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 4

Cx43 colocalizes with large dyadic RyR2 superclusters and forms microdomains at the perinexus.

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Cx43 colocalizes with large dyadic RyR2 superclusters and forms microdom...
(A) 2D SMLM images of a murine cardiomyocyte (top) and cardiomyocyte cell pair (bottom), triple stained for Cx43 (red), RyR2 (green), and Cav1.2 (blue). Scale bar: 10 μm. (B) Straightened region of interest (from yellow boxes in A) of Cx43, RyR2, and Cav1.2 at different subcellular domains. Scale bar: 2 μm. (C) Heatmap of RyR2 cluster density, number of molecules, and colocalization with Cx43 at different subcellular domains (n = 5, n = 42 single cardiomyocytes, 16 cardiomyocyte cell pairs). RyR2 clusters were classified as dyadic or extradyadic based on the proximity of Cav1.2 clusters, RyR2 clusters occurring less than 250 nm from a Cav1.2 cluster were categorized as dyadic. (D) Heatmap of RyR2 supercluster abundance, size, and colocalization with Cx43 at different subcellular domains (n = 5, n = 42 single cardiomyocytes, 16 cardiomyocyte cell pairs). (E) Relative localization overview in left ventricular mouse cardiomyocyte cell pairs. Dyadic Cav1.2 clusters were categorized as perinexal or distant based on edge distance 200 nm or less or greater than 200 nm from edge of Cx43 cluster, respectively (n = 5, n = 16 cardiomyocyte cell pairs). (F) EM images of an SR cistern forming a dyadic cleft at the perinexus in mouse ventricular myocardium. Left image shows an EM overview of a murine ventricular intercalated disc. Scale bar: 500 nm. White box is enlarged on the right. Arrows indicate electron dense particles, likely ryanodine receptors. Scale bars: 100 nm. Pn, perinexus.