RyR2 regulates Cx43 hemichannel intracellular Ca2+-dependent activation in cardiomyocytes

Connexin-based gap junctions are crucial for electrical communication in the heart; they are each composed of two docked hemichannels (HCs), supplied as unpaired channels via the sarcolemma. When open, an unpaired HC forms a large pore, high-conductance and Ca2+-permeable membrane shunt pathway that may disturb cardiomyocyte function. HCs composed of connexin 43 (Cx43), a major cardiac connexin, can be opened by electrical stimulation but only by very positive membrane potentials. Here, we investigated the activation of Cx43 HCs in murine ventricular cardiomyocytes voltage-clamped at -70 mV.Using whole-cell patch-clamp, co-immunoprecipitation, western blot analysis, immunocytochemistry, proximity ligation assays, and protein docking studies, we found that stimulation of ryanodine receptors (RyRs) triggered unitary currents with a single-channel conductance of ∼220 pS, which were strongly reduced by Cx43 knockdown. Recordings under Ca2+-clamp conditions showed that both RyR activation and intracellular Ca2+ elevation were necessary for HC opening. Proximity ligation studies indicated close Cx43-RyR2 apposition (<40 nm), and both proteins co-immunoprecipitated indicating physical interaction. Molecular modelling suggested a strongly conserved RyR-mimicking peptide sequence (RyRHCIp), which inhibited RyR/Ca2+ HC activation but not voltage-triggered activation. The peptide also slowed down action potential repolarization. Interestingly, alterations in the concerned RyR sequence are known to be associated with primary familial hypertrophic cardiomyopathy.Our results demonstrate that Cx43 HCs are intimately linked to RyRs, allowing them to open at negative diastolic membrane potential in response to RyR activation.