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Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a next-generation morpholino: arginine-rich, cell-penetrating peptide-conjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMDJ) dog model of DMD. A PPMO cocktail designed to skip dystrophin exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMDJ dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMDJ dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart.

There is a persistent, aberrant accumulation of V0/V1 versican in skeletal muscles from patients with Duchenne muscular dystrophy and in diaphragm muscles from mdx mice. Versican is a provisional matrix protein implicated in fibrosis and inflammation in various disease states, yet its role in the pathogenesis of muscular dystrophy is not known. Here, female mdx and male hdf mice (haploinsufficient for the versican allele) were bred. In the resulting F1 mdx -hdf male pups, V0/V1 versican expression in diaphragm muscles was decreased by 50% compared to mdx littermates at 20–26 weeks of age. In mdx -hdf mice, spontaneous physical activity increased by 17% and there was a concomitant decrease in total energy expenditure and whole-body glucose oxidation. Versican reduction improved the ex vivo strength and endurance of diaphragm muscle strips. These changes in diaphragm contractile properties in mdx -hdf mice were associated with decreased monocyte and macrophage infiltration and a reduction in the proportion of fibres expressing the slow type I myosin heavy chain isoform. Given the high metabolic cost of inflammation in dystrophy, an attenuated inflammatory response may contribute to the effects of versican reduction on whole-body metabolism. Altogether, versican reduction ameliorates the dystrophic pathology of mdx -hdf mice as evidenced by improved diaphragm contractile function and increased physical activity.

Duchenne muscular dystrophy (DMD) is caused by an X-linked mutation that leads to the absence of dystrophin, resulting in life-threatening arrhythmogenesis and associated heart failure. We targeted the gap junction protein connexin43 (Cx43) responsible for maintaining cardiac conduction. In mild mdx and severe mdx:utr mouse models of DMD and human DMD tissues, Cx43 was found to be pathologically mislocalized to lateral sides of cardiomyocytes. In addition, overall Cx43 protein levels were markedly increased in mouse and human DMD heart tissues examined. Electrocardiography on isoproterenol challenged mice showed that both models developed arrhythmias and died within 24 hours, while wild-type mice were free of pathology. Administering peptide mimetics to inhibit lateralized Cx43 function prior to challenge protected mdx mice from arrhythmogenesis and death, while mdx:utr mice displayed markedly improved ECG scores. These findings suggest that Cx43 lateralization contributes significantly to DMD arrhythmogenesis and that selective inhibition may provide substantial benefit.

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. 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. 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.

Increased utrophin expression is known to reduce pathology in dystrophin-deficient skeletal muscles. Transgenic over-expression of PGC-1α has been shown to increase levels of utrophin mRNA and improve the histology of mdx muscles. Other reports have shown that PGC-1α signaling can lead to increased oxidative capacity and a fast to slow fiber type shift. Given that it has been shown that slow fibers produce and maintain more utrophin than fast skeletal muscle fibers, we hypothesized that over-expression of PGC-1α in post-natal mdx mice would increase utrophin levels via a fiber type shift, resulting in more slow, oxidative fibers that are also more resistant to contraction-induced damage. To test this hypothesis, neonatal mdx mice were injected with recombinant adeno-associated virus (AAV) driving expression of PGC-1α. PGC-1α over-expression resulted in increased utrophin and type I myosin heavy chain expression as well as elevated mitochondrial protein expression. Muscles were shown to be more resistant to contraction-induced damage and more fatigue resistant. Sirt-1 was increased while p38 activation and NRF-1 were reduced in PGC-1α over-expressing muscle when compared to control. We also evaluated if the use a pharmacological PGC-1α pathway activator, resveratrol, could drive the same physiological changes. Resveratrol administration (100 mg/kg/day) resulted in improved fatigue resistance, but did not achieve significant increases in utrophin expression. These data suggest that the PGC-1α pathway is a potential target for therapeutic intervention in dystrophic skeletal muscle.

The GRMD (Golden retriever muscular dystrophy) dog has been widely used in pre-clinical trials targeting DMD (Duchenne muscular dystrophy), using in many cases a concurrent immune-suppressive treatment. The aim of this study is to assess if such a treatment could have an effect on the disease course of these animals. Seven GRMD dogs were treated with an association of cyclosporine A (immunosuppressive dosage) and prednisolone (2 mg/kg/d) during 7 months, from 2 to 9 months of age. A multi-parametric evaluation was performed during this period which allowed us to demonstrate that this treatment had several significant effects on the disease progression. The gait quality as assessed by 3D-accelerometry was dramatically improved. This was consistent with the evolution of other parameters towards a significant improvement, such as the clinical motor score, the post-tetanic relaxation and the serum CK levels. In contrast the isometric force measurement as well as the histological evaluation argued in favor of a more severe disease progression. In view of the disease modifying effects which have been observed in this study it should be concluded that immunosuppressive treatments should be used with caution when carrying out pre-clinical studies in this canine model of DMD. They also highlight the importance of using a large range of multi-parametric evaluation tools to reliably draw any conclusion from trials involving dystrophin-deficient dogs, which reproduce the complexity of the human disease.

Duchenne muscular dystrophy (DMD) is a debilitating X-linked disorder that is fatal. DMD patients lack the expression of the structural protein dystrophin caused by mutations within the DMD gene. The absence of functional dystrophin protein results in excessive damage from normal muscle use due to the compromised structural integrity of the dystrophin associated glycoprotein complex. As a result, DMD patients exhibit ongoing cycles of muscle destruction and regeneration that promote inflammation, fibrosis, mitochondrial dysfunction, satellite cell (SC) exhaustion and loss of skeletal and cardiac muscle function. The nuclear receptor REV-ERB suppresses myoblast differentiation and recently we have demonstrated that the REV-ERB antagonist, SR8278, stimulates muscle regeneration after acute injury. Therefore, we decided to explore whether the REV-ERB antagonist SR8278 could slow the progression of muscular dystrophy. In mdx mice SR8278 increased lean mass and muscle function, and decreased muscle fibrosis and muscle protein degradation. Interestingly, we also found that SR8278 increased the SC pool through stimulation of Notch and Wnt signaling. These results suggest that REV-ERB is a potent target for the treatment of DMD.

A large number of human diseases are caused by chronic tissue injury with fibrosis potentially leading to organ failure. There is a need for more effective anti-fibrotic therapies. Congenital muscular dystrophy type 1A (MDC1A) is a devastating form of muscular dystrophy caused by laminin α2 chain-deficiency. It is characterized with early inflammation and build-up of fibrotic lesions, both in patients and MDC1A mouse models (e.g. dy 3K / dy 3K ). Despite the enormous impact of inflammation on tissue remodelling in disease, the inflammatory response in MDC1A has been poorly described. Consequently, a comprehensive understanding of secondary mechanisms (impaired regeneration, enhanced fibrosis) leading to deterioration of muscle phenotype in MDC1A is missing. We have monitored inflammatory processes in dy 3K / dy 3K muscle and created mice deficient in laminin α2 chain and osteopontin or galectin-3, two pro-inflammatory and pro-fibrotic molecules drastically increased in dystrophic muscle. Surprisingly, deletion of osteopontin worsened the phenotype of dy 3K / dy 3K mice and loss of galectin-3 did not reduce muscle pathology. Our results indicate that osteopontin could even be a beneficial immunomodulator in MDC1A. This knowledge is essential for the design of future therapeutic interventions for muscular dystrophies that aim at targeting inflammation, especially that osteopontin inhibition has been suggested for Duchenne muscular dystrophy therapy.

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation. We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice. These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.

The mdx mouse, a mouse model of Duchenne muscular dystrophy, carries a loss-of-function mutation in dystrophin, a component of the membrane-associated dystrophin-glycoprotein complex. Unlike humans, mdx mice rarely display cardiac abnormalities and exhibit dystrophic changes only in a small number of heavily used skeletal muscle groups. By contrast, mdx:MyoD-/-mice lacking dystrophin and the skeletal muscle-specific bHLH transcription factor MyoD display a severe skeletal myopathy leading to widespread dystrophic changes in skeletal muscle and premature death around 1 year of age. The severely increased phenotype of mdx:MyoD-/-muscle is a consequence of impaired muscle regeneration caused by enhanced satellite cell self-renewal. Here we report that mdx:MyoD-/-mice developed a severe cardiac myopathy with areas of necrosis associated with hypertrophied myocytes. Moreover, heart tissue from mdx:MyoD-/-mice exhibited constitutive activation of stress-activated signaling components, similar to in vitro models of cardiac myocyte adaptation. Taken together, these results support the hypothesis that the progression of skeletal muscle damage is a significant contributing factor leading to development of cardiomyopathy.