Mitochondrial connexin43 and mitochondrial K_ATP channels modulate triggered arrhythmias in mouse ventricular muscle

Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial K_ATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43^−/−) mice, Cx43^flox/flox mice were crossed with α-MHC (Myh6)-cre^+/− mice. The resulting offspring, Cx43^flox/flox/Myh6-cre^+/− mice (cCx43^−/− mice) and their littermates (cCx43^+/+ mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca²⁺ concentration ([Ca²⁺]_o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2′,7′-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca²⁺ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca²⁺ with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial K_ATP. Most of cCx43^−/− mice died suddenly within 8 weeks. Cx43 was present in the inner mitochondrial membrane in cCx43^+/+ mice but not in cCx43^−/− mice. In cCx43^−/− mice, the [Ca²⁺]_o,min was lower, and Ca²⁺ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43^−/− mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43^−/− mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca²⁺]_o,min. These results suggest that Cx43 deficiency activates Ca²⁺ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca²⁺, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial K_ATP channels in mouse hearts.