Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial Kchannels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43) mice, Cx43mice were crossed with α-MHC (Myh6)-cremice. The resulting offspring, Cx43/Myh6-cremice (cCx43mice) and their littermates (cCx43mice), 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 Caconcentration ([Ca]), 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 Caspark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Cawith rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial K. Most of cCx43mice died suddenly within 8 weeks. Cx43 was present in the inner mitochondrial membrane in cCx43mice but not in cCx43mice. In cCx43mice, the [Ca]was lower, and Caspark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca]. These results suggest that Cx43 deficiency activates Caleak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial Kchannels in mouse hearts.