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Skeletal muscle regeneration that follows acute injury is strongly influenced by interactions with immune cells that invade and proliferate in the damaged tissue. Discoveries over the past 20 years have identified many of the key mechanisms through which myeloid cells, especially macrophages, regulate muscle regeneration. In addition, lymphoid cells that include CD8+ T-cells and regulatory T-cells also significantly affect the course of muscle regeneration. During aging, the regenerative capacity of skeletal muscle declines, which can contribute to progressive loss of muscle mass and function. Those age-related reductions in muscle regeneration are accompanied by systemic, age-related changes in the immune system, that affect many of the myeloid and lymphoid cell populations that can influence muscle regeneration. In this review, we present recent discoveries that indicate that aging of the immune system contributes to the diminished regenerative capacity of aging muscle. Intrinsic, age-related changes in immune cells modify their expression of factors that affect the function of a population of muscle stem cells, called satellite cells, that are necessary for normal muscle regeneration. For example, age-related reductions in the expression of growth differentiation factor-3 (GDF3) or CXCL10 by macrophages negatively affect adult myogenesis, by disrupting regulatory interactions between macrophages and satellite cells. Those changes contribute to a reduction in the numbers and myogenic capacity of satellite cells in old muscle, which reduces their ability to restore damaged muscle. In addition, aging produces changes in the expression of molecules that regulate the inflammatory response to injured muscle, which also contributes to age-related defects in muscle regeneration. For example, age-related increases in the production of osteopontin by macrophages disrupts the normal inflammatory response to muscle injury, resulting in regenerative defects. These nascent findings represent the beginning of a newly-developing field of investigation into mechanisms through which aging of the immune system affects muscle regeneration.

Muscle aging is associated with changes in myeloid cell phenotype that may influence age-related changes in muscle structure. We tested whether preventing age-related reductions in muscle neuronal nitric oxide synthase (nNOS) would obviate age-related changes in myeloid cells in muscle. Our findings show that muscle aging is associated with elevations of anti-inflammatory M2a macrophages that can increase muscle fibrosis. Expression of a muscle-specific nNOS transgene in mice prevented age-related increases in M2a macrophages. Transgene expression also reduced expression of collagens and decreased muscle fibrosis. The nNOS transgene prevented age-related increases in arginase-1 but did not influence TGFβ expression, indicating that the transgene may prevent age-related muscle fibrosis by inhibiting the arginase-dependent profibrotic pathway. Although aged satellite cells or fibro-adipogenic precursor (FAPs) cells also promote fibrosis, transgene expression had no effect on the expression of key signaling molecules that regulate fibrogenic activity of those cells. Finally, we tested whether increases in M2a macrophages and the associated increase in fibrosis were attributable to aging of myeloid lineage cells. Young bone marrow cells (BMCs) were transplanted into young or old mice, and muscles were collected 8 months later. Muscles of young mice receiving young BMCs showed no effect on M2a macrophage number or collagen accumulation compared to age-matched, nontransplanted controls. However, muscles of old mice receiving young BMCs showed fewer M2a macrophages and less accumulation of collagen. Thus, the age-related increase in M2a macrophages in aging muscle and the associated muscle fibrosis are determined in part by the age of bone marrow cells.

The secondary loss of neuronal nitric oxide synthase (nNOS) that occurs in dystrophic muscle is the basis of numerous, complex and interacting features of the dystrophic pathology that affect not only muscle itself, but also influence the interaction of muscle with other tissues. Many mechanisms through which nNOS deficiency contributes to misregulation of muscle development, blood flow, fatigue, inflammation and fibrosis in dystrophic muscle have been identified, suggesting that normalization in NO production could greatly attenuate diverse aspects of the pathology of muscular dystrophy through multiple regulatory pathways. However, the relative importance of the loss of nNOS from the sarcolemma versus the importance of loss of total nNOS from dystrophic muscle remains unknown. Although most current evidence indicates that nNOS localization at the sarcolemma is not required to achieve NO-mediated reductions of pathology in muscular dystrophy, the question remains open concerning whether membrane localization would provide a more efficient rescue from features of the dystrophic phenotype.

Sarcopenia, the age-related loss of muscle mass, is a highly-debilitating consequence of aging. In this investigation, we show sarcopenia is greatly reduced by muscle-specific over-expression of calpastatin, the endogenous inhibitor of calcium-dependent proteases (calpains). Further, we show that calpain cleavage of specific structural and regulatory proteins in myofibrils is prevented by covalent modification of calpain by nitric oxide (NO) through S-nitrosylation. We find that calpain in adult, non-sarcopenic muscles is S-nitrosylated but that aging leads to loss of S-nitrosylation, suggesting that reduced S-nitrosylation during aging leads to increased calpain-mediated proteolysis of myofibrils. Further, our data show that muscle aging is accompanied by loss of neuronal nitric oxide synthase (nNOS), the primary source of muscle NO, and that expression of a muscle-specific nNOS transgene restores calpain S-nitrosylation in aging muscle and prevents sarcopenia. Together, the findings show that in vivo reduction of calpain S-nitrosylation in muscle may be an important component of sarcopenia, indicating that modulation of NO can provide a therapeutic strategy to slow muscle loss during old age.

Duchenne muscular dystrophy (DMD) involves a complex pathophysiology that is not easily explained by the loss of the protein dystrophin, the primary defect in DMD. Instead, many features of the pathology are attributable to the secondary loss of neuronal nitric oxide synthase (nNOS) from dystrophin-deficient muscle. In this investigation, we tested whether the loss of nNOS contributes to the increased fatigability of mdx mice, a model of DMD. Our findings show that the expression of a muscle-specific, nNOS transgene increases the endurance of mdx mice and enhances glycogen metabolism during treadmill-running, but did not affect vascular perfusion of muscles. We also find that the specific activity of phosphofructokinase (PFK; the rate limiting enzyme in glycolysis) is positively affected by nNOS in muscle; PFK-specific activity is significantly reduced in mdx muscles and the muscles of nNOS null mutants, but significantly increased in nNOS transgenic muscles and muscles from mdx mice that express the nNOS transgene. PFK activity measured under allosteric conditions was significantly increased by nNOS, but unaffected by endothelial NOS or inducible NOS. The specific domain of nNOS that positively regulates PFK activity was assayed by cloning and expressing different domains of nNOS and assaying their effects on PFK activity. This approach yielded a polypeptide that included the flavin adenine dinucleotide (FAD)-binding domain of nNOS as the region of the molecule that promotes PFK activity. Smaller peptides in this domain were then synthesized and used in activity assays that showed a 36-amino acid peptide in the FAD-binding domain in which most of the positive allosteric activity of nNOS for PFK resides. Mapping this peptide onto the structure of nNOS shows that the peptide is exposed on the surface, readily available for binding. Collectively, these findings indicate that defects in glycolytic metabolism and increased fatigability in dystrophic muscle may be caused in part by the loss of positive allosteric interactions between nNOS and PFK.

The immune response to dystrophin-deficient muscle promotes the pathology of Duchenne muscular dystrophy (DMD) and the mdx mouse model of DMD. In this investigation, we find that the release of major basic protein (MBP) by eosinophils is a prominent feature of DMD and mdx dystrophy and that eosinophils lyse muscle cells in vitro by the release of MBP-1. We also show that eosinophil depletions of mdx mice by injections of anti-chemokine receptor-3 reduce muscle cell lysis, although lysis of mdx muscle membranes is not reduced by null mutation of MBP-1 in vivo. However, ablation of MBP-1 expression in mdx mice produces other effects on muscular dystrophy. First, fibrosis of muscle and hearts, a major cause of mortality in DMD, is greatly reduced by null mutation of MBP-1 in mdx mice. Furthermore, either ablation of MBP-1 or eosinophil depletion causes large increases in cytotoxic T-lymphocytes (CTLs) in mdx muscles. The increase in CTLs in MBP-1-null mice does not reflect a general shift toward a Th1 inflammatory response, because the mutation had no significant effect on the expression of interferon-gamma, inducible nitric oxide synthase or tumor necrosis factor. Rather, MBP-1 reduces the activation and proliferation of splenocytes in vitro, indicating that MBP-1 acts in a more specific immunomodulatory role to affect the inflammatory response in muscular dystrophy. Together, these findings show that eosinophil-derived MBP-1 plays a significant role in regulating muscular dystrophy by attenuating the cellular immune response and promoting tissue fibrosis that can eventually contribute to increased mortality.

Null mutation of any one of several members of the dystrophin protein complex can cause progressive, and possibly fatal, muscle wasting. Although these muscular dystrophies arise from mutation of a single gene that is expressed primarily in muscle, the resulting pathology is complex and multisystemic, which shows a broader disruption of homeostasis than would be predicted by deletion of a single-gene product. Before the identification of the deficient proteins that underlie muscular dystrophies, such as Duchenne muscular dystrophy (DMD), oxidative stress was proposed as a major cause of the disease. Now, current knowledge supports the likelihood that interactions between the primary genetic defect and disruptions in the normal production of free radicals contribute to the pathophysiology of muscular dystrophies. In this review, we focus on the pathophysiology that results from dystrophin deficiency in humans with DMD and the mdx mouse model of DMD. Current evidence indicates three general routes through which free radical production can be disrupted in dystrophin deficiency to contribute to the ensuing pathology. First, constitutive differences in free radical production can disrupt signaling processes in muscle and other tissues and thereby exacerbate pathology. Second, tissue responses to the presence of pathology can cause a shift in free radical production that can promote cellular injury and dysfunction. Finally, behavioral differences in the affected individual can cause further changes in the production and stoichiometry of free radicals and thereby contribute to disease. Unfortunately, the complexity of the free radical-mediated processes that are perturbed in complex pathologies such as DMD will make it difficult to develop therapeutic approaches founded on systemic administration of antioxidants. More mechanistic knowledge of the specific disruptions of free radicals that underlie major features of muscular dystrophy is needed to develop more targeted and successful therapeutic approaches.

Duchenne muscular dystrophy (DMD) is a progressive, lethal, muscle wasting disease that affects 1 of 3500 boys born worldwide. The disease results from mutation of the dystrophin gene that encodes a cytoskeletal protein associated with the muscle cell membrane. Although gene therapy will likely provide the cure for DMD, it remains on the distant horizon, emphasizing the need for more rapid development of palliative treatments that build on improved understanding of the complex pathology of dystrophin deficiency. In this review, we have focused on therapeutic strategies that target downstream events in the pathologic progression of DMD. Much of this work has been developed initially using the dystrophin-deficient mdx mouse to explore basic features of the pathophysiology of dystrophin deficiency and to test potential therapeutic interventions to slow, reverse, or compensate for functional losses that occur in muscular dystrophy. In some cases, the initial findings in the mdx model have led to clinical treatments for DMD boys that have produced improvements in muscle function and quality of life. Many of these investigations have concerned interventions that can affect protein balance in muscle, by inhibiting specific proteases implicated in the DMD pathology, or by providing anabolic factors or depleting catabolic factors that can contribute to muscle wasting. Other investigations have exploited the use of anti-inflammatory agents that can reduce the contribution of leukocytes to promoting secondary damage to dystrophic muscle. A third general strategy is designed to increase the regenerative capacity of dystrophic muscle and thereby help retain functional muscle mass. Each of these general approaches to slowing the pathology of dystrophin deficiency has yielded encouragement and suggests that targeting downstream events in dystrophinopathy can yield worthwhile, functional improvements in DMD.

Dystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (NOS), which suggests that NO deficiency may influence the dystrophic pathology. Because NO can function as an antiinflammatory and cytoprotective molecule, we propose that the loss of NOS from dystrophic muscle exacerbates muscle inflammation and fiber damage by inflammatory cells. Analysis of transgenic mdx mice that were null mutants for dystrophin, but expressed normal levels of NO in muscle, showed that the normalization of NO production caused large reductions in macrophage concentrations in the mdx muscle. Expression of the NOS transgene in mdx muscle also prevented the majority of muscle membrane injury that is detectable in vivo, and resulted in large decreases in serum creatine kinase concentrations. Furthermore, our data show that mdx muscle macrophages are cytolytic at concentrations that occur in dystrophic, NOS-deficient muscle, but are not cytolytic at concentrations that occur in dystrophic mice that express the NOS transgene in muscle. Finally, our data show that antibody depletions of macrophages from mdx mice cause significant reductions in muscle membrane injury. Together, these findings indicate that macrophages promote injury of dystrophin-deficient muscle, and the loss of normal levels of NO production by dystrophic muscle exacerbates inflammation and membrane injury in muscular dystrophy.

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.

Mechanical stimuli can cause changes in muscle mass and structure which indicate that mechanisms exist for transducing mechanical stimuli into signals that influence gene expression. Myotendinous junctions show adaptations to modified muscle loading which suggest that these are transcriptionally distinct domains in muscle fibers that may experience local regulation of expression of structural proteins that are concentrated at these sites. Vinculin and talin are cytoskeletal proteins that are highly enriched at myotendinous junctions that we hypothesize to be subject to local transcriptional regulation. Our findings show that mechanical stimulation of muscle cells in vivo and in vitro causes an increase in the expression of vinculin and talin that is mediated by nitric oxide. Furthermore, nitric oxide-stimulated increases in vinculin and talin expression occur through a protein kinase G-dependent pathway and therefore differ from other mechanisms through which nitric oxide has been shown previously to modulate transcription. Analysis of vinculin mRNA distribution in mechanically stimulated muscle fibers shows that the mRNA is highly concentrated at myotendinous junctions, which supports the hypothesis that myotendinous junctions are distinct domains in which the expression of cytoskeletal proteins is modulated by mechanical stimuli through a nitric oxide and protein kinase G-dependent pathway.

Survival of dystrophin/utrophin double-knockout (dko) mice was increased by muscle-specific expression of a neuronal nitric oxide synthase (nNOS) transgene. Dko mice expressing the transgene (nNOS TG+/dko) experienced delayed onset of mortality and increased life-span. The nNOS TG+/dko mice demonstrated a significant decrease in the concentration of CD163+, M2c macrophages that can express arginase and promote fibrosis. The decrease in M2c macrophages was associated with a significant reduction in fibrosis of heart, diaphragm and hindlimb muscles of nNOS TG+/dko mice. The nNOS transgene had no effect on the concentration of cytolytic, CD68+, M1 macrophages. Accordingly, we did not observe any change in the extent of muscle fiber lysis in the nNOS TG+/dko mice. These findings show that nNOS/NO (nitric oxide)-mediated decreases in M2c macrophages lead to a reduction in the muscle fibrosis that is associated with increased mortality in mice lacking dystrophin and utrophin. Interestingly, the dramatic and beneficial effects of the nNOS transgene were not attributable to localization of nNOS protein at the cell membrane. We did not detect any nNOS protein at the sarcolemma in nNOS TG+/dko muscles. This important observation shows that sarcolemmal localization is not necessary for nNOS to have beneficial effects in dystrophic tissue and the presence of nNOS in the cytosol of dystrophic muscle fibers can ameliorate the pathology and most importantly, significantly increase life-span.

Background Duchenne muscular dystrophy (DMD) is the most common, lethal disease of childhood. One of 3500 new-born males suffers from this universally-lethal disease. Other than the use of corticosteroids, little is available to affect the relentless progress of the disease, leading many families to use dietary supplements in hopes of reducing the progression or severity of muscle wasting. Arginine is commonly used as a dietary supplement and its use has been reported to have beneficial effects following short-term administration to mdx mice, a genetic model of DMD. However, the long-term effects of arginine supplementation are unknown. This lack of knowledge about the long-term effects of increased arginine metabolism is important because elevated arginine metabolism can increase tissue fibrosis, and increased fibrosis of skeletal muscles and the heart is an important and potentially life-threatening feature of DMD. Methodology We use both genetic and nutritional manipulations to test whether changes in arginase metabolism promote fibrosis and increase pathology in mdx mice. Our findings show that fibrotic lesions in mdx muscle are enriched with arginase-2-expressing macrophages and that muscle macrophages stimulated with cytokines that activate the M2 phenotype show elevated arginase activity and expression. We generated a line of arginase-2-null mutant mdx mice and found that the mutation reduced fibrosis in muscles of 18-month-old mdx mice, and reduced kyphosis that is attributable to muscle fibrosis. We also observed that dietary supplementation with arginine for 17-months increased mdx muscle fibrosis. In contrast, arginine-2 mutation did not reduce cardiac fibrosis or affect cardiac function assessed by echocardiography, although 17-months of dietary supplementation with arginine increased cardiac fibrosis. Long-term arginine treatments did not decrease matrix metalloproteinase-2 or -9 or increase the expression of utrophin, which have been reported as beneficial effects of short-term treatments. Conclusions/Significance Our findings demonstrate that arginine metabolism by arginase promotes fibrosis of muscle in muscular dystrophy and contributes to kyphosis. Our findings also show that long-term, dietary supplementation with arginine exacerbates fibrosis of dystrophic heart and muscles. Thus, commonly-practiced dietary supplementation with arginine by DMD patients has potential risk for increasing pathology when performed for long periods, despite reports of benefits acquired with short-term supplementation.

Null mutation of dystrophin causes the lethal pathology of Duchenne muscular dystrophy (DMD) in which there is progressive pathology of skeletal and cardiac muscles. A large proportion of DMD patient deaths are attributable to cardiac dysfunction associated with ventricular fibrosis, arrhythmias and conduction abnormalities, although the relationships between the dystrophin mutation and the cardiac defects are unknown. Here, we tested whether cardiac pathology in dystrophin-deficient mdx mice can be corrected by the elevated production of nitric oxide (NO) by the myocardium. Dystrophin-deficient mdx mice were produced in which there was myocardial expression of a neuronal nitric oxide synthase (nNOS) transgene. Expression of the transgene prevented the progressive ventricular fibrosis of mdx mice and greatly reduced myocarditis. Electrocardiographs (ECG) attained by radiotelemetry of freely ambulatory mice showed that mdx mice displayed cardiac abnormalities that are characteristic of DMD patients, including deep Q-waves, diminished S:R ratios, polyphasic R-waves and frequent premature ventricular contractions. All of these ECG abnormalities in mdx mice were improved or corrected by nNOS transgene expression. In addition, defects in mdx cardiac autonomic function, which were reflected by decreased heart rate variability, were significantly reduced by nNOS transgene expression. These findings indicate that increasing NO production by dystrophic hearts may have therapeutic value.

Muscular dystrophies that arise from mutations of genes that encode proteins in the dystrophin-glycoprotein complex (DGC) frequently involve defects in the structure of neuromuscular junctions (NMJs). DGC mutations that cause NMJ defects typically cause a secondary loss of neuronal nitric oxide synthase (nNOS) from the post-synaptic membrane. We tested the hypothesis that reduction of muscle-derived NO production causes NMJ defects in DGC mutants by analyzing the effect of modulating muscle NO production on NMJ structure in mutant and wild-type muscles. We found that nNOS null mutants, dystrophin-deficient mdx mice and alpha-syntrophin null mutants showed reductions in the concentration of acetylcholine receptors (AChRs) at the post-synaptic membrane. Also, expression of a muscle-specific NOS transgene increased AChR concentration, which reflected an increase in both AChR expression and clustering. NOS transgene expression also increased the size of NMJs, and partially corrected defects in normal NMJ architecture that were observed in mdx and alpha-syntrophin null muscles. In addition, stimulation of AChR clustering in vitro by application of laminin or VVA B4 lectin induced a 3-4-fold increase in NOS activity and increased AChR clustering that could be prevented by NOS inhibition. However, the partial rescue of NMJ structure by expression of a NOS transgene required the expression of alpha- or beta1-syntrophin at the NMJ; partial NMJ rescue was seen in the muscles of alpha-syntrophin mutants that expressed beta1-syntrophin, but no rescue was observed in muscles of alpha-syntrophin mutants that also lacked beta1-syntrophin. These findings show that NO promotes AChR expression and clustering in vivo and contributes to normal NMJ architecture. The results suggest that defects in NMJ structure that occur in some DGC mutants can result from the secondary loss of NOS from muscle.

Muscular dystrophy that is caused by mutation of the membrane-associated, cytoskeletal protein called dystrophin, is accompanied by loss of a dystrophin-associated protein complex (DPC) that includes neuronal nitric oxide synthase (nNOS). Previous work showed that expression of a nNOS transgene in the dystrophin-deficient, mdx mouse greatly reduces muscle membrane damage. In this investigation, we test whether expression of a nNOS transgene in wild-type or mdx muscle increases expression of DPC proteins, or functionally related proteins in the integrin complex that are upregulated in dystrophin-deficiency, or affects expression of the dystrophin homolog, utrophin. Many members of the DPC are enriched in Western blots of cell membranes isolated from NOS transgenic muscle, compared to wild-type. Similarly, alpha7-integrin and the associated cytoskeletal proteins talin and vinculin are increased in NOS transgenic, non-dystrophic muscle. However, utrophin expression is unaffected by elevated NOS expression in healthy muscle. A similar trend in mRNA levels for these proteins was observed by expression profiling. Analysis of membrane preparations from mdx mice and NOS transgenic mdx mice shows that expression of the NOS transgene causes significant reductions in utrophin, talin, and vinculin. Expression profiling of mRNA from mdx and NOS transgenic mdx muscles also shows reduced expression of talin. Immunohistochemistry of mdx and NOS transgenic mdx muscle indicates that reduction in utrophin in NOS transgenic mdx muscle results from a decrease in regenerative fibers that express high levels of utrophin. Together, these findings indicate that the NOS transgene does not reduce dystrophinopathy by increasing the expression of compensatory, structural proteins.

The hypothesis that changes in muscle activation and loading regulate the expression and activity of neuronal nitric oxide (NO) synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNA concentration in soleus muscles, which returned to control concentrations after return to weight bearing. Similarly, the concentration of nNOS in cultured myotubes increased by application of cyclic loading for 2 days. NO release from excised soleus muscles was increased significantly by a single passive stretch of 20% or by submaximal activation at 2 Hz, although the increases were not additive when both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on the presence of extracellular calcium. Cyclic loading of cultured myotubes also resulted in a significant increase in NO release. Together, these findings show that activity of muscle influences NO production in the short term, by regulating NOS activity, and in the long term, by regulating nNOS expression.

Previous findings have led to speculations that decreased concentration of nNOS (neuronal nitric oxide synthase) may underlie some aspects of the pathophysiology of dystrophic muscle. We have tested whether the sparing of extraocular muscles (EOM) in muscular dystrophy is attributable to the presence of normal nNOS concentration and distribution in these muscles. Measurements of total nNOS concentration in control muscle showed that total nNOS comprises approximately 0.05% of total muscle protein, indicating a molar stoichiometry of approximately 60 and 20 to total dystrophin and syntrophin, respectively. Thus, most muscle nNOS is either not associated with the dystrophin complex, or binds to yet unidentified sites in the complex. nNOS concentration was at least two-fold greater in C57 EOM and tibialis anterior (TA) compared with mdx samples. No significant differences in nNOS concentration in EOM versus TA in either mdx or C57 mice were observed. nNOS was concentrated at the sarcolemma of all C57 samples, while mdx nNOS displayed a cytosolic distribution, except in fibers that reverted to express dystrophin. These data show that mdx EOM are spared by a mechanism other than normalized concentration and location of nNOS.

Myostatin, a TGF-β family member, is a negative regulator of muscle growth. Here, we generated transgenic mice that expressed myostatin mutated at its cleavage site under the control of a muscle specific promoter creating a dominant negative myostatin. These mice exhibited a significant (20–35%) increase in muscle mass that resulted from myofiber hypertrophy and not from myofiber hyperplasia. We also evaluated the role of myostatin in muscle degenerative states, such as muscular dystrophy, and found significant downregulation of myostatin. Thus, further inhibition of myostatin may permit increased muscle growth in muscle degenerative disorders.