The Ras Antagonist,Farnesylthiosalicylic Acid (FTS), Decreases Fibrosis and Improves Muscle Strength in dy2J/dy2J Mouse Model of Muscular Dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy^2J/dy^2J mouse model of merosin deficient congenital muscular dystrophy. The dy^2J/dy^2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy^2J/dy^2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy^2J/dy^2J mouse model of congenital muscular dystrophy.     

Muscular Dystrophy in mdx Mice Despite Lack of Neuronal Nitric Oxide Synthase

Abstract: Neuronal nitric oxide synthase (nNOS) is a component of the dystrophin complex in skeletal muscle. The absence of dystrophin protein in Duchenne muscular dystrophy and in mdx mouse causes a redistribution of nNOS from the plasma membrane to the cytosol in muscle cells. Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers. To test the hypothesis that derangements in nNOS disposition mediate muscle damage in Duchenne dystrophy, we bred dystrophin-deficient mdx male mice and female mdx heterozygote mice that lack nNOS. We found that genetic deletion of nNOS does not itself cause detectable pathology and that removal of nNOS does not influence the extent of increased sarcolemmal permeability in dystrophin-deficient mice. Thus, histological analyses of nNOS-dystrophin double mutants show pathological changes similar to the dystrophin mutation alone. Taken together, nNOS defects alone do not produce muscular dystrophy in the mdx model.     

Sarcoglycan complex: a muscular supporter of dystroglycan-dystrophin interplay?

In striated muscle, the cytoskeletal protein dystrophin, the protein product of the Duchenne muscular dystrophy gene, is associated with a number of sarcolemmal glycoproteins to form a large oligomeric complex, the dystrophin-glycoprotein complex (DGC). Over the last 10 years, four of these sarcolemmal glycoproteins, alpha-, beta-, gamma- and delta-sarcoglycans, have been shown to form a distinct subcomplex, the sarcoglycan complex, in the DGC. Furthermore, the genetic defects of alpha-, beta-, gamma- and delta-sarcoglycans have been identified as the causes of four distinct forms of muscular dystrophies, which are now collectively called sarcoglycanopathy. Current studies are beginning to focus on the biological functions of the sarcoglycan complex and the molecular mechanism by which its dysfunction leads to muscle cell degeneration.     

Several dystrophin-glycoprotein complex members are present in crude surface membranes but they are sodium dodecyl sulphate invisible in KCl-washed microsomes from <Emphasis Type="Italic">mdx</Emphasis> mouse muscle

The dystrophin-glycoprotein complex (DGC) is a large trans-sarcolemmal complex that provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. In skeletal muscle, it consists of the dystroglycan, sarcoglycan and cytoplasmic complexes, with dystrophin forming the core protein. The DGC has been described as being absent or greatly reduced in dystrophin-deficient muscles, and this lack is considered to be involved in the dystrophic phenotype. Such a decrease in the DGC content was observed in dystrophin-deficient muscle from humans with muscular dystrophy and in mice with X-linked muscular dystrophy (mdx mice). These deficits were observed in total muscle homogenates and in partially membrane-purified muscle fractions, the so-called KCl-washed microsomes. Here, we report that most of the proteins of the DGC are actually present at normal levels in the mdx mouse muscle plasma membrane. The proteins are detected in dystrophic animal muscles when the immunoblot assay is performed with crude surface membrane fractions instead of the usually employed KCl-washed microsomes. We propose that these proteins form SDS-insoluble membrane complexes when dystrophin is absent.     

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca²⁺ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies

Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.     

Phospholipase A2-derived lysophosphatidylcholine triggers Ca entry in dystrophic skeletal muscle fibers

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca²⁺ entry through sarcolemmal cationic channels. Using the Mn²⁺ quench method, we show here that Mn²⁺ entry triggered by Ca²⁺ store depletion but not basal Mn²⁺ entry relies on Ca²⁺-independent PLA₂ (iPLA₂) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx^5cv mouse. iPLA₂ was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA₂ lipid product lysophosphatidylcholine was found to trigger Ca²⁺ entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA₂ and lysophospholipid production may be of interest to reduce Ca²⁺ entry and subsequent degeneration of dystrophic muscle.     

Pharmacological rescue of the dystrophin-glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment

In this report, we have developed a novel method to identify compounds that rescue the dystrophin-glycoprotein complex (DGC) in patients with Duchenne or Becker muscular dystrophy. Briefly, freshly isolated skeletal muscle biopsies (termed skeletal muscle explants) from patients with Duchenne or Becker muscular dystrophy were maintained under defined cell culture conditions for a 24-h period in the absence or presence of a specific candidate compound. Using this approach, we have demonstrated that treatment with a well-characterized proteasome inhibitor, MG-132, is sufficient to rescue the expression of dystrophin, -dystroglycan, and -sarcoglycan in skeletal muscle explants from patients with Duchenne or Becker muscular dystrophy. These data are consistent with our previous findings regarding systemic treatment with MG-132 in a dystrophin-deficient mdx mouse model (Bonuccelli G, Sotgia F, Schubert W, Park D, Frank PG, Woodman SE, Insabato L, Cammer M, Minetti C, and Lisanti MP. Am J Pathol 163: 1663–1675, 2003). Our present results may have important new implications for the possible pharmacological treatment of Duchenne or Becker muscular dystrophy in humans. muscular dystrophy; membrane proteins; MG-132     

Immunologic function and cell surface antigen expression of lymphocytes of dystrophic mice

Previous studies have reported abnormalities of thymic histology and cell numbers in 129/ ReJ-dy homozygous dystrophic mice, suggesting an association between murine muscular dystrophy and disorders of the immune system. The present study of C57BL/6J-dy^2J and 129/ReJ-dy homozygous dystrophic mice included a thorough analysis of thymic development and histology, of T-cell function demonstrated by mitogen stimulation, mixed-leukocyte culture, and graft-vs-host assays, and of surface antigen expression as measured by flow microfluorometry. Although sporadic differences can be seen in some dystrophic mice, we find no evidence of consistent abnormalities of the immune system in murine muscular dystrophy. It does not seem possible, therefore, to study either the dy or the dy^2J defect through analysis of lymphocytes. The feasibility of elucidating metabolic or membrane defects by utilizing cell populations other than those most conspicuously affected by a mutation with multisystem effects is discussed and our coincidental finding of a subpopulation of T cells with unusual antigenic properties is described.     

Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice

Mdx mouse, the animal model of Duchenne muscular dystrophy, lacks dystrophin and develops an X-linked recessive inflammatory myopathy characterized by degeneration of skeletal muscle fibers and connective tissue replacement. The present work aimed to assess whether gender dimorphism in mdx mice would influence skeletal muscle pathology at ages corresponding to main histological changes in the microenvironment of muscular tissue: myonecrosis, regeneration, and fibrosis. At the height of myonecrosis (6 weeks postnatal), skeletal muscles of male mdx mice showed increased sarcolemmal permeability, numerous inflammatory foci, and marked deposition of the extracellular matrix components (ECM) type I collagen and laminin. In contrast, age-matched mdx females showed mild ECM deposition, discrete myonecrosis, but increased numbers of regenerating fibers expressing the satellite cell marker NCAM. In contrast ovariectomized mdx females showed decreased numbers of regenerating fibers. Older (24 and 48 weeks postnatal) mdx females showed extensive fibrosis with increased sarcolemmal permeability and marked deposition of ECM components than corresponding males. These results suggest a role for female hormones in the control of myonecrosis probably by promoting regeneration of muscular tissue and mitigating inflammation especially at ages under the critical influence of sex hormones.     

Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice

Mdx mouse, the animal model of Duchenne muscular dystrophy, lacks dystrophin and develops an X-linked recessive inflammatory myopathy characterized by degeneration of skeletal muscle fibers and connective tissue replacement. The present work aimed to assess whether gender dimorphism in mdx mice would influence skeletal muscle pathology at ages corresponding to main histological changes in the microenvironment of muscular tissue: myonecrosis, regeneration, and fibrosis. At the height of myonecrosis (6 weeks postnatal), skeletal muscles of male mdx mice showed increased sarcolemmal permeability, numerous inflammatory foci, and marked deposition of the extracellular matrix components (ECM) type I collagen and laminin. In contrast, age-matched mdx females showed mild ECM deposition, discrete myonecrosis, but increased numbers of regenerating fibers expressing the satellite cell marker NCAM. In contrast ovariectomized mdx females showed decreased numbers of regenerating fibers. Older (24 and 48 weeks postnatal) mdx females showed extensive fibrosis with increased sarcolemmal permeability and marked deposition of ECM components than corresponding males. These results suggest a role for female hormones in the control of myonecrosis probably by promoting regeneration of muscular tissue and mitigating inflammation especially at ages under the critical influence of sex hormones.     

Amelioration of muscular dystrophy by transgenic expression of Niemann-Pick C1

Duchenne muscular dystrophy (DMD) and other types of muscular dystrophies are caused by the loss or alteration of different members of the dystrophin protein complex. Understanding the molecular mechanisms by which dystrophin-associated protein abnormalities contribute to the onset of muscular dystrophy may identify new therapeutic approaches to these human disorders. By examining gene expression alterations in mouse skeletal muscle lacking α-dystrobrevin (Dtna^−/−), we identified a highly significant reduction of the cholesterol trafficking protein, Niemann-Pick C1 (NPC1). Mutations in NPC1 cause a progressive neurodegenerative, lysosomal storage disorder. Transgenic expression of NPC1 in skeletal muscle ameliorates muscular dystrophy in the Dtna^−/− mouse (which has a relatively mild dystrophic phenotype) and in the mdx mouse, a model for DMD. These results identify a new compensatory gene for muscular dystrophy and reveal a potential new therapeutic target for DMD.     

Omigapil Treatment Decreases Fibrosis and Improves Respiratory Rate in dy2J Mouse Model of Congenital Muscular Dystrophy

Introduction

Congenital muscular dystrophy is a distinct group of diseases presenting with weakness in infancy or childhood and no current therapy. One form, MDC1A, is the result of laminin alpha-2 deficiency and results in significant weakness, respiratory insufficiency and early death. Modification of apoptosis is one potential pathway for therapy in these patients.

Methods

dy^2J mice were treated with vehicle, 0.1 mg/kg or 1 mg/kg of omigapil daily via oral gavage over 17.5 weeks. Untreated age matched BL6 mice were used as controls. Functional, behavioral and histological measurements were collected.

Results

dy^2J mice treated with omigapil showed improved respiratory rates compared to vehicle treated dy^2J mice (396 to 402 vs. 371 breaths per minute, p<0.03) and similar to control mice. There were no statistical differences in normalized forelimb grip strength between dy^2J and controls at baseline or after 17.5 weeks and no significant differences seen among the dy^2J treatment groups. At 30–33 weeks of age, dy^2J mice treated with 0.1 mg/kg omigapil showed significantly more movement time and less rest time compared to vehicle treated. dy^2J mice showed normal cardiac systolic function throughout the trial. dy^2J mice had significantly lower hindlimb maximal (p<0.001) and specific force (p<0.002) compared to the control group at the end of the trial. There were no statistically significant differences in maximal or specific force among treatments. dy^2J mice treated with 0.1 mg/kg/day omigapil showed decreased percent fibrosis in both gastrocnemius (p<0.03) and diaphragm (p<0.001) compared to vehicle, and in diaphragm (p<0.013) when compared to 1 mg/kg/day omigapil treated mice. Omigapil treated dy^2J mice demonstrated decreased apoptosis.

Conclusion

Omigapil therapy (0.1 mg/kg) improved respiratory rate and decreased skeletal and respiratory muscle fibrosis in dy^2J mice. These results support a putative role for the use of omigapil in laminin deficient congenital muscular dystrophy patients.     

Sarcospan-deficient mice maintain normal muscle function

Sarcospan is an integral membrane component of the dystrophin-glycoprotein complex (DGC) found at the sarcolemma of striated and smooth muscle. The DGC plays important roles in muscle function and viability as evidenced by defects in components of the DGC, which cause muscular dystrophy. Sarcospan is unique among the components of the complex in that it contains four transmembrane domains with intracellular N- and C-terminal domains and is a member of the tetraspan superfamily of proteins. Sarcospan is tightly linked to the sarcoglycans, and together these proteins form a subcomplex within the DGC. Stable expression of sarcospan at the sarcolemma is dependent upon expression of the sarcoglycans. Here we describe the generation and analysis of mice carrying a null mutation in the Sspn gene. Surprisingly, the Sspn-deficient muscle maintains expression of other components of the DGC at the sarcolemma, and no gross histological abnormalities of muscle from the mice are observed. The Sspn-deficient muscle maintains sarcolemmal integrity as determined by serum creatine kinase and Evans blue uptake assays, and the Sspn-deficient muscle maintains normal force and power generation capabilities. These data suggest either that sarcospan is not required for normal DGC function or that the Sspn-deficient muscle is compensating for the absence of sarcospan, perhaps by utilizing another protein to carry out its function.     

Phosphorylation of the carboxyl terminal region of dystrophin by mitogen-activated protein (MAP) kinase

Dystrophin is the 427-kDa protein product of the Duchenne muscular dystrophy gene (DMD). The function of this protein remains to be elucidated. We have recently reported that dystrophin is phosphorylated,in vivo, in rat skeletal muscle primary cell culture (RE Milner, JL Busaan, CFB Holmes, JH Wang, M Michalak (1993) J Biol Chem 268: 21901–21905). This observation suggests that protein phosphorylation may have some role in modulating the function of dystrophin or its interaction with membrane associate dystroglycan. We report here that the carboxyl-terminal of dystrophin is phosphorylated by the MAP kinase p44^mpk (mitogen-activated protein kinase), from the sea star oocytes and by soluble extracts of rabbit skeletal muscle. Importantly we showed that native dystrophin in isolated sarcolemmal vesicles is phosphorylated by sea star p44^mpk. Partial purification and immunological analysis show that a mammalian kinase related to p44^mpk is present in the skeletal muscle extracts and that it contributes to phosphorylation of the carboxyl-terminal of dystrophin. This kinase phosphorylates dystrophin on a threonine residue(s). We conclude that phosphorylation of dystrophin may play an important role in the function of this cytoskeletal protein.Abbreviations MAP kinase mitogen-activated protein kinase - DMD Duchenne muscular dystrophy - GST Glutathione S-transferase - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - MOPS 4-morpholinepropanesulfonic acid     

Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene

Duchenne muscular dystrophy (DMD) patients and mdx mice are characterized by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoproteins, including dystroglycan which provides a linkage to the extarcellular matrix component, laminin. The finding that all of the dystrophin-associated proteins (DAPs) are drastically reduced in DMD and mdx skeletal muscle supports the primary function of dystrophin as an anchor of the sarcolemmal glycoprotein complex to the subsarcolemmal cytoskeleton. These findings indicate that the efficacy of dystrophin gene therapy will depend not only on replacing dystrophin but also on restoring all of the DAPs in the sarcolemma. Here we have investigated the status of the DAPs in the skeletal muscle of mdx mice transgenic for the dystrophin gene. Our results demonstrate that transfer of dystrophin gene restores all of the DAPs together with dystrophin, suggesting that dystrophin gene therapy should be effective in restoring the entire dystrophin-glycoprotein complex.     

Role of gelatinases in pathological and physiological processes involving the dystrophin–glycoprotein complex

Dystrophin is a cytosolic protein belonging to a membrane-spanning glycoprotein complex, called dystrophin–glycoprotein complex (DGC) that is expressed in many tissues, especially in skeletal muscle and in the nervous system. The DGC connects the cytoskeleton to the extracellular matrix and, although none of the proteins of the DGC displays kinase or phosphatase activity, it is involved in many signal transduction pathways. Mutations in some components of the DGC are linked to many forms of inherited muscular dystrophies. In particular, a mutation in the dystrophin gene, leading to a complete loss of the protein, provokes one of the most prominent muscular dystrophies, the Duchenne muscular dystrophy, which affects 1 out of 3500 newborn males. What is observed in these circumstances, is a dramatic alteration of the expression levels of a multitude of metalloproteinases (MMPs), a family of extracellular Zn²⁺-dependent endopeptidases, in particular of MMP-2 and MMP-9, also called gelatinases. Indeed, the enzymatic activity of MMP-2 and MMP-9 on dystroglycan, an important member of the DGC, plays a significant role also in physiological processes taking place in the central and peripheral nervous system. This mini-review discusses the role of MMP-2 and MMP-9, in physiological as well as pathological processes involving members of the DGC.     

Whole Body Periodic Acceleration Is an Effective Therapy to Ameliorate Muscular Dystrophy in mdx Mice

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 Ca²⁺ 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 Ca²⁺ and Na⁺ overload, diminished abnormal sarcolemmal Ca²⁺ 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.     

Evaluation of Skeletal and Cardiac Muscle Function after Chronic Administration of Thymosin β-4 in the Dystrophin Deficient Mouse

Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tβ4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8–10 weeks old, were treated with 150 µg of Tβ4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tβ4 and amount of fibrosis were quantified using immunohistochemistry and Gomori''s tri-chrome staining, respectively. Mdx mice treated with Tβ4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tβ4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tβ4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.