Phosphorylation kinetics of cardiac gap junction regulation during stress

The coordinated contraction of the heart occurs because of the propagation of action potential between the cardiomyocytes. Gap junctions consisting primarily of connexin43 (Cx43) connect cardiomyocytes at regions of contact between cells known as the intercalated disc to facilitate cellular coupling. Cardiac pathologies frequently manifest with disrupted gap junctional intercellular communication which can generate potentially fatal arrhythmias, thus, it is essential to elucidate mechanisms underlying Cx43 regulation and altered intercellular communication. Phosphorylation of residues in the Cx43 carboxyl terminus can alter the subcellular localization, channel gating, and internalization of Cx43. The channel open probability of gap junctions is regulated, in part, by the phosphorylation of S368. Phosphorylation of S365 and S373 have been reported to exert gatekeeper effects on the phosphorylation of S368 and these phosphorylation events further affect protein interactions with 14-3-3 and zonula occludens-1 (ZO-1). While it is established that pS365 creates a conformational change preventing pS368, it is currently unclear precisely how pS373 regulates pS368. Further, it is unclear if alterations to these residues might impact protein binding and pathological cardiac remodeling during stress. Utilizing an ex vivo ischemia model, we find by immunofluorescent confocal microscopy that wildtype Cx43 hearts exhibit significantly decreased Cx43/N-cadherin colocalization during ischemia, while phospho-null mutant hearts retain Cx43/N-cadherin colocalization. Triton X-100 solubility assay indicates S365A/S373A mice have increased junctional Cx43 during ischemia. Additionally, we show that pS368 decay is more rapid in S373A mutants than wildtype suggesting, for the first time, that pS373 may prevent dephosphorylation at Cx43-S368 rather than promote Cx43-pS368. This knowledge could highlight potential therapies for prevention of cardiac remodeling and arrhythmogenesis.