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Limb girdle muscular dystrophy 2C is caused by mutations in the gamma-sarcoglycan gene (gsg) that results in loss of this protein, and disruption of the sarcoglycan (SG) complex. Signal transduction after mechanical perturbation is mediated, in part, through the SG complex and leads to phosphorylation of tyrosines on the intracellular portions of the sarcoglycans. This study tested if the Tyr(6) in the intracellular region of gamma-sarcoglycan protein (gamma-SG) was necessary for proper localization of the protein in skeletal muscle membranes or for the normal pattern of ERK1/2 phosphorylation after eccentric contractions. Viral mediated gene transfer of wild type gsg (WTgsg) and mutant gsg lacking Tyr(6) (Y6Agsg) was performed into the muscles of gsg(-/-) mice. Muscles were examined for production and stability of the gamma-SG, as well as the level of ERK1/2 phosphorylation before and after eccentric contraction. Sarcolemmal localization of gamma-SG was achieved regardless of which construct was expressed. However, only expression of WTgsg corrected the aberrant ERK1/2 phosphorylation associated with the absence of gamma-SG, whereas Y6Agsg failed to have any effect. This study shows that localization of gamma-SG does not require Tyr(6), but localization alone is insufficient for restoration of normal signal transduction patterns after mechanical perturbation.

The endoplasmic reticulum chaperone GRP94 is essential for early embryonic development and in particular affects differentiation of muscle lineages. To determine why an ubiquitously expressed protein has such a specific effect, we investigated the function of GRP94 in the differentiation of established myogenic cell lines in culture. Using both genetic suppression of expression, via RNA interference, and inhibition of function, via specific chemical inhibitors, we show that GRP94 expression and activity are needed for the in vitro fusion of myoblasts precursors into myotubes and the expression of contractile proteins that mark terminal differentiation. The inhibition can be complemented by addition of insulin-like growth factors to the cultures. GRP94 is not needed for the initial steps of myogenesis, only for the steps downstream of MyoD up-regulation, coinciding with the known need for synergistic input from growth factor signaling. Indeed, GRP94 is needed for the production of insulin-like growth factors I and II (IGF-I and IGF-II) by the differentiating cells. Moreover, the depletion of the chaperone does not increase the rate of apoptosis that always accompanies myogenic differentiation. Thus, the major effect of GRP94 on muscle differentiation is mediated by its regulation of IGF production.

Objective To determine if gender distinctions of force generating capacity existed in murine masticatory muscles. Design In order to investigate the effect of sex on force generating capacity in this muscle group, an isolated muscle preparation was developed utilising the murine anterior deep masseter. Age-matched male and female mice were utilized to assess function, muscle fibre type and size in this muscle. Results Maximum isometric force production was not different between age-matched male and female mice. However, the rate of force generation and relaxation was slower in female masseter muscles. Assessment of fibre type distribution by immunohistochemistry revealed a three-fold decrease in the proportion of myosin heavy chain 2b positive fibres in female masseters, which correlated with the differences in contraction kinetics. Conclusions These results provide evidence that masticatory muscle strength in mice is not affected by sex, but there are significant distinctions in kinetics associated with force production between males and females.

Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from

Skeletal muscle and bone rely on a number of growth factors to undergo development, modulate growth, and maintain physiological strength. A major player in these actions is insulin-like growth factor I (IGF-I). However, because this growth factor can directly enhance muscle mass and bone density, it alters the state of the musculoskeletal system indirectly through mechanical crosstalk between these two organ systems. Thus, there are clearly synergistic actions of IGF-I that extend beyond the direct activity through its receptor. This review will cover the production and signaling of IGF-I as it pertains to muscle and bone, the chemical and mechanical influences that arise from IGF-I activity, and the potential for therapeutic strategies based on IGF-I. This article is part of a Special Issue entitled "Muscle Bone Interactions".

Myostatin is a secreted signaling molecule that normally acts to limit muscle growth. As a result, there is extensive effort directed at developing drugs capable of targeting myostatin to treat patients with muscle loss. One potential concern with this therapeutic approach in patients with muscle degenerative diseases like muscular dystrophy is that inducing hypertrophy may increase stress on dystrophic fibers, thereby accelerating disease progression. To investigate this possibility, we examined the effect of blocking the myostatin pathway in dysferlin-deficient (Dysf(-/-)) mice, in which membrane repair is compromised, either by transgenic expression of follistatin in skeletal muscle or by systemic administration of the soluble form of the activin type IIB receptor (ACVR2B/Fc). Here, we show that myostatin inhibition by follistatin transgene expression in Dysf(-/-) mice results in early improvement in histopathology but ultimately exacerbates muscle degeneration; this effect was not observed in dystrophin-deficient (mdx) mice, suggesting that accelerated degeneration induced by follistatin transgene expression is specific to mice lacking dysferlin. Dysf(-/-) mice injected with ACVR2B/Fc showed significant increases in muscle mass and amelioration of fibrotic changes normally seen in 8-month-old Dysf(-/-) mice. Despite these potentially beneficial effects, ACVR2B/Fc treatment caused increases in serum CK levels in some Dysf(-/-) mice, indicating possible muscle damage induced by hypertrophy. These findings suggest that depending on the disease context, inducing muscle hypertrophy by myostatin blockade may have detrimental effects, which need to be weighed against the potential gains in muscle growth and decreased fibrosis.

Loss of the dystrophin glycoprotein complex (DGC) or a subset of its components can lead to muscular dystrophy. However, the patterns of symptoms differ depending on which proteins are affected. Absence of dystrophin leads to loss of the entire DGC and is associated with susceptibility to contractile injury. In contrast, muscles lacking γ-sarcoglycan (γ-SG) display little mechanical fragility and still develop severe pathology. Animals lacking dystrophin or γ-SG were used to identify DGC components critical for sensing dynamic mechanical load. Extensor digitorum longus muscles from 7-wk-old normal (C57), dystrophin- null ( mdx), and γ-SG-null ( gsg−/−) mice were subjected to a series of eccentric contractions, after which ERK1/2 phosphorylation levels were determined. At rest, both dystrophic strains had significantly higher ERK1 phosphorylation, and gsg−/− muscle also had heightened ERK2 phosphorylation compared with wild-type controls. Eccentric contractions produced a significant and transient increase in ERK1/2 phosphorylation in normal muscle, whereas the mdx strain displayed no significant proportional change of ERK1/2 phosphorylation after eccentric contraction. Muscles from gsg−/− mice had no significant increase in ERK1 phosphorylation; however, ERK2 phosphorylation was more robust than in C57 controls. The reduction in mechanically induced ERK1 phosphorylation in gsg−/− muscle was not dependent on age or severity of phenotype, because muscle from both young and old (age 20 wk) animals exhibited a reduced response. Immunoprecipitation experiments revealed that γ-SG was phosphorylated in normal muscle after eccentric contractions, indicating that members of the DGC are modified in response to mechanical perturbation. This study provides evidence that the SGs are involved in the transduction of mechanical information in skeletal muscle, potentially unique from the entire DGC.

GH and IGF-I are potent regulators of muscle growth and function. Although IGF-I is known to mediate many of the effects of GH, it is not yet clear whether all effects of GH are completely dependent on the IGF-I system. To evaluate the biological effects of the GH/IGF-I axis on muscle growth, we administrated recombinant human GH to mice, which lack IGF-I function specifically in skeletal muscle, due to the overexpression of a dominant-negative IGF-I receptor in this tissue (MKR mice). GH treatment significantly increased the levels of hepatic IGF-I mRNA and serum IGF-I levels in both wild-type (WT) and MKR mice. These GH-induced effects were paralleled by increases in body weight and in the weights of most GH-responsive organs in both groups of mice. Interestingly, unlike WT mice, GH treatment had no effect on skeletal muscle weight in MKR mice. GH treatment failed to reverse the impaired muscle function in MKR mice. Furthermore, MKR mice exhibited no effects of GH on the cross-sectional area of myofibers and the proliferation of satellite cells. Taken together, these data suggest that the in vivo effects of GH on muscle mass and strength are primarily mediated by activation of the IGF-I receptor.

Muscle hypertrophy is the product of increased drive through protein synthetic pathways and the incorporation of newly divided satellite cells. Gains in muscle mass and strength can be achieved through exercise regimens that include resistance training. Increased insulin-like growth factor-I (IGF-I) can also promote hypertrophy through increased protein synthesis and satellite cell proliferation. However, it is not known whether the combined effect of IGF-I and resistance training results in an additive hypertrophic response. Therefore, rats in which viral administration of IGF-I was directed to one limb were subjected to ladder climbing to test the interaction of each intervention on muscle mass and strength. After 8 wk of resistance training, a 23.3% increase in muscle mass and a 14.4% increase in peak tetanic tension (Po) were observed in the flexor hallucis longus (FHL). Viral expression of IGF-I without resistance training produced a 14.8% increase in mass and a 16.6% increase in Po in the FHL. The combined interventions produced a 31.8% increase in muscle mass and a 28.3% increase in Po in the FHL. Therefore, the combination of resistance training and overexpression of IGF-I induced greater hypertrophy than either treatment alone. The effect of increased IGF-I expression on the loss of muscle mass associated with detraining was also addressed. FHL muscles treated with IGF-I lost only 4.8% after detraining, whereas the untreated FHL lost 8.3% muscle mass. These results suggest that a combination of resistance training and overexpression of IGF-I could be an effective measure for attenuating the loss of training-induced adaptations.

Successful gene therapy for most inherited diseases will require stable expression of the therapeutic gene. This can be addressed with integrating or self-replicating viruses by targeting postmitotic cells that have a long lifetime or stem cells that can replenish defective tissue with corrected cells. In this study, we explore the possibility of targeting a muscle stem cell population in situ through in vivo administration of vector. To develop this concept, we selected a mouse model of muscular dystrophy (mdx mice) that undergoes rapid turnover of muscle fibers. In vivo targeting of muscle progenitor cells, notably satellite cells, with a pseudotyped lentiviral vector encoding the minidystrophin restores dystrophin expression and provides functional correction in skeletal muscle of mdx mice. This study shows that progenitor cells can be genetically engineered in vivo and subsequently proliferate into terminally differentiated tissue carrying the genetic graft in a way that stably corrects function.

Skeletal muscle size is modulated by a number of factors, including muscle load, utilization, and regenerative capacity. Surprisingly, actions that can promote muscle growth do not necessarily prevent the loss of muscle mass, or atrophy. This suggests that divergent mechanisms are important for the maintenance of muscle mass in different contexts. In acute atrophy, muscles rapidly lose mass when load is lacking, and this response seems to involve active elimination of myonuclei. In contrast, chronic atrophy, such as loss of muscle mass related to aging, is associated with impairments in muscle repair. In this review, two contexts in which muscle mass is lost are explored to determine if similar processes are involved.

Mice and cattle with mutations in the myostatin (GDF8) gene show a marked increase in body weight and muscle mass, indicating that this new member of the TGF-beta superfamily is a negative regulator of skeletal muscle growth. Inhibition of the myostatin gene product is predicted to increase muscle mass and improve the disease phenotype in a variety of primary and secondary myopathies. We tested the ability of inhibition of myostatin in vivo to ameliorate the dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). Blockade of endogenous myostatin by using intraperitoneal injections of blocking antibodies for three months resulted in an increase in body weight, muscle mass, muscle size and absolute muscle strength in mdx mouse muscle along with a significant decrease in muscle degeneration and concentrations of serum creatine kinase. The functional improvement of dystrophic muscle by myostatin blockade provides a novel, pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD, and circumvents the major problems associated with conventional gene therapy in these disorders.

Duchenne muscular dystrophy is an X-linked degenerative disorder of muscle caused by the absence of the protein dystrophin. A major consequence of muscular dystrophy is that the normal regenerative capacity of skeletal muscle cannot compensate for increased susceptibility to damage, leading to repetitive cycles of degeneration–regeneration and ultimately resulting in the replacement of muscle fibers with fibrotic tissue. Because insulin-like growth factor I (IGF-I) has been shown to enhance muscle regeneration and protein synthetic pathways, we asked whether high levels of muscle-specific expression of IGF-I in mdx muscle could preserve muscle function in the diseased state. In transgenic mdx mice expressing mIgf-I (mdx:mIgf+/+), we showed that muscle mass increased by at least 40% leading to similar increases in force generation in extensor digitorum longus muscles compared with those from mdx mice. Diaphragms of transgenic mdx:mIgf+/+ exhibited significant hypertrophy and hyperplasia at all ages observed. Furthermore, the IGF-I expression significantly reduced the amount of fibrosis normally observed in diaphragms from aged mdx mice. Decreased myonecrosis was also observed in diaphragms and quadriceps from mdx:mIgf+/+ mice when compared with age-matched mdx animals. Finally, signaling pathways associated with muscle regeneration and protection against apoptosis were significantly elevated. These results suggest that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin.

Latent transforming growth factor-β (TGFβ) binding proteins (LTBPs) bind to inactive TGFβ in the extracellular matrix. In mice, muscular dystrophy symptoms are intensified by a genetic polymorphism that changes the hinge region of LTBP, leading to increased proteolytic susceptibility and TGFβ release. We have found that the hinge region of human LTBP4 was also readily proteolysed and that proteolysis could be blocked by an antibody to the hinge region. Transgenic mice were generated to carry a bacterial artificial chromosome encoding the human LTBP4 gene. These transgenic mice displayed larger myofibers, increased damage after muscle injury, and enhanced TGFβ signaling. In the mdx mouse model of Duchenne muscular dystrophy, the human LTBP4 transgene exacerbated muscular dystrophy symptoms and resulted in weaker muscles with an increased inflammatory infiltrate and greater LTBP4 cleavage in vivo. Blocking LTBP4 cleavage may be a therapeutic strategy to reduce TGFβ release and activity and decrease inflammation and muscle damage in muscular dystrophy.

Loss of gamma-sarcoglycan (γ-SG) induces muscle degeneration and signaling defects in response to mechanical load, and its absence is common to both Duchenne and limb girdle muscular dystrophies. Growing evidence suggests that aberrant signaling contributes to the disease pathology; however, the mechanisms of γ-SG-mediated mechanical signaling are poorly understood. To uncover γ-SG signaling pathway components, we performed yeast two-hybrid screens and identified the muscle-specific protein archvillin as a γ-SG and dystrophin interacting protein. Archvillin protein and message levels were significantly upregulated at the sarcolemma of murine γ-SG-null (gsg(-/-)) muscle but delocalized in dystrophin-deficient mdx muscle. Similar elevation of archvillin protein was observed in human quadriceps muscle lacking γ-SG. Reintroduction of γ-SG in gsg(-/-) muscle by rAAV injection restored archvillin levels to that of control C57 muscle. In situ eccentric contraction of tibialis anterior (TA) muscles from C57 mice caused ERK1/2 phosphorylation, nuclear activation of P-ERK1/2 and stimulus-dependent archvillin association with P-ERK1/2. In contrast, TA muscles from gsg(-/-) and mdx mice exhibited heightened P-ERK1/2 and increased nuclear P-ERK1/2 localization following eccentric contractions, but the archvillin-P-ERK1/2 association was completely ablated. These results position archvillin as a mechanically sensitive component of the dystrophin complex and demonstrate that signaling defects caused by loss of γ-SG occur both at the sarcolemma and in the nucleus.

Background The dystrophin glycoprotein complex (DGC) is located at the sarcolemma of muscle fibers, providing structural integrity. Mutations in and loss of DGC proteins cause a spectrum of muscular dystrophies. When only the sarcoglycan subcomplex is absent, muscles display severe myofiber degeneration, but little susceptibility to contractile damage, suggesting that disease occurs not by structural deficits but through aberrant signaling, namely, loss of normal mechanotransduction signaling through the sarcoglycan complex. We extended our previous studies on mechanosensitive, γ-sarcoglycan-dependent ERK1/2 phosphorylation, to determine whether additional pathways are altered with the loss of γ-sarcoglycan. Methods We examined mechanotransduction in the presence and absence of γ-sarcoglycan, using C2C12 myotubes, and primary cultures and isolated muscles from C57Bl/6 (C57) and γ-sarcoglycan-null (γ-SG-/-) mice. All were subjected to cyclic passive stretch. Signaling protein phosphorylation was determined by immunoblotting of lysates from stretched and non-stretched samples. Calcium dependence was assessed by maintaining muscles in calcium-free or tetracaine-supplemented Ringer’s solution. Dependence on mTOR was determined by stretching isolated muscles in the presence or absence of rapamycin. Results C2C12 myotube stretch caused a robust increase in P-p70S6K, but decreased P-FAK and P-ERK2. Neither Akt nor ERK1 were responsive to passive stretch. Similar but non-significant trends were observed in C57 primary cultures in response to stretch, and γ-SG-/- cultures displayed no p70S6K response. In contrast, in isolated muscles, p70S6K was mechanically responsive. Basal p70S6K activation was elevated in muscles of γ-SG-/- mice, in a calcium-independent manner. p70S6K activation increased with stretch in both C57 and γ-SG-/- isolated muscles, and was sustained in γ-SG-/- muscles, unlike the transient response in C57 muscles. Rapamycin treatment blocked all of p70S6K activation in stretched C57 muscles, and reduced downstream S6RP phosphorylation. However, even though rapamycin treatment decreased p70S6K activation in stretched γ-SG-/- muscles, S6RP phosphorylation remained elevated. Conclusions p70S6K is an important component of γ-sarcoglycan-dependent mechanotransduction in skeletal muscle. Our results suggest that loss of γ-sarcoglycan uncouples the response of p70S6K to stretch and implies that γ-sarcoglycan is important for inactivation of this pathway. Overall, we assert that altered load-sensing mechanisms exist in muscular dystrophies where the sarcoglycans are absent.

Duchenne muscular dystrophy (DMD) is a progressive, fatal, muscle wasting disease caused by a dystrophin deficiency. The benefits of exercise for DMD patients are unclear and advanced disease progr...

Aging skeletal muscles suffer a steady decline in mass and functional performance, and compromised muscle integrity as fibrotic invasions replace contractile tissue, accompanied by a characteristic loss in the fastest, most powerful muscle fibers. The same programmed deficits in muscle structure and function are found in numerous neurodegenerative syndromes and disease-related cachexia. We have generated a model of persistent, functional myocyte hypertrophy using a tissue-restricted transgene encoding a locally acting isoform of insulin-like growth factor-1 that is expressed in skeletal muscle (mIgf-1). Transgenic embryos developed normally, and postnatal increases in muscle mass and strength were not accompanied by the additional pathological changes seen in other Igf-1 transgenic models. Expression of GATA-2, a transcription factor normally undetected in skeletal muscle, marked hypertrophic myocytes that escaped age-related muscle atrophy and retained the proliferative response to muscle injury characteristic of younger animals. The preservation of muscle architecture and age-independent regenerative capacity through localized mIgf-1 transgene expression suggests clinical strategies for the treatment of age or disease-related muscle frailty.

In humans, a subset of cases of Limb-girdle muscular dystrophy (LGMD) arise from mutations in the genes encoding one of the sarcoglycan (alpha, beta, gamma, or delta) subunits of the dystrophin-glycoprotein complex. While adeno-associated virus (AAV) is a potential gene therapy vector for these dystrophies, it is unclear if AAV can be used if a diseased muscle is undergoing rapid degeneration and necrosis. The skeletal muscles of mice lacking gamma-sarcoglycan (gsg-/- mice) differ from the animal models that have been evaluated to date in that the severity of the skeletal muscle pathology is much greater and more representative of that of humans with muscular dystrophy. Following direct muscle injection of a recombinant AAV [in which human gamma-sarcoglycan expression is driven by a truncated muscle creatine kinase (MCK) promoter/enhancer], we observed significant numbers of muscle fibers expressing gamma-sarcoglycan and an overall improvement of the histologic pattern of dystrophy. However, these results could be achieved only if injections into the muscle were prior to the development of significant fibrosis in the muscle. The results presented in this report show promise for AAV gene therapy for LGMD, but underscore the need for intervention early in the time course of the disease process.

Insulin-like growth factor I (IGF-I) is critical in promoting growth of skeletal muscle. When IGF-I is introduced into mouse hindlimb muscles by viral-mediated gene transfer, local overexpression of IGF-I produces significant increases in muscle mass and strength compared with untreated controls (Barton-Davis et al. 1998). We have proposed that this functional hypertrophy is primarily owing to the activation of satellite cells which leads to increased muscle regeneration. In order to test if satellite cells are essential in mediating the hypertrophic effects of IGF-I, we used gamma radiation to destroy the proliferative capacity of satellite cells. The right hindlimbs of adult C57BL/6 male mice were subjected to one of the following treatments: (1) 2,500 rad gamma radiation only, (2) viral-mediated gene transfer of IGF-I only, (3) 2,500 rad gamma radiation plus viral-mediated gene transfer of IGF-I, or (4) no intervention as a control. Approximately 4 months after treatment, the extensor digitorum longus muscles (EDL) from both hindlimbs were removed for mechanical and morphological measurements. Treatment with gamma radiation significantly prevented normal growth of the muscle. When combined with IGF-I treatment, approximately half of the IGF-I effect was prevented by gamma radiation treatment. This suggests that the remaining half of IGF-I induced hypertrophy is owing to paracrine/autocrine effects on the adult myofibres. Thus, these data are consistent with a mechanism by which IGF-I induced muscle hypertrophy via a combination of satellite cell activation and increasing protein synthesis in differentiated myofibres.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse — an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD. J. Clin. Invest. 104:375–381 (1999).

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca(2+) and Na(+) overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca(2+) and Na(+) overload, diminished abnormal sarcolemmal Ca(2+) entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway.

Contains fulltext : 136666.pdf (Publisher’s version ) (Closed access) Collagen VI-related myopathies are disorders of connective tissue presenting with an overlap phenotype combining clinical involvement from the muscle and from the connective tissue. Not all patients displaying related overlap phenotypes between muscle and connective tissue have mutations in collagen VI. Here, we report a homozygous recessive loss of function mutation and a de novo dominant mutation in collagen XII (COL12A1) as underlying a novel overlap syndrome involving muscle and connective tissue. Two siblings homozygous for a loss of function mutation showed widespread joint hyperlaxity combined with weakness precluding independent ambulation, while the patient with the de novo missense mutation was more mildly affected, showing improvement including the acquisition of walking. A mouse model with inactivation of the Col12a1 gene showed decreased grip strength, a delay in fiber-type transition and a deficiency in passive force generation while the muscle seems more resistant to eccentric contraction induced force drop, indicating a role for a matrix-based passive force-transducing elastic element in the generation of the weakness. This new muscle connective tissue overlap syndrome expands on the emerging importance of the muscle extracellular matrix in the pathogenesis of muscle disease.

Background Histological assessment of skeletal muscle tissue is commonly applied to many areas of skeletal muscle physiological research. Histological parameters including fiber distribution, fiber type, centrally nucleated fibers, and capillary density are all frequently quantified measures of skeletal muscle. These parameters reflect functional properties of muscle and undergo adaptation in many muscle diseases and injuries. While standard operating procedures have been developed to guide analysis of many of these parameters, the software to freely, efficiently, and consistently analyze them is not readily available. In order to provide this service to the muscle research community we developed an open source MATLAB script to analyze immunofluorescent muscle sections incorporating user controls for muscle histological analysis. Results The software consists of multiple functions designed to provide tools for the analysis selected. Initial segmentation and fiber filter functions segment the image and remove non-fiber elements based on user-defined parameters to create a fiber mask. Establishing parameters set by the user, the software outputs data on fiber size and type, centrally nucleated fibers, and other structures. These functions were evaluated on stained soleus muscle sections from 1-year-old wild-type and mdx mice, a model of Duchenne muscular dystrophy. In accordance with previously published data, fiber size was not different between groups, but mdx muscles had much higher fiber size variability. The mdx muscle had a significantly greater proportion of type I fibers, but type I fibers did not change in size relative to type II fibers. Centrally nucleated fibers were highly prevalent in mdx muscle and were significantly larger than peripherally nucleated fibers. Conclusions The MATLAB code described and provided along with this manuscript is designed for image processing of skeletal muscle immunofluorescent histological sections. The program allows for semi-automated fiber detection along with user correction. The output of the code provides data in accordance with established standards of practice. The results of the program have been validated using a small set of wild-type and mdx muscle sections. This program is the first freely available and open source image processing program designed to automate analysis of skeletal muscle histological sections.

SummaryEx vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3β inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.

IGF-I is a key regulator of muscle development and growth. The pre-pro-peptide produced by the Igf1gene undergoes several posttranslational processing steps to result in a secreted mature protein, which is thought to be the obligate ligand for the IGF-I receptor (IGF-IR). The goals of this study were to determine what forms of IGF-I exist in skeletal muscle, and whether the mature IGF-I protein was the only form able to activate the IGF-IR. We measured the proportion of IGF-I species in murine skeletal muscle and found that the predominant forms were nonglycosylated pro-IGF-I and glycosylated pro-IGF-I, which retained the C-terminal E peptide extension, instead of mature IGF-I. These forms were validated using samples subjected to viral expression of IGF-I combined with furin and glycosidase digestion. To determine whether the larger molecular weight IGF-I forms were also ligands for the IGF-IR, we generated each specific form through transient transfection of 3T3 cells and used the enriched media to perform kinase receptor activation assays. Compared with mature IGF-I, nonglycosylated pro-IGF-I had similar ability to activate the IGF-IR, whereas glycosylation of pro-IGF-I significantly reduced receptor activation. Thus, it is important to understand not only the quantity, but also the proportion of IGF-I forms produced, to evaluate the true biological activity of this growth factor.

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were ∼30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.—Barton, E. R., Park, S., James, J. K., Makarewich, C. A., Philippou, A., Eletto, D., Lei, H., Brisson, B., Ostrovsky, O., Li, Z., Argon, Y. Deletion of muscle GRP94 impairs both muscle and body growth by inhibiting local IGF production.

Insulin-like growth factor-I (IGF-I) is an essential growth factor that regulates the processes necessary for cell proliferation, differentiation, and survival. The Igf1 gene encodes mature IGF-I and a carboxy-terminal extension called the E-peptide. In rodents, alternative splicing and post-translational processing produce two E-peptides (EA and EB). EB has been studied extensively and has been reported to promote cell proliferation and migration independently of IGF-I and its receptor (IGF-IR), but the mechanism by which EB causes these actions has not been identified. Further, the properties of EA have not been evaluated. Therefore, the goals of this study were to determine if EA and EB possessed similar activity and if these actions were IGF-IR independent. We utilized synthetic peptides for EA, EB, and a scrambled control to examine cellular responses. Both E-peptides increased MAPK signaling, which was blocked by pharmacologic IGF-IR inhibition. Although the E-peptides did not directly induce IGF-IR phosphorylation, the presence of either E-peptide increased IGF-IR activation by IGF-I, and this was achieved through enhanced cell surface bioavailability of the receptor. To determine if E-peptide biological actions required the IGF-IR, we took advantage of the murine C2C12 cell line as a platform to examine the key steps of skeletal muscle proliferation, migration and differentiation. EB increased myoblast proliferation and migration while EA delayed differentiation. The proliferation and migration effects were inhibited by MAPK or IGF-IR signaling blockade. Thus, in contrast to previous studies, we find that E-peptide signaling, mitogenic, and motogenic effects are dependent upon IGF-IR. We propose that the E-peptides have little independent activity, but instead affect growth via modulating IGF-I signaling, thereby increasing the complexity of IGF-I biological activity.

Insulin-like growth factor I (IGF-I) is a growth hormone-dependent growth-promoting peptide. It cooperates with several additional growth factors to coordinate myoblast proliferation and differentiation, muscle growth, and the regeneration of issues after injury. The potent actions of IGF-I are tightly controlled at many levels, because not only can it cause growth of muscle, it can cause growth of almost any cell in the body. This chapter will introduce the signaling pathways associated with IGF-I activity and how activity is tuned. Next, regulation of IGF-I during transcription and translation and post-translational processing will be described. Finally, the prospects for IGF-I based therapies for both healthy and diseased muscle will be discussed, as well as where these therapeutic strategies may lead in the future.

Anchorage to matrix is mediated for many cells not only by integrin-based focal adhesions but also by a parallel assembly of integral and peripheral membrane proteins known as the Dystroglycan Complex. Deficiencies in either dystrophin (mdx mice) or γ-sarcoglycan (γSG(-/-) mice) components of the Dystroglycan Complex lead to upregulation of numerous focal adhesion proteins, and the phosphoprotein paxillin proves to be among the most prominent. In mdx muscle, paxillin-Y31 and Y118 are both hyper-phosphorylated as are key sites in focal adhesion kinase (FAK) and the stretch-stimulatable pro-survival MAPK pathway, whereas γSG(-/-) muscle exhibits more erratic hyper-phosphorylation. In cultured myotubes, cell tension generated by myosin-II appears required for localization of paxillin to adhesions while vinculin appears more stably integrated. Overexpression of wild-type (WT) paxillin has no obvious effect on focal adhesion density or the physical strength of adhesion, but WT and a Y118F mutant promote contractile sarcomere formation whereas a Y31F mutant shows no effect, implicating Y31 in striation. Self-peeling of cells as well as Atomic Force Microscopy (AFM) probing of cells with or without myosin-II inhibition indicate an increase in cell tension within paxillin-overexpressing cells. However, prednisolone, a first-line glucocorticoid for muscular dystrophies, decreases cell tension without affecting paxillin at adhesions, suggesting a non-linear relationship between paxillin and cell tension. Hypertension that results from upregulation of integrin adhesions is thus a natural and treatable outcome of Dystroglycan Complex down-regulation.

Calpain activation has been implicated in the disease pathology of Duchenne muscular dystrophy. Inhibition of calpain has been proposed as a promising therapeutic target, which could lessen the protein degradation and prevent progressive fibrosis. At the same time, there are conflicting reports as to whether elevation of calpastatin, an endogenous calpain inhibitor, alters pathology. We compared the effects of pharmacological calpain inhibition in the mdx mouse using leupeptin and a proprietary compound (C101) that linked the inhibitory portion of leupeptin to carnitine (to increase uptake into muscle). Administration of C101 for 4 wk did not improve muscle histology, function, or serum creatine kinase levels in mdx mice. Mdx mice injected daily with leupeptin (36 mg/kg) for 6 mo also failed to show improved muscle function, histology, or creatine kinase levels. Biochemical analysis revealed that leupeptin administration caused an increase in m-calpain autolysis and proteasome activity, yet calpastatin levels were similar between treated and untreated mdx mice. These data demonstrate that pharmacological inhibition of calpain is not a promising intervention for the treatment of Duchenne muscular dystrophy due to the ability of skeletal muscle to counter calpain inhibitors by increasing multiple degradative pathways.

Modulation of transforming growth factor-β (TGF-β) signaling to promote muscle growth holds tremendous promise for the muscular dystrophies and other disorders involving the loss of functional muscle mass. Previous studies have focused on the TGF-β family member myostatin and demonstrated that inhibition of myostatin leads to muscle growth in normal and dystrophic mice. We describe a unique method of systemic inhibition of activin IIB receptor signaling via adeno-associated virus (AAV)-mediated gene transfer of a soluble form of the extracellular domain of the activin IIB receptor to the liver. Treatment of mdx mice with activin IIB receptor blockade led to increased skeletal muscle mass, increased force production in the extensor digitorum longus (EDL), and reduced serum creatine kinase. No effect on heart mass or function was observed. Our results indicate that activin IIB receptor blockade represents a novel and effective therapeutic strategy for the muscular dystrophies.

In a previous study investigating the effects of low temperature on skeletal muscle differentiation, we demonstrated that C2C12 mouse myoblasts cultured at 30°C do not express myogenin, a myogenic regulatory factor (MRF), or fuse into multinucleated myotubes. At this low temperature, the myoblasts continuously express Id3, a negative regulator of MRFs, and do not upregulate muscle-specific microRNAs. In this study, we examined if insulin-like growth factor-I (IGF-I) and a stable form of vitamin C (L-ascorbic acid phosphate) could alleviate the low temperature-induced inhibition of myogenic differentiation in C2C12 cells. Although the addition of either IGF-I or vitamin C alone could promote myogenin expression in C2C12 cells at 30°C, elongated multinucleated myotubes were not formed unless both IGF-I and vitamin C were continuously administered. In human skeletal muscle cells, low temperature-induced blockage of myogenic differentiation was also ameliorated by exogenous IGF-I and vitamin C. In addition, we demonstrated that satellite cells of IGF-I overexpressing transgenic mice in single-fiber culture expressed myogenin at a higher level than those of wild-type mice at 30°C. This study suggests that body temperature plays an important role in myogenic differentiation of endotherms, but the sensitivity to low temperature could be buffered by certain factors in vivo, such as IGF-I and vitamin C.

Mouse lines with dysferlin deficiency are accepted animal models for limb girdle muscular dystrophy 2B and Miyoshi myopathy, yet slow progression of pathology prevents rapid screening of potential therapies for this disease. Our goal was to define a functional signature for skeletal muscles that lack dysferlin. Force generation and susceptibility to eccentric contractile injury measurements were performed in isolated limb muscles and the diaphragm from 10- and 36-week-old A/J and age-matched control mice. Limb muscles had normal specific force at both 10 and 36 weeks, whereas the diaphragm had significant deficits in both specific force and susceptibility to eccentric contractile injury. Membrane ruptures in the diaphragm during eccentric contractions occurred predominantly in myosin heavy chain 2A-expressing fibers. Dysferlin content did not vary significantly between wildtype muscles, suggesting that there was no correlation between disease severity and normal endogenous levels of the protein. These studies show that, unlike limb muscles, the diaphragm from the A/J mouse displays early deficits in function that may lower the age needed for evaluating potential therapies for dysferlinopathies.

Although the precise pathophysiological mechanism of muscle damage in dystrophin-deficient muscle remains disputed, calcium appears to be a critical mediator of the dystrophic process. Duchenne muscular dystrophy patients and mouse models of dystrophin deficiency exhibit extensive abnormalities of calcium homeostasis, which we hypothesized would be mitigated by increased expression of the sarcoplasmic reticulum calcium pump. Neonatal adeno-associated virus gene transfer of sarcoplasmic reticulum ATPase 1a to the mdx diaphragm decreased centrally located nuclei and resulted in reduced susceptibility to eccentric contraction-induced damage at 6 months of age. As the diaphragm is the mouse muscle most representative of human disease, these results provide impetus for further investigation of therapeutic strategies aimed at enhanced cytosolic calcium removal.

Insulin-like growth factor I (IGF-I) coordinates proliferation and differentiation in a wide variety of cell types. The igf1 gene not only produces IGF-I, but also generates multiple carboxy-terminal extensions, the E-peptides, through alternative splicing leading to different isoforms. It is not known if the IGF-I isoforms share a common pathway for their actions, or if there are specific actions of each protein. Viral administration of murine IGF-IA, IGF-IB, and mature IGF, which lacked an E-peptide extension, was utilized to identify IGF-I isoform-specific responsive genes in muscles of young growing mice. Microarray analysis revealed responses that were driven by increased IGF-I regardless of the presence of E-peptide, such as Bcl-XL. In contrast, distinct expression patterns were observed after viral delivery of IGF-IA or IGF-IB, which included matrix metalloproteinase 13 (MMP13). Expression of Bcl-XL was prevented when viral administration of the IGF-I isoforms was performed into muscles of MKR mice, which lack functional IGF-I receptors on the muscle fibers. However, MMP13 expression persisted under the same conditions after viral injection of IGF-IB. At 4 mo after viral delivery, expression of IGF-IA or IGF-IB promoted muscle hypertrophy, but viral delivery of mature IGF-I failed to increase muscle mass. These studies provide evidence that local production of IGF-I requires the E-peptides to drive hypertrophy in growing muscle and that both common and unique pathways exist for the IGF-I isoforms to promote biological effects.

Background Myostatin inhibition is a promising therapeutic strategy to maintain muscle mass in a variety of disorders, including the muscular dystrophies, cachexia, and sarcopenia. Previously described approaches to blocking myostatin signaling include injection delivery of inhibitory propeptide domain or neutralizing antibodies. Methodology/Principal Findings Here we describe a unique method of myostatin inhibition utilizing recombinant adeno-associated virus to overexpress a secretable dominant negative myostatin exclusively in the liver of mice. Systemic myostatin inhibition led to increased skeletal muscle mass and strength in control C57 Bl/6 mice and in the dystrophin-deficient mdx model of Duchenne muscular dystrophy. The mdx soleus, a mouse muscle more representative of human fiber type composition, demonstrated the most profound improvement in force production and a shift toward faster myosin-heavy chain isoforms. Unexpectedly, the 11-month-old mdx diaphragm was not rescued by long-term myostatin inhibition. Further, mdx mice treated for 11 months exhibited cardiac hypertrophy and impaired function in an inhibitor dose–dependent manner. Conclusions/Significance Liver-targeted gene transfer of a myostatin inhibitor is a valuable tool for preclinical investigation of myostatin blockade and provides novel insights into the long-term effects and shortcomings of myostatin inhibition on striated muscle.

Muscle cells contain a membrane-spanning complex of proteins that are associated with dystrophin, which is a spectrin-related protein of the muscle membrane cytoskeleton (1). The members of this complex include membrane-spanning subunits, such as β-dystroglycan and the sarcoglycans, as well as strictly intracellular and extracellular components. As diagrammed in Fig. 1, the complex connects the cortical actin cytoskeleton (via dystrophin interactions) to the extracellular matrix (via interactions with laminin). Considerable work has focused on identifying the members of this complex and delineating the functions of the individual parts and the complex as a whole. Attention directed at this complex, and indeed the discovery of dystrophin (2), largely stems from the fact that the loss of dystrophin in humans results in loss of the entire protein complex shown in Fig. 1 and causes a progressive, lethal muscle-wasting disease, known as Duchenne muscular dystrophy (DMD). Figure 1 Schematic representation of the dystrophin-associated glycoprotein complex. The N-terminal, actin-binding domain of dystrophin in purple is associated with the cortical actin. The C-terminal domain associates with β-dystroglycan and with α- and β-syntrophin and dystrobrevin. nNOS is known to interact with the syntrophins as well as with caveolin. Two papers in this issue of PNAS address unresolved issues concerning the function of dystrophin and the dystrophin-associated proteins. First, the paper by Wang et al. (3) evaluates the ability of dystrophin molecules that have large segments deleted to rescue skeletal muscles of the mouse model of DMD ( mdx mouse). The motivation for this study is to design a functional dystrophin molecule that can fit into the most promising vector for skeletal muscle-directed gene therapy, recombinant adeno-associated virus. However, this and other such studies also will help to elucidate the functional roles of the dystrophin domains. The paper by Sander et al. (4) focuses on another member of the …

Insulin-like growth factor (IGF)-I is a critical protein for cell development and growth. Alternative splicing of the igf1 gene gives rise to multiple isoforms. In rodents, proIGF-IA and proIGF-IB have different carboxy-terminal extensions called the E-peptides (EA and EB) and upon further posttranslational processing, produce the identical mature IGF-I protein. Rodent EB has been reported to have mitogenic and motogenic effects independent of IGF-I. However, effects of EA or EB on mature IGF-I, or whether proIGF-IA and proIGF-IB have different properties, have not been addressed. To determine whether the presence of EA or EB affected the distribution and stability of mature IGF-I protein, transient transfections of cDNAs encoding murine IGF-IA, IGF-IB, and mature IGF-I were performed in C2C12 cells, a skeletal muscle cell line. IGF-I secretion was measured by enzyme-linked immunosorbent assay of the media, and did not differ between expression of proIGF-IA, proIGF-IB, or mature IGF-I expression. Next, epitope-tagged constructs were transfected to determine cellular distribution of IGF-I, EA, and EB in the cells throughout the culture. IGF-I was detected in significantly fewer nontransfected cells in cultures transfected with mature IGF-I compared with transfection of proIGF-IA or proIGF-IB. These results demonstrate that EA and EB are not required for IGF-I secretion but that they increase cell entry of IGF-I from the media. This study provides evidence that the EA and EB may modulate IGF-I in addition to having independent activity.

Distinctions between craniofacial and axial muscles exist from the onset of development and throughout adulthood. The masticatory muscles are a specialized group of craniofacial muscles that retain embryonic fiber properties in the adult, suggesting that the developmental origin of these muscles may govern a pattern of expression that differs from limb muscles. To determine the extent of these differences, expression profiling of total RNA isolated from the masseter and tibialis anterior (TA) muscles of adult female mice was performed, which identified transcriptional changes in unanticipated functional classes of genes in addition to those attributable to fiber type. In particular, the masseters displayed a reduction of transcripts associated with contractile and cytoskeletal load-sensing and anabolic processes, and heightened expression of genes associated with stress. Associated with these observations was a significantly smaller fiber cross-sectional area in masseters, significantly elevated load-sensing signaling (phosphorylated focal adhesion kinase), and increased apoptotic index in masseters compared with TA muscles. Based on these results, we hypothesize that masticatory muscles may have a fundamentally different strategy for muscle design, compared with axial muscles. Specifically there are small diameter fibers that have an attenuated ability to hypertrophy, but an increased propensity to undergo apoptosis. These results may provide insight into the molecular basis for specific muscle-related pathologies associated with masticatory muscles.

Insulin-like growth factor I (IGF-I) plays a critical role in the growth and development of many tissues in the body. It is a key regulator of skeletal muscle development, and continues to enhance the ability for muscle to grow and undergo repair throughout life. Although the focus of research has been on the molecular actions and physiological impact of IGF-I, there has also been a growing undercurrent of studies geared toward the characterization of additional potentially active peptides produced by the igf1 gene. Alternative splicing of the gene results in multiple isoforms that retain the identical sequence for mature IGF-I, but also give rise to divergent C-terminal peptides. The peptides might modulate the actions, stability, or bioavailability of IGF-I, or they might have independent activity. These possibilities have gained the attention of the skeletal muscle field, where novel actions of IGF-I could have significant impact on muscle mass, strength, and repair.