Rationale Post-translational phosphorylation of connexin43 (Cx43) has been proposed as a key regulatory event in normal cardiac gap junction expression and pathologic gap junction remodeling. Nonetheless, the role of Cx43 phosphorylation in the context of the intact organism is poorly understood. Objective To establish whether specific connexin43 phosphorylation events influence gap junction expression and pathologic remodeling. Methods and Results We generated Cx43 germline knock-in mice in which serines 325/328/330 were replaced with phosphomimetic glutamic acids (S3E) or non-phosphorylatable alanines (S3A). The S3E mice were resistant to acute and chronic pathologic gap junction remodeling (GJR) and displayed diminished susceptibility to the induction of ventricular arrhythmias. Conversely, the S3A mice showed deleterious effects on cardiac gap junction formation and function, developed electrical remodeling and were highly susceptible to inducible arrhythmias. Conclusions These data demonstrate a mechanistic link between post-translational phosphorylation of Cx43 and gap junction formation, remodeling and arrhythmic susceptibility.

Loss of connexin43 (Cx43) gap junction channels in the heart results in a marked increase in the incidence of spontaneous and inducible polymorphic ventricular tachyarrhythmias (PVTs). The mechanisms resulting in this phenotype remain unclear. We hypothesized that uncoupling promotes regional ion channel remodeling, thereby increasing electrical heterogeneity and facilitating the development of PVT. In isolated-perfused control hearts, programmed electrical stimulation elicited infrequent monomorphic ventricular tachyarrhythmias (MVT), and dominant frequencies (DFs) during MVT were similar in the right ventricle (RV) and left ventricle (LV). Moreover, conduction properties, action potential durations (APDs), and repolarizing current densities were similar in RV and LV myocytes. In contrast, PVT was common in Cx43 conditional knockout (OCKO) hearts, and arrhythmias were characterized by significantly higher DFs in the RV compared to the LV. APDs in OCKO myocytes were significantly shorter than those from chamber-matched controls, with RV OCKO myocytes being most affected. APD shortening was associated with higher levels of sustained current in myocytes from both chambers as well as higher levels of the inward rectifier current only in RV myocytes. Thus, alterations in cell-cell coupling lead to regional changes in potassium current expression, which in this case facilitates the development of reentrant arrhythmias. We propose a new mechanistic link between electrical uncoupling and ion channel remodeling. These findings may be relevant not only in cardiac tissue but also to other organ systems where gap junction remodeling is known to occur.

Abstract-Impulse propagation in cardiac tissue is a complex process in which intercellular coupling through gap junction channels is a critical component. Connexin40 (Cx40) is an abundant gap junction protein that is expressed in atrial myocytes. Alterations in the expression of Cx40 have been implicated in atrial arrhythmogenesis. The purpose of the current study was to assess the role of Cx40 in atrial impulse propagation. High-resolution optical mapping was used to study conduction in the right and left atrial appendages of isolated Langendorff-perfused murine hearts. Wild-type (Cx40 ϩ/ϩ ), heterozygous (Cx40 ϩ/Ϫ ), and knockout (Cx40 Ϫ/Ϫ ) mice, both adult and embryonic, were studied to assess the effects of reduced Cx40 expression on sinus node function and conduction velocity at different pacing cycle lengths (100 and 60 ms). In both adult and late-stage embryonic Cx40 ϩ/ϩ mice, heterogeneity in CV was found between the right and left atrial appendages. Either partial (Cx40 ϩ/Ϫ ) or complete (Cx40 Ϫ/Ϫ ) deletion of Cx40 was associated with the loss of conduction heterogeneity in both adult and embryonic mice. Additionally, sinus node impulse initiation was found to be ectopic in Cx40 Ϫ/Ϫ mice at 15.5 days postcoitus, whereas Cx40 ϩ/ϩ mice showed normal activation occurring near the crista terminalis. Our findings suggest that Cx40 plays an essential role in establishing interatrial conduction velocity heterogeneity in the murine model. Additionally, we describe for the first time a developmental requirement for Cx40 in normal sinus node impulse initiation at 15. Key Words: arrhythmia Ⅲ conduction velocity Ⅲ Cx40 Ⅲ optical mapping Ⅲ sinus node C ardiac myocytes are connected electrically and metabolically through gap junction channels. 1 A complete channel is composed of 2 opposing hexameric structures or connexons, each consisting of 6 protein subunits called connexins. 2 At least 3 connexins are expressed in cardiac myocytes: connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45), each forming channels with unique electrophysiological and biophysical properties. 3,4 Within the adult heart, connexin isoforms have distinct patterns of expression, which are generally well preserved among different species. Cx40 is expressed predominantly in atrial myocytes and in the ventricular conduction system; Cx43 is expressed in both atrial and ventricular myocytes; Cx45 is expressed mainly in the sinoatrial node, anteroventral node, and in the ventricular conduction system, with lower levels reported in the atrial myocardium.Alterations in the expression of connexin proteins have been described in a variety of pathological conditions in both animal 5 and human models. 6,7 However, most studies that have investigated the importance of connexins have focused on ventricular conduction, whereas relatively few studies are available directly addressing their role in mediating atrial impulse initiation and propagation. Studies investigating the role of Cx40 in atrial arrhythmogenesis have found highly variable levels of...