The two myostatin genes of Atlantic salmon (Salmo salar) are expressed in a variety of tissues.

Two myostatin isoforms were identified in Atlantic salmon (Salmo salar) by RT-PCR, and genomic sequences encoding this negative muscle growth factor were for the first time isolated from a nonmammalian species. Salmon myostatin isoform I is transcribed in white skeletal muscle as a 2346-nucleotide mRNA species that encodes a precursor protein of 373 amino acids. Salmon myostatin I shows 93% sequence identity with isoform II which was isolated from white muscle as a partial cDNA sequence of 1409 nucleotides. In contrast to the restricted gene expression of myostatin in mammals, salmon myostatin I and II mRNAs were identified by RT-PCR in multiple tissues, including white muscle, intestine, brain, gills, tongue and eye. In addition, isoform I mRNA was found in red skeletal muscle, heart, spleen, and ovarian tissue. Using polyclonal antibodies against both isoforms, a 55-kDa precursor protein was detected by Western blot analysis in the red and white skeletal muscle, heart, intestine, and brain. Immunoreactive peptides of 35-40 kDa were identified in the gills, tongue, spleen, and head kidney, while the 25-kDa mature myostatin was found in the eye and serum, and in vitro expressed in rabbit reticulocyte lysate. Salmon myostatin was immunohistochemically localized in the sarcoplasma of red and white muscle fibres, in intestinal epithelial cells, at the basis of the branchial primary lamellae, and in odontoblasts and ameloblasts of the tongue teeth. The results indicate that the role of fish myostatin may not be restricted to muscle growth regulation, but may have additional functions similar to the growth/differentiation factor-11 in mammals.     

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-β (TGF-β) 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.     

Relative abundance of mature myostatin rather than total myostatin is negatively associated with bone mineral density in Chinese

Myostatin is mainly secreted by skeletal muscle and negatively regulates skeletal muscle growth. However, the roles of myostatin on bone metabolism are still largely unknown. Here, we recruited two large populations containing 6308 elderly Chinese and conducted comprehensive statistical analyses to evaluate the associations among lean body mass (LBM), plasma myostatin, and bone mineral density (BMD). Our data revealed that total myostatin in plasma was mainly determined by LBM. The relative abundance of mature myostatin (mature/total) was significantly lower in high versus low BMD subjects. Moreover, the relative abundance of mature myostatin was positively correlated with bone resorption marker. Finally, we carried out in vitro experiments and found that myostatin has inhibitory effects on the proliferation and differentiation of human osteoprogenitor cells. Taken together, our results have demonstrated that the relative abundance of mature myostatin in plasma is negatively associated with BMD, and the underlying functional mechanism for the association is most likely through inhibiting osteoblastogenesis and promoting osteoclastogenesis.     

Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin

Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.     

Identification of a novel pool of extracellular pro-myostatin in skeletal muscle

Myostatin, a transforming growth factor-beta superfamily ligand, negatively regulates skeletal muscle growth. Generation of the mature signaling peptide requires cleavage of pro-myostatin by a proprotein convertase, which is thought to occur constitutively in the Golgi apparatus. In serum, mature myostatin is found in an inactive, non-covalent complex with its prodomain. We find that in skeletal muscle, unlike serum, myostatin is present extracellularly as uncleaved pro-myostatin. In cultured cells, co-expression of pro-myostatin and latent transforming growth factor-beta-binding protein-3 (LTBP-3) sequesters pro-myostatin in the extracellular matrix, and secreted pro-myostatin can be cleaved extracellularly by the proprotein convertase furin. Co-expression of LTBP-3 with myostatin reduces phosphorylation of Smad2, and ectopic expression of LTBP-3 in mature mouse skeletal muscle increases fiber area, consistent with reduction of myostatin activity. We propose that extracellular pro-myostatin constitutes the major pool of latent myostatin in muscle. Post-secretion activation of this pool by furin family proprotein convertases may therefore represent a major control point for activation of myostatin in skeletal muscle.     

Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp and 667 bp, respectively. A microsatellite sequence, CA₃₀ and CA₂₆ separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues 1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR, residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin gene was expressed not only in the skeletal muscle, but also in the other tissues.     

Molecular characterization of myostatin in black seabream, Acanthopagrus schlegelii.

Myostatin is a negative regulator of skeletal muscle growth and has a potential application in aquaculture. The black seabream myostatin gene was cloned and sequenced. It had three exons encoding a protein of 382 amino acids. A 90 bp 5'-untranslated region (UTR) and a 536 bp 3'-UTR were obtained by RACE. Four microsatellite sequences, a (CAG)9, a (TC)12, a (CA)16 repeat and an "imperfect" (CA)25 microsatellite, were found in the myostatin. Two introns were 329 and 742 bp in length, respectively. The deduced amino acid sequence of the myostatin had a putative amino terminal signal sequence, a TGF-beta propeptide domain, a RXXR proteolytic processing site, a TGF-beta domain, and 12 conserved cysteine residues. The myostatin gene was expressed in four of the examined ten tissues and organs. The expression of myostatin was the strongest in the skeletal muscle and brain, intermediate in the eye, and low in the heart.     

Myostatin maps to porcine chromosome 15 by linkage and physical analyses

Myostatin belongs to the transforming growth factor-β superfamily, and is expressed specifically in developing and mature skeletal muscle. Myostatin appears to act as a negative regulator of muscle development, since mice with targeted disruption of this gene display a large increase in muscle mass. In this study, the porcine myostatin gene was mapped to chromosome 15q2·3 by fluorescence in situ hybridization. Myostatin was also positioned within the chromosome 15 linkage group using both a polymorphism located in the second intron and an associated microsatellite. The development of highly polymorphic markers associated with myostatin will support population studies to identify alleles of this gene that affects muscle mass and/or fat deposition in swine.     

Inhibition of myostatin in adult mice increases skeletal muscle mass and strength

A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.     

Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia

Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.     

Effects of myostatin deletion in aging mice

Inhibitors of myostatin, a negative regulator of skeletal muscle mass, are being developed to mitigate aging-related muscle loss. Knock-out (KO) mouse studies suggest myostatin also affects adiposity, glucose handling and cardiac growth. However, the cardiac consequences of inhibiting myostatin remain unclear. Myostatin inhibition can potentiate cardiac growth in specific settings ( Morissette et al., 2006) , a concern because of cardiac hypertrophy is associated with adverse clinical outcomes. Therefore, we examined the systemic and cardiac effects of myostatin deletion in aged mice (27–30 months old). Heart mass increased comparably in both wild-type (WT) and KO mice. Aged KO mice maintained twice as much quadriceps mass as aged WT; however, both groups lost the same percentage (36%) of adult muscle mass. Dual-energy X-ray absorptiometry revealed increased bone density, mineral content, and area in aged KO vs. aged WT mice. Serum insulin and glucose levels were lower in KO mice. Echocardiography showed preserved cardiac function with better fractional shortening (58.1% vs. 49.4%, P  = 0.002) and smaller left ventricular diastolic diameters (3.41 vs. 2.71, P  = 0.012) in KO vs. WT mice. Phospholamban phosphorylation was increased 3.3-fold in KO hearts ( P  < 0.05), without changes in total phospholamban, sarco(endo)plasmic reticulum calcium ATPase 2a or calsequestrin. Aged KO hearts showed less fibrosis by Masson's Trichrome staining. Thus, myostatin deletion does not affect aging-related increases in cardiac mass and appears beneficial for bone density, insulin sensitivity and heart function in senescent mice. These results suggest that clinical interventions designed to inhibit skeletal muscle mass loss with aging could have beneficial effects on other organ systems as well.     

The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum

Myostatin, also known as growth and differentiation factor 8, is a member of the transforming growth factor beta superfamily that negatively regulates skeletal muscle mass (1). Recent experiments have shown that myostatin activity is detected in serum by a reporter gene assay only after activation by acid, suggesting that native myostatin circulates as a latent complex (2). We have used a monoclonal myostatin antibody, JA16, to isolate the native myostatin complex from normal mouse and human serum. Analysis by mass spectrometry and Western blot shows that circulating myostatin is bound to at least two major proteins, the myostatin propeptide and the follistatin-related gene (FLRG). The myostatin propeptide is known to bind and inhibit myostatin in vitro (3). Here we show that this interaction is relevant in vivo, with a majority (>70%) of myostatin in serum bound to its propeptide. Studies with recombinant V5-His-tagged FLRG protein confirm a direct interaction between mature myostatin and FLRG. Functional studies show that FLRG inhibits myostatin activity in a reporter gene assay. These experiments suggest that the myostatin propeptide and FLRG are major negative regulators of myostatin in vivo.     

Myostatin regulates glucose metabolism via the AMP-activated protein kinase pathway in skeletal muscle cells

Myostatin (Mstn) is a secreted growth factor predominately expressed in skeletal muscle that negatively regulates skeletal muscle mass. Recent studies have indicated that loss function of myostatin not only increases muscle mass but also improves insulin sensitivity in vivo. In the present report, we demonstrated that myostatin regulates glucose metabolism by promoting glucose consumption and glucose uptake, increasing glycolysis, and inhibiting glycogen synthesis in skeletal muscle cells. Microarray analysis revealed that myostatin upregulates several genes involved in regulating glucose metabolism such as Glut1, Glut4, Hk2, and IL-6. Further investigation of the molecular basis of these phenomena revealed that AMP-activated protein kinase (AMPK), a key component for maintaining energy homeostasis, was activated by myostatin for promotion of glycolysis. Taken together, these findings provide the first experimental evidence that myostatin regulates glucose metabolism through the AMPK signal pathway in muscle cells. Importantly, our findings highlight that continued investigation of the metabolic function of myostatin is necessary for a comprehensive understanding of its active role in the regulation of skeletal muscle energy metabolism.     

Characterization of a myostatin-like gene from the bay scallop, Argopecten irradians

A complete cDNA was cloned from the bay scallop (Argopecten irradians) that codes for a 382-amino-acid myostatin-like protein (sMSTN). The sMSTN sequence is most similar to mammalian myostatin (MSTN), containing a conserved proteolytic cleavage site (RXXR) and conserved cysteine residues in the C-terminus. Based on quantitative RT-PCR, the sMSTN gene is predominantly expressed in the adductor muscle, with limited expression in other tissues. Using the sMSTN sequence, a Ciona MSTN-like gene was also identified from the Ciona intestinalis genome. These findings indicate that the MSTN gene has been conserved throughout evolution and suggests that MSTN could play a major role in muscle growth and development in invertebrates, as it does in mammals.     

Modulation of myostatin expression during modified muscle use.

Previous findings have provided strong evidence that myostatin functions as a negative regulator of muscle mass during development and growth. In the present study, we test the hypothesis that myostatin may serve a similar function in fully differentiated muscle experiencing modified loading. Our findings show that myostatin expression can be modulated in fully differentiated, nonpathological skeletal muscle in a manner that is inversely related to changes in muscle mass. Atrophy of rat hind limb muscles induced by 10 days of unloading resulted in a 16% decrease in plantaris mass, a 110% increase in myostatin mRNA, and a 37% increase in myostatin protein. Immunohistochemical observations showed a detectable increase in myostatin concentration at myotendinous junctions during muscle unloading. The concentration of myostatin mRNA and protein returned to values not significantly different from ambulatory controls after 4 days of reloading, during which time plantaris mass also returned to control values. However, the results also show that periods of 30 min of daily muscle loading during the unloading period were sufficient to prevent significant losses of muscle mass caused by unloading, although myostatin mRNA still showed a 55% increase in concentration. Thus, significant increases in myostatin expression are not sufficient for muscle mass loss, although muscle mass loss during unloading is accompanied by increases in myostatin.     

Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects

Since its identification in 1997, myostatin has been considered as a novel and unique negative regulator of muscle growth, as mstn-/- mice display a dramatic and widespread increase in skeletal muscle mass. Myostatin also appears to be involved in muscle homeostasis in adults as its expression is regulated during muscle atrophy. Moreover, deletion of the myostatin gene seems to affect adipose tissue mass in addition to skeletal muscle mass. Natural myostatin gene mutations occur in cattle breeds such as Belgian Blue, exhibiting an obviously increased muscle mass, but also in humans, as has recently been demonstrated. Here we review these natural mutations and their associated phenotypes as well as the physiological influence of the alterations in myostatin expression and the physiopathological consequences of changes in myostatin expression, especially with regard to satellite cells. Interestingly, studies have demonstrated some rescue effects of myostatin in muscular pathologies such as myopathies, providing a novel pharmacological strategy for treatment. Furthermore, the myostatin pathway is now better understood thanks to in vitro studies and it consists of inhibition of myoblast progression in the cell cycle, inhibition of myoblast terminal differentiation, in both cases associated to protection from apoptosis. The molecular pathway driving the myogenic myostatin influence is currently under extensive study and many molecular partners of myostatin have been identified, suggesting novel potent muscle growth enhancers for both human and agricultural applications.     

Characterization of gene expression in double-muscled and normal-muscled bovine embryos

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth. Cattle with mutations that inactivate myostatin exhibit a remarkable increase in mass of skeletal muscle called double muscling that is accompanied by an equally remarkable decrease in carcass fat. Although a mouse knockout model has been created which results in mice with a 200% increase in skeletal muscle mass, molecular mechanisms whereby myostatin regulates skeletal muscle and fat mass are not fully understood. Using suppressive subtractive hybridization, genes that were differentially expressed in double-muscled vs. normal-muscled cattle embryos were identified. Genetic variation at other loci was minimized by using embryonic samples collected from related Piedmontese x Angus dams or Belgian Blue x Hereford dams bred to a single sire of the same breed composition. Embryos were collected at 31-33 days of gestation, which is 2-4 days after high-level expression of myostatin in the developing bovine embryo. The suppressive subtraction resulted in 30 clones that were potentially differentially expressed, 19 of which were confirmed by macroarray analysis. Several of these genes have biological functions that suggest that they are directly involved in myostatin's regulation of skeletal muscle development. Furthermore, several of these genes map to quantitative trait loci known to interact with variation in the myostatin gene.