Abstract Body (Do not enter title and authors here): Connexin-43 (Cx43) plays a critical role in the propagation of action potentials among cardiomyocytes and proper cardiac contractility. In healthy cardiomyocytes, Cx43 is located at the intercalated disk; however, Cx43 remodeling is observed in cardiac pathologies and is linked with arrhythmogenesis and sudden cardiac death. Utilizing a mouse model of Duchenne muscular dystrophy (DMD^mdx), we have previously demonstrated that cardiac Cx43 is laterally localized, forming undocked hemichannels that activate via S-nitrosylation in response to isoproterenol-evoked cardiac stress. This activation leads to the disruption of cardiac membrane permeability, triggered activity, and deadly arrhythmogenic behaviors. To establish the direct role of S-nitrosylated Cx43 in DMD cardiomyopathy, we developed a specific knock-in mouse line in which the single Cx43 site for S-nitrosylation, cysteine 271 (Cys271), was substituted with a serine (C271S^+/-). Here, we developed a DMD^mdx:C271S^+/- line (4–6 months old), exhibiting reduced levels of S-nitrosylated Cx43 after crossing DMDmdx mice and C271S^+/- mouse lines to assess the effect of β-adrenergic stimulation-induced cardiac stress and heart dysfunction. We show that cardiac Cx43 remodeling was not prevented in DMD^mdx:C271S^+/-, similar to what was shown in DMD^mdx mice via immunofluorescence analysis. In addition, DMD^mdx mice displayed an increased number of deadly arrhythmogenic events, increased Ca²⁺ signaling, and prolonged action potentials in Langendorff-perfused whole hearts via optical mapping, compared to wild-type and DMD^mdx:C271S^+/- mice. Similarly, isoproterenol treatment evoked severe myocardial injury, increased levels of plasmatic cardiac troponin I (cTnI), and 40% mortality in DMD^mdx mice. Notably, DMD^mdx:C271S^+/- mice, similar to DMD^mdx mice treated with the Cx43 hemichannel blocker Gap19, exhibited cardioprotection compared to the cardiac dysfunction observed in DMD^mdx mice. Therefore, these findings strongly suggest that S-nitrosylation of Cx43 proteins at site Cys271 represents a fundamental NO-mediated mechanism involved in the induction of arrhythmias and myocardial injury in DMD^mdx after β-adrenergic stress.