Potential limitations of microdystrophin gene therapy for Duchenne muscular dystrophy.

Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (microdystrophins) are underway for Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with 4 microdystrophin constructs (3 in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using AAV doses comparable with those in clinical trials. We achieved high levels of microdystrophin expression in striated muscles with cardiac expression approximately 10-fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease occurred with 2 of the microdystrophins. The detrimental cardiac effect appears to be caused by variable competition (dependent on microdystrophin design and expression level) between microdystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-microdystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, these findings suggest that microdystrophin treatments need to avoid excessively high levels of expression in the heart and that cardiac function should be carefully monitored in these patients.

Evaluation of the DBA/2J mouse as a potential background strain for genetic models of cardiomyopathy.

The potential use of the D2.mdx mouse (the mdx mutation on the DBA/2J genetic background) as a preclinical model of the cardiac aspects of Duchenne muscular dystrophy (DMD) has been criticized based on speculation that the DBA/2J genetic background displays an inherent hypertrophic cardiomyopathy (HCM) phenotype. Accordingly, the goal of the current study was to further examine the cardiac status of this mouse strain over a 12-month period to determine if observable signs of HCM develop, including histopathology and pathological enlargement of the myocardium. Previous reports have documented heightened TGFß signaling in the DBA2/J striated muscles, as compared to the C57 background, which, as expected, is manifested as increased cardiomyocyte size, wall thickness, and heart mass as compared to the C57 background. While normalized heart mass is larger in the DBA/2J mice, compared to age-matched C57/BL10 mice, both strains similarly increase in size from 4 to 12 months of age. We also report that DBA/2J mice contain equivalent amounts of left ventricular collagen as healthy canine and human samples. In a longitudinal echocardiography study, neither sedentary nor exercised DBA/2J mice demonstrated left ventricular wall thickening or cardiac functional deficits. In summary, we find no evidence of HCM, nor any other cardiac pathology, and thus propose that it is an appropriate background strain for genetic modeling of cardiac diseases, including the cardiomyopathy associated with DMD.

Filopodia powered by class x myosin promote fusion of mammalian myoblasts.

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.

The D2.mdx mouse as a preclinical model of the skeletal muscle pathology associated with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle degenerative disease caused by loss of dystrophin protein. DMD has no cure and few treatment options. Preclinical efforts to identify potential DMD therapeutics have been hampered by lack of a small animal model that recapitulates key features of the human disease. While the dystrophin-deficient mdx mouse on the C57BL/10 genetic background (B10.mdx) is mildly affected, a more severe muscle disease is observed when the mdx mutation is crossed onto the DBA/2J genetic background (D2.mdx). In this study, the functional and histological progression of the D2.mdx skeletal muscle pathology was evaluated to determine the distinguishing features of disease. Data herein details the muscular weakness and wasting exhibited by D2.mdx skeletal muscle, as well as severe histopathological features, which include the rapid progression of fibrosis and calcifications in the diaphragm and progressive fibrosis accumulation in limb muscles. Furthermore, a timeline of D2.mdx progression is provided that details distinct stages of disease progression. These data support the D2.mdx as a superior small animal model for DMD, as compared to the B10.mdx model. The insights provided in this report should facilitate the design of preclinical evaluations for potential DMD therapeutics.

Glucocorticoids counteract hypertrophic effects of myostatin inhibition in dystrophic muscle.

Duchenne muscular dystrophy (DMD) is a devastating genetic muscle disease resulting in progressive muscle degeneration and wasting. Glucocorticoids, specifically prednisone/prednisolone and deflazacort, are commonly used by DMD patients. Emerging DMD therapeutics include those targeting the muscle-wasting factor, myostatin (Mstn). The aim of this study was to investigate how chronic glucocorticoid treatment impacts the efficacy of Mstn inhibition in the D2.mdx mouse model of DMD. We report that chronic treatment of dystrophic mice with prednisolone (Pred) causes significant muscle wasting, entailing both activation of the ubiquitin-proteasome degradation pathway and inhibition of muscle protein synthesis. Combining Pred with Mstn inhibition, using a modified Mstn propeptide (dnMstn), completely abrogates the muscle hypertrophic effects of Mstn inhibition independently of Mstn expression or SMAD3 activation. Transcriptomic analysis identified that combining Pred with dnMstn treatment affects gene expression profiles associated with inflammation, metabolism, and fibrosis. Additionally, we demonstrate that Pred-induced muscle atrophy is not prevented by Mstn ablation. Therefore, glucocorticoids interfere with potential muscle mass benefits associated with targeting Mstn, and the ramifications of glucocorticoid use should be a consideration during clinical trial design for DMD therapeutics. These results have significant implications for past and future Mstn inhibition trials in DMD.