Tirasemtiv enhances submaximal muscle tension in an Acta1:p.Asp286Gly mouse model of nemaline myopathy.

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.

Diaphragm Fatigue in SMNΔ7 Mice and Its Molecular Determinants: An Underestimated Issue.

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.

Structural and functional impairments of skeletal muscle in patients with postacute sequelae of SARS-CoV-2 infection.

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 (V̇o2) kinetics were determined by moderate-intensity exercises. Muscle oxidative capacity (k) was assessed by near-infrared spectroscopy. Histochemical analysis, O2 flux (JO2) 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. V̇o2peak was lower in PASC than CTRL (24.7 ± 5.0 vs. 32.9 ± 7.4 mL·min-1·kg-1, respectively, P < 0.05). V̇o2 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 JO2 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.

Acute and chronic tirasemtiv treatment improves in vivo and in vitro muscle performance in actin-based nemaline myopathy mice.

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.

Exercise Preconditioning Blunts Early Atrogenes Expression and Atrophy in Gastrocnemius Muscle of Hindlimb Unloaded Mice.

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.

Voluntary physical activity counteracts Chronic Heart Failure progression affecting both cardiac function and skeletal muscle in the transgenic Tgαq*44 mouse model.

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.

Exercise training in Tgαq*44 mice during the progression of chronic heart failure: cardiac vs. peripheral (soleus muscle) impairments to oxidative metabolism.

Cardiac function, skeletal (soleus) muscle oxidative metabolism, and the effects of exercise training were evaluated in a transgenic murine model (Tgαq*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 Tgαq*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 Tgαq*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 O2 delivery, were mainly responsible for the functional improvement.

Structure and function of human muscle fibres and muscle proteome in physically active older men.

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.

FoxO-dependent atrogenes vary among catabolic conditions and play a key role in muscle atrophy induced by hindlimb suspension.

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.

Myosin content of single muscle fibers following short-term disuse and active recovery in young and old healthy men.

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.

Human skeletal muscle fibre contractile properties and proteomic profile: adaptations to 3 weeks of unilateral lower limb suspension and active recovery.

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.

The role of alterations in mitochondrial dynamics and PGC-1α over-expression in fast muscle atrophy following hindlimb unloading.

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.

Quantitative and qualitative adaptations of muscle fibers to glucocorticoids.

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.

PGC1-α over-expression prevents metabolic alterations and soleus muscle atrophy in hindlimb unloaded mice.

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.

The time course of the adaptations of human muscle proteome to bed rest and the underlying mechanisms.

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.

Statin or fibrate chronic treatment modifies the proteomic profile of rat skeletal muscle.

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.

Redox homeostasis, oxidative stress and disuse muscle atrophy.

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.

Antioxidant treatment of hindlimb-unloaded mouse counteracts fiber type transition but not atrophy of disused muscles.

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.

Is oxidative stress a cause or consequence of disuse muscle atrophy in mice? A proteomic approach in hindlimb-unloaded mice.

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.