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Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin-positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present. Stem Cells 2017;35:597-610.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder that affects males. However, 8% of female carriers are symptomatic and underrepresented in research due to the lack of animal models. We generated a symptomatic mouse model of DMD carriers via injection of mdx (murine DMD) embryonic stem cells (ESCs) into wild-type (WT) blastocysts (mdx/WT chimera). mdx/WT chimeras developed cardiomyopathic features and dystrophic skeletal muscle phenotypes including elevated mononuclear invasion, central nucleation, fibrosis and declined forelimb grip strength. The disease was accompanied by connexin-43 (Cx43) aberrantly enhanced in both cardiac and skeletal muscles and remodeled in the heart. Genetic reduction of Cx43-copy number in mdx/WT-Cx43(+/−) chimeras protected them from both cardiac and skeletal muscle fiber damage. In dystrophic skeletal muscle, Cx43 expression was not seen in the fibers but in adjacent F4/80+ mononuclear cells. Ethidium Bromide uptake in purified F4/80+/CD11b+ mdx macrophages revealed functional activity of Cx43, which was inhibited by administration of Gap19 peptide mimetic, a Cx43 hemichannel-specific inhibitor. Thus, we suggest that Cx43 reduction in symptomatic DMD carrier mice leads to prevention of Cx43 remodeling in the heart and prevention of aberrant Cx43 hemichannel activity in the skeletal muscle macrophages neighboring Cx43 non-expressing fibers. Duchenne muscular dystrophy (DMD) is an incurable X-linked recessive disorder characterized by mutations in the dystrophin gene. The dystrophin protein is an essential part of the dystrophin-glycoprotein complex (DGC), which provides structure and mechanically-induced signal propagation between intracellular and extracellular environments 1. X-linked disorders only affect male patients; however, a percentage of female DMD heterozygotes are symptomatic. Symptomatic female carriers consist of about 8-40% of definite DMD carriers and begin to present symptoms between the ages of 2-47 years old 2,3. Patients display a range of severe to less severe phenotypes. It is generally accepted to affect 8% of female carriers, yet this statistic aligns with those affected by dilated cardiomyopathy. A higher percentage (17%) suffer from muscle weakness 3-10. Heart complications can be further expanded to include left-ventricle dilation (40%) 10. Research on symptomatic carriers has been confined to clinical observations largely due to the absence of a representative animal model 2,3,9,11,12. These epidemiological studies are subject to a larger margin of error from confounding variables, necessitating a DMD animal model that successfully recapitulates the symptomatic carrier phenotypes and mosaicism. X-linked muscular dystrophy mice (mdx) 13 heterozygotes, mdx(+/−), show normal muscle histology and do not develop cardiomyopathy 11,12. Recently, we described the generation of a model to study symptomatic DMD carriers that faithfully recapitulates the pathology and symptoms (mdx/WT mouse chimeras) 14. Symptomatic mdx mouse chimeras were ...