Muscle hypertrophy driven by myostatin blockade does not require stem/precursor-cell activity

Myostatin, a member of the TGF-beta family, has been identified as a powerful inhibitor of muscle growth. Absence or blockade of myostatin induces massive skeletal muscle hypertrophy that is widely attributed to proliferation of the population of muscle fiber-associated satellite cells that have been identified as the principle source of new muscle tissue during growth and regeneration. Postnatal blockade of myostatin has been proposed as a basis for therapeutic strategies to combat muscle loss in genetic and acquired myopathies. But this approach, according to the accepted mechanism, would raise the threat of premature exhaustion of the pool of satellite cells and eventual failure of muscle regeneration. Here, we show that hypertrophy in the absence of myostatin involves little or no input from satellite cells. Hypertrophic fibers contain no more myonuclei or satellite cells and myostatin had no significant effect on satellite cell proliferation in vitro, while expression of myostatin receptors dropped to the limits of detectability in postnatal satellite cells. Moreover, hypertrophy of dystrophic muscle arising from myostatin blockade was achieved without any apparent enhancement of contribution of myonuclei from satellite cells. These findings contradict the accepted model of myostatin-based control of size of postnatal muscle and reorient fundamental investigations away from the mechanisms that control satellite cell proliferation and toward those that increase myonuclear domain, by modulating synthesis and turnover of structural muscle fiber proteins. It predicts too that any benefits of myostatin blockade in chronic myopathies are unlikely to impose any extra stress on the satellite cells.

Myostatin is a key mediator between energy metabolism and endurance capacity of skeletal muscle

Myostatin (Mstn) participates in the regulation of skeletal muscle size and has emerged as a regulator of muscle metabolism. Here, we hypothesized that lack of myostatin profoundly depresses oxidative phosphorylation-dependent muscle function. Toward this end, we explored Mstn/ mice as a model for the constitutive absence of myostatin and AAV-mediated overexpression of myostatin propeptide as a model of myostatin blockade in adult wild-type mice. We show that muscles from Mstn/ mice, although larger and stronger, fatigue extremely rapidly. Myostatin deficiency shifts muscle from aerobic toward anaerobic energy metabolism, as evidenced by decreased mitochondrial respiration, reduced expression of PPAR transcriptional regulators, increased enolase activity, and exercise-induced lactic acidosis. As a consequence, constitutively reduced myostatin signaling diminishes exercise capacity, while the hypermuscular state of Mstn/ mice increases oxygen consumption and the energy cost of running. We wondered whether these results are the mere consequence of the congenital fiber-type switch toward a glycolytic phenotype of constitutive Mstn/ mice. Hence, we overexpressed myostatin propeptide in adult mice, which did not affect fiber-type distribution, while nonetheless causing increased muscle fatigability, diminished exercise capacity, and decreased Pparb/d and Pgc1a expression. In conclusion, our results suggest that myostatin endows skeletal muscle with high oxidative capacity and low fatigability, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.

Lack of myostatin results in excessive muscle growth but impaired force generation

The lack of myostatin promotes growth of skeletal muscle, and blockade of its activity has been proposed as a treatment for various muscle-wasting disorders. Here, we have examined two independent mouse lines that harbor mutations in the myostatin gene, constitutive null (Mstn(-/-)) and compact (Berlin High Line, BEH(c/c)). We report that, despite a larger muscle mass relative to age-matched wild types, there was no increase in maximum tetanic force generation, but that when expressed as a function of muscle size (specific force), muscles of myostatin-deficient mice were weaker than wild-type muscles. In addition, Mstn(-/-) muscle contracted and relaxed faster during a single twitch and had a marked increase in the number of type IIb fibers relative to wild-type controls. This change was also accompanied by a significant increase in type IIB fibers containing tubular aggregates. Moreover, the ratio of mitochondrial DNA to nuclear DNA and mitochondria number were decreased in myostatin-deficient muscle, suggesting a mitochondrial depletion. Overall, our results suggest that lack of myostatin compromises force production in association with loss of oxidative characteristics of skeletal muscle.

Blockade of ActRIIB signaling triggers muscle fatigability and metabolic myopathy

Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here we show in mice that four months of pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling down-regulates Porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phophorus Magnetic Resonance Spectroscopy (31P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparβ, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.

Morphology and myofiber composition of skeletal musculature of the forelimb in young and aged wild type and myostatin null mice

Most current research into therapeutic approaches to muscle diseases involves the use of the mouse as an experimental model. Furthermore, a major strategy to alleviate myopathic symptoms through enhancing muscle growth and regeneration is to inhibit the action of myostatin (Mstn), a transforming growth factor-beta (TGF-beta) family member that inhibits muscle growth. Presently, however, no study has expanded the morphological analysis of mouse skeletal muscle beyond a few individual muscles of the distal hindlimb, through which broad conclusions have been based. Therefore, we have initially undertaken an expansive analysis of the skeletal musculature of the mouse forelimb and highlighted the species-specific differences between equivalent muscles of the rat, another prominently used experimental model. Subsequently, we examined the musculature of the forelimb in both young and old adult wild-type (mstn(+/+)) and myostatin null (mstn(-/-)) mice and assessed the potential beneficial and detrimental effects of myostatin deletion on muscle morphology and composition during the aging process. We showed that: (1) the forelimb muscles of the mouse display a more glycolytic phenotype than those of the rat; (2) in the absence of myostatin, the induced myofiber hyperplasia, hypertrophy, and glycolytic conversion all occur in a muscle-specific manner; and, importantly, (3) the loss of myostatin significantly alters the dynamics of postnatal muscle growth and impairs age-related oxidative myofiber conversion.

Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear: cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice. (C) 2009 Elsevier B.V. All rights reserved.

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation

Objectives: Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes. Methods: We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We. determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved. Results: Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2. Conclusions: We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All fights reserved.

Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis

Follistatin is known to antagonise the function of several members of the TGF-beta family of secreted signalling factors, including Myostatin, the most powerful inhibitor of muscle growth characterised to date. In this study, we compare the expression of Myostatin and Follistatin during chick development and show that they are expressed in the vicinity or in overlapping domains to suggest possible interaction during muscle development. We performed yeast and mammalian two-hybrid studies and show that Myostatin and Follistatin interact directly. We further show that single modules of the Follistatin protein cannot associate with Myostatin suggesting that the entire protein is required for the interaction. We analysed the interaction kinetics of the two proteins and found that Follistatin binds Myostatin with a high affinity of 5.84 x 10(-10) M. We next tested whether Follistatin suppresses Myostatin activity during muscle development. We confirmed our previous observation that treatment of chick limb buds with Myostatin results in a severe decrease in the expression of two key myogenic regulatory genes Pax-3 and MyoD. However, in the presence of Follistatin, the Myostatin-mediated inhibition of Pax-3 and MyoD expression is blocked. We additionally show that Myostatin inhibits terminal differentiation of muscle cells in high-density cell cultures of limb mesenchyme (micromass) and that Follistatin rescues muscle differentiation in a concentration-dependent manner. In summary, our data suggest that Follistatin antagonises Myostatin by direct protein interaction, which prevents Myostatin from executing its inhibitory effect on muscle development. (C) 2004 Elsevier Inc. All rights reserved.

Myostatin regulates cardiomyocyte growth through modulation of Akt signaling

Myostatin is a highly conserved, potent negative regulator of skeletal muscle hypertrophy in many species, from rodents to humans, although its mechanisms of action are incompletely understood. Transcript profiling of hearts from a genetic model of cardiac hypertrophy revealed dramatic upregulation of myostatin, not previously recognized to play a role in the heart. Here we show that myostatin abrogates the cardiomyocyte growth response to phenylephrine in vitro through inhibition of p38 and the serine - threonine kinase Akt, a critical determinant of cell size in many species from drosophila to mammals. Evaluation of male myostatin-null mice revealed that their cardiomyocytes and hearts overall were slightly smaller at baseline than littermate controls but exhibited more exuberant growth in response to chronic phenylephrine infusion. The increased cardiac growth in myostatin-null mice corresponded with increased p38 phosphorylation and Akt activation in vivo after phenylephrine treatment. Together, these data demonstrate that myostatin is dynamically regulated in the heart and acts more broadly than previously appreciated to regulate growth of multiple types of striated muscle.

Characterisation of connective tissue from the hypertrophic skeletal muscle of myostatin null mice

Myostatin is a potent inhibitor of muscle development. Genetic deletion of myostatin in mice results in muscle mass increase, with muscles often weighing three times their normal values. Contracting muscle transfers tension to skeletal elements through an elaborate connective tissue network. Therefore, the connective tissue of skeletal muscle is an integral component of the contractile apparatus. Here we examine the connective tissue architecture in myostatin null muscle. We show that the hypertrophic muscle has decreased connective tissue content compared with wild-type muscle. Secondly, we show that the hypertrophic muscle fails to show the normal increase in muscle connective tissue content during ageing. Therefore, genetic deletion of myostatin results in an increase in contractile elements but a decrease in connective tissue content. We propose a model based on the contractile profile of muscle fibres that reconciles this apparent incompatible tissue composition phenotype.

Food restriction reverses the hyper-muscular phenotype and force generation capacity deficit of the myostatin null mouse

Food restriction has a great impact on skeletal muscle mass by inducing muscle protein breakdown to provide substrates for energy production through gluconeogenesis. Genetic models of hyper-muscularity interfere with the normal balance between protein synthesis and breakdown which eventually results in extreme muscle growth. Mutations or deletions in the myostatin gene result in extreme muscle mass. Here we evaluated the impact of food restriction for a period of 5 weeks on skeletal muscle size (i.e., fibre cross-sectional area), fibre type composition and contractile properties (i.e., tetanic and specific force) in myostatin null mice. We found that this hyper-muscular model was more susceptible to catabolic processes than wild type mice. The mechanism of skeletal muscle mass loss was examined and our data shows that the myostatin null mice placed on a low calorie diet maintained the activity of molecules involved in protein synthesis and did not up-regulate the expression of genes pivotal in ubiquitin-mediated protein degradation. However, we did find an increase in the expression of genes associated with autophagy. Surprisingly, the reduction on muscle size was followed by improved tetanic and specific force in the null mice compared to wild type mice. These data provide evidence that food restriction may revert the hyper-muscular phenotype of the myostatin null mouse restoring muscle function.

Adeno-associated virus-8-Mediated intravenous transfer of myostatin propeptide leads to systemic functional improvements of slow but not fast muscle

Myostatin is a member of the transformating growth factor-_ (TGF-_) superfamily of proteins and is produced almost exclusively in skeletal muscle tissue, where it is secreted and circulates as a serum protein. Myostatin acts as a negative regulator of muscle mass through the canonical SMAD2/3/4 signaling pathway. Naturally occurring myostatin mutants exhibit a ‘double muscling’ phenotype in which muscle mass is dramatically increased as a result of both hypertrophy and hyperplasia. Myostatin is naturally inhibited by its own propeptide; therefore, we assessed the impact of adeno associated virus-8 (AAV8) myostatin propeptide vectors when systemically introduced in MF-1 mice. We noted a significant systemic increase in muscle mass in both slow and fast muscle phenotypes, with no evidence of hyperplasia; however, the nuclei-to- cytoplasm ratio in all myofiber types was significantly reduced. An increase in muscle mass in slow (soleus) muscle led to an increase in force output; however, an increase in fast (extensor digitorum longus [EDL]) muscle mass did not increase force output. These results suggest that the use of gene therapeutic regimens of myostatin inhibition for age-related or disease-related muscle loss may have muscle-specific effects.

Molecular requirements for L-type Ca2+ channel blockade by testosterone

Despite being generally perceived as detrimental to the cardiovascular system, testosterone has marked beneficial vascular effects; most notably it acutely and directly causes vasodilatation. Indeed, men with hypotestosteronaemia can present with myocardial ischemia and angina which can be rapidly alleviated by infusion of testosterone. To date, however, in vitro studies have failed to provide a convincing mechanism to account for this clinically important effect. Here, using whole-cell patch-clamp recordings to measure current flow through recombinant human L-type Ca2+ channel alpha(1C) subunits (Ca(v)1.2), we demonstrate that testosterone inhibits such currents in a concentration-dependent manner. Importantly, this occurs over the physiological range of testosterone concentrations (IC50 34 nM), and is not mimicked by the metabolite 5alpha-androstan-17beta-ol-3-one (DHT), nor by progesterone or estradiol, even at high (10 microM) concentration. L-type Ca2+ channels in the vasculature are also important clinical targets for vasodilatory dihydropyridines. A single point mutation (T1007Y) almost completely abolishes nifedipine sensitivity in our recombinant expression system. Crucially, the same mutation renders the channels insensitive to testosterone. Our data strongly suggest, for the first time, the molecular requirements for testosterone binding to L-type Ca2+ channels, thereby supporting its beneficial role as an endogenous Ca2+ channel antagonist in the treatment of cardiovascular disease.

Antisense-induced myostatin exon skipping leads to muscle hypertrophy in mice following Octa‑guanidine morpholino oligomer treatment

Myostatin is a negative regulator of muscle mass, and several strategies are being developed to knockdown its expression to improve muscle-wasting conditions. Strategies using antimyostatin-blocking antibodies, inhibitory-binding partners, signal transduction blockers, and RNA interference system (RNAi)-based knockdown have yielded promising results and increased muscle mass in experimental animals. These approaches have, however, a number of disadvantages such as transient effects or adverse immune complications. We report here the use of antisense oligonucleotides (AOs) to manipulate myostatin pre-mRNA splicing and knockdown myostatin expression. Both 2’O-methyl phosphorothioate RNA (2’OMePS) and phosphorodiamidate morpholino oligomers (PMO) led to efficient exon skipping in vitro and in vivo and knockdown of myostatin at the transcript level. The substantial myostatin exon skipping observed after systemic injection of Vivo-PMO into normal mice led to a significant increase in soleus muscle mass as compared to the controls injected with normal saline suggesting that this approach could be feasible to ameliorate muscle-wasting pathologies.

Voltage-gated sodium (NaV) channel blockade by plant cannabinoids does not confer anticonvulsant effects per se

Cannabidiol (CBD) is a non-psychoactive, well-tolerated, anticonvulsant plant cannabinoid, although its mechanism(s) of seizure suppression remains unknown. Here, we investigate the effect of CBD and the structurally similar cannabinoid, cannabigerol (CBG), on voltage-gated Na+ (NaV) channels, a common anti-epileptic drug target. CBG’s anticonvulsant potential was also assessed in vivo. CBD effects on NaV channels were investigated using patch-clamp recordings from rat CA1 hippocampal neurons in brain slices, human SH-SY5Y (neuroblastoma) cells and mouse cortical neurons in culture. CBG effects were also assessed in SH-SY5Y cells and mouse cortical neurons. CBD and CBG effects on veratridine-stimulated human recombinant NaV1.1, 1.2 or 1.5 channels were assessed using a membrane potential-sensitive fluorescent dye high-throughput assay. The effect of CBG on pentyleneterazole-induced (PTZ) seizures was assessed in rat. CBD (10M) blocked NaV currents in SH-SY5Y cells, mouse cortical neurons and recombinant cell lines, and affected spike parameters in rat CA1 neurons; CBD also significantly decreased membrane resistance. CBG blocked NaV to a similar degree to CBD in both SH-SY5Y and mouse recordings, but had no effect (50-200mg/kg) on PTZ-induced seizures in rat. CBD and CBG are NaV channel blockers at micromolar concentrations in human and murine neurons and recombinant cells. In contrast to previous reports investigating CBD, CBG had no effect upon PTZ-induced seizures in rat, indicating that NaV blockade per se does not correlate with anticonvulsant effects.