Cx43 hemichannel microdomain signaling at the intercalated disc enhances cardiac excitability
Maarten A.J. De Smet, … , Karin R. Sipido, Luc Leybaert
Maarten A.J. De Smet, … , Karin R. Sipido, Luc Leybaert
Published February 23, 2021
Citation Information: J Clin Invest. 2021;131(7):e137752. https://doi.org/10.1172/JCI137752.
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Research Article Cardiology Cell biology

Cx43 hemichannel microdomain signaling at the intercalated disc enhances cardiac excitability

Abstract

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.

Authors

Maarten A.J. De Smet, Alessio Lissoni, Timur Nezlobinsky, Nan Wang, Eef Dries, Marta Pérez-Hernández, Xianming Lin, Matthew Amoni, Tim Vervliet, Katja Witschas, Eli Rothenberg, Geert Bultynck, Rainer Schulz, Alexander V. Panfilov, Mario Delmar, Karin R. Sipido, Luc Leybaert

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Figure 6

Cx43 hemichannel opening during adrenergic stimulation modulates spontaneous Ca2+ release from the sarcoplasmic reticulum and arrhythmogenic afterdepolarizations.

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Cx43 hemichannel opening during adrenergic stimulation modulates spontan...
(A) Freshly isolated mouse and pig left ventricular cardiomyocytes were subjected to voltage clamp experiments while [Ca2+]i was simultaneously monitored. Top trace shows experimental protocol: cells were paced to steady state for 2 minutes at 1 Hz and then clamped to –70 mV. Middle and bottom traces depict resulting current and [Ca2+]i signals: final 3 paced Ca2+ transients and accompanying currents followed by 15-second rest period showing spontaneous diastolic Ca2+ release with resulting NCX current. Protocols were repeated at 2 Hz with and without isoproterenol. Example traces were recorded in pig cardiomyocytes. (B) In a subset of experiments, we switched to current clamp mode following steady-state pacing in voltage clamp. Example traces, recorded in the same pig cardiomyocyte, without and with TAT-Gap19 were recorded in current clamp mode following 2-minute pacing to steady state at 2 Hz with isoproterenol (in voltage clamp mode). Black arrows indicate DADs, red arrow indicates a triggered action potential. (C) Summary dot plots (nested 1-way ANOVA; N/nmouse = 23/75 for voltage clamp experiments and N/nmouse = 5/45 for current clamp experiments) illustrating increased frequency and amplitude of diastolic Ca2+ release with increased resulting NCX current and membrane depolarization during adrenergic stimulation (2 Hz + ISO) compared with baseline. tAP, triggered action potential. Similar results were obtained in pig cardiomyocytes (not shown). (D) Summary data showing the impact of different interventions on diastolic Ca2+ release and resulting NCX currents and membrane depolarization (nested 1-way ANOVA; N/nmouse = 5–11/15–24 per condition, N/npig = 5/15–20 per condition). Values reported as differences from the control condition.