistent with age-related NMJ instability in all the older groups. The iMUNE was lower in all older groups (P < 0.0001), confirming age-related loss of MUs. An age-related increase in MU potential complexity was also observed. These observations were accompanied by increased muscle denervation and axonal damage, evinced by the increase in neural cell adhesion molecule-positive fibres (Y vs. NS: P < 0.0001; Y vs. S: P = 0.02) and the increase in serum concentration of neurofilament light chain (P < 0.0001), respectively. Notably, most of these MU and NMJ parameters did not differ when comparing older individuals with or without sarcopenia., Conclusions: Alterations in MU properties, axonal damage, an altered innervation profile and NMJ instability are prominent features of the ageing of the neuromuscular system. These neuromuscular alterations are accompanied by muscle wasting and weakness; however, they appear to precede clinically diagnosed sarcopenia, as they are already detectable in older NS individuals., (© 2024 The Author(s). Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

Nemaline myopathies are the most common form of congenital myopathies. Variants in ACTA1 (NEM3) comprise 15-25% of all nemaline myopathy cases. Patients harboring variants in ACTA1 present with a heterogeneous disease course characterized by stable or progressive muscle weakness and, in severe cases, respiratory failure and death. To date, no specific treatments are available. Since NEM3 is an actin-based thin filament disease, we tested the ability of tirasemtiv, a fast skeletal muscle troponin activator, to improve skeletal muscle function in a mouse model of NEM3, harboring the patient-based p.Asp286Gly variant in Acta1. Acute and long-term tirasemtiv treatment significantly increased muscle contractile capacity at submaximal stimulation frequencies in both fast-twitch extensor digitorum longus and gastrocnemius muscle, and intermediate-twitch diaphragm muscle in vitro and in vivo. Additionally, long-term tirasemtiv treatment in NEM3 mice resulted in a decreased respiratory rate with preserved minute volume, suggesting more efficient respiration. Altogether, our data support the therapeutic potential of fast skeletal muscle troponin activators in alleviating skeletal muscle weakness in a mouse model of NEM3 caused by the Acta1:p.Asp286Gly variant., (© 2024 Galli et al.)

Spinal muscular atrophy (SMA) is a genetic disorder characterized by the loss of spinal motor neurons leading to muscle weakness and respiratory failure. Mitochondrial dysfunctions are found in the skeletal muscle of patients with SMA. For obvious ethical reasons, the diaphragm muscle is poorly studied, notwithstanding the very important role that respiratory involvement plays in SMA mortality. The main goal of this study was to investigate diaphragm functionality and the underlying molecular adaptations in SMNΔ7 mice, a mouse model that exhibits symptoms similar to that of patients with intermediate type II SMA. Functional, biochemical, and molecular analyses on isolated diaphragm were performed. The obtained results suggest the presence of an intrinsic energetic imbalance associated with mitochondrial dysfunction and a significant accumulation of reactive oxygen species (ROS). In turn, ROS accumulation can affect muscle fatigue, cause diaphragm wasting, and, in the long run, respiratory failure in SMNΔ7 mice. Exposure to the antioxidant molecule ergothioneine leads to the functional recovery of the diaphragm, confirming the presence of mitochondrial impairment and redox imbalance. These findings suggest the possibility of carrying out a dietary supplementation in SMNΔ7 mice to preserve their diaphragm function and increase their lifespan.

Following acute coronavirus disease 2019 (COVID-19), a substantial proportion of patients showed symptoms and sequelae for several months, namely the postacute sequelae of COVID-19 (PASC) syndrome. Major phenomena are exercise intolerance, muscle weakness, and fatigue. We aimed to investigate the physiopathology of exercise intolerance in patients with PASC syndrome by structural and functional analyses of skeletal muscle. At least 3 mo after infection, nonhospitalized patients with PASC ( n = 11, age: 54 ± 11 yr; PASC) and patients without long-term symptoms ( n = 12, age: 49 ± 9 yr; CTRL) visited the laboratory on four nonconsecutive days. Spirometry, lung diffusion capacity, and quality of life were assessed at rest. A cardiopulmonary incremental exercise test was performed. Oxygen consumption (V̇o 2 ) kinetics were determined by moderate-intensity exercises. Muscle oxidative capacity ( k ) was assessed by near-infrared spectroscopy. Histochemical analysis, O 2 flux ( J O 2 ) by high-resolution respirometry, and quantification of key molecular markers of mitochondrial biogenesis and dynamics were performed in vastus lateralis biopsies. Pulmonary and cardiac functions were within normal range in all patients. V̇o 2peak was lower in PASC than CTRL (24.7 ± 5.0 vs. 32.9 ± 7.4 mL·min -1 ·kg -1 , respectively, P < 0.05). V̇o 2 kinetics was slower in PASC than CTRL (41 ± 12 vs. 30 ± 9 s -1 , P < 0.05). k was lower in PASC than CTRL (1.54 ± 0.49 vs. 2.07 ± 0.51 min -1 , P < 0.05). Citrate synthase, peroxisome proliferator-activated receptor-γ coactivator (PGC)1α, and J O 2 for mitochondrial complex II were significantly lower in PASC vs. CTRL (all P values <0.05). In our cohort of patients with PASC, we showed limited exercise tolerance mainly due to "peripheral" determinants. Substantial reductions were observed for biomarkers of mitochondrial function, content, and biogenesis. PASC syndrome, therefore, appears to negatively impact skeletal muscle function, although the disease is a heterogeneous condition. NEW & NOTEWORTHY Several months after mild acute SARS-CoV-2 infection, a substantial proportion of patients present persisting, and often debilitating, symptoms and sequelae. These patients show reduced quality of life due to exercise intolerance, muscle weakness, and fatigue. The present study supports the hypothesis that "peripheral" impairments at skeletal muscle level, namely, reduced mitochondrial function and markers of mitochondrial biogenesis, are major determinants of exercise intolerance and fatigue, "central" phenomena at respiratory, and cardiac level being less relevant.

Physical inactivity (PI) is a major risk factor of chronic diseases. A major aspect of PI is loss of muscle mass and strength. The latter phenomenon significantly impacts daily life and represent a major issue for global health. Understandably, skeletal muscle itself has been the major focus of studies aimed at understanding the mechanisms underlying loss of mass and strength. Relatively lesser attention has been given to the contribution of alterations in somatomotor control, despite the fact that these changes can start very early and can occur at multiple levels, from the cortex down to the neuromuscular junction (NMJ). It is well known that exposure to chronic inactivity or immobilization causes a disproportionate loss of force compared to muscle mass, i.e. a loss of specific or intrinsic whole muscle force. The latter phenomenon may be partially explained by the loss of specific force of individual muscle fibres, but several other players are very likely to contribute to such detrimental phenomenon. Irrespective of the length of the disuse period, the loss of force is, in fact, more than two-fold greater than that of muscle size. It is very likely that somatomotor alterations may contribute to this loss in intrinsic muscle force. Here we review evidence that alterations of one component of somatomotor control, namely the neuromuscular junction, occur in disuse. We also discuss some of the novel players in NMJ stability (e.g., homer, bassoon, pannexin) and the importance of new established and emerging molecular markers of neurodegenerative processes in humans such as agrin, neural-cell adhesion molecule and light-chain neurofilaments., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Incomplete functional recovery following traumatic peripheral nerve injury is common, mainly because not all axons successfully regenerate and reinnervate target muscles. Exercise can improve functional outcomes increasing the terminal sprouting during the muscle reinnervation. However, exercise is not a panacea per se. Indeed, the type of exercise adopted dramatically impacts the outcomes of rehabilitation therapy. To gain insight into the therapeutic effects of different exercise regimens on reinnervation following traumatic nerve lesion, we evaluated the impact of different clinically transferable exercise protocols (EPs) on metabolic and functional muscle recovery following nerve crush. Methods: The reinnervation of soleus muscle in adult nerve-crushed rats was studied following 6 days of different patterns (continuous or intermittent) and intensities (slow, mid, and fast) of treadmill running EPs. The effects of EPs on muscle fiber multiple innervation, contractile properties, metabolic adaptations, atrophy, and autophagy were assessed using functional and biochemical approaches. Results: Results showed that an intermittent mid-intensity treadmill EP improves soleus muscle reinnervation, whereas a slow continuous running EP worsens the functional outcome. However, the mid-intensity intermittent EP neither enhanced the critical mediators of exercise-induced metabolic adaptations, namely, PGC-1α, nor improved muscle atrophy. Conversely, the autophagy-related marker LC3 increased exclusively in the mid-intensity intermittent EP group. Conclusion: Our results demonstrated that an EP characterized by a mid-intensity intermittent activity enhances the functional muscle recovery upon a nerve crush, thus representing a promising clinically transferable exercise paradigm to improve recovery in humans following peripheral nerve injuries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Di Palma, Ambrogini, Lattanzi, Brocca, Bottinelli, Cuppini, Pellegrino and Sartini.)

A large set of FoxOs-dependent genes play a primary role in controlling muscle mass during hindlimb unloading. Mitochondrial dysfunction can modulate such a process. We hypothesized that endurance exercise before disuse can protect against disuse-induced muscle atrophy by enhancing peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) expression and preventing mitochondrial dysfunction and energy-sensing AMP-activated protein kinase (AMPK) activation. We studied cross sectional area (CSA) of muscle fibers of gastrocnemius muscle by histochemistry following 1, 3, 7, and 14 days of hindlimb unloading (HU). We used Western blotting and qRT-PCR to study mitochondrial dynamics and FoxOs-dependent atrogenes' expression at 1 and 3 days after HU. Preconditioned animals were submitted to moderate treadmill exercise for 7 days before disuse. Exercise preconditioning protected the gastrocnemius from disuse atrophy until 7 days of HU. It blunted alterations in mitochondrial dynamics up to 3 days after HU and the expression of most atrogenes at 1 day after disuse. In preconditioned mice , the activation of atrogenes resumed 3 days after HU when mitochondrial dynamics, assessed by profusion and pro-fission markers (mitofusin 1, MFN1, mitofusin 2, MFN2, optic atrophy 1, OPA1, dynamin related protein 1, DRP1 and fission 1, FIS1), PGC1α levels, and AMPK activation were at a basal level. Therefore, the normalization of mitochondrial dynamics and function was not sufficient to prevent atrogenes activation just a few days after HU. The time course of sirtuin 1 (SIRT1) expression and content paralleled the time course of atrogenes' expression. In conclusion, seven days of endurance exercise counteracted alterations of mitochondrial dynamics and the activation of atrogenes early into disuse. Despite the normalization of mitochondrial dynamics, the effect on atrogenes' suppression died away within 3 days of HU. Interestingly, muscle protection lasted until 7 days of HU. A longer or more intense exercise preconditioning may prolong atrogenes suppression and muscle protection.

Nemaline myopathy, a disease of the actin-based thin filament, is one of the most frequent congenital myopathies. To date, no specific therapy is available to treat muscle weakness in nemaline myopathy. We tested the ability of tirasemtiv, a fast skeletal troponin activator that targets the thin filament, to augment muscle force-both in vivo and in vitro-in a nemaline myopathy mouse model with a mutation (H40Y) in Acta1. In Acta1H40Y mice, treatment with tirasemtiv increased the force response of muscles to submaximal stimulation frequencies. This resulted in a reduced energetic cost of force generation, which increases the force production during a fatigue protocol. The inotropic effects of tirasemtiv were present in locomotor muscles and, albeit to a lesser extent, in respiratory muscles, and they persisted during chronic treatment, an important finding as respiratory failure is the main cause of death in patients with congenital myopathy. Finally, translational studies on permeabilized muscle fibers isolated from a biopsy of a patient with the ACTA1H40Y mutation revealed that at physiological Ca2+ concentrations, tirasemtiv increased force generation to values that were close to those generated in muscle fibers of healthy subjects. These findings indicate the therapeutic potential of fast skeletal muscle troponin activators to improve muscle function in nemaline myopathy due to the ACTA1H40Y mutation, and future studies should assess their merit for other forms of nemaline myopathy and for other congenital myopathies., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Physical activity is emerging as an alternative nonpharmaceutical strategy to prevent and treat a variety of cardiovascular diseases due to its cardiac and skeletal muscle beneficial effects. Oxidative stress occurs in skeletal muscle of chronic heart failure (CHF) patients with possible impact on muscle function decline. We determined the effect of voluntary-free wheel running (VFWR) in preventing protein damage in Tgαq*44 transgenic mice (Tg) characterized by a delayed CHF progression. In the early (6 months) and transition (12 months) phase of CHF, VFWR increased the daily mean distance covered by Tg mice eliminating the difference between Tg and WT present before exercise at 12 months of age (WT Pre-EX 3.62 ± 1.66 vs. Tg Pre-EX 1.51 ± 1.09 km, P < 0.005; WT Post-EX 5.72 ± 3.42 vs. Tg Post-EX 4.17 ± 1.8 km, P > 0.005). This effect was concomitant with an improvement of in vivo cardiac performance [(Cardiac Index (mL/min/cm 2 ): 6 months, untrained-Tg 0.167 ± 0.005 vs. trained-Tg 0.21 ± 0.003, P < 0.005; 12 months, untrained-Tg 0.1 ± 0.009 vs. trained-Tg 0.133 ± 0.005, P < 0.005]. Such effects were associated with a skeletal muscle antioxidant response effective in preventing oxidative damage induced by CHF at the transition phase (untrained-Tg 0.438 ± 0.25 vs. trained-Tg 0.114 ± 0.010, P < 0.05) and with an increased expression of protein control markers (MuRF-1, untrained-Tg 1.12 ± 0.29 vs. trained-Tg 14.14 ± 3.04, P < 0.0001; Atrogin-1, untrained-Tg 0.9 ± 0.38 vs. trained-Tg 7.79 ± 2.03, P < 0.01; Cathepsin L, untrained-Tg 0.91 ± 0.27 vs. trained-Tg 2.14 ± 0.55, P < 0.01). At the end-stage of CHF (14 months), trained-Tg mice showed a worsening of physical performance (decrease in daily activity and weekly distance and time of activity) compared to trained age-matched WT in association with oxidative protein damage of a similar level to that of untrained-Tg mice (untrained-Tg 0.62 ± 0.24 vs. trained-Tg 0.64 ± 0.13, P > 0.05). Prolonged voluntary physical activity performed before the onset of CHF end-stage, appears to be a useful tool to increase cardiac function and to reduce skeletal muscle oxidative damage counteracting physical activity decline., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Rationale: The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency and increases morbidity, duration of hospital stay, and health care costs. The mechanisms underlying diaphragm weakness are unknown, but might include mitochondrial dysfunction and oxidative stress., Objectives: We hypothesized that weakness of diaphragm muscle fibers in critically ill patients is accompanied by impaired mitochondrial function and structure, and by increased markers of oxidative stress., Methods: To test these hypotheses, we studied contractile force, mitochondrial function, and mitochondrial structure in diaphragm muscle fibers. Fibers were isolated from diaphragm biopsies of 36 mechanically ventilated critically ill patients and compared with those isolated from biopsies of 27 patients with suspected early-stage lung malignancy (control subjects)., Measurements and Main Results: Diaphragm muscle fibers from critically ill patients displayed significant atrophy and contractile weakness, but lacked impaired mitochondrial respiration and increased levels of oxidative stress markers. Mitochondrial energy status and morphology were not altered, despite a lower content of fusion proteins., Conclusions: Critically ill patients have manifest diaphragm muscle fiber atrophy and weakness in the absence of mitochondrial dysfunction and oxidative stress. Thus, mitochondrial dysfunction and oxidative stress do not play a causative role in the development of atrophy and contractile weakness of the diaphragm in critically ill patients.

Cardiac function, skeletal (soleus) muscle oxidative metabolism, and the effects of exercise training were evaluated in a transgenic murine model (Tgα q *44) of chronic heart failure during the critical period between the occurrence of an impairment of cardiac function and the stage at which overt cardiac failure ensues (i.e., from 10 to 12 mo of age). Forty-eight Tgα q *44 mice and 43 wild-type FVB controls were randomly assigned to control groups and to groups undergoing 2 mo of intense exercise training (spontaneous running on an instrumented wheel). In mice evaluated at the beginning and at the end of training we determined: exercise performance (mean distance covered daily on the wheel); cardiac function in vivo (by magnetic resonance imaging); soleus mitochondrial respiration ex vivo (by high-resolution respirometry); muscle phenotype [myosin heavy chain (MHC) isoform content; citrate synthase (CS) activity]; and variables related to the energy status of muscle fibers [ratio of phosphorylated 5'-AMP-activated protein kinase (AMPK) to unphosphorylated AMPK] and mitochondrial biogenesis and function [peroxisome proliferative-activated receptor-γ coactivator-α (PGC-1α)]. In the untrained Tgα q *44 mice functional impairments of exercise performance, cardiac function, and soleus muscle mitochondrial respiration were observed. The impairment of mitochondrial respiration was related to the function of complex I of the respiratory chain, and it was not associated with differences in CS activity, MHC isoforms, p-AMPK/AMPK, and PGC-1α levels. Exercise training improved exercise performance and cardiac function, but it did not affect mitochondrial respiration, even in the presence of an increased percentage of type 1 MHC isoforms. Factors "upstream" of mitochondria were likely mainly responsible for the improved exercise performance. NEW & NOTEWORTHY Functional impairments in exercise performance, cardiac function, and soleus muscle mitochondrial respiration were observed in transgenic chronic heart failure mice, evaluated in the critical period between the occurrence of an impairment of cardiac function and the terminal stage of the disease. Exercise training improved exercise performance and cardiac function, but it did not affect the impaired mitochondrial respiration. Factors "upstream" of mitochondria, including an enhanced cardiovascular O 2 delivery, were mainly responsible for the functional improvement., (Copyright © 2017 the American Physiological Society.)

Key Points: Loss of muscle mass and strength in the growing population of elderly people is a major health concern for modern societies. This condition, termed sarcopenia, is a major cause of falls and of the subsequent increase in morbidity and mortality. Despite numerous studies on the impact of ageing on individual muscle fibres, the contribution of single muscle fibre adaptations to ageing-induced atrophy and functional impairment is still unsettled. The level of physical function and disuse is often associated with ageing. We studied relatively healthy older adults in order to understand the effects of ageing per se without the confounding impact of impaired physical function. We found that in healthy ageing, structural and functional alterations of muscle fibres occur. Protein post-translational modifications, oxidation and phosphorylation contribute to such alterations more than loss of myosin and other muscle protein content., Abstract: Contradictory results have been reported on the impact of ageing on structure and functions of skeletal muscle fibres, likely to be due to a complex interplay between ageing and other phenomena such as disuse and diseases. Here we recruited healthy, physically and socially active young (YO) and elderly (EL) men in order to study ageing per se without the confounding effects of impaired physical function. In vivo analyses of quadriceps and in vitro analyses of vastus lateralis muscle biopsies were performed. In EL subjects, our results show that (i) quadriceps volume, maximum voluntary contraction isometric torque and patellar tendon force were significantly lower; (ii) muscle fibres went through significant atrophy and impairment of specific force (isometric force/cross-sectional area) and unloaded shortening velocity; (iii) myosin/actin ratio and myosin content in individual muscle fibres were not altered; (iv) the muscle proteome went through quantitative adaptations, namely an up-regulation of the content of several groups of proteins among which were myofibrillar proteins and antioxidant defence systems; (v) the muscle proteome went through qualitative adaptations, namely phosphorylation of several proteins, including myosin light chain-2 slow and troponin T and carbonylation of myosin heavy chains. The present results indicate that impairment of individual muscle fibre structure and function is a major feature of ageing per se and that qualitative adaptations of muscle proteome are likely to be more involved than quantitative adaptations in determining such a phenomenon., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Key Points: Muscle atrophy is a debilitating condition that affects a high percentage of the population with a negative impact on quality of life. Dissecting the molecular level of the atrophy process, and the similarities/dissimilarities among different catabolic conditions, is a necessary step for designing specific countermeasures to attenuate/prevent muscle loss. The FoxO family transcription factors represent one of the most important regulators of atrophy programme stimulating the expression of many atrophy-related genes. The findings of the present study clearly indicate that the signalling network controlling the atrophy programme is specific for each catabolic condition., Abstract: Muscle atrophy is a complex process that is in common with many different catabolic diseases including disuse/inactivity and ageing. The signalling pathways that control the atrophy programme in the different disuse/inactivity conditions have not yet been completely dissected. The inhibition of FoxO is considered to only partially spare muscle mass after denervation. The present study aimed: (i) to determine the involvement of FoxOs in hindlimb suspension disuse model; (ii) to define whether the molecular events of protein breakdown are shared among different unloaded muscles; and finally (iii) to compare the data obtained in this model with another model of inactivity such as denervation. Both wild-type and muscle-specific FoxO1,3,4 knockout (FoxO1,3,4 -/- ) mice were unloaded for 3 and 14 days and muscles were characterized by functional, morphological, biochemical and molecular assays. The data obtained show that FoxOs are required for muscle loss and force drop during unloading. Moreover, we found that FoxO-dependent atrogenes vary in different unloaded muscles and that they diverge from denervation. The findings of the present study clearly indicate that the signalling network that controls the atrophy programme is specific for each catabolic condition., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Short-term disuse and subsequent recovery affect whole muscle and single myofiber contractile function in young and old. While the loss and recovery of single myofiber specific force (SF) following disuse and rehabilitation has been shown to correlate with alterations in myosin concentrations in young, it is unknown whether similar relationships exist in old. Therefore, the purpose of the present study was to examine the effect of 14days lower limb disuse followed by 28days of active recovery on single muscle fiber myosin content in old (68yrs) and young (24yrs) recreationally physically active healthy men. Following disuse, myosin content decreased (p<0.05) in MHC 1 (young -28%, old -19%) and 2a fibers (young -23%, old -32%). In old, myosin content decreased more (p<0.05) in MHC 2a vs 1 fibers. Following recovery, myosin content increased (p<0.05) and returned to pre-disuse levels for both young and old in both fiber types, with MHC 2a fibers demonstrating an overshooting in young (+31%, p<0.05) but not old. Strong correlations were observed between myosin content and single fiber SF in both young and old, with greater slope steepness in MHC 2a vs 1 fibers indicating an enhanced intrinsic contractile capacity of MHC 2a fibers. In conclusion, adaptive changes in myofiber myosin content appear to occur rapidly following brief periods of disuse (2wks) and after subsequent active recovery (4wks) in young and old, which contribute to alterations in contractile function at the single muscle fiber level. Changes in myosin content appear to occur independently of age, while influenced by fiber type (MHC isoform) in young but not old., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Key Points: It is generally assumed that muscle fibres go through atrophy following disuse with a loss of specific force and an increase in unloaded shortening velocity. However, the underlying mechanisms remain to be clarified. Most studies have focused on events taking place during the development of disuse, whereas the subsequent recovery phase, which is equally important, has received little attention. Our findings support the hypotheses that the specific force of muscle fibres decreased following unilateral lower limb suspension (ULLS) and returned to normal after 3 weeks of active recovery as a result of a loss and recovery of myosin and actin content. Furthermore, muscle fibres went through extensive qualitative changes in muscle protein pattern following ULLS, and these were reversed by active recovery. Resistance training was very effective in restoring both muscle mass and qualitative muscle changes, indicating that long-term ULLS did not prevent the positive effect of exercise on human muscle., Abstract: Following disuse, muscle fibre function goes through adaptations such as a loss of specific force (PO /CSA) and an increase in unloaded shortening velocity, which could be a result of both quantitative changes (i.e. atrophy) and qualitative changes in protein pattern. The underlying mechanisms remain to be clarified. In addition, little is known about the recovery of muscle mass and strength following disuse. In the present study, we report an extensive dataset describing, in detail,the functional and protein content adaptations of skeletal muscle in response to both disuse and re-training. Eight young healthy subjects were subjected to 3 weeks of unilateral lower limb suspension (ULLS), a widely used human model of disuse skeletal muscle atrophy. Needle biopsies samples were taken from the vastus lateralis muscle Pre-ULLS, Post-ULLS and after 3 weeks of recovery during which heavy resistance training was performed. After disuse, cross-sectional area (CSA), PO /CSA and myosin concentration (MC) decreased in both type 1 and 2A skinned muscle fibres. After recovery, CSA and MC returned to levels comparable to those observed before disuse, whereas Po/CSA and unloaded shortening velocity reached a higher level. Myosin heavy chain isoform composition of muscle samples did not differ among the experimental groups. To study the mechanisms underlying such adaptations, a two-dimensional proteomic analysis was performed. ULLS induced a reduction of myofibrillar, metabolic (glycolytic and oxidative) and anti-oxidant defence system protein content. Resistance training was very effective in counteracting ULLS-induced alterations, indicating that long-term ULLS did not prevent the positive effect of exercise on human muscle., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Introduction: The aim of this study was to understand the effects of short-term glucocorticoid administration in healthy subjects., Methods: Five healthy men received dexamethasone (8 mg/day) for 7 days. Vastus lateralis muscle biopsy and knee extension torque measurement were performed before and after administration. A large number of individual muscle fibers were dissected from the biopsy samples (pre-administration: n = 165, post-administration: n = 177)., Results: Maximal knee extension torque increased after administration (∼ 13%), whereas both type 1 and type 2A fibers had decreased cross-sectional area (type 1: ∼ 11%, type 2A: ∼ 17%), myosin loss (type 1: ∼ 18%, type 2A: ∼ 32%), and loss of specific force (type 1: ∼ 24%, type 2A: ∼ 33%), which were preferential for fast fibers., Conclusion: Short-term dexamethasone administration in healthy subjects elicits quantitative and qualitative adaptations of muscle fibers that precede (and may predict) the clinical appearance of myopathy in glucocorticoid-treated subjects., (© 2015 Wiley Periodicals, Inc.)

Key Points: Skeletal muscle atrophy occurs as a result of disuse. Although several studies have established that a decrease in protein synthesis and increase in protein degradation lead to muscle atrophy, little is known about the triggers underlying such processes. A growing body of evidence challenges oxidative stress as a trigger of disuse atrophy; furthermore, it is also becoming evident that mitochondrial dysfunction may play a causative role in determining muscle atrophy. Mitochondrial fusion and fission have emerged as important processes that govern mitochondrial function and PGC-1α may regulate fusion/fission events. Although most studies on mice have focused on the anti-gravitary slow soleus muscle as it is preferentially affected by disuse atrophy, several fast muscles (including gastrocnemius) go through a significant loss of mass following unloading. Here we found that in fast muscles an early down-regulation of pro-fusion proteins, through concomitant AMP-activated protein kinase (AMPK) activation, can activate catabolic systems, and ultimately cause muscle mass loss in disuse. Elevated muscle PGC-1α completely preserves muscle mass by preventing the fall in pro-fusion protein expression, AMPK and catabolic system activation, suggesting that compounds inducing PGC-1α expression could be useful to treat and prevent muscle atrophy., Abstract: The mechanisms triggering disuse muscle atrophy remain of debate. It is becoming evident that mitochondrial dysfunction may regulate pathways controlling muscle mass. We have recently shown that mitochondrial dysfunction plays a major role in disuse atrophy of soleus, a slow, oxidative muscle. Here we tested the hypothesis that hindlimb unloading-induced atrophy could be due to mitochondrial dysfunction in fast muscles too, notwithstanding their much lower mitochondrial content. Gastrocnemius displayed atrophy following both 3 and 7 days of unloading. SOD1 and catalase up-regulation, no H2 O2 accumulation and no increase of protein carbonylation suggest the antioxidant defence system efficiently reacted to redox imbalance in the early phases of disuse. A defective mitochondrial fusion (Mfn1, Mfn2 and OPA1 down-regulation) occurred together with an impairment of OXPHOS capacity. Furthermore, at 3 days of unloading higher acetyl-CoA carboxylase (ACC) phosphorylation was found, suggesting AMP-activated protein kinase (AMPK) pathway activation. To test the role of mitochondrial alterations we used Tg-mice overexpressing PGC-1α because of the known effect of PGC-1α on stimulation of Mfn2 expression. PGC-α overexpression was sufficient to prevent (i) the decrease of pro-fusion proteins (Mfn1, Mfn2 and OPA1), (ii) activation of the AMPK pathway, (iii) the inducible expression of MuRF1 and atrogin1 and of authopagic factors, and (iv) any muscle mass loss in response to disuse. As the effects of increased PGC-1α activity were sustained throughout disuse, compounds inducing PGC-1α expression could be useful to treat and prevent muscle atrophy also in fast muscles., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Prolonged skeletal muscle inactivity causes muscle fibre atrophy. Redox imbalance has been considered one of the major triggers of skeletal muscle disuse atrophy, but whether redox imbalance is actually the major cause or simply a consequence of muscle disuse remains of debate. Here we hypothesized that a metabolic stress mediated by PGC-1α down-regulation plays a major role in disuse atrophy. First we studied the adaptations of soleus to mice hindlimb unloading (HU) in the early phase of disuse (3 and 7 days of HU) with and without antioxidant treatment (trolox). HU caused a reduction in cross-sectional area, redox status alteration (NRF2, SOD1 and catalase up-regulation), and induction of the ubiquitin proteasome system (MuRF-1 and atrogin-1 mRNA up-regulation) and autophagy (Beclin1 and p62 mRNA up-regulation). Trolox completely prevented the induction of NRF2, SOD1 and catalase mRNAs, but not atrophy or induction of catabolic systems in unloaded muscles, suggesting that oxidative stress is not a major cause of disuse atrophy. HU mice showed a marked alteration of oxidative metabolism. PGC-1α and mitochondrial complexes were down-regulated and DRP1 was up-regulated. To define the link between mitochondrial dysfunction and disuse muscle atrophy we unloaded mice overexpressing PGC-1α. Transgenic PGC-1α animals did not show metabolic alteration during unloading, preserving muscle size through the reduction of autophagy and proteasome degradation. Our results indicate that mitochondrial dysfunction plays a major role in disuse atrophy and that compounds inducing PGC-1α expression could be useful to treat/prevent muscle atrophy., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

In order to get a comprehensive picture of the complex adaptations of human skeletal muscle to disuse and further the understanding of the underlying mechanisms, we participated in two bed rest campaigns, one lasting 35 days and one 24 days. In the first bed rest (BR) campaign, myofibrillar proteins, metabolic enzymes and antioxidant defence systems were found to be down-regulated both post-8 days and post-35 days BR by proteomic analysis of vastus lateralis muscle samples from nine subjects. Such profound alterations occurred early (post-8 days BR), before disuse atrophy developed, and persisted through BR (post-35 days BR). To understand the mechanisms underlying the protein adaptations observed, muscle biopsies from the second bed rest campaign (nine subjects) were used to evaluate the adaptations of master controllers of the balance between muscle protein breakdown and muscle protein synthesis (MuRF-1 and atrogin-1; Akt and p70S6K), of autophagy (Beclin-1, p62, LC3, bnip3, cathepsin-L), of expression of antioxidant defence systems (NRF2) and of energy metabolism (PGC-1α, SREBP-1, AMPK). The results indicate that: (i) redox imbalance and remodelling of muscle proteome occur early and persist through BR; (ii) impaired energy metabolism is an early and persistent phenomenon comprising both the oxidative and glycolytic one; (iii) although both major catabolic systems, ubiquitin proteasome and autophagy, could contribute to the progression of atrophy late into BR, a decreased protein synthesis cannot be ruled out; (iv) a decreased PGC-1α, with the concurrence of SREBP-1 up-regulation, is a likely trigger of metabolic impairment, whereas the AMPK pathway is unaltered.

A pivotal role has been ascribed to oxidative stress in determining the imbalance between protein synthesis and degradation leading to muscle atrophy in many pathological conditions and in disuse. However, a large variability in disuse-induced alteration of redox homeostasis through muscles, models and species emerges from the literature. Whereas the causal role of oxidative stress appears well established in the mechanical ventilation model, findings are less compelling in the hindlimb unloaded mice and very limited in humans. The mere coexistence of muscle atrophy, indirect indexes of increased reactive oxygen species (ROS) production and impairment of antioxidant defence systems, in fact, does not unequivocally support a causal role of oxidative stress in the phenomenon. We hypothesise that in some muscles, models and species only, due to a large redox imbalance, the leading phenomena are activation of proteolysis and massive oxidation of proteins, which would become more susceptible to degradation. In other conditions, due to a lower extent and variable time course of ROS production, different ROS-dependent, but also -independent intracellular pathways might dominate determining the variable extent of atrophy and even dispensable protein oxidation. The ROS production and removal are complex and finely tuned phenomena. They are indeed important intracellular signals and redox balance maintains normal muscle homeostasis and can underlie either positive or negative adaptations to exercise. A precise approach to determine the levels of ROS in living cells in various conditions appears to be of paramount importance to define and support such hypotheses.

Statins and fibrates can cause myopathy. To further understand the causes of the damage we performed a proteome analysis in fast-twitch skeletal muscle of rats chronically treated with different hypolipidemic drugs. The proteomic maps were obtained from extensor digitorum longus (EDL) muscles of rats treated for 2-months with 10mg/kg atorvastatin, 20 mg/kg fluvastatin, 60 mg/kg fenofibrate and control rats. The proteins differentially expressed were identified by mass spectrometry and further analyzed by immunoblot analysis. We found a significant modification in 40 out of 417 total spots analyzed in atorvastatin treated rats, 15 out of 436 total spots in fluvastatin treated rats and 21 out of 439 total spots in fenofibrate treated rats in comparison to controls. All treatments induced a general tendency to a down-regulation of protein expression; in particular, atorvastatin affected the protein pattern more extensively with respect to the other treatments. Energy production systems, both oxidative and glycolytic enzymes and creatine kinase, were down-regulated following atorvastatin administration, whereas fenofibrate determined mostly alterations in glycolytic enzymes and creatine kinase, oxidative enzymes being relatively spared. Additionally, all treatments resulted in some modifications of proteins involved in cellular defenses against oxidative stress, such as heat shock proteins, and of myofibrillar proteins. These results were confirmed by immunoblot analysis. In conclusions, the proteomic analysis showed that either statin or fibrate administration can modify the expression of proteins essential for skeletal muscle function suggesting potential mechanisms for statin myopathy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The aim of the present study was to define the chronic effects of neuromuscular electrical stimulation (NMES) on the neuromuscular properties of human skeletal muscle. Eight young healthy male subjects were subjected to 25 sessions of isometric NMES of the quadriceps muscle over an 8-wk period. Needle biopsies were taken from the vastus lateralis muscle before and after training. The training status, myosin heavy chain (MHC) isoform distribution, and global protein pattern, as assessed by proteomic analysis, widely varied among subjects at baseline and prompted the identification of two subgroups: an "active" (ACT) group, which performed regular exercise and had a slower MHC profile, and a sedentary (SED) group, which did not perform any exercise and had a faster MHC profile. Maximum voluntary force and neural activation significantly increased after NMES in both groups (+∼30% and +∼10%, respectively). Both type 1 and 2 fibers showed significant muscle hypertrophy. After NMES, both groups showed a significant shift from MHC-2X toward MHC-2A and MHC-1, i.e., a fast-to-slow transition. Proteomic maps showing ∼500 spots were obtained before and after training in both groups. Differentially expressed proteins were identified and grouped into functional categories. The most relevant changes regarded 1) myofibrillar proteins, whose changes were consistent with a fast-to-slow phenotype shift and with a strengthening of the cytoskeleton; 2) energy production systems, whose changes indicated a glycolytic-to-oxidative shift in the metabolic profile; and 3) antioxidant defense systems, whose changes indicated an enhancement of intracellular defenses against reactive oxygen species. The adaptations in the protein pattern of the ACT and SED groups were different but were, in both groups, typical of both resistance (i.e., strength gains and hypertrophy) and endurance (i.e., a fast-to-slow shift in MHC and metabolic profile) training. These training-induced adaptations can be ascribed to the peculiar motor unit recruitment pattern associated with NMES.

The review first briefly summarizes how myosin isoforms have been identified as the major determinant of the functional variability among skeletal muscle fibres. The latter feature is a major characteristic of muscle fibres and a major basis of skeletal muscle heterogeneity and plasticity in vivo. Then, evidence is reported, which indicates that the properties of muscle fibres can vary with no change in the myosin isoform they express. Moreover, the physiological and pathological conditions (ageing, disuse, exercise training, muscular dystrophy) in which such myosin isoform independent change in functional properties occurs and the possible underlying mechanisms are considered. Finally, the known molecular bases of the functional differences among slow and fast isoforms are briefly dealt with.

Oxidative stress was proposed as a trigger of muscle impairment in various muscle diseases. The hindlimb-unloaded (HU) rodent is a model of disuse inducing atrophy and slow-to-fast transition of postural muscles. Here, mice unloaded for 14 days were chronically treated with the selective antioxidant trolox. After HU, atrophy was more pronounced in the slow-twitch soleus muscle (Sol) than in the fast-twitch gastrocnemius and tibialis anterior muscles, and was absent in extensor digitorum longus muscle. In accord with the phenotype transition, HU Sol showed a reduced expression of myosin heavy chain type 2A (MHC-2A) and increase in MHC-2X and MHC-2B isoforms. In parallel, HU Sol displayed an increased sarcolemma chloride conductance related to an increased expression of ClC-1 channels, changes in excitability parameters, a positive shift of the mechanical threshold, and a decrease of the resting cytosolic calcium concentration. Moreover, the level of lipoperoxidation increased proportionally to the degree of atrophy of each muscle type. As expected, trolox treatment fully prevented oxidative stress in HU mice. Atrophy was not prevented but the drug significantly attenuated Sol phenotypic transition and excitability changes. Trolox treatment had no effect on control mice. These results suggest possible benefits of antioxidants in protecting muscle against disuse., ((c) 2010 Elsevier Ltd. All rights reserved.)

Since the middle of the 1980s, it was understood that myosin, the motor of contraction, can be expressed in several isoforms. The isoforms of the myosin heavy-chain (MHC) portion of the molecule were found to be mostly responsible for the diversity in the contractile and energetic properties of muscle fibers. In humans, three MHC isoforms are expressed in limb muscles (MHC-1, MHC-2A and MHC-2X) and they generate three pure fiber types (types 1, 2A and 2X) and two hybrid types (types 1-2A and -2AX). Type 1, 2A and 2X fibers widely differ with respect to most of their contractile and energetic properties, and a change in their relative distribution within muscles is known to modulate their functional properties in vivo through a "qualitative" mechanism. On the basis of the MHC regulation of muscle fibers properties, it is expected that a given fiber type develops the same force and shortens at the same speed regardless of the physiologic and pathologic conditions under which the muscle works. Surprisingly, several evidences have been accumulating to show that in aging and disuse, the properties of a muscle fiber type can change with no change in its myosin isoform content. This short review considers the latter phenomenon and the possible underlying mechanisms.

Two-dimensional proteomic maps of soleus (Sol), a slow oxidative muscle, and gastrocnemius (Gas), a fast glycolytic muscle of control mice (CTRL), of mice hindlimb unloaded for 14 days (HU mice) and of HU mice treated with trolox (HU-TRO), a selective and potent antioxidant, were compared. The proteomic analysis identified a large number of differentially expressed proteins in a pool of approximately 800 proteins in both muscles. The protein pattern of Sol and Gas adapted very differently to hindlimb unloading. The most interesting adaptations related to the cellular defense systems against oxidative stress and energy metabolism. In HU Sol, the antioxidant defense systems and heat shock proteins were downregulated, and protein oxidation index and lipid peroxidation were higher compared with CTRL Sol. In contrast, in HU Gas the antioxidant defense systems were upregulated, and protein oxidation index and lipid peroxidation were normal. Notably, both Sol and Gas muscles and their muscle fibres were atrophic. Antioxidant administration prevented the impairment of the antioxidant defense systems in Sol and further enhanced them in Gas. Accordingly, it restored normal levels of protein oxidation and lipid peroxidation in Sol. However, muscle and muscle fibre atrophy was not prevented either in Sol or in Gas. A general downsizing of all energy production systems in Sol and a shift towards glycolytic metabolism in Gas were observed. Trolox administration did not prevent metabolic adaptations in either Sol or Gas. The present findings suggest that oxidative stress is not a major determinant of muscle atrophy in HU mice.

Biopsy samples were taken from the vastus lateralis muscle of seven male subjects pre- and post-35 days bed rest (BR). The myosin heavy chain (MHC) isoform distribution of the samples was determined by densitometry of MHC bands separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Individual muscle fibers were dissected from biopsy samples pre-BR (n=143) and post-BR (n=144). They were studied as regards cross-sectional area (CSA), myosin content by quantitative electrophoresis and myosin actin (M/A) ratio by densitometry of myosin and actin bands of individual muscle fibers. All fibers were typed according to their MHC isoform content determined by SDS-PAGE. A decrease in MHC-1 relative content and an increase in MHC-2X content of whole muscle samples were found, suggesting a slow to fast shift in muscle phenotype. Consistently, fiber type distribution was shifted toward type 2X and 2AX fibers. Muscle fiber atrophy occurred at variable extent among fiber types. Myosin concentration was significantly lower in type 1 and type 2A muscle fibers post-BR than pre-BR, whereas M/A ratio did not vary. The latter findings indicate a disproportionate loss of myosin compared with fiber CSA and a proportional loss of myosin and actin.

We aimed to assess whether a specific mixture of amino acid (AA) supplements counteracts the metabolic and functional changes in the streptozotocin (STZ)-induced diabetic rat heart model. Adult male Wistar rats were divided into 6 groups (n = 10 each) and treated for 43 days: nondiabetic controls, nondiabetic rats given an AA mixture (0.1 g/kg per day), diabetic rats (induced with 65 mg/kg STZ given intraperitoneally), diabetic rats given AAs, diabetic rats given insulin (5 IU/day given subcutaneously), and diabetic rats given insulin plus AAs. During treatment, glycemia and insulinemia levels were measured in all groups. Changes in enzyme (reduced nicotinamide adenine dinucleotide-dehydrogenase, cytochrome c oxidase) activities and myosin heavy chain (MHC) composition were measured in the left ventricle. In 5 rats contractile function was assessed by measuring maximal shortening velocity of skinned ventricular trabeculae and the expression of translational regulator mammalian target of rapamycin (mTOR) was also found. STZ-induced diabetes was associated with reduced myocardial contractility, overall loss of oxidative capacity, a shift toward a slower MHC phenotype, and decreased mTOR tissue content. All of these changes appeared to be reversible with insulin. AA supplements partially restored the myocardial and oxidative dysfunction and also increased mTOR tissue content. The combination of insulin and AAs did not have a synergistic effect on either enzymatic or functional profiles. We conclude that AA supplements may contribute to restoring the oxidative and contractile dysfunction of diabetic rat hearts, probably through an mTOR-insulin independent mechanism.

Oxidative stress plays an important role in the pathogenesis of diabetic complications. We investigated the effects of a specific oral mixture of amino acid (AA) supplements on the antioxidant defense system, superoxide dismutase (SOD), and heat shock proteins (HSPs: HspB1, similar to Hsp 20 kDa, and HspB7) in the soleus muscle of streptozotocin (STZ)-diabetic mice by bidimensional electrophoresis and mass spectrometry. Four groups of 5 mice were considered: nondiabetic control mice, nondiabetic mice given AA supplements (0.1 g/kg per day for 15 days), diabetic mice (induced with STZ 65 mg/kg), and diabetic mice given AAs. AA supplements in the nondiabetic animals were associated with a statistical increase of SOD and no changes in expression of HSPs. Diabetes mellitus decreased antioxidant SOD and increased cellular stress as demonstrated by the overall upregulated HSPs. Administration of AAs counteracted the effects of diabetes, producing upregulation of SOD and downregulation of HSPs. These data suggest a role for AA supplements in controlling the antioxidant defense system and reducing the oxidative stress in diabetic skeletal muscle.

The impact of ageing on force and velocity of human skeletal muscle fibres has been extensively studied. As discrepancies have been reported, it is still unclear whether or not a deterioration of the capacity of muscle fibres to develop force and shortening is involved in determining weakness and decrease in shortening velocity of skeletal muscle of elderly people. We compared myosin heavy chain (MHC) isoform distribution of vastus lateralis muscle, and specific force (Po/CSA) and maximum shortening velocity (Vo) of skeletal muscle fibres among one population of young controls (CTRL) and three populations of elderly (EL) subjects with very variable levels of physical activity: sedentary (EL-SED, n = 3); controls (EL-CTRL, n = 4); endurance trained (EL-END, n = 3). Muscle phenotype was progressively faster in the order EL-END --> CTRL --> EL-CTRL --> EL-SED. Po/CSA and Vo also varied among the different populations of elderly subjects generally showing a decreasing deterioration with increasing activity levels. The results suggest that discrepancies observed so far in age-induced deterioration of contractile properties of muscle fibres could depend on the different activity levels of the populations of elderly subjects enrolled in the studies.

The aim of the present study was to assess age-dependent changes of proteins in the vastus lateralis muscle of physically active elderly and young subjects by a combination of two-dimensional difference gel electrophoresis, SDS-PAGE and ESI-MS/MS. The differences observed in the elderly group included down-regulation of regulatory myosin light chains, particularly the phosphorylated isoforms, a higher proportion of myosin heavy chain isoforms 1 and 2A, and enhanced oxidative and reduced glycolytic capacity.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is not due to a classical mutation within a protein-coding gene. Instead, almost all FSHD patients carry deletions of an integral number of tandem 3.3-kilobase repeat units, termed D4Z4, located on chromosome 4q35 (ref. 3). D4Z4 contains a transcriptional silencer whose deletion leads to inappropriate overexpression in FSHD skeletal muscle of 4q35 genes located upstream of D4Z4 (ref. 4). To identify the gene responsible for FSHD pathogenesis, we generated transgenic mice selectively overexpressing in skeletal muscle the 4q35 genes FRG1, FRG2 or ANT1. We find that FRG1 transgenic mice develop a muscular dystrophy with features characteristic of the human disease; by contrast, FRG2 and ANT1 transgenic mice seem normal. FRG1 is a nuclear protein and several lines of evidence suggest it is involved in pre-messenger RNA splicing. We find that in muscle of FRG1 transgenic mice and FSHD patients, specific pre-mRNAs undergo aberrant alternative splicing. Collectively, our results suggest that FSHD results from inappropriate overexpression of FRG1 in skeletal muscle, which leads to abnormal alternative splicing of specific pre-mRNAs.

A combination of in vivo and in vitro analyses was performed to investigate muscular and neural adaptations of the weaker (nondominant) quadriceps femoris muscle of one healthy individual to short-term electrostimulation resistance training. The increase in maximal voluntary strength (+12%) was accompanied by neural (cross-education effect and increased muscle activation) and muscle adaptations (impairment of whole-muscle contractile properties). Significant changes in myosin heavy chain (MHC) isoforms relative content (+22% for MHC-2A and -28% for MHC-2X), single-fiber cross-sectional area (+27% for type 1 and +6% for type 2A muscle fibers), and specific tension of type 1 (+67%) but not type 2A fibers were also observed after training. Plastic changes in neural control confirm the possible involvement of both spinal and supraspinal structures to electrically evoked contractions. Changes at the single muscle fiber level induced by electrostimulation resistance training were significant and preferentially affected slow, type 1 fibers.

Needle biopsy samples were taken from vastus lateralis muscle (VL) of five male body builders (BB, age 27.4+/-0.93 years; mean+/-s.e.m.), who had being performing hypertrophic heavy resistance exercise (HHRE) for at least 2 years, and from five male active, but untrained control subjects (CTRL, age 29.9+/-2.01 years). The following determinations were performed: anatomical cross-sectional area and volume of the quadriceps and VL muscles in vivo by magnetic resonance imaging (MRI); myosin heavy chain isoform (MHC) distribution of the whole biopsy samples by SDS-PAGE; cross-sectional area (CSA), force (Po), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n=524) of single skinned muscle fibres classified on the basis of MHC isoform composition by SDS-PAGE; actin sliding velocity (Vf) on pure myosin isoforms by in vitro motility assays. In BB a preferential hypertrophy of fast and especially type 2X fibres was observed. The very large hypertrophy of VL in vivo could not be fully accounted for by single muscle fibre hypertrophy. CSA of VL in vivo was, in fact, 54% larger in BB than in CTRL, whereas mean fibre area was only 14% larger in BB than in CTRL. MHC isoform distribution was shifted towards 2X fibres in BB. Po/CSA was significantly lower in type 1 fibres from BB than in type 1 fibres from CTRL whereas both type 2A and type 2X fibres were significantly stronger in BB than in CTRL. Vo of type 1 fibres and Vf of myosin 1 were significantly lower in BB than in CTRL, whereas no difference was observed among fast fibres and myosin 2A. The findings indicate that skeletal muscle of BB was markedly adapted to HHRE through extreme hypertrophy, a shift towards the stronger and more powerful fibre types and an increase in specific force of muscle fibres. Such adaptations could not be fully accounted for by well known mechanisms of muscle plasticity, i.e. by the hypertrophy of single muscle fibre (quantitative mechanism) and by a regulation of contractile properties of muscle fibres based on MHC isoform content (qualitative mechanism). Two BB subjects took anabolic steroids and three BB subjects did not. The former BB differed from the latter BB mostly for the size of their muscles and muscle fibres.

In rat skeletal muscle the unloaded shortening velocity (Vo) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit Vo. However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5 M KCl, 22 degrees C for kinetic assays of myosin, 200 mM ionic strength, 12 degrees C to measure Vo). Here, a method was developed to prepare heavy meromyosin (HMM) from pure myosin isoforms isolated from single muscle fibres and to study k(-AD) (determined from the affinity of the acto-myosin complex for ADP, KAD) and the rate of ATP-induced acto-HMM dissociation (controlled by K1k+2) under the same experimental condition used to measure Vo). In fast-muscle myosin isolated from a wide range of mammalian muscles, k(-AD) was found to be too fast to limit Vo, whereas K1k+2 was of the right magnitude for ATP-induced dissociation of the cross-bridge to limit shortening velocity. The result was unexpected and prompted further experiments using the stopped-flow approach on myosin subfragment-1 (S1) and HMM obtained from bulk preparations of rabbit and rat muscle. These confirmed that the rate of cross-bridge dissociation by ATP limits the velocity of contraction for fast myosin II isoforms at 12 degrees C, while k(-AD) limits the velocity of slow myosin II isoforms. Extrapolating our data to 37 degrees C suggests that at physiological temperature the rate of ADP dissociation may limit Vo for both isoforms.

It is generally believed that the maximum shortening velocity (V(o)) of a skeletal muscle fiber type does not vary unless a change in myosin heavy chain (MHC) isoform composition occurs. However, recent findings have shown that V(o) of a given fiber type can change after training, suggesting the hypothesis that the function of myosin can vary without a change in isoform. The present study addressed the latter hypothesis by studying the function of isolated myosin isoforms by the use of the in vitro motility assay (IVMA) technique. Four young (age 23-29 yr, YO) and four elderly men (age 68-82 yr, EL) underwent a 12-wk progressive resistance training program of the knee extensor muscles and to one pre- and one posttraining biopsy of the vastus lateralis muscle. The significant increase in one-repetition maximum posttraining in both YO and EL indicated that training was effective. After training, MHC isoform composition showed a shift from MHC(2X) toward MHC(2A) in YO and no shift in EL. The velocity of sliding (V(f)) of actin filaments on pure myosin isoforms extracted from single fibers was studied in IVMA. One hundred sixty IVMA samples were prepared from 480 single fibers, and at least 50 filaments were analyzed in each experiment. Whereas no training-induced change was observed in V(f) of myosin isoform 1 either in YO or in EL, a significant increase in V(f) of myosin isoform 2A after training was observed in both YO (18%) and EL (19%). The results indicate that resistance training can change the velocity of the myosin molecule.