Anchored-PKA regulates connexin-43 gap junction communication in the heart

Abstract

Cardiac connexin-43 (Cx43) forms gap junction (GJ) channels at the intercalated discs (ID) of cardiomyocytes ensuring anisotropic action potential (AP) propagation. This process is essential for sequential and coordinated contraction of the heart. Stimulation of β-adrenergic receptors activates protein kinase A (PKA), which in turn increases electrical conduction velocity. Myocardial infarction (MI) is characterized by a prolonged ischemia leading to irreversible ventricular cardiomyocytes death and impaired cardiac function. To compensate this loss, chronic activation of PKA signaling is established. In addition, post-MI, Cx43 subcellular localization is altered affecting GJ communication and AP propagation leading to uncoordinated contractions and fatal arrhythmias. Considering the key role of Cx43 and PKA signaling in AP propagation, we investigated the potential regulation of GJ communication by PKA under cardiac physiological condition and post-MI. First, we assessed Cx43 phosphorylation with PKA specific inhibition and with a peptide displacing PKA anchoring from A-Kinase Anchoring Protein (AKAPs). Then, we identified the ezrin as the AKAP mediated the regulation of Cx43 phosphorylation by immunoprecipitation, immunofluorescence and siRNA mediated knockdown experiments. We assessed by functional GAP-FLIP experiments the key role of ezrin-PKA complex in the regulation of intercellular communication. Finally, we generated a left anterior descending coronary artery model of MI in rats to evaluate by proximity ligation assay and western blot experiments the Cx43-ezrin-PKA complex localization and expression. We point that ezrin forms a supramolecular complex with PKA and Cx43 at the ID of cardiomyocytes. Ezrin regulates Cx43 phosphorylation and GJ communication in cardiomyocytes. Interestingly, post-MI we noticed a significant reduction in Cx43 level and phosphorylation associated with a decrease in proximity with ezrin at the ID. Our data identified a cardiac PKA-ezrin-Cx43 complex regulating Cx43 phosphorylation, and therefore, GJ communication. In addition, we reported a loss of proximity between ezrin and Cx43 post-MI. We proposed that this may explain partly an impaired AP propagation leading to uncoordinated contractions post MI.