Search

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.)

Background: Branched chain amino acid (BCAA) supplementation is a recently identified strategy to promote longevity in mice. A proteomic approach was used to identify proteins which are differentially expressed in the sera of mice following supplementation with selected branched chain amino acid enriched mixture (BCAAem)., Findings: 12 male mice (C57Bl6, 9 months-old) were randomly assigned to unsupplemented (Control, n = 6) and supplemented (BCAA, n = 6, 0.1 mg/gr/day in drink water for 4 weeks). At the end of treatment total plasma samples from Control and BCAAem mice were separated by two-dimensional gel electrophoresis (2-DE). After staining, the gels were imaged and differential protein expression patterns were interrogated using image analysis software. Spots showing a different expression level were identified through a comparison with 2D maps found in databases officially recognized (ExPASy).Master gels of Control and BCAA mice exhibited slightly different 2-DE patterns as only 10 spots out of 500 appeared differentially expressed: 8 were upregulated (corresponding to Apolipoprotein A-I (APOA1), Complement factor B, Complement C3, Immunoglobulin light chain) and 2 appeared downregulated (Alpha-1-antitrypsin and unknown)., Conclusions: Supplementation with BCAAem in mice results in a slight perturbation of the host serum proteome. Of particular interest is the increased Apolipoprotein A-I (APOAI) following treatment.

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.

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.)

The involvement of ATP-sensitive K(+) (K(ATP)) channels in the atrophy of slow-twitch (MHC-I) soleus (SOL) and fast-twitch (MHC-IIa) flexor digitorum brevis (FDB) muscles was investigated in vivo in 14-day-hindlimb-unloaded (14-HU) rats, an animal model of disuse, and in vitro in drug-induced muscle atrophy. Patch-clamp and gene expression experiments were performed in combination with measurements of fibre diameters used as an index of atrophy, and with MHC labelling in 14-HU rats and controls. A down-regulation of K(ATP) channel subunits Kir6.2, SUR1 and SUR2B with marked atrophy and incomplete phenotype transition were observed in SOL of 14-HU rats. The observed changes in K(ATP) currents were well correlated with changes in fibre diameters and SUR1 expression, as well as with MHC-IIa expression. Half of the SOL fibres of 14-HU rats had reduced diameter and K(ATP) currents and were labelled by MHC-I antibodies. Non-atrophic fibres were labelled by MHC-IIa (22%) antibodies and had enhanced K(ATP) currents, or were labelled by MHC-I (28%) antibodies but had normal current. FDB was not affected in 14-HU rats and this is related to the high expression/activity of Kir6.2/SUR1 subunits characterizing this muscle phenotype. The long-term incubation of the control muscles in vitro with the K(ATP) channel blocker glibenclamide (10(6)m) reduced the K(ATP) currents with atrophy and these effects were prevented by the K(ATP) channel opener diazoxide (10(4)m). The in vivo down-regulation of SUR1, and possibly of Kir6.2 and SUR2B, or their in vitro pharmacological blockade activates atrophic signalling in skeletal muscle. All these findings suggest a new role for the K(ATP) channel as a molecular sensor of atrophy.

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.

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.

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.

We investigated the effects of a specific mixture of amino acid (AA) supplements on the adaptation changes induced by aging in the soleus muscle of rats. Male Wistar rats were divided into 3 groups (n = 5 each): young control (YO), 3 months of age; elderly control (EL), 18 months of age; and elderly orally supplemented with an AA mixture (EL-AA), 18 months of age, given as 0.1 g/kg per day in drinking water for 8 weeks. Myosin heavy chain (MHC) composition was analyzed in all muscles. The total fiber number and fiber cross-sectional area of types 1 and 2A fibers were also measured in immunostained sections of the soleus muscle. The ratios between the sarcomere volume (Vsar) and the total volume (Vtot) and single muscle fibers were studied by electron microscopy. The expression of total and phosphorylated serine/threonine protein kinase mammalian target of rapamycin (mTOR), a potent regulator of messenger RNA translation initiation, was also determined in all groups. Aging was associated with an overall shift toward the expression of a slower MHC phenotype, atrophy of fast and slow fibers, a significant decrease in Vtot/Vsar, and no changes in total fiber number. AA supplementation antagonized the effects of aging. A shift toward the expression of faster MHC isoforms was observed. Fiber atrophy appeared to be partly counteracted by the AA supplements; we noted an increase in cross-sectional area fibers and Vtot/Vsar in EL-AAs. Total and phosphorylated mTOR expression appeared to decrease in EL and was restored by the AA supplements. Collectively, these results suggest that aging-induced muscle adaptations can be partly restored by AA supplementation. An mTOR signal pathway may mediate the effects on fiber trophism.

We recently generated a mouse model of facioscapulohumeral muscular dystrophy (FSHD) by selectively overexpressing FRG1, a candidate gene for FSHD, in skeletal muscle. The muscles of the FRG-1 mice did not show any plasmamembrane defect suggesting a novel pathogenetic mechanism for FSHD. Here, we study structure and function of muscle fibres from three lines of mice overexpressing FRG1 at different levels: FRG1-low, FRG1-med, FRG1-high. Cross-sectional area (CSA), specific force (Po/CSA) and maximum shortening velocity (V(o)) of identified types of muscle fibres from FRG1-low and FRG1-med mice were analysed and found to be lower than in WT mice. Fast fibres and especially type 2B fibres (the fastest type) were preferentially involved in the dystrophic process showing a much larger force deficit than type 1 (slow) fibres. Consistent with the latter observation, the MHC isoform distribution of several muscles of the three FRG1 lines showed a shift towards slower MHC isoforms in comparison to WT muscle. Moreover, fast muscles showed a more evident histological deterioration, a larger atrophy and a higher percentage of centrally nucleated fibres than the soleus, the slowest muscle in mice. Interestingly, loss in CSA, Po/CSA and V(o) of single muscle fibres and MHC isoform shift towards a slower phenotype can be considered early signs of muscular dystrophy (MD). They were, in fact, found also in FRG1-low mice which did not show any impairment of function in vivo and of muscle size in vitro and in soleus muscles, which had a completely preserved morphology. This study provides a detailed characterization of structure and function of muscle fibres in a novel murine model of one of the main human MDs and suggests that fundamental features of the dystrophic process, common to most MDs, such as the intrinsic loss of contractile strength of muscle fibres, the preferential involvement of fast fibres and the shift towards a slow muscle phenotype can occur independently from obvious alterations of the plasma membrane.

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.

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.