Increased Myostatin Expression Research Articles

Inflammatory Cytokine-Induced Muscle Atrophy and Weakness Can Be Ameliorated by an Inhibition of TGF-β-Activated Kinase-1.

Chronic inflammation causes muscle wasting. Because most inflammatory cytokine signals are mediated via TGF-β-activated kinase-1 (TAK1) activation, inflammatory cytokine-induced muscle wasting may be ameliorated by the inhibition of TAK1 activity. The present study was undertaken to clarify whether TAK1 inhibition can ameliorate inflammation-induced muscle wasting. SKG/Jcl mice as an autoimmune arthritis animal model were treated with a small amount of mannan as an adjuvant to enhance the production of TNF-α and IL-1β. The increase in these inflammatory cytokines caused a reduction in muscle mass and strength along with an induction of arthritis in SKG/Jcl mice. Those changes in muscle fibers were mediated via the phosphorylation of TAK1, which activated the downstream signaling cascade via NF-κB, p38 MAPK, and ERK pathways, resulting in an increase in myostatin expression. Myostatin then reduced the expression of muscle proteins not only via a reduction in MyoD1 expression but also via an enhancement of Atrogin-1 and Murf1 expression. TAK1 inhibitor, LL-Z1640-2, prevented all the cytokine-induced changes in muscle wasting. Thus, TAK1 inhibition can be a new therapeutic target of not only joint destruction but also muscle wasting induced by inflammatory cytokines.

Effects of elastase-induced emphysema on muscle and bone in mice.

Chronic obstructive pulmonary disease (COPD) causes sarcopenia and osteoporosis. However, the mechanisms underlying muscle and bone loss as well as the interactions between muscle and bone in the COPD state remain unclear. Therefore, we herein investigated the effects of the COPD state on muscle and bone in mice intratracheally administered porcine pancreatic elastase (PPE). The intratracheal administration of PPE to mice significantly reduced trabecular bone mineral density (BMD), trabecular bone volume, trabecular number, cortical BMD and cortical area. It also significantly decreased grip strength, but did not affect muscle mass or the expression of myogenic differentiation-, protein degradation- or autophagy-related genes in the soleus and gastrocnemius muscles. Among the myokines examined, myostatin mRNA levels in the soleus muscles were significantly elevated in mice treated with PPE, and negatively related to grip strength, but not bone parameters, in mice treated with or without 2 U PPE in simple regression analyses. Grip strength positively related to bone parameters in mice treated with or without PPE. In conclusion, we showed that a PPE model of COPD in mice exerts dominant effects on bone rather than skeletal muscles. Increased myostatin expression in the soleus muscles of mice in the COPD state may negatively relate to a reduction in grip strength, but not bone loss.

The expression of myogenic gene markers during the embryo‐larval‐transition in Pikeperch ( Sander lucioperca )

Pikeperch (Sander lucioperca) has become a species of interest in aquaculture. It is a popular and economically valuable food fish and can produce high numbers of offspring. However, during early development, there are transition phases when high mortality rates concur with growth changes, vital organ transformations and a limited energy budget. Up to now, no study focused on the developmental adaption of muscle tissue in pikeperch, regardless of muscle tissue influencing essential traits such as locomotion and thus the competence to hunt prey and avoid predators. In the present study, therefore, the developmental myogenesis of pikeperch was analysed using specimens from early embryonic to larval development. Myogenic and developmental genes were utilized to gain insights into transcriptomic regulation during these stages by applying a nanofluidic qPCR approach. Result, three phases of myogenic gene expression, during somitogenesis, during the late embryonic development and during the larval development were detected. Increased myostatin expression showed an interim arrest of muscle formation between embryonic and larval myogenesis. Expression patterns of satellite cell gene markers indicated an accumulation of stem cells before myogenesis interruption. The here gained data will help to broaden the knowledge on percid myogenesis and can support pikeperch rearing in aquaculture.

Research Note: Increased myostatin expression and decreased expression of myogenic regulatory factors in embryonic ages in a quail line with muscle hypoplasia

Genetic selection of quail for a low body weight for more than 80 generations established a low-weight (LW) Japanese quail line that has been previously characterized to have a muscle hypoplasia phenotype. The aim of this study is to investigate the relationship of temporal expression levels of myostatin (Mstn) and myogenic regulatory factors (MRFs) with hypoplastic muscle growth in the LW line. During embryonic day (E) 13 to 15, gain of embryo weight was 2-fold lower (P < 0.001) in the LW line than that in the random bred control (CON). Gains in body weight and pectoralis muscle weight from hatch to posthatch day (P) 28 were also significantly lower (P < 0.01) in the LW line but increased by 4-fold (P < 0.05) during P42 to P75. PCR analysis showed that expression levels of Mstn were greater in the LW at embryonic stage (E12 to E14, P < 0.05), but there was no difference after hatch. In addition, expression levels of Pax7 and myogenin (MyoG) at E12 were 23-fold (P < 0.05) and 3.4-fold (P < 0.05) lesser in the LW line, respectively. At E14, expression of Pax3, Pax7, and MyoG gene was 3.5-fold (P < 0.05), 6.5-fold (P = 0.065), and 4.4-fold (P < 0.01) less than that in the CON. Taken together, high expression levels of Mstn and low expression of MRFs during embryonic stages can be associated with development of muscle hypoplasia and delayed muscle growth in the LW quail line. These data provide evidence that genetic selection for a low body weight resulting in an avian model with muscle hypoplasia has altered the expression profiles of myogenic factors.

Sarcopenia in chronic liver disease: mechanisms and countermeasures.

Sarcopenia, a condition of low muscle mass, quality, and strength, is commonly found in patients with cirrhosis and is associated with adverse clinical outcomes including reduction in quality of life, increased mortality, and posttransplant complications. In chronic liver disease (CLD), sarcopenia is most commonly defined through the measurement of the skeletal muscle index of the third lumbar spine. A major contributor to sarcopenia in CLD is the imbalance in muscle protein turnover, which likely occurs due to a decrease in muscle protein synthesis and an elevation in muscle protein breakdown. This imbalance is assumed to arise due to several factors including accelerated starvation, hyperammonemia, amino acid deprivation, chronic inflammation, excessive alcohol intake, and physical inactivity. In particular, hyperammonemia is a key mediator of the liver-gut axis and is known to contribute to mitochondrial dysfunction and an increase in myostatin expression. Currently, the use of nutritional interventions such as late-evening snacks, branched-chain amino acid supplementation, and physical activity have been proposed to help the management and treatment of sarcopenia. However, little evidence exists to comprehensively support their use in clinical settings. Several new pharmacological strategies, including myostatin inhibition and the nutraceutical Urolithin A, have recently been proposed to treat age-related sarcopenia and may also be of use in CLD. This review highlights the potential molecular mechanisms contributing to sarcopenia in CLD alongside a discussion of existing and potential new treatment strategies.

Ferulic Acid Promotes Growth of Both Fast Glycolytic and Slow Oxidative Skeletal Muscles in Corticosteroid-Induced Rat Myopathy

AbstractBackground: Glucocorticoids (GCs) are wide spread anti-inflammatory supplementation associated with various side effects especially in long term administration. Currently, there is no universally accepted prophylactic treatment for patients commencing long-term steroids.Aim of Study: This research aims to study the possible beneficial role of Ferulic Acid (FA), as a naturally existing antioxidant and anti-inflammatory, in preventing muscle atrophy and promoting growth of fast glycolytic and slow oxidative skeletal muscle fibers in rat model of glucocorticoid induced myopathy.Material and Methods: 48 rats were divided into 4 groups: Control, Ferulic acid supplemented group, Dexamethasone (Dex) supplemented group, rats supplemented with FA and Dex. After 2 weeks, all rats were sacrificed after taking blood samples. Left legs were preserved for histopathological and RT- PCR examinations. While, the right legs were kept for recording of isometric muscle contractility.Results: Dex group showed marked decrease in muscle mass, isometric contraction forces, antioxidant enzymes and Mechano-Growth Factor (MGF) expression together with increase in myostatin expression and malondehyde. FA ad-ministration improved all these deleterious effects of Dex on both skeletal muscle fibers. This beneficial effect of FA was proved by histopathological improvement of sarcoplasmic degeneration, inflammatory cells infiltration and Zenker's necrosis of muscle bundle which featured rats' muscles of Dex group.Conclusion: Ferulic acid, as an antioxidant and anti-inflammatory, can be used as an adjuvant therapy with anti-inflammatory corticosteroids to augment its therapeutic effect and reduce its myotrophic changes.

Influence of feeding a grass hay diet during the early stage of the fattening period on growth performance, carcass characteristics, and meat production in Japanese Black steers.

The present study investigated the influence of feeding a large amount of grass hay to steers from the early to middle fattening period on growth, carcass characteristics, and meat characteristics. Steers were randomly divided into grass hay‐fed (GHF, n = 6) and concentrate‐fed (CF, n = 6) groups. The dressed weight of the GHF steers was lower than that of the CF steers, but the final body weight was not significantly different between the groups. The GHF steers had decreased subcutaneous fat and rib thickness compared with the CF steers. Lipid content, monounsaturated fatty acids, and drip loss in the muscles were lower in the GHF steers than in the CF steers. Furthermore, n‐3 polyunsaturated fatty acids were higher in the GHF steers compared with the CF steers. The GHF steers had lower body weight during the middle fattening stage, which may have occurred as a result of muscle growth suppression caused by increased Myostatin expression; an increase in daily gain during the finishing period may have occurred as a result of muscle growth activation caused by decreased Myostatin expression. Feeding steers a grass hay‐based diet during the early fattening period possibly maintains the quantitative productivity of beef similarly to feeding a concentrate‐based diet.

Glucocorticoid-induced CREB activation and myostatin expression in C2C12 myotubes involves phosphodiesterase-3/4 signaling

Muscle atrophy in metabolic conditions like chronic kidney disease (CKD) and diabetes are associated with glucocorticoid production, dysfunctional insulin/Akt/FoxO3 signaling and increased myostatin expression. We recently found that CREB, a transcription factor proposed to regulate myostatin expression, is highly phosphorylated in some wasting conditions. Based on a novel Akt-PDE3/4 signaling paradigm, we hypothesized that reduced Akt signaling contributes to CREB activation and myostatin expression. C2C12 myotubes were incubated with dexamethasone (Dex), an atrophy-inducing synthetic glucocorticoid. Akt/CREB signaling and myostatin expression were evaluated by immunoblot and qPCR analyses. Inhibitors of Akt, phosphodiesterase (PDE)-3/4, and protein kinase A (PKA) signaling were used to test our hypothesis. Incubating myotubes with Dex for 3–24 h inhibited Akt phosphorylation and enhanced CREB phosphorylation as well as myostatin mRNA and protein. Inhibition of PI3K/Akt signaling with LY294002 similarly increased CREB phosphorylation. Isobutyl-methylxanthine (IBMX, a pan PDE inhibitor), milrinone (PDE3 inhibitor) and rolipram (PDE4 inhibitor) augmented CREB phosphorylation and myostatin expression. Inhibition of protein kinase A by PKI reverted Dex- or IBMX-induced CREB phosphorylation and myostatin expression. Our study provides evidence supporting a newly identified mechanism by which a glucocorticoid-related reduction in Akt signaling contributes to myostatin expression via CREB activation.

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.

Glucocorticoid‐induced inhibition of AKT leads to CREB phosphorylation and increased myostatin expression via a PDE/cAMP/PKA pathway in skeletal muscle

Muscle atrophy in diseases like chronic kidney disease and diabetes is associated with dysfunctional regulation of insulin/Akt/FoxO3 and myostatin‐SMAD3 signaling pathways. We recently found CREB, a transcription factor that regulates maintenance and regeneration of skeletal muscle, is phosphorylated in wasting conditions. In silico analysis identifies a CREB binding site in the myostatin promoter. This study investigated the mechanism by which atrophy‐inducing conditions regulate CREB phosphorylation and its impact on myostatin expression. Differentiated C2C12 myotubes were incubated with combinations of dexamethasone (Dex), LY294002 (PI‐3 kinase inhibitor), H89 (protein kinase A inhibitor), isobutylmethyl xanthine (IBMX, a general phosphodiesterase (PDE) inhibitor), milrinone (PDE3 inhibitor), or rolipram (PDE4 inhibitor) for up to 24 h. Cells were harvested for analysis of proteins or mRNAs. Treatment of myotubes with Dex for 6–24h increased CREB phosphorylation (pCREB) within 3 h and sustained the increase over 24h. Dex also inhibited Akt phosphorylation (pAkt) over a similar time frame. Inhibition of pAkt by LY294002 mimicked Dex by increasing pCREB; the actions of LY294002 and Dex on pCREB were not additive. Since Akt can activate PDE3/PDE4 and reduce cAMP/PKA signaling in non‐muscle cells, we tested whether Dex‐induced inhibition of PDE3/4 increases pCREB. Without Dex, IBMX, milrinone, and rolipram increased pCREB. The Dex‐induced increase in pCREB was attenuated by the PKA inhibitor H89. When added individually, Dex, milrinone, rolipram and IBMX increase myostatin mRNA and protein significantly. Our study identifies a new mechanism by which atrophy‐inducing conditions activate CREB and contribute to myostatin expression. Signals that attenuate insulin/Akt signaling reduce the PDE3/4 activity. This leads to an increase in cAMP, PKA activity, CREB phosphorylation and myostatin expression.Support or Funding InformationFunded by NIH RO1 DK95610 and VA Merit X01BX001456

Skeletal muscle myopenia in mice model of bile duct ligation and carbon tetrachloride-induced liver cirrhosis.

Skeletal muscle myopathy is universal in cirrhotic patients, however, little is known about the main mechanisms involved. The study aims to investigate skeletal muscle morphological, histological, and functional modifications in experimental models of cirrhosis and the principal molecular pathways responsible for skeletal muscle myopathy. Cirrhosis was induced by bile duct ligation (BDL) and carbon tetrachloride (CCl4) administration in mice. Control animals (CTR) underwent bile duct exposure or vehicle administration only. At sacrifice, peripheral muscles were dissected and weighed. Contractile properties of extensor digitorum longus (EDL) were studied in vitro. Muscle samples were used for histological and molecular analysis. Quadriceps muscle histology revealed a significant reduction in cross‐sectional area of muscle and muscle fibers in cirrhotic mice with respect to CTR. Kinetic properties of EDL in both BDL and CCl4 were reduced with respect to CTR; BDL mice also showed a reduction in muscle force and a decrease in the resistance to fatigue. Increase in myostatin expression associated with a decrease in AKT‐mTOR expressions was observed in BDL mice, together with an increase in LC3 protein levels. Upregulation of the proinflammatory citochines TNF‐a and IL6 and an increased expression of NF‐kB and MuRF‐1 were observed in CCl4 mice. In conclusion, skeletal muscle myopenia was present in experimental models of BDL and CCl4‐induced cirrhosis. Moreover, reduction in protein synthesis and activation of protein degradation were the main mechanisms responsible for myopenia in BDL mice, while activation of ubiquitin‐pathway through inflammatory cytokines seems to be the main potential mechanism involved in CCl4 mice.

Inhibition of Stat3 signaling ameliorates atrophy of the soleus muscles in mice lacking the vitamin D receptor

BackgroundAlthough skeletal muscle wasting has long been observed as a clinical outcome of impaired vitamin D signaling, precise molecular mechanisms that mediate the loss of muscle mass in the absence of vitamin D signaling are less clear. To determine the molecular consequences of vitamin D signaling, we analyzed the role of signal transducer and activator of transcription 3 (Stat3) signaling, a known contributor to various muscle wasting pathologies, in skeletal muscles.MethodsWe isolated soleus (slow) and tibialis anterior (fast) muscles from mice lacking the vitamin D receptor (VDR−/−) and used western blot analysis, quantitative RTPCR, and pharmacological intervention to analyze muscle atrophy in VDR−/− mice.ResultsWe found that slow and fast subsets of muscles of the VDR−/− mice displayed elevated levels of phosphorylated Stat3 accompanied by an increase in Myostatin expression and signaling. Consequently, we observed reduced activity of mammalian target of rapamycin (mTOR) signaling components, ribosomal S6 kinase (p70S6K) and ribosomal S6 protein (rpS6), that regulate protein synthesis and cell size, respectively. Concomitantly, we observed an increase in atrophy regulators and a block in autophagic gene expression. An examination of the upstream regulation of Stat3 levels in VDR−/− muscles revealed an increase in IL-6 protein expression in the soleus, but not in the tibialis anterior muscles. To investigate the involvement of satellite cells (SCs) in atrophy in VDR−/− mice, we found that there was no significant deficit in SC numbers in VDR−/− muscles compared to the wild type. Unlike its expression within VDR−/− fibers, Myostatin levels in VDR−/− SCs from bulk muscles were similar to those of wild type. However, VDR−/− SCs induced to differentiate in culture displayed increased p-Stat3 signaling and Myostatin expression. Finally, VDR−/− mice injected with a Stat3 inhibitor displayed reduced Myostatin expression and function and restored active p70S6K and rpS6 levels, resulting in an amelioration of loss of muscle mass in the soleus muscles.ConclusionsThe loss of muscle mass in slow muscles in the absence of vitamin D signaling is due to elevated levels of phosphorylated Stat3 that leads to an increase in Myostatin signaling, which in turn decreases protein synthesis and fiber size through the phosphorylation of p70S6K and rpS6, respectively.

Pivotal role of glutamine synthetase in ammonia detoxification.

Hepatic GS contributes to both enteral and systemic ammonia detoxification. Glutamine synthesis in the periphery (including that in pericentral hepatocytes) and glutamine catabolism in (periportal) hepatocytes represents the high-affinity ammonia-detoxifying system of the body. The dependence of glutamine-bound ammonia disposal to urea on the rate of glutamine synthesis suggests that enhancing peripheral glutamine synthesis is a promising strategy to treat hyperammonemia. Because total urea synthesis does not depend on glutamine synthesis, we hypothesize that glutamate dehydrogenase complements mitochondrial ammonia production. (Hepatology 2017;65:281-293).

Ammonia elicits a different myogenic response in avian and murine myotubes

Increased myostatin expression, resulting in muscle loss, has been associated with hyperammonemia in mammalian models of cirrhosis. However, there is evidence that hyperammonemia in avian embryos results in a reduction of myostatin expression, suggesting a proliferative myogenic environment. The present in vitro study examines species differences in myotube and liver cell response to ammonia using avian and murine-derived cells. Primary myoblasts and liver cells were isolated from embryonic day 15 and 17 chick embryos to be compared with mouse myoblasts (C2C12) and liver (AML12) cells. Cells were exposed to varying concentrations of ammonium acetate (AA; 2.5, 5, or 10mM) to determine the effects of ammonia on the cells. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly increased in AA (10mM) treated C2C12 myotubes compared to both ages of chick embryonic myotube cultures after 48h (P<0.02). Western blot analysis of myostatin protein confirmed an increase in myostatin expression in AA-treated C2C12 myotubes compared to the sodium acetate (SA) controls, while myostatin expression was decreased in the chick embryonic myotube cultures when treated with AA. Myotube diameter was significantly decreased in AA-treated C2C12 myotubes compared to controls, while avian myotube diameter increased with AA treatment (P<0.001). There were no significant differences between avian and murine liver cell viability, assessed using 2', 7'- bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein, acetoxymethyl ester, when treated with AA. However, after 24h, AA-treated avian myotubes showed a significant increase in cell viability compared to the C2C12 myotubes (P<0.05). Overall, it appears that there is a positive myogenic response to hyperammonemia in avian myotubes compared to murine myotubes, which supports a proliferative myogenic environment.

A comparative examination of cortisol effects on muscle myostatin and HSP90 gene expression in salmonids

Cortisol, the primary corticosteroid in teleost fishes, is released in response to stressors to elicit local functions, however little is understood regarding muscle-specific responses to cortisol in these fishes. In mammals, glucocorticoids strongly regulate the muscle growth inhibitor, myostatin, via glucocorticoid response elements (GREs) leading to muscle atrophy. Bioinformatics methods suggest that this regulatory mechanism is conserved among vertebrates, however recent evidence suggests some fishes exhibit divergent regulation. Therefore, the aim of this study was to evaluate the conserved actions of cortisol on myostatin and hsp90 expression to determine if variations in cortisol interactions have emerged in salmonid species. Representative salmonids; Chinook salmon (Oncorhynchus tshawytscha), cutthroat trout (Oncorhynchus clarki), brook trout (Salvelinus fontinalis), and Atlantic salmon (Salmo salar); were injected intraperitoneally with a cortisol implant (50μg/g body weight) and muscle gene expression was quantified after 48h. Plasma glucose and cortisol levels were significantly elevated by cortisol in all species, demonstrating physiological effectiveness of the treatment. HSP90 mRNA levels were elevated by cortisol in brook trout, Chinook salmon, and Atlantic salmon, but were decreased in cutthroat trout. Myostatin mRNA levels were affected in a species, tissue (muscle type), and paralog specific manner. Cortisol treatment increased myostatin expression in brook trout (Salvelinus) and Atlantic salmon (Salmo), but not in Chinook salmon (Oncorhynchus) or cutthroat trout (Oncorhynchus). Interestingly, the VC alone increased myostatin mRNA expression in Chinook and Atlantic salmon, while the addition of cortisol blocked the response. Taken together, these results suggest that cortisol affects muscle-specific gene expression in species-specific manners, with unique Oncorhynchus-specific divergence observed, that are not predictive solely based upon mammalian stress responses.

Depleting extracellular vesicles from fetal bovine serum alters proliferation and differentiation of skeletal muscle cells in vitro.

BackgroundFetal bovine serum (FBS) contains a wide range of growth factors, hormones, vitamins, amino acids, fatty acids and trace elements required for cell growth. It was shown that animal sera contain also extracellular vesicles (EVs) with important biological properties; thus we wondered whether EVs present in FBS would influence muscle cell phenotype.EVs were removed from sera by ultracentrifugation (18 h). C2C12, L6 and human primary myoblasts, were grown either in classical media (CM) or in EVs-depleted media. Differentiation was induced by replacing the culture medium either with CM or EV-depleted media. qRT-PCR of relevant genes and miRNA involved in proliferation, differentiation, energy metabolism and EVs formation and secretion were performed.ResultsGrowth of myoblasts in EV-free media during proliferation produces the most unfavorable situation for proper myotube formation, when considering C212 and human myoblasts. Removing EVs from serum committed myoblasts to differentiate precociously (induction of myogenin and decreased expression of myomiR involved in myogenesis). C2C12 and human myoblasts, grown constantly in EV-depleted media during proliferation and differentiation, formed less myotubes than in CM. They had a reduced level of myogenin and a strong increase in myostatin expression, a negative regulator of muscle cell differentiation that affects myotube size. This situation was not reversed when confluent myoblasts were switched to CM for differentiation. Like C2C12 and human cells, L6 formed less myotubes in EVs-depleted media. However, as they do not express myostatin, L6 myotubes were larger and expressed higher level of CKTM2 compared to myotubes grown in CM suggesting that they had reached a higher level of differentiation.ConclusionsResearchers studying the role of muscle EVs in culture conditions should consider that depleting EVs from serum alters the phenotype of muscle cells. Interestingly, the cross-talk between myoblasts and myotubes during myogenesis (Forterre 2014, PLoS One. 2014 Jan 2;9(1):e84153) can be recapitulate by using FBS-EVs as well. This implies that EVs can transfer specific signals to cells from unrelated species and that part of serum EV composition is evolutionarily conserved (e.g.; myomiR are detected in FBS-EVs). EVs in body fluids could have an unsuspected function during embryogenesis and in regulation of cellular processes such as hypertrophy and hyperplasia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0262-0) contains supplementary material, which is available to authorized users.

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

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. 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). 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. 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.

Myostatin induces interstitial fibrosis in the heart via TAK1 and p38.

Myostatin, a member of the TGF-β superfamily of secreted growth factors, is a negative regulator of skeletal muscle growth. In the heart, it is expressed at lower levels compared to skeletal muscle but up-regulated under disease conditions. Cre recombinase-mediated inactivation of myostatin in adult cardiomyocytes leads to heart failure and increased mortality but cardiac function of surviving mice is restored after several weeks probably due to compensatory expression in non-cardiomyocytes. To study long-term effects of increased myostatin expression in the heart and to analyze the putative crosstalk between cardiomyocytes and fibroblasts, we overexpressed myostatin in cardiomyocytes. Increased expression of myostatin in heart muscle cells caused interstitial fibrosis via activation of the TAK-1-MKK3/6-p38 signaling pathway, compromising cardiac function in older mice. Our results uncover a novel role of myostatin in the heart and highlight the necessity for tight regulation of myostatin to maintain normal heart function.

Impaired musculoskeletal response to age and exercise in PPARβ(-/-) diabetic mice.

Fragility fractures are recognized complication of diabetes, but yet the underlying mechanisms remain poorly understood. This is particularly pronounced in type 2 diabetes in which the propensity to fall is increased but bone mass is not necessarily low. Thus, whether factors implicated in the development of insulin resistance and diabetes directly impact on the musculoskeletal system remains to be investigated. PPARβ−/− mice have reduced metabolic activity and are glucose intolerant. We examined changes in bone and muscle in PPARβ−/− mice and investigated both the mechanism behind those changes with age as well as their response to exercise. Compared with their wild type, PPARβ−/− mice had an accelerated and parallel decline in both muscle and bone strength with age. These changes were accompanied by increased myostatin expression, low bone formation, and increased resorption. In addition, mesenchymal cells from PPARβ−/− had a reduced proliferation capacity and appeared to differentiate into more of an adipogenic phenotype. Concomitantly we observed an increased expression of PPARγ, characteristic of adipocytes. The anabolic responses of muscle and bone to exercise were also diminished in PPARβ−/− mice. The periosteal bone formation response to direct bone compression was, however, maintained, indicating that PPARβ controls periosteal bone formation through muscle contraction and/or metabolism. Taken together, these data indicate that PPARβ deficiency leads to glucose intolerance, decreased muscle function, and reduced bone strength. On a molecular level, PPARβ appears to regulate myostatin and PPARγ expression in muscle and bone, thereby providing potential new targets to reverse bone fragility in patients with metabolic disturbances.

Absence of Hyperplasia in Gasp-1 Overexpressing Mice is Dependent on Myostatin Up-Regulation

Background/Aims: Overexpression of Gasp-1, an inhibitor of myostatin, leads to a hypermuscular phenotype due to hypertrophy rather than hyperplasia in mice. However to date, the cellular and molecular mechanisms underlying this phenotype are not investigated. Methods: Skeletal muscles of overexpressing Gasp-1 mice, called Tg(Gasp-1) mice, were analyzed by histological methods. Satellite cell-derived myoblasts from these mice were used to investigate the molecular mechanisms. Results: We demonstrated that hypertrophy in Tg(Gasp-1) mice was related to a myonuclear accretion during the first 3 postnatal weeks and an activation of the pro-hypertrophic Akt/mTORC/p70S6K signaling. In accordance with these results, we showed that overexpressing Gasp-1 primary myoblasts proliferated faster and myonuclei average per myotube was increased during differentiation. Molecular analysis revealed that Gasp-1 overexpression resulted in increased myostatin expression related to its auto-regulation. Despite its inhibition, myostatin led to Pax7 deregulation through its non-canonical Erk1/2 signaling pathway. Consistent with this, inhibition of Erk1/2 signaling pathway as well as neutralization of secreted myostatin rescue the Pax7 expression in overexpressing Gasp-1 myoblasts. Conclusion: Our study shows that myostatin is able to act independently of its canonical pathway to regulate the Pax7 expression. Altogether, our results indicate that myostatin could regulate muscle development despite its protein inhibition.