Cx43 hemichannel microdomain signaling at the intercalated disc enhances cardiac excitability
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
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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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 5

Discrete sites of Cx43 hemichannel activation at the intercalated disc during Ca2+ release from the sarcoplasmic reticulum.

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Discrete sites of Cx43 hemichannel activation at the intercalated disc d...
(A) Transmitted light images of a single cardiomyocyte (top) and cardiomyocyte cell pair (bottom). Triangle, square, and circle symbols indicate cell-attached macropatch (Rp = ~2 MΩ, ~2 μm pipette inner diameter) recording positions at the lateral membrane and cell end of single cardiomyocytes and intercalated disc of cardiomyocyte cell pairs, respectively. (B) Example traces showing single channel currents recorded at the lateral membrane, cell end, or intercalated disc. Traces recorded in mouse cardiomyocytes during caffeine superfusion (10 mM, 8 seconds) at indicated membrane potentials. (C) IV plots depicting linear current-voltage relationship with slope conductance of approximately 220 pS and Erev ≈ 0 mV (N/nmouse = 5/10–18 patches per condition, N/npig = 5/15–20 patches per condition). (D) Summary dot plots and transition histograms indicate recording of approximately 220 pS single channel currents at the cell end of single cardiomyocytes, but not at the lateral membrane, with significantly increased open probability at the intercalated disc of cardiomyocyte cell pairs. Comparative statistics with nested 1-way ANOVA. Heatmap summarizes single channel open probability at the cell end or at the intercalated disc under conditions of Cx43 knockdown or in the presence of TAT-Gap19, inactive TAT-Gap19I130A, TAT-CT9, or 10Panx1 (N/nmouse = 5/10–18 patches per condition, N/npig = 5/15–20 patches per condition). (E) SICM-generated membrane topology of the cell end of a mouse left ventricular cardiomyocyte. Pipette indicates the recording position distally of the last Z-line. (F) Example trace recorded at –70 mV during caffeine superfusion. (G) Transition histogram from all experiments (n = 5, n = 35) showing a fully open state at approximately 220 pS and a substate at approximately 110 pS.