Myostatin deficiency not only prevents muscle wasting but also improves survival in septic mice.

Sepsis remains a leading cause of mortality in critically ill patients. Muscle wasting is a major complication of sepsis and negatively affects clinical outcomes. Despite intense investigation for many years, the molecular mechanisms underlying sepsis-related muscle wasting are not fully understood. In addition, a potential role of muscle wasting in disease development of sepsis has not been studied. Myostatin is a myokine that downregulates skeletal muscle mass. We studied the effects of myostatin deficiency on muscle wasting and other clinically relevant outcomes, including mortality and bacterial clearance, in mice. Myostatin deficiency prevented muscle atrophy along with inhibition of increases in muscle-specific RING finger protein 1 (MuRF-1) and atrogin-1 expression and phosphorylation of signal transducer and activator of transcription protein 3 (STAT3; major players of muscle wasting) in septic mice. Moreover, myostatin deficiency improved survival and bacterial clearance of septic mice. Sepsis-induced liver dysfunction, acute kidney injury, and neutrophil infiltration into the liver and kidney were consistently mitigated by myostatin deficiency, as indicated by plasma concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and neutrophil gelatinase-associated lipocalin (NGAL) and myeloperoxidase activity in the organs. Myostatin deficiency also inhibited sepsis-induced increases in plasma high-mobility group protein B1 (HMGB1) and macrophage inhibitory cytokine (MIC)-1/growth differentiation factor (GDF)-15 concentrations. These results indicate that myostatin plays an important role not only in muscle wasting but also in other clinically relevant outcomes in septic mice. Furthermore, our data raise the possibility that muscle wasting may not be simply a complication, but myostatin-mediated muscle cachexia and related changes in muscle may actually drive the development of sepsis as well.NEW & NOTEWORTHY Muscle wasting is a major complication of sepsis, but its role in the disease development is not known. Myostatin deficiency improved bacterial clearance and survival and mitigated damage in the liver and kidney in septic mice, which paralleled prevention of muscle wasting. These results raise the possibility that muscle wasting may not simply be a complication of sepsis, but myostatin-mediated cachexic changes may have a role in impaired bacterial clearance and mortality in septic mice.

Muscle differentiation induced by p53 signaling pathway-related genes in myostatin-knockout quail myoblasts.

The myostatin (MSTN) gene is of interest in the livestock industry because mutations in this gene are closely related to growth performance and muscle differentiation. Thus, in this study, we established MSTN knockout (KO) quail myoblasts (QM7) and investigated the regulatory pathway of the myogenic differentiation process. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate MSTN KO QM7 cells and subsequently isolated a single cell-derived MSTN KO QM7 subline with 10- and 16-nucleotide deletions that induced translational frameshift mutations. The differentiation capacity and proliferation rate of MSTN KO QM7 cells were enhanced. We conducted next-generation-sequencing (NGS) analysis to compare the global gene expression profiles of wild-type (WT) QM7 and MSTN KO QM7 cells. Intriguingly, NGS expression profiles showed different expression patterns of p21 and p53 in MSTN KO QM7 cells. Moreover, we identified downregulated expression patterns of leukemia inhibitory factor and DNA Damage Inducible Transcript 4, which are genes in the p53 signaling pathway. Using quantitative RT-PCR (qRT-PCR) analysis and western blotting, we concluded that p53-related genes promote the cell cycle by upregulating p21 and enhancing muscle differentiation in MSTN KO QM7 cells. These results could be applied to improve economic traits in commercial poultry by regulating MSTN-related networks.

Regulation of the dystrophin-associated glycoprotein complex composition by the metabolic properties of muscle fibres.

The dystrophin-glycoprotein complex (DGC) links the muscle cytoskeleton to the extracellular matrix and is responsible for force transduction and protects the muscle fibres from contraction induced damage. Mutations in components of the DGC are responsible for muscular dystrophies and congenital myopathies. Expression of DGC components have been shown to be altered in many myopathies. In contrast we have very little evidence of whether adaptive changes in muscle impact on DGC expression. In this study we investigated connection between muscle fibre phenotype and the DGC. Our study reveals that the levels of DGC proteins at the sarcolemma differ in highly glycolytic muscle compared to wild-type and that these changes can be normalised by the super-imposition of an oxidative metabolic programme. Importantly we show that the metabolic properties of the muscle do not impact on the total amount of DGC components at the protein level. Our work shows that the metabolic property of a muscle fibre is a key factor in regulating the expression of DGC proteins at the sarcolemma.

The Possible Role of Complete Loss of Myostatin in Limiting Excessive Proliferation of Muscle Cells (C2C12) via Activation of MicroRNAs.

Myostatin (MSTN) is a member of the TGF-ß superfamily that negatively regulates skeletal muscle growth and differentiation. However, the mechanism by which complete MSTN deletion limits excessive proliferation of muscle cells remains unclear. In this study, we knocked out MSTN in mouse myoblast lines using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system and sequenced the mRNA and miRNA transcriptomes. The results show that complete loss of MSTN upregulates seven miRNAs targeting an interaction network composed of 28 downregulated genes, including TGFB1, FOS and RB1. These genes are closely associated with tumorigenesis and cell proliferation. Our study suggests that complete loss of MSTN may limit excessive cell proliferation via activation of miRNAs. These data will contribute to the treatment of rhabdomyosarcoma (RMS).

Enhanced exercise and regenerative capacity in a mouse model that violates size constraints of oxidative muscle fibres.

A central tenet of skeletal muscle biology is the existence of an inverse relationship between the oxidative fibre capacity and its size. However, robustness of this relationship is unknown. We show that superimposition of Estrogen-related receptor gamma (Errγ) on the myostatin (Mtn) mouse null background (Mtn(-/-)/Errγ(Tg/+)) results in hypertrophic muscle with a high oxidative capacity thus violating the inverse relationship between fibre size and oxidative capacity. We also examined the canonical view that oxidative muscle phenotype positively correlate with Satellite cell number, the resident stem cells of skeletal muscle. Surprisingly, hypertrophic fibres from Mtn(-/-)/Errγ(Tg/+) mouse showed satellite cell deficit which unexpectedly did not affect muscle regeneration. These observations 1) challenge the concept of a constraint between fibre size and oxidative capacity and 2) indicate the important role of the microcirculation in the regenerative capacity of a muscle even when satellite cell numbers are reduced.

Pluripotent Conversion of Muscle Stem Cells Without Reprogramming Factors or Small Molecules.

Muscle derived stem cells (MDSCs) are multipotent stem cells that can differentiate into several lineages including skeletal muscle precursor cells. Here, we show that MDSCs from myostatin null mice (Mstn (-/-) ) can be readily induced into pluripotent stem cells without using reprogramming factors. Microarray studies revealed a strong upregulation of markers like Leukemia Inhibitory factor (LIF) and Leukemia Inhibitory factor receptor (LIFR) in Mstn (-/-) MDSCs as compared to wild type MDSCs (WT-MDSCs). Furthermore when cultured in mouse embryonic stem cell media with LIF for 95 days, Mstn (-/-) MDSCs formed embryonic stem cell (ES) like colonies. We termed such ES like cells as the culture-induced pluripotent stem cells (CiPSC). CiPSCs from Mstn (-/-) MDSCs were phenotypically similar to ESCs, expressed high levels of Oct4, Nanog, Sox2 and SSEA-1, maintained a normal karyotype. Furthermore, CiPSCs formed embryoid bodies and teratomas when injected into immunocompromised mice. In addition, CiPSCs differentiated into somatic cells of all three lineages. We further show that culturing in ES cell media, resulted in hypermethylation and downregulation of BMP2 in Mstn(-/-) MDSCs. Western blot further confirmed a down regulation of BMP2 signaling in Mstn (-/-) MDSCs in supportive of pluripotent reprogramming. Given that down regulation of BMP2 has been shown to induce pluripotency in cells, we propose that lack of myostatin epigenetically reprograms the MDSCs to become pluripotent stem cells. Thus, here we report the successful establishment of ES-like cells from adult stem cells of the non-germline origin under culture-induced conditions without introducing reprogramming genes.

Myostatin dysfunction impairs force generation in extensor digitorum longus muscle and increases exercise-induced protein efflux from extensor digitorum longus and soleus muscles.

Myostatin dysfunction promotes muscle hypertrophy, which can complicate assessment of muscle properties. We examined force generating capacity and creatine kinase (CK) efflux from skeletal muscles of young mice before they reach adult body and muscle size. Isolated soleus (SOL) and extensor digitorum longus (EDL) muscles of Berlin high (BEH) mice with dysfunctional myostatin, i.e., homozygous for inactivating myostatin mutation, and with a wild-type myostatin (BEH+/+) were studied. The muscles of BEH mice showed faster (P < 0.01) twitch and tetanus contraction times compared with BEH+/+ mice, but only EDL displayed lower (P < 0.05) specific force. SOL and EDL of age-matched but not younger BEH mice showed greater exercise-induced CK efflux compared with BEH+/+ mice. In summary, myostatin dysfunction leads to impairment in muscle force generating capacity in EDL and increases susceptibility of SOL and EDL to protein loss after exercise.

Lack of myostatin reduces MyoD induced myogenic potential of primary muscle fibroblasts.

Conversion of skin fibroblasts into myoblasts by transducing the cells with myogenic master regulator MyoD has been in practice for more than two decades. The purpose of such conversion is due to scarcity of muscle biopsies during muscle wasting, hence conversion of fibroblasts to myogenic lineage from various genetic backgrounds offers a great alternative for cell therapies. Here, we have investigated if eliminating Myostatin, a potent negative regulator of myogenesis, could improve the myogenic conversion of fibroblasts. In the present study, we have isolated primary muscle fibroblasts from the skeletal muscles of wild-type (WT) and myostatin null (Mstn(-/-)) mice and transduced the muscle fibroblasts with MyoD using adenoviral, lentiviral transduction, and electroporation methods. In contrast to what we predicted, it is only in WT muscle fibroblasts we detected significant ectopic expression of MyoD, and myogenic conversion. Muscle fibroblasts from Mstn(-/-) genotype failed to take up as much MyoD using the three methods and, therefore, failed to form myotubes. The aforesaid condition of greater MyoD uptake by WT muscle fibroblasts was attributed to the presence of adenoviral receptors, which facilitated adenoviral transduction. However, in Mstn(-/-) fibroblasts we detected negligible levels of adenovirus receptors. Moreover, we also detected significantly higher levels of MyoD antagonists, c-Fos, c-Jun, and cyclin D1 in Mstn(-/-) muscle fibroblasts. Taken together, our results demonstrate that lack of myostatin reduces myogenic potential of muscle fibroblasts by inhibiting MyoD function.

Muscle atrophy reversed by growth factor activation of satellite cells in a mouse muscle atrophy model.

Muscular dystrophies comprise a large group of inherited disorders that lead to progressive muscle wasting. We wanted to investigate if targeting satellite cells can enhance muscle regeneration and thus increase muscle mass. We treated mice with hepatocyte growth factor and leukemia inhibitory factor under three conditions: normoxia, hypoxia and during myostatin deficiency. We found that hepatocyte growth factor treatment led to activation of the Akt/mTOR/p70S6K protein synthesis pathway, up-regulation of the myognic transcription factors MyoD and myogenin, and subsequently the negative growth control factor, myostatin and atrophy markers MAFbx and MuRF1. Hypoxia-induced atrophy was partially restored by hepatocyte growth factor combined with leukemia inhibitory factor treatment. Dividing satellite cells were three-fold increased in the treatment group compared to control. Finally, we demonstrated that myostatin regulates satellite cell activation and myogenesis in vivo following treatment, consistent with previous findings in vitro. Our results suggest, not only a novel in vivo pharmacological treatment directed specifically at activating the satellite cells, but also a myostatin dependent mechanism that may contribute to the progressive muscle wasting seen in severely affected patients with muscular dystrophy and significant on-going regeneration. This treatment could potentially be applied to many conditions that feature muscle wasting to increase muscle bulk and strength.

Elevated expression of activins promotes muscle wasting and cachexia.

In models of cancer cachexia, inhibiting type IIB activin receptors (ActRIIBs) reverse muscle wasting and prolongs survival, even with continued tumor growth. ActRIIB mediates signaling of numerous TGF-ß proteins; of these, we demonstrate that activins are the most potent negative regulators of muscle mass. To determine whether activin signaling in the absence of tumor-derived factors induces cachexia, we used recombinant serotype 6 adeno-associated virus (rAAV6) vectors to increase circulating activin A levels in C57BL/6 mice. While mice injected with control vector gained ~10% of their starting body mass (3.8±0.4 g) over 10 wk, mice injected with increasing doses of rAAV6:activin A exhibited weight loss in a dose-dependent manner, to a maximum of -12.4% (-4.2±1.1 g). These reductions in body mass in rAAV6:activin-injected mice correlated inversely with elevated serum activin A levels (7- to 24-fold). Mechanistically, we show that activin A reduces muscle mass and function by stimulating the ActRIIB pathway, leading to deleterious consequences, including increased transcription of atrophy-related ubiquitin ligases, decreased Akt/mTOR-mediated protein synthesis, and a profibrotic response. Critically, we demonstrate that the muscle wasting and fibrosis that ensues in response to excessive activin levels is fully reversible. These findings highlight the therapeutic potential of targeting activins in cachexia.

Exercise training attenuates the hypermuscular phenotype and restores skeletal muscle function in the myostatin null mouse.

Myostatin regulates both muscle mass and muscle metabolism. The myostatin null (MSTN(-/-)) mouse has a hypermuscular phenotype owing to both hypertrophy and hyperplasia of the myofibres. The enlarged muscles display a reliance on glycolysis for energy production; however, enlarged muscles that develop in the absence of myostatin have compromised force-generating capacity. Recent evidence has suggested that endurance exercise training increases the oxidative properties of muscle. Here, we aimed to identify key changes in the muscle phenotype of MSTN(-/-) mice that can be induced by training. To this end, we subjected MSTN(-/-) mice to two different forms of training, namely voluntary wheel running and swimming, and compared the response at the morphological, myocellular and molecular levels. We found that both regimes normalized changes of myostatin deficiency and restored muscle function. We showed that both exercise training regimes increased muscle capillary density and the expression of Ucp3, Cpt1α, Pdk4 and Errγ, key markers for oxidative metabolism. Cross-sectional area of hypertrophic myofibres from MSTN(-/-) mice decreased towards wild-type values in response to exercise and, in this context, Bnip3, a key autophagy-related gene, was upregulated. This reduction in myofibre size caused an increase of the nuclear-to-cytoplasmic ratio towards wild-type values. Importantly, both training regimes increased muscle force in MSTN(-/-) mice. We conclude that impaired skeletal muscle function in myostatin-deficient mice can be improved through endurance exercise-mediated remodelling of muscle fibre size and metabolic profile.

Modulation of reactive oxygen species in skeletal muscle by myostatin is mediated through NF-κB.

Abnormal levels of reactive oxygen species (ROS) and inflammatory cytokines have been observed in the skeletal muscle during muscle wasting including sarcopenia. However, the mechanisms that signal ROS production and prolonged maintenance of ROS levels during muscle wasting are not fully understood. Here, we show that myostatin (Mstn) is a pro-oxidant and signals the generation of ROS in muscle cells. Myostatin, a transforming growth factor-ß (TGF-ß) family member, has been shown to play an important role in skeletal muscle wasting by increasing protein degradation. Our results here show that Mstn induces oxidative stress by producing ROS in skeletal muscle cells through tumor necrosis factor-α (TNF-α) signaling via NF-κB and NADPH oxidase. Aged Mstn null (Mstn(-/-) ) muscles, which display reduced sarcopenia, also show an increased basal antioxidant enzyme (AOE) levels and lower NF-κB levels indicating efficient scavenging of excess ROS. Additionally, our results indicate that both TNF-α and hydrogen peroxide (H(2) O(2) ) are potent inducers of Mstn and require NF-κB signaling for Mstn induction. These results demonstrate that Mstn and TNF-α are components of a feed forward loop in which Mstn triggers the generation of second messenger ROS, mediated by TNF-α and NADPH oxidase, and the elevated TNF-α in turn stimulates Mstn expression. Higher levels of Mstn in turn induce muscle wasting by activating proteasomal-mediated catabolism of intracellular proteins. Thus, we propose that inhibition of ROS induced by Mstn could lead to reduced muscle wasting during sarcopenia.

Myostatin from the heart: local and systemic actions in cardiac failure and muscle wasting.

A significant proportion of heart failure patients develop skeletal muscle wasting and cardiac cachexia, which is associated with a very poor prognosis. Recently, myostatin, a cytokine from the transforming growth factor-ß (TGF-ß) family and a known strong inhibitor of skeletal muscle growth, has been identified as a direct mediator of skeletal muscle atrophy in mice with heart failure. Myostatin is mainly expressed in skeletal muscle, although basal expression is also detectable in heart and adipose tissue. During pathological loading of the heart, the myocardium produces and secretes myostatin into the circulation where it inhibits skeletal muscle growth. Thus, genetic elimination of myostatin from the heart reduces skeletal muscle atrophy in mice with heart failure, whereas transgenic overexpression of myostatin in the heart is capable of inducing muscle wasting. In addition to its endocrine action on skeletal muscle, cardiac myostatin production also modestly inhibits cardiomyocyte growth under certain circumstances, as well as induces cardiac fibrosis and alterations in ventricular function. Interestingly, heart failure patients show elevated myostatin levels in their serum. To therapeutically influence skeletal muscle wasting, direct inhibition of myostatin was shown to positively impact skeletal muscle mass in heart failure, suggesting a promising strategy for the treatment of cardiac cachexia in the future.

Myostatin-deficient mice exhibit reduced insulin resistance through activating the AMP-activated protein kinase signalling pathway.

AIMS/HYPOTHESIS: Myostatin-null mice (Mstn(-/-)) have reduced body fat and increased tolerance to glucose. To date the molecular mechanisms through which myostatin regulates body fat content and insulin sensitivity are not known. Therefore, the aim of the current study was to identify signalling pathways through which myostatin regulates insulin sensitivity. METHODS: Wild-type (WT) mice and Mstn(-/-) mice were fed either a control chow diet or a high fat diet (HFD) for 12 weeks. Glucose tolerance testing and insulin stimulated glucose uptake by M. extensor digitorum longus (EDL) were used as variables to determine insulin sensitivity. Quantitative PCR, Western blotting and enzyme assays were used to monitor AMP-activated protein kinase (AMPK) levels and activity. RESULTS: Mstn(-/-) mice exhibited reduced fat accumulation and peripheral insulin resistance when compared with WT mice, even when they were fed an HFD. Furthermore, treatment with a myostatin antagonist also increased insulin sensitivity during HFD. Consistent with increased insulin sensitivity, we also detected elevated levels of GLUT4, AKT, p-AKT and insulin receptor substrate-1 in Mstn(-/-) muscles. Molecular analysis showed that there is increased expression and activity of AMPK in Mstn(-/-) muscles. Furthermore, we also observed an increase in the AMPK downstream target genes, Sirt1 and Pgc-1α (also known as Ppargc1a), in skeletal muscle of Mstn(-/-) mice. CONCLUSIONS/INTERPRETATION: We conclude that myostatin inactivation leads to increased AMPK levels and activity resulting in increased insulin sensitivity of skeletal muscle. We propose that, by regulating AMPK in skeletal muscle and adipose tissues, myostatin plays a major role in regulating insulin signalling.

Myostatin knockout mice increase oxidative muscle phenotype as an adaptive response to exercise.

Myostatin-deficient mice (MSTN (-/-)) display excessive muscle mass and this is associated with a profound loss of oxidative metabolic properties. In this study we analysed the effect of two endurance-based exercise regimes, either a forced high-impact swim training or moderate intensity voluntary wheel running on the adaptive properties of the tibialis anterior and plantaris muscle from MSTN (-/-) mice. MSTN (-/-) and wild type (MSTN (+/+)) animals had comparable performances in the wheel running regime in terms of distance, average speed and time, but MSTN (-/-) mice showed a reduced ability to sustain a high-impact activity via swimming. Swim training elicited muscle specific adaptations on fibre type distribution in MSTN (-/-); the tibialis anterior displaying a partial transformation in contrast to the plantaris which showed no change. Conversely, wheel running induced similar changes in fibre type composition of both muscles, favouring transitions from IIB-to-IIA. Succinate dehydrogenase activity, an indicator of mitochondrial oxidative potential was increased in response to either exercise regime, with wheel running eliciting more robust changes in the MSTN (-/-) muscles. Examination of the cross sectional area of individual fibre types showed genotype-specific responses with MSTN (-/-) mice exhibiting an incapability of fibre enlargement following the wheel running regime, as opposed to MSTN (+/+) mice and a greater susceptibility to muscle fibre area loss following swimming. In conclusion, the muscle fibre hypertrophy, oxidative capacity and glycolytic phenotype of myostatin deficient muscle can be altered with endurance exercise regimes.

Role of myostatin (GDF-8) signaling in the human anterior cruciate ligament.

Myostatin, also referred to as growth and differentiation factor-8 (GDF-8), is expressed in muscle tissue where it functions to suppress myoblast proliferation and myofiber hypertrophy. Recently, myostatin and its receptor, the type IIB activin receptor (ActRIIB), were detected in the leg tendons of mice, and recombinant myostatin was shown to increase cellular proliferation and the expression of type 1 collagen in primary fibroblasts from mouse tendons. We sought to determine whether myostatin and its receptor were present in human anterior cruciate ligament (ACL) tissue, and if myostatin treatment had any effect on primary ACL fibroblasts. ACL tissue samples were obtained from material discarded during ACL reconstruction surgery. Real-time PCR and immunohistochemistry demonstrate that both myostatin and its receptor are abundant in the human ACL. Primary fibroblasts isolated from human ACL specimens were treated with recombinant myostatin, and myostatin treatment increased fibroblast proliferation as well as the expression of tenascin C (TNC), type 1 collagen, and transforming growth factor-beta1. Real-time PCR analysis of TNC and type 1 collagen expression in ACL specimens from normal mice and mice lacking myostatin supported these findings by showing that both TNC and type 1 collagen were downregulated in ACL tissue from myostatin-deficient mice. Together, these data suggest that myostatin is a pro-fibrogenic factor that enhances cellular proliferation and extracellular matrix synthesis by ACL fibroblasts. Recombinant myostatin may therefore have therapeutic applications in the area of tendon and ligament engineering and regeneration.