Role of Connexin43 in the heart and skeletal muscle in a model for Duchenne muscular dystrophy symptomatic carriers

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SUMMARY Duchenne Muscular Dystrophy (DMD) is a severe x-linked neuromuscular disorder that affects male patients. The culprit is a mutation in dystrophin. Absence of dystrophin at the sarcolemma of cardiomyocytes and skeletal muscle fibers leads to fragility of the cell membrane due to mechanical-induced damage. A fraction of DMD female carriers are also vulnerable to dystrophin loss. These symptomatic DMD female carriers develop a range of skeletal weakness, cardiomyopathy, and electrocardiographic abnormalities. Many therapeutics for male DMD are being investigated in the mdx mouse model. However, research on DMD symptomatic female carriers lags behind, partly because there was no faithful mouse model of DMD symptomatic carriers to be tested. Recently, we created the first symptomatic mouse model of DMD female carriers. We developed mosaic mice by injecting mdx (murine DMD) embryonic stem cells (ESCs) into wild-type (WT) blastocysts (mdx/WT chimera). mdx/WT mice develop cardiac and skeletal muscle abnormalities that model the characteristics of symptomatic DMD female carriers. Recent discoveries in our lab showed an important role for connexin-43 (Cx43). In DMD patients and mdx mice, Cx43 is pathologically upregulated and remodeled away from the ID and is likely responsible for the cardiomyopathy and arrhythmias patients experience. In DMD, Cx43 exhibits a distinct phosphorylation pattern in a triplet of serine residues. We have initiated studies using mutant knock-in mice harboring a phospho-mimetic form of Cx43 where the triplet has been mutated to glutamic acids (Cx43S3E). The mutation was incorporated into mdx mice, and Cx43 was retained to the ID. Importantly, all the cardiac defects were rescued. In contrast to the heart, Cx43 is not expressed in the skeletal muscle fibers. This is because the fibers form a syncytium, and thus, do not need connectors. However, we found exacerbated Cx43 expression in mononuclear cells between the dystrophic skeletal muscle fibers. Our preliminary results indicate that reduction of Cx43 copy number eliminates pathology in the heart and the skeletal muscle of mdx/WT DMD female carriers. This leads to prevention of Cx43 remodeling in the heart. Based on our results, we hypothesize that regulation of Cx43 remodeling and levels in the heart and in the skeletal muscle improves DMD manifestation in mdx/WT female carriers. We will first determine whether a modified form of Cx43, unable to remodel, overcomes development of cardiomyopathy in mdx/WT chimeric mice. We will generate mdx/WT:Cx43(S3E) mice by injecting mdx ESCs into WT:Cx43(S3E) blastocysts (Aim1). We will also determine whether normalization of Cx43 levels by gene-copy number reduction overcomes development of skeletal muscle pathology in mdx/WT chimeric mice. Cx43 is not expressed in the skeletal fibers but expressed in the mononuclear cells between fibers, and higher Cx43 protein levels are observed in DMD skeletal muscle. Chimeric experiments in-vivo will be complemented by co-culture experiments, where adult WT and mdx skeletal muscle fibers will be cultured in the presence of mdx macrophages (or their secreted components) treated or not with Cx43 hemichannel inhibitor Gap19 (Aim2). Because peptide mimetic inhibitors of Cx43 are being tested in clinical trials, with toxicological profiles in place, this training opportunity will open new therapeutic venues to address cardiac and skeletal muscle pathology in understudied symptomatic DMD carriers.