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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. [ABSTRACT FROM AUTHOR]

Abstract: 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, 20mg/kg fluvastatin, 60mg/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 &y& Elsevier]

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 ∼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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The aims of the present study were as follows: (1) to examine the adaptational changes to chronic endurance voluntary exercise and (2) to investigate the effects of amino acid supplementation on the adaptational changes induced by endurance training in hindlimb (gastrocnemius, tibialis, soleus) and respiratory (diaphragm) muscles of mice. Male C57Bl6 mice were divided in four groups: control sedentary, sedentary supplemented with amino acid mixture (BigOne, 1.5 mg g day(-1) in drinking water for 8 weeks), running (free access to running wheels for 8 weeks), and running supplemented with amino acid mixture. Myosin heavy chain (MHC) isoform distribution was determined in all muscles considered. Fiber cross-sectional area (CSA) was measured in the soleus muscle. In all muscles except the tibialis, endurance training was associated with an overall shift towards the expression of slower MHC isoforms. Amino acid supplementation produced a shift towards the expression of faster MHC isoforms in the soleus and diaphragm muscles, and partially antagonized the effects of training. Immunohistochemical analysis of CSA of individual muscle fibers from the soleus muscle suggests that voluntary running produced a decrease in the size of type 1 fibers, and amino acid supplementation during training resulted in an increase in size in both type 1 and type 2A fibers. Collectively, these results suggest that the endurance adaptations induced by voluntary running depend on the muscle type, and that amino acid supplementation is able to modulate both fiber size and MHC isoform composition of skeletal muscles in sedentary and exercised mice. [ABSTRACT FROM AUTHOR]

Beta-agonists and glucocorticoids are frequently coprescribed for chronic asthma treatment. In this study the effects of 4 week treatment with beta-agonist clenbuterol (CL) and glucocorticoid dexamethasone (DEX) on respiratory (diaphragm and parasternal) and limb (soleus and tibialis) muscles of the mouse were studied. Myosin heavy chain (MHC) distribution, fibres cross sectional area (CSA), glycolytic (phosphofructokinase, PFK; lactate dehydrogenase, LDH) and oxidative enzyme (citrate synthase, CS; cytochrome oxidase, COX) activities were determined. Muscle samples were obtained from four groups of adult C57/B16 mice: (1) Control (2) Mice receiving CL (CL, 1.5 mg kg-1 day-1 in drinking water) (3) Mice receiving DEX (DEX, 5.7 mg kg-1 day-1 S.C.) (4) Mice receiving both treatments (DEX + CL). As a general rule, CL and DEX showed opposite effects on CSA, MHC distribution, glycolytic and mitochondrial enzyme activities: CL alone stimulated a slow-to-fast transition of MHCs, an increase of PFK and LDH and an increase of muscle weight and fibre CSA; DEX produced an opposite (fast-to-slow transition) change of MHC distribution, a decrease of muscle weight and fibre CSA and in some case an increase of CS. The response varied from muscle to muscle with mixed muscles, as soleus and diaphragm, being more responsive than fast muscles, as tibialis and parasternal. In combined treatments (DEX + CL), the changes induced by DEX or CL alone were generally minimized: in soleus, however, the effects of CL predominated over those of DEX, whereas in diaphragm DEX prevailed over CL. Taken together the results suggest that CL might counteract the unwanted effects on skeletal muscles of chronic treatment with glucocorticoids. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo, is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca2+ activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single-fibre gel electrophoresis. Fibres were activated by using a new set-up that allows development of most of the tension following a temperature jump from 0-1°C to the test temperature (∼12°C). In this way we could prevent the development of sarcomere nonuniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40-70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force-generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin-myosin interactions under stretch become independent of the MHC isoform. [ABSTRACT FROM AUTHOR]

Aging is associated with a progressive loss of muscle mass and strength, defined as sarcopenia. Neuromuscular junction (NMJ) deterioration plays a key role in agerelated musculoskeletal impairment. Moreover, loss of nitric oxide synthase in skeletal muscle is associated with defects at NMJ, 1 and, together with a reduced age-related nitric oxide bioavailability, is causative of muscle wasting and harmed mitochondria. Here we investigated the ability of chronic nitrate supplementation to counteract neuromuscular alterations during aging. 22-months-old C57BL/6 male mice were assigned as follows: old controls (Old-CTRL) and old supplemented with 1.5 mM inorganic nitrate in drinking water for two months (Old-N). All mice were sacrificed at 24 months and skeletal muscles dissected for ex-vivo determinations. A cohort of young male mice (7 months) was also evaluated to quantify age-related changes. Compared to Old-CTRL, treated mice showed a positive remodelling of the NMJ morphology (evaluated through the confocal microscopy) and an improvement of fibers innervation status supported by a decreased postsynaptic fragmentation and a reduced number of NCAM-positive fibers, respectively. Moreover, Old-N mice showed an improvement in mitochondrial respiratory function (assessed through the High-Resolution Respirometry), despite no change in mitochondrial dynamics. Furthermore, nitrate treatment induced an anabolic response through higher activation of factors involved in protein synthesis (P70S6K and S6), consistent with higher Gastrocnemius fibers crosssectional area found in treated animals in comparison to Old-CTRL. The present data indicate that nitrate supplementation has the potential to counteract agerelated neuromuscular deterioration. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 14 days lower limb disuse followed by 28 days of active recovery on single muscle fiber myosin content in old (68 yrs) and young (24 yrs) 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 (2 wks) and after subsequent active recovery (4 wks) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 H2O2 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. [ABSTRACT FROM AUTHOR]

Key points Oxidative stress is widely considered a major cause of muscle loss not only in disuse but also in most chronic diseases, triggering carbonylation of proteins and activation of catabolic pathways involved in their degradation., Here we show that administration of an antioxidant prevents redox imbalance, but does not prevent activation of catabolic pathways and muscle atrophy., We indicate that alterations of oxidative metabolism, occurring in slow soleus muscle, are not just a consequence of disuse, but a major cause of activation of catabolic pathways and loss of mass., This conclusion is confirmed by the observation that muscle-specific overexpression of PGC-1α, a master regulator of mitochondrial biogenesis, prevents activation of catabolic systems and disuse muscle atrophy., These findings contribute to a better mechanistic understanding of disuse muscle loss., Abstract 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. [ABSTRACT FROM AUTHOR]

Key points It is still debated whether an imbalance between production and removal of reactive oxygen species is a major trigger of disuse skeletal muscle atrophy in human limb muscles and what the underlying mechanisms are., In the bed rest model of human disuse, redox imbalance, impairment of antioxidant defence systems and metabolic derangement occurred early, before vastus lateralis muscle atrophy developed, and persisted through 35 days of bed rest., Down-regulation of PGC-1α, a master controller of muscle metabolism, and up-regulation of SREBP-1, a master controller of lipid synthesis, are likely to have triggered disuse adaptations through mitochondrial dysfunction, whereas AMP kinase, an energy sensor pathway, was unaltered., The present and previous results on the same subjects suggest a causal link between muscle atrophy, impaired skeletal muscle metabolism, impaired whole body oxidative metabolism, and insulin sensitivity and moderate inflammation, which are major risk factors of physical inactivity related diseases., Abstract 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

In rat skeletal muscle the unloaded shortening velocity (V o) 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 V o. However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5M KCl, 22°C for kinetic assays of myosin, 200mM ionic strength, 12°C to measure V o). 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, K AD) and the rate of ATP-induced acto-HMM dissociation (controlled by K 1 k +2) under the same experimental condition used to measure V o. In fast-muscle myosin isolated from a wide range of mammalian muscles, k −AD was found to be too fast to limit V o, whereas K 1 k +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°C, while k −AD limits the velocity of slow myosin II isoforms. Extrapolating our data to 37°C suggests that at physiological temperature the rate of ADP dissociation may limit V o for both isoforms. [Copyright &y& Elsevier]

Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. Here we report that a subpopulation of circulating cells expressing AC133, a well-characterized marker of hematopoietic stem cells, also expresses early myogenic markers. Freshly isolated, circulating AC133+ cells were induced to undergo myogenesis when cocultured with myogenic cells or exposed to Wnt-producing cells in vitro and when delivered in vivo through the arterial circulation or directly into the muscles of transgenic scid/mdx mice (which allow survival of human cells). Injected cells also localized under the basal lamina of host muscle fibers and expressed satellite cell markers such as M-cadherin and MYPS. Furthermore, functional tests of injected muscles revealed a substantial recovery of force after treatment. As these cells can be isolated from the blood, manipulated in vitro, and delivered through the circulation, they represent a possible tool for future cell therapy applications in DMD disease or other muscular dystrophies. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. Here we report that a subpopulation of circulating cells expressing AC133, a well-characterized marker of hematopoietic stem cells, also expresses early myogenic markers. Freshly isolated, circulating AC133(+) cells were induced to undergo myogenesis when cocultured with myogenic cells or exposed to Wnt-producing cells in vitro and when delivered in vivo through the arterial circulation or directly into the muscles of transgenic scid/mdx mice (which allow survival of human cells). Injected cells also localized under the basal lamina of host muscle fibers and expressed satellite cell markers such as M-cadherin and MYF5. Furthermore, functional tests of injected muscles revealed a substantial recovery of force after treatment. As these cells can be isolated from the blood, manipulated in vitro, and delivered through the circulation, they represent a possible tool for future cell therapy applications in DMD disease or other muscular dystrophies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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/cm2): 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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]