Myostatin gene invalidation does not prevent skeletal muscle mass loss during experimental sepsis in mice.

Loss of muscle mass and function induced by sepsis contributes to physical inactivity and disability in intensive care unit patients. Limiting skeletal muscle deconditioning may thus be helpful in reducing the long-term effect of muscle wasting in patients. We tested the hypothesis that invalidation of the myostatin gene, which encodes a powerful negative regulator of skeletal muscle mass, could prevent or attenuate skeletal muscle wasting and improve survival of septic mice. Sepsis was induced by caecal ligature and puncture (CLP) in 13-week-old C57BL/6J wild-type and myostatin knock-out male mice. Survival rates were similar in wild-type and myostatin knock-out mice seven days after CLP. Loss in muscle mass was also similar in wild-type and myostatin knock-out mice 4 and 7 days after CLP. The loss in muscle mass was molecularly supported by an increase in the transcript level of E3-ubiquitin ligases and autophagy-lysosome markers. This transcriptional response was blunted in myostatin knock-out mice. No change was observed in the protein level of markers of the anabolic insulin/IGF1-Akt-mTOR pathway. Muscle strength was similarly decreased in wild-type and myostatin knock-out mice 4 and 7 days after CLP. This was associated with a modified expression of genes involved in ion homeostasis and excitation-contraction coupling, suggesting that a long-term functional recovery following experimental sepsis may be impaired by a dysregulated expression of molecular determinants of ion homeostasis and excitation-contraction coupling. In conclusion, myostatin gene invalidation does not provide any benefit in preventing skeletal muscle mass loss and strength in response to experimental sepsis. KEY POINTS: Survival rates are similar in wild-type and myostatin knock-out mice seven days after the induction of sepsis. Loss in muscle mass and muscle strength are similar in wild-type and myostatin knock-out mice 4 and 7 days after the induction of an experimental sepsis. Despite evidence of a transcriptional regulation, the protein level of markers of the anabolic insulin/IGF1-Akt-mTOR pathway remained unchanged. RT-qPCR analysis of autophagy-lysosome pathway markers indicates that activity of the pathway may be altered by experimental sepsis in wild-type and myostatin knock-out mice. Experimental sepsis induces greater variations in the mRNA levels of wild-type mice than those of myostatin knock-out mice, without providing any significant catabolic resistance or functional benefits.

Molecular mechanisms of skeletal muscle atrophy in a mouse model of cerebral ischemia.

BACKGROUND AND PURPOSE: Loss of muscle mass and function is a severe complication in patients with stroke that contributes to promoting physical inactivity and disability. The deleterious consequences of skeletal muscle mass loss underline the necessity to identity the molecular mechanisms involved in skeletal muscle atrophy after cerebral ischemia. METHODS: Transient focal cerebral ischemia (60 minutes) was induced by occlusion of the right middle cerebral artery in C57BL/6J male mice. Skeletal muscles were removed 3 days later and analyzed for the regulation of critical determinants of muscle mass homeostasis (Akt/mammalian target of rapamycin pathway, myostatin-Smad2/3 and bone morphogenetic protein-Smad1/5/8 signaling pathways, ubiquitin-proteasome and autophagy-lysosome proteolytic pathways). RESULTS: Cerebral ischemia induced severe sensorimotor deficits associated with muscle mass loss of the paretic limbs. Mechanistically, cerebral ischemia repressed Akt/mammalian target of rapamycin pathway and increased expression of key players of ubiquitin-proteasome pathway (MuRF1 [muscle RING finger-1], MAFbx [muscle atrophy F-box], Musa1 [muscle ubiquitin ligase of SCF complex in atrophy-1]), together with a marked increase in myostatin expression, in both paretic and nonparetic skeletal muscles. The Smad1/5/8 pathway was also activated. CONCLUSIONS: Our data fit with a model in which a repression of Akt/mammalian target of rapamycin pathway and an increase in the expression of key players of ubiquitin-proteasome pathway are critically involved in skeletal muscle atrophy after cerebral ischemia. Cerebral ischemia also caused an activation of bone morphogenetic protein-Smad1/5/8 signaling pathway, suggesting that compensatory mechanisms are also concomitantly activated to limit the extent of skeletal muscle atrophy.

Detailed characterization of a long-term rodent model of critical illness and recovery.

OBJECTIVE: To characterize a long-term model of recovery from critical illness, with particular emphasis on cardiorespiratory, metabolic, and muscle function. DESIGN: Randomized controlled animal study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Intraperitoneal injection of the fungal cell wall constituent, zymosan or n-saline. MEASUREMENTS AND MAIN RESULTS: Following intervention, rats were followed for up to 2 weeks. Animals with zymosan peritonitis reached a clinical and biochemical nadir on day 2. Initial reductions were seen in body weight, total body protein and fat, and muscle mass. Leg muscle fiber diameter remained subnormal at 14 days with evidence of persisting myonecrosis, even though gene expression of regulators of muscle mass (e.g., MAFbx, MURF1, and myostatin) had peaked on days 2-4 but normalized by day 7. Treadmill exercise capacity, forelimb grip strength, and in vivo maximum tetanic force were also reduced. Food intake was minimal until day 4 but increased thereafter. This did not relate to appetite hormone levels with early (6 hr) rises in plasma insulin and leptin followed by persisting subnormal levels; ghrelin levels did not change. Serum interleukin-6 level peaked at 6 hours but had normalized by day 2, whereas interleukin-10 remained persistently elevated and high-density lipoprotein cholesterol persistently depressed. There was an early myocardial depression and rise in core temperature, yet reduced oxygen consumption and respiratory exchange ratio with a loss of diurnal rhythmicity that showed a gradual but incomplete recovery by day 7. CONCLUSIONS: This detailed physiological, metabolic, hormonal, functional, and histological muscle characterization of a model of critical illness and recovery reproduces many of the findings reported in human critical illness. It can be used to assess putative therapies that may attenuate loss, or enhance recovery, of muscle mass and function.

A centronuclear myopathy--dynamin 2 mutation impairs autophagy in mice.

Dynamin 2 (Dnm2) is involved in endocytosis and intracellular membrane trafficking through its function in vesicle formation from distinct membrane compartments. Heterozygous (HTZ) mutations in the DNM2 gene cause dominant centronuclear myopathy or Charcot-Marie-Tooth neuropathy. We generated a knock-in Dnm2R465W mouse model expressing the most frequent human mutation and recently reported that HTZ mice progressively developed a myopathy. We investigated here the cause of neonatal lethality occurring in homozygous (HMZ) mice. We show that HMZ mice present at birth with a reduced body weight, hypoglycemia, increased liver glycogen content and hepatomegaly, in agreement with a defect in neonatal autophagy. In vitro studies performed in HMZ embryonic fibroblasts point out to a decrease in the autophagy flux prior to degradation at the autolysosome. We show that starved HMZ cells have a higher number of immature autophagy-related structures probably due to a defect of acidification. Our results highlight the role of Dnm2 in the cross talk between endosomal and autophagic pathways and evidence a new role of Dnm2-dependent membrane trafficking in autophagy which may be relevant in DNM2-related human diseases.

Hypothalamic-pituitary-adrenal axis activation and glucocorticoid-responsive gene expression in skeletal muscle and liver of Apc mice.

BACKGROUND: Cancer patients at advanced stages experience a severe depletion of skeletal muscle compartment together with a decrease in muscle function, known as cancer cachexia. Cachexia contributes to reducing quality of life, treatment efficiency, and lifespan of cancer patients. However, the systemic nature of the syndrome is poorly documented. Here, we hypothesize that glucocorticoids would be important systemic mediators of cancer cachexia. METHODS: To explore the role of glucocorticoids during cancer cachexia, biomolecular analyses were performed on several tissues (adrenal glands, blood, hypothalamus, liver, and skeletal muscle) collected from ApcMin/+ male mice, a mouse model of intestine and colon cancer, aged of 13 and 23 weeks, and compared with wild type age-matched C57BL/6J littermates. RESULTS: Twenty-three-week-old Apc mice recapitulated important features of cancer cachexia including body weight loss (-16%, P < 0.0001), muscle atrophy (gastrocnemius muscle: -53%, P < 0.0001), and weakness (-50% in tibialis anterior muscle force, P < 0.0001), increased expression of atrogens (7-fold increase in MuRF1 transcript level, P < 0.0001) and down-regulation of Akt-mTOR pathway (3.3-fold increase in 4EBP1 protein content, P < 0.0001), together with a marked transcriptional rewiring of hepatic metabolism toward an increased expression of gluconeogenic genes (Pcx: +90%, Pck1: +85%), and decreased expression of glycolytic (Slc2a2: -40%, Gk: -30%, Pklr: -60%), ketogenic (Hmgcs2: -55%, Bdh1: -80%), lipolytic/fatty oxidation (Lipe: -50%, Mgll: -60%, Cpt2: -60%, Hadh: -30%), and lipogenic (Acly: -30%, Acacb: -70%, Fasn: -45%) genes. The hypothalamic pituitary-adrenal axis was activated, as evidenced by the increase in the transcript levels of genes encoding corticotropin-releasing hormone in the hypothalamus (2-fold increase, P < 0.01), adrenocorticotropic hormone receptor (3.4-fold increase, P < 0.001), and steroid biosynthesis enzymes (Cyp21a1, P < 0.0001, and Cyp11b1, P < 0.01) in the adrenal glands, as well as by the increase in corticosterone level in the serum (+73%, P < 0.05), skeletal muscle (+17%, P < 0.001), and liver (+24%, P < 0.05) of cachectic 23-week-old Apc mice. A comparative transcriptional analysis with dexamethasone-treated C57BL/6J mice indicated that the activation of the hypothalamic-pituitary-adrenal axis in 23-week-old ApcMin/+ mice was significantly associated with the transcription of glucocorticoid-responsive genes in skeletal muscle (P < 0.05) and liver (P < 0.001). The transcriptional regulation of glucocorticoid-responsive genes was also observed in the gastrocnemius muscle of Lewis lung carcinoma tumour-bearing mice and in KPC mice (tibialis anterior muscle and liver). CONCLUSIONS: These findings highlight the role of the hypothalamic-pituitary-adrenal-glucocorticoid pathway in the transcriptional regulation of skeletal muscle catabolism and hepatic metabolism during cancer cachexia. They also provide the paradigm for the design of new therapeutic strategies.

Effects of compression stockings during exercise and recovery on blood lactate kinetics.

This study aimed to investigate if wearing compression stockings (CS) during exercise and recovery could affect lactate profile in sportsmen. Eight young healthy trained male subjects performed two maximal exercise tests on a cycle ergometer on two different occasions performed randomly: CS during both exercise and recovery, and no CS. Blood lactate concentration was taken during exercise and at 0, 3, 5, 10, 15, 30 and 60 min post-exercise. The individual blood lactate recovery curves were fitted to a biexponential time function: La(t) = La(0) + A1(1 - e(-gamma1t)) + A2(1 - e(-gamma2t)), where gamma(1) and gamma(2) denote the abilities to exchange lactate between the previously active muscles and the blood and to remove lactate from the organism, respectively. A significantly higher blood lactate value at the end of the maximal exercise was found (12.1 +/- 0.5 vs. 10.8 +/- 0.5 mmol l(-1)) wearing CS as compared to no CS (P < 0.05). Lower gamma(1) and higher gamma(2) values were observed with CS during recovery, as compared to no CS. It was concluded that CS during graded exercise leads to a significant higher blood lactate value at exhaustion. Since lactate exchanges were expected to be decreased during exercise due to CS, this result was likely attributable to a higher lactate accumulation related to a greater overall contribution of anaerobic glycolysis. Although the lactate removal ability was significantly improved when wearing CS during recovery, its efficacy in promoting blood lactate clearance after high-intensity exercise is limited.

High intensity aerobic exercise training improves chronic intermittent hypoxia-induced insulin resistance without basal autophagy modulation.

Chronic intermittent hypoxia (IH) associated with obstructive sleep apnea (OSA) is a major risk factor for cardiovascular and metabolic diseases (insulin resistance: IR). Autophagy is involved in the pathophysiology of IR and high intensity training (HIT) has recently emerged as a potential therapy. We aimed to confirm IH-induced IR in a tissue-dependent way and to explore the preventive effect of HIT on IR-induced by IH. Thirty Swiss 129 male mice were randomly assigned to Normoxia (N), Intermittent Hypoxia (IH: 21-5% FiO2, 30 s cycle, 8 h/day) or IH associated with high intensity training (IH HIT). After 8 days of HIT (2*24 min, 50 to 90% of Maximal Aerobic Speed or MAS on a treadmill) mice underwent 14 days IH or N. We found that IH induced IR, characterized by a greater glycemia, an impaired insulin sensitivity and lower AKT phosphorylation in adipose tissue and liver. Nevertheless, MAS and AKT phosphorylation were greater in muscle after IH. IH associated with HIT induced better systemic insulin sensitivity and AKT phosphorylation in liver. Autophagy markers were not altered in both conditions. These findings suggest that HIT could represent a preventive strategy to limit IH-induced IR without change of basal autophagy.

Pharmacological inhibition of myostatin improves skeletal muscle mass and function in a mouse model of stroke.

In stroke patients, loss of skeletal muscle mass leads to prolonged weakness and less efficient rehabilitation. We previously showed that expression of myostatin, a master negative regulator of skeletal muscle mass, was strongly increased in skeletal muscle in a mouse model of stroke. We therefore tested the hypothesis that myostatin inhibition would improve recovery of skeletal muscle mass and function after cerebral ischemia. Cerebral ischemia (45 minutes) was induced by intraluminal right middle cerebral artery occlusion (MCAO). Swiss male mice were randomly assigned to Sham-operated mice (n = 10), MCAO mice receiving the vehicle (n = 15) and MCAO mice receiving an anti-myostatin PINTA745 (n = 12; subcutaneous injection of 7.5 mg.kg-1 PINTA745 immediately after surgery, 3, 7 and 10 days after MCAO). PINTA745 reduced body weight loss and improved body weight recovery after cerebral ischemia, as well as muscle strength and motor function. PINTA745 also increased muscle weight recovery 15 days after cerebral ischemia. Mechanistically, the better recovery of skeletal muscle mass in PINTA745-MCAO mice involved an increased expression of genes encoding myofibrillar proteins. Therefore, an anti-myostatin strategy can improve skeletal muscle recovery after cerebral ischemia and may thus represent an interesting strategy to combat skeletal muscle loss and weakness in stroke patients.

Regulation of Akt-mTOR, ubiquitin-proteasome and autophagy-lysosome pathways in locomotor and respiratory muscles during experimental sepsis in mice.

Sepsis induced loss of muscle mass and function contributes to promote physical inactivity and disability in patients. In this experimental study, mice were sacrificed 1, 4, or 7 days after cecal ligation and puncture (CLP) or sham surgery. When compared with diaphragm, locomotor muscles were more prone to sepsis-induced muscle mass loss. This could be attributed to a greater activation of ubiquitin-proteasome system and an increased myostatin expression. Thus, this study strongly suggests that the contractile activity pattern of diaphragm muscle confers resistance to atrophy compared to the locomotor gastrocnemius muscle. These data also suggest that a strategy aimed at preventing the activation of catabolic pathways and preserving spontaneous activity would be of interest for the treatment of patients with sepsis-induced neuromyopathy.

Effect of endurance training on the VO2-work rate relationship in normoxia and hypoxia.

PURPOSE: We postulated that the relationship between VO2 and work rate (VO2-WR relationship) during incremental exercise is dependent on O2 availability, and that training-induced adaptations alter this relationship. We therefore studied the effect of endurance training on VO2 response during incremental exercise in normoxia and hypoxia (FIO2=0.134). METHODS: Before and after training (6 d.wk, 4 wk), eight subjects performed incremental exercises under normoxia and hypoxia and one constant-work rate exercise in normoxia at 80% of pretraining VO2max. The slopes of the VO2-WR relationship during incremental exercise were calculated using all the points (whole slope) or only points before the lactate threshold (pre-LT slope). The difference between VO2max measured and VO2max expected from the pre-LT slope (DeltaVO2) was determined, as was the difference between VO2 at minute 10 and VO2 at minute 4 during the constant-work rate exercise (DeltaVO2(10'-4')). RESULTS: In normoxia, training induced a significant decrease in the whole slope (11.0+/-1.0 vs 9.9+/-0.4 mL.min.W, P<0.05). In hypoxia, training induced a significant increase in the pre-LT slope (8.7+/-1.2 vs 9.8+/-0.7 mL.min.W; P<0.05) and the whole slope (8.5+/-1.2 vs 9.4+/-0.5 mL.min.W; P<0.05). A significant correlation between the decrease of DeltaVO2 and the decrease of DeltaVO2(10'-4') with training was found in normoxia (P<0.01, r=0.79). CONCLUSIONS: Taken together, these results indicate that adaptations induced by endurance training are associated with more efficient incremental and constant-workload exercise performed in normoxia. Moreover, training contributes to improved O2 delivery during moderate exercise performed in hypoxia, and to enhanced near-maximal exercise tolerance.

Trichostatin A, a histone deacetylase inhibitor, modulates unloaded-induced skeletal muscle atrophy.

Skeletal muscle atrophy is commonly associated with immobilization, ageing, and catabolic diseases such as diabetes and cancer cachexia. Epigenetic regulation of gene expression resulting from chromatin remodeling through histone acetylation has been implicated in muscle disuse. The present work was designed to test the hypothesis that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, would partly counteract unloading-induced muscle atrophy. Soleus muscle atrophy (-38%) induced by 14 days of rat hindlimb suspension was reduced to only 25% under TSA treatment. TSA partly prevented the loss of type I and IIa fiber size and reversed the transitions of slow-twitch to fast-twitch fibers in soleus muscle. Unloading or TSA treatment did not affect myostatin gene expression and follistatin protein. Soleus protein carbonyl content remained unchanged, whereas the decrease in glutathione vs. glutathione disulfide ratio and the increase in catalase activity (biomarkers of oxidative stress) observed after unloading were abolished by TSA treatment. The autophagy-lysosome pathway (Bnip3 and microtubule-associated protein 1 light chain 3 proteins, Atg5, Gabarapl1, Ulk1, and cathepsin B and L mRNA) was not activated by unloading or TSA treatment. However, TSA suppressed the rise in muscle-specific RING finger protein 1 (MuRF1) caused by unloading without affecting the forkhead box (Foxo3) transcription factor. Prevention of muscle atrophy by TSA might be due to the regulation of the skeletal muscle atrophy-related MuRF1 gene. Our findings suggest that TSA may provide a novel avenue to treat unloaded-induced muscle atrophy.

Impaired exercise training-induced muscle fiber hypertrophy and Akt/mTOR pathway activation in hypoxemic patients with COPD.

Exercise training (ExTr) is largely used to improve functional capacity in patients with chronic obstructive pulmonary disease (COPD). However, ExTr only partially restores muscle function in patients with COPD, suggesting that confounding factors may limit the efficiency of ExTr. In the present study, we hypothesized that skeletal muscle adaptations triggered by ExTr could be compromised in hypoxemic patients with COPD. Vastus lateralis muscle biopsies were obtained from patients with COPD who were either normoxemic (n = 15, resting arterial Po2 = 68.5 ± 1.5 mmHg) or hypoxemic (n = 8, resting arterial Po2 = 57.0 ± 1.0 mmHg) before and after a 2-mo ExTr program. ExTr induced a significant increase in exercise capacity both in normoxemic and hypoxemic patients with COPD. However, ExTr increased citrate synthase and lactate dehydrogenase enzyme activities only in skeletal muscle of normoxemic patients. Similarly, muscle fiber cross-sectional area and capillary-to-fiber ratio were increased only in patients who were normoxemic. Expression of atrogenes (MuRF1, MAFbx/Atrogin-1) and autophagy-related genes (Beclin, LC3, Bnip, Gabarapl) remained unchanged in both groups. Phosphorylation of Akt (Ser473), GSK-3ß (Ser9), and p70S6k (Thr389) was nonsignificantly increased in normoxemic patients in response to ExTr, but it was significantly decreased in hypoxemic patients. We further showed on C2C12 myotubes that hypoxia completely prevented insulin-like growth factor-1-induced phosphorylation of Akt, GSK-3ß, and p70S6K. Together, our observations suggest a role for hypoxemia in the adaptive response of skeletal muscle of patients with COPD in an ExTr program.

A methodology to assess the accuracy of a portable metabolic system (VmaxST).

PURPOSE: Validity of a portable metabolic system (VmaxST) was investigated during gas exchanges simulations by a mechanical system (GESS) and during human exercise. METHODS: Three tests were conducted while gas exchanges were measured continuously by VmaxST. Test 1 was composed of six simulations of gas exchanges during steady-state exercise (20 min at V̇E = 80 L.min-1). Test 2 was composed of seven simulations of gas exchanges during incremental exercise (V̇O(2) from 300 to 5600 mL.min-1). In the human trial, 11 subjects performed an incremental running exercise on a treadmill while gas exchanges were measured at the end of each stage with the Douglas bag method (DBM). RESULTS: Test 1 showed that the VmaxST measurements were stable, despite inaccurate measurements of gas concentrations at the start of the test. During test 2, the mean error (difference between measured and predicted value) and the upper and lower limits of agreement were -8.0%, -12.6%, and -3.4% for V̇O(2); -4.6%, -12.0%, and +2.8% for V̇CO(2); and -0.7%, -4.7%, and +3.3% for V̇E. During the human trial, no significant difference was shown between V̇O(2) measured by VmaxST and by DBM at any stage of exercise. The mean difference and the upper and lower limits of agreement between the VmaxST and the DBM measurements were -0.5%, -14.3%, and +13.3% for V̇O(2); -6.3%, -20.9%, and +8.3% for V̇CO(2); and -9.9%, -25.5%, and +5.7% for V̇E. CONCLUSIONS: The use of GESS showed that measurements of V̇O(2) by VmaxST could be biased in a standardized condition. In more realistic condition of use, this bias was lower but the accuracy of measurements was impaired.

Early changes in costameric and mitochondrial protein expression with unloading are muscle specific.

We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P < 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (-23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

Post-transcriptional regulation of autophagy in C2C12 myotubes following starvation and nutrient restoration.

In skeletal muscle, autophagy is activated in multiple physiological and pathological conditions, notably through the transcriptional regulation of autophagy-related genes by FoxO3. However, recent evidence suggests that autophagy could also be regulated by post-transcriptional mechanisms. The purpose of the study was therefore to determine the temporal regulation of transcriptional and post-transcriptional events involved in the control of autophagy during starvation (4h) and nutrient restoration (4h) in C2C12 myotubes. Starvation was associated with an activation of autophagy (decrease in mTOR activity, increase in AMPK activity and Ulk1 phosphorylation on Ser467), an increase in autophagy flux (increased LC3B-II/LC3B-I ratio, LC3B-II level and LC3B-positive punctate), and an increase in the content of autophagy-related proteins (Ulk1, Atg13, Vps34, and Atg5-Atg12 conjugate). Our data also indicated that the content of autophagy-related proteins was essentially maintained when nutrient sufficiency was restored. By contrast, mRNA level of Ulk1, Atg5, Bnip3, LC3B and Gabarapl1 did not increase in response to starvation. Accordingly, binding of FoxO3 transcription factor on LC3B promoter was only increased at the end of the starvation period, whereas mRNA levels of Atrogin1/MAFbx and MuRF1, two transcriptional targets of FoxO involved in ubiquitin-proteasome pathway, were markedly increased at this time. Together, these data provide evidence that target genes of FoxO3 are differentially regulated during starvation and that starvation of C2C12 myotubes is associated with a post-transcriptional regulation of autophagy.

Myostatin gene inactivation prevents skeletal muscle wasting in cancer.

Cachexia is a muscle-wasting syndrome that contributes significantly to morbidity and mortality of many patients with advanced cancers. However, little is understood about how the severe loss of skeletal muscle characterizing this condition occurs. In the current study, we tested the hypothesis that the muscle protein myostatin is involved in mediating the pathogenesis of cachexia-induced muscle wasting in tumor-bearing mice. Myostatin gene inactivation prevented the severe loss of skeletal muscle mass induced in mice engrafted with Lewis lung carcinoma (LLC) cells or in Apc(Min) (/+) mice, an established model of colorectal cancer and cachexia. Mechanistically, myostatin loss attenuated the activation of muscle fiber proteolytic pathways by inhibiting the expression of atrophy-related genes, MuRF1 and MAFbx/Atrogin-1, along with autophagy-related genes. Notably, myostatin loss also impeded the growth of LLC tumors, the number and the size of intestinal polyps in Apc(Min) (/+) mice, thus strongly increasing survival in both models. Gene expression analysis in the LLC model showed this phenotype to be associated with reduced expression of genes involved in tumor metabolism, activin signaling, and apoptosis. Taken together, our results reveal an essential role for myostatin in the pathogenesis of cancer cachexia and link this condition to tumor growth, with implications for furthering understanding of cancer as a systemic disease.

Regulation of Akt-mTOR, ubiquitin-proteasome and autophagy-lysosome pathways in response to formoterol administration in rat skeletal muscle.

Administration of ß2-agonists triggers skeletal muscle anabolism and hypertrophy. We investigated the time course of the molecular events responsible for rat skeletal muscle hypertrophy in response to 1, 3 and 10 days of formoterol administration (i.p. 2000µg/kg/day). A marked hypertrophy of rat tibialis anterior muscle culminated at day 10. Phosphorylation of Akt, ribosomal protein S6, 4E-BP1 and ERK1/2 was increased at day 3, but returned to control level at day 10. This could lead to a transient increase in protein translation and could explain previous studies that reported increase in protein synthesis following ß2-agonist administration. Formoterol administration was also associated with a significant reduction in MAFbx/atrogin-1 mRNA level (day 3), suggesting that formoterol can also affect protein degradation of MAFbx/atrogin1 targeted substrates, including MyoD and eukaryotic initiation factor-3f (eIF3-f). Surprisingly, mRNA level of autophagy-related genes, light chain 3 beta (LC3b) and gamma-aminobutyric acid receptor-associated protein-like 1 (Gabarapl1), as well as lysosomal hydrolases, cathepsin B and cathepsin L, was significantly and transiently increased after 1 and/or 3 days, suggesting that autophagosome formation would be increased in response to formoterol administration. However, this has to be relativized since the mRNA level of Unc-51-like kinase1 (Ulk1), BCL2/adenovirus E1B interacting protein3 (Bnip3), and transcription factor EB (TFEB), as well as the protein content of Ulk1, Atg13, Atg5-Atg12 complex and p62/Sqstm1 remained unchanged or was even decreased in response to formoterol administration. These results demonstrate that the effects of formoterol are mediated, in part, through the activation of Akt-mTOR pathway and that other signaling pathways become more important in the regulation of skeletal muscle mass with chronic administration of ß2-agonists.

Energy cost and mechanical work of walking during load carriage in soldiers.

UNLABELLED: In the military context, soldiers carry equipments of total mass often exceeding 30%-40% of their body mass (BM) and complexly distributed around their body (backpack, weapons, electronics, protections, etc.), which represents severe load carrying conditions. PURPOSE: This study aimed to better understand the effects of load carriage on walking energetics and mechanics during military-type walking. METHODS: Ten male infantrymen recently retired from the French Foreign Legion performed 3-min walking trials at a constant speed of 4 km·h(-1) on an instrumented treadmill, during which walking pattern spatiotemporal parameters, energy cost (C(W)), external mechanical work (W(ext)), and the work done by one leg against the other during the double-contact period (W(int,dc)) were specifically assessed. Three conditions were tested: (i) light sportswear (SP, reference condition considered as unloaded), (ii) battle equipment (BT, ∼22 kg, ∼27% of subjects' BM, corresponding to a military intermediate load), and (iii) road march equipment (RM, ∼38 kg, ∼46% of subjects' BM, corresponding to a military high load). RESULTS: Repeated-measures ANOVA showed that military equipment carriage significantly (i) altered the spatiotemporal pattern of walking (all P < 0.01), (ii) increased absolute gross and net CW (P < 0.0001), and (iii) increased both absolute and mass-relative W(ext) (P < 0.01) and W(int,dc) (P < 0.0001) but did not alter the inverted pendulum recovery or locomotor efficiency. CONCLUSIONS: Military equipments carriage induced significant changes in walking mechanics and energetics, but these effects appeared not greater than those reported with loads carried around the waist and close to the center of mass. This result was not expected because the latter has been hypothesized to be the optimal method of load carriage from a metabolic standpoint.

Sirtuin 1 regulates SREBP-1c expression in a LXR-dependent manner in skeletal muscle.

Sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase, has emerged as a main determinant of whole body homeostasis in mammals by regulating a large spectrum of transcriptional regulators in metabolically relevant tissue such as liver, adipose tissue and skeletal muscle. Sterol regulatory element binding protein (SREBP)-1c is a transcription factor that controls the expression of genes related to fatty acid and triglyceride synthesis in tissues with high lipid synthesis rates such as adipose tissue and liver. Previous studies indicate that SIRT1 can regulate the expression and function of SREBP-1c in liver. In the present study, we determined whether SIRT1 regulates SREBP-1c expression in skeletal muscle. SREBP-1c mRNA and protein levels were decreased in the gastrocnemius muscle of mice harboring deletion of the catalytic domain of SIRT1 (SIRT1(Δex4/Δex4) mice). By contrast, adenoviral expression of SIRT1 in human myotubes increased SREBP-1c mRNA and protein levels. Importantly, SREBP-1c promoter transactivation, which was significantly increased in response to SIRT1 overexpression by gene electrotransfer in skeletal muscle, was completely abolished when liver X receptor (LXR) response elements were deleted. Finally, our in vivo data from SIRT1(Δex4/Δex4) mice and in vitro data from human myotubes overexpressing SIRT1 show that SIRT1 regulates LXR acetylation in skeletal muscle cells. This suggests a possible mechanism by which the regulation of SREBP-1c gene expression by SIRT1 may require the deacetylation of LXR transcription factors.

Evidence for mitochondrial respiratory deficiency in rat rhabdomyosarcoma cells.

BACKGROUND: Mitochondria can sense signals linked to variations in energy demand to regulate nuclear gene expression. This retrograde signaling pathway is presumed to be involved in the regulation of myoblast proliferation and differentiation. Rhabdomyosarcoma cells are characterized by their failure to both irreversibly exit the cell cycle and complete myogenic differentiation. However, it is currently unknown whether mitochondria are involved in the failure of rhabdomyosarcoma cells to differentiate. METHODOLOGY/PRINCIPAL FINDINGS: Mitochondrial biogenesis and metabolism were studied in rat L6E9 myoblasts and R1H rhabdomyosacoma cells during the cell cycle and after 36 hours of differentiation. Using a combination of flow cytometry, polarographic and molecular analyses, we evidenced a marked decrease in the cardiolipin content of R1H cells cultured in growth and differentiation media, together with a significant increase in the content of mitochondrial biogenesis factors and mitochondrial respiratory chain proteins. Altogether, these data indicate that the mitochondrial inner membrane composition and the overall process of mitochondrial biogenesis are markedly altered in R1H cells. Importantly, the dysregulation of protein-to-cardiolipin ratio was associated with major deficiencies in both basal and maximal mitochondrial respiration rates. This deficiency in mitochondrial respiration probably contributes to the inability of R1H cells to decrease mitochondrial H2O2 level at the onset of differentiation. CONCLUSION/SIGNIFICANCE: A defect in the regulation of mitochondrial biogenesis and mitochondrial metabolism may thus be an epigenetic mechanism that may contribute to the tumoral behavior of R1H cells. Our data underline the importance of mitochondria in the regulation of myogenic differentiation.