Impaired S-nitrosylation of Cx43 prevents arrhythmogenicity and myocardial injury upon cardiac stress in Duchenne Muscular Dystrophy

Connexin-43 (Cx43) plays a critical role in the propagation of action potentials and cardiac contractility. In healthy cardiomyocytes, Cx43 is mainly located at the intercalated disk; however, Cx43 remodeling is observed in cardiac pathologies and is linked with arrhythmogenesis and sudden cardiac death. Using a mouse model of Duchenne muscular dystrophy (DMD), we previously demonstrated that Cx43 localizes to the lateral side of dystrophic cardiomyocytes, forming undocked hemichannels. β-adrenergic signaling-induced cardiac stress promotes S-nitrosylation and the opening of undocked Cx43 hemichannels leading to disrupted cardiac membrane excitability and deadly arrhythmogenic behaviors. To establish the direct role of S-nitrosylated Cx43 in DMD cardiomyopathy, we generated knockin DMD^mdx mice with reduced levels of S-nitrosylated Cx43, by replacing cysteine 271 with a serine in one Cx43 of the unique site for S-nitrosylation of Cx43 (DMD^mdx:C271S^+/-). Immunofluorescence analysis revealed that cardiac Cx43 lateralization in DMD^mdx:C271S^+/- mice was similar to DMD^mdx mice, indicating that the genetic modification did not prevent Cx43 remodeling. Upon isoproterenol treatment, DMD^mdx mice displayed a higher incidence of arrhythmogenic events when compared to DMD^mdx:C271S^+/- mice, which more closely resemble wild-type mice. Optical mapping imaging in isolated hearts showed that DMD^mdx mice displayed aberrant Ca²⁺ signaling and prolonged action potentials, which is restored in DMD^mdx:C271S^+/- mice. Isoproterenol treatment evoked severe myocardial injury in DMD^mdx mice, which was significantly attenuated in DMD^mdx:C271S^+/- mice. Notably, DMD^mdx mice treated with Gap19, a Cx43 hemichannel blocker, exhibited cardioprotection against myocardial injury. We concluded that S-nitrosylation of Cx43 proteins is a fundamental NO-mediated mechanism involved in arrhythmias and myocardial injury in DMD^mdx, occurring through the opening of hemichannels following β-adrenergic stress.