Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca²⁺ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic Ca²⁺ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel Ca²⁺ entry and coupling to Ca²⁺ release microdomains at the intercalated disc, resulting in enhanced Ca²⁺ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca²⁺ release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure.

Connexins are crucial cardiac proteins that form hemichannels and gap junctions. Gap junctions are responsible for the propagation of electrical and chemical signals between myocardial cells and cells of the specialized conduction system in order to synchronize the cardiac cycle and steer cardiac pump function. Gap junctions are normally open, while hemichannels are closed, but pathological circumstances may close gap junctions and open hemichannels, thereby perturbing cardiac function and homeostasis. Current evidence demonstrates an emerging role of hemichannels in myocardial ischemia and arrhythmia, and tools are now available to selectively inhibit hemichannels without inhibiting gap junctions as well as to stimulate hemichannel incorporation into gap junctions. We review available experimental evidence for hemichannel contributions to cellular pro-arrhythmic events in ventricular and atrial cardiomyocytes, and link these to insights at the level of molecular control of connexin-43–based hemichannel opening. We conclude that a double-edged approach of both preventing hemichannel opening and preserving gap junctional function will be key for further research and development of new connexin-based experimental approaches for treating heart disease.

Connexin-based gap junctions are crucial for electrical communication in the heart; they are each composed of two docked hemichannels, supplied as unpaired channels via the sarcolemma. When open, an unpaired hemichannel forms a large pore, high-conductance and Ca²⁺-permeable membrane shunt pathway that may disturb cardiomyocyte function. Hemichannels composed of connexin43, a major cardiac connexin, can be opened by electrical stimulation but only by very positive membrane potentials. Here, we investigated the activation of connexin43 hemichannels in murine ventricular cardiomyocytes voltage-clamped at -70 mV. Using whole-cell patch clamp, Co-IP, 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 connexin43 knock-down. Recordings under Ca²⁺-clamp conditions showed that both RyR activation and intracellular Ca²⁺ elevation were necessary for hemichannel opening. Proximity ligation studies indicated close connexin43-RyR2 apposition (<40 nm), and both proteins co-immunoprecipitated indicating physical interaction. Molecular modeling suggested a strongly conserved RyR-mimicking peptide sequence (RyRHCIp), which inhibited RyR/Ca²⁺ hemichannel activation but not voltage-triggered activation. The peptide also slowed down action potential repolarization. Our results demonstrate that connexin43 hemichannels are intimately linked to RyRs, allowing them to open at negative diastolic membrane potential in response to RyR activation. We here provide detailed evidence that Cx43 hemichannels can be opened by concomitant RyR activation and [Ca²⁺]_i elevation, two fundamental players in cardiomyocyte excitation-contraction coupling. Recent work in a plakophillin-2 deficient mouse model for ARVC indicated RyR2-linked Ca²⁺ dysregulations that were suppressed by the Cx43 hemichannel inhibitor Gap19. Our present work delineates the necessary conditions to activate Cx43 hemichannels in RyR2- and Ca²⁺-dependent manners and provides a new peptide tool, RyRHCIp, that inhibits hemichannel opening by acting at this crucial RyR-hemichannel axis.

Cx43 hemichannels (HCs) are electrically and chemically gated transmembrane pores with low open probability and multiple conductance states, which makes kinetic studies of channel gating in large datasets challenging. Here, we developed open access software, named HemiGUI, to analyze HC gating transitions and investigated voltage-induced HC opening based on up to ≈4000 events recorded in HeLa-Cx43-overexpressing cells. We performed a detailed characterization of Cx43 HC gating profiles and specifically focused on the role of the C-terminal tail (CT) domain by recording the impact of adding an EGFP tag to the Cx43 CT end (Cx43-EGFP) or by supplying the Cx43 HC-inhibiting peptide Gap19 that interferes with CT interaction with the cytoplasmic loop (CL). We found that Gap19 not only decreased HC opening activity to the open state (≈217 pS) but also increased the propensity of subconductance (≈80 pS) transitions that additionally became slower as compared to the control. The work demonstrates that large sample transition analysis allows detailed investigations on Cx43 HC gating and shows that Gap19 acts as a HC gating modifier by interacting with the CT that forms a crucial gating element.

Cx43 hemichannels (HCs) and Panx1 channels are two genetically distant protein families. Despite the lack of sequence homology, Cx43 and Panx1 channels have been the subject of debate due to their overlapping expression and the fact that both channels present similarities in terms of their membrane topology and electrical properties. Using the mimetic peptides Gap19 and ¹⁰Panx1, this study aimed to investigate the cross-effects of these peptides on Cx43 HCs and Panx1 channels. The single-channel current activity from stably expressing HeLa-Cx43 and C6-Panx1 cells was recorded using patch-clamp experiments in whole-cell voltage-clamp mode, demonstrating 214 pS and 68 pS average unitary conductances for the respective channels. Gap19 was applied intracellularly while ¹⁰Panx1 was applied extracellularly at different concentrations (100, 200 and 500 μM) and the average nominal open probability (NP_o) was determined for each testing condition. A concentration of 100 µM Gap19 more than halved the NP_o of Cx43 HCs, while 200 µM ¹⁰Panx1 was necessary to obtain a half-maximal NP_o reduction in the Panx1 channels. Gap19 started to significantly inhibit the Panx1 channels at 500 µM, reducing the NP_o by 26% while reducing the NP_o of the Cx43 HCs by 84%. In contrast ¹⁰Panx1 significantly reduced the NP_o of the Cx43 HCs by 37% at 100 µM and by 83% at 200 µM, a concentration that caused the half-maximal inhibition of the Panx1 channels. These results demonstrate that ¹⁰Panx1 inhibits Cx43 HCs over the 100–500 µM concentration range while 500 µM intracellular Gap19 is necessary to observe some inhibition of Panx1 channels.