Abstract Introduction Connexin43 (Cx43) is a connexin that forms gap junctions in the hearts and also exits as hemichannels in the inner mitochondrial membrane (mCx43). In our previous study, deficiency of Cx43 increased mitochondrial Ca2+ (mCa2+). It has not yet been established, however, how mCx43 are involved in arrhythmogenesis. Purpose To examine how mCa2+ changes with deficiency of Cx43 and what roles mitochondrial KATP (mKATP) channels play in the changes in mCa2+ and arrhythmogenesis. Methods Cx43flox/flox mice were crossed with α-myosin heavy chain cre+/- mice, and cardiac-specific Cx43-deficient (Cx43-/-) mice were generated. The resulting offspring, Cx43-/- mice and their littermates (Cx43+/+ mice) were used. Trabeculae were dissected from their right ventricles, and force was measured with a strain gauge. To estimate arrhythmogenesis, the minimal extracellular Ca2+ (Cao), at which arrhythmias were induced by electrical stimulation (0.3-s stimulus intervals for 30 s), was determined (Caomin). In digitonin-skinned trabeculae, mCa2+ was estimated using rhod-2, and mitochondrial membrane potential using JC-1 fluorescence ratio (Red/Green). The spatial distribution of rhod-2 and JC-1 was similar to that of MitoTracker Green, meaning that rhod-2 and JC-1 are mainly loaded within the mitochondria. To investigate the roles of mKATP channels and mCx43, 100 µM diazoxide (DZX), a mKATP channel opener, 200 μM 5-hydroxydecanoic acid (5-HD), a mKATP channel inhibitor, and 5 μM carbenoxolone, a Cx43 inhibitor, were used. Results Cx43 was present in the inner mitochondrial membrane in Cx43+/+ mice but not in Cx43-/- mice. Most of Cx43-/- mice died within 8 weeks (p<0.01). The Cao min in Cx43-/- mice was lower than that in Cx43+/+ mice (n=5, p<0.01) and was increased by DZX (n=4, p<0.05), suggesting that increased arrhythmogenesis in Cx43-/- mice is attenuated by DZX. Rhod-2 fluorescence in Cx43-/- mice was higher than that in Cx43+/+ mice (n=7, p<0.01) and was decreased by DZX at Cao from 0.2 to 6 mM (n=7, p<0.05, 2-way ANOVA). Besides, in digitonin-skinned trabeculae, rhod-2 fluorescence in Cx43-/- mice was higher than that in Cx43+/+ mice (n=5, p<0.05) and was decreased by DZX at Ca2+ from 80 to 2700 nM (n=5, p<0.01, 2-way ANOVA). Conversely, rhod-2 fluorescence in Cx43+/+ mice was increased by CBX and 5-HD in skinned trabeculae at Ca2+ from 80 to 2700 nM (n=7, p<0.05, 2-way ANOVA). In skinned trabeculae, JC-1 fluorescence ratio was increased only in Cx43-/- mice depending on the increase in Ca2+ from 80 to 2700 nM (n=5, p<0.01), and this increase was suppressed by DZX, meaning that mitochondrial membrane potential is hyperpolarized with deficiency of mCx43, and that this hyperpolarization is reversed with activation of mKATP channels. Conclusions In the myocardium with deficiency of Cx43, increased arrhythmogenesis is attenuated by activation of mKATP channels probably through a decrease in mCa2+ and normalization of mitochondrial membrane potential.