Role of S-nitrosylated Cx43 in normal cardiac contractility

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Modulation of cardiac function by sympathetic activity, which occurs during exercise and emotional excitement, is thought to promote an augmentation of cell-to-cell electrical coupling in the heart. This is necessary to increase the strength of cardiac muscle contraction. Electrical coupling in the heart is mediated by specialized structures at the intercalated discs of cardiomyocytes, referred to as gap junction channels (GJCs). These are intercellular channels formed by connexin proteins, with connexin 43 (Cx43) being the most abundant in the heart. While is known that Cx43 GJCs are vital to sustain the propagation of electrical impulses and coordinated heart contraction, the mechanisms by which cardiac GJC coupling is regulated remains poorly understood. It has recently been proposed that β-adrenergic stimulation, which is used to mimic sympathetic activity, induces nitric oxide (NO) production and S-nitrosylation of cardiac proteins and this may account for one third of the inotropic effects in the heart. In this context, we found Cx43 at the intercalated disk is heavily S-nitrosylated upon β-adrenergic stimulation. In addition, we found that NO increases coupling mediated by Cx43 GJCs in a heterologous expression system. Accordingly, we hypothesize that S-nitrosylated Cx43 GJCs enhances electrical coupling between cardiomyocytes, which is critical for concerted contractility. To test our hypothesis, we identified the S-nitrosylated site and created a knockin mouse line where this site (C271) was replaced by a serine, and which we will use to assess cardiac function in vivo. In addition, we will perform biophysical studies to unveil the molecular mechanisms by which NO induce increased GJC coupling in the cardiomyocytes. The proposed research is innovative, as it is designed to reveal novel biophysical properties of S-nitrosylated Cx43 GJCs and provide mechanistic understanding on cardiac conduction and contractility.