Deficiency of connexin43 increases arrhythmias due to increases in Ca2+ and ROS within the mitochondria

Abstract Introduction Connexin43 (Cx43) is a major connexin that forms gap junctions in the hearts and plays important roles in the occurrence of reentrant arrhythmias. Cx43 also exits as hemichannels in the inner mitochondrial membrane (mCx43), but it has not yet been established whether mCx43 are involved in the occurrence of arrhythmias. Purpose To examine what roles mCx43 plays in the occurrence of arrhythmias and whether mitochondrial Ca2+ and ROS are involved in their occurrence. Methods To generate cardiac-specific Cx43-deficient (Cx43-/-) mice, Cx43flox/flox mice were crossed with α-myosin heavy chain cre+/- mice. The resulting offspring, Cx43-/- mice and their littermates (Cx43+/+ mice) were used. Trabeculae were dissected from right ventricles of mouse hearts. Force was measured with a strain gauge, mitochondrial Ca2+ with rhod-2, and mitochondrial ROS with MitoSox Red. The spatial distribution of rhod-2 and MitoSox Red was imaged using confocal microscopy, and it was similar to that of MitoTracker Green within trabeculae, meaning that rhod-2 and MitoSox Red are mainly loaded within the mitochondria. To determine the amount of ROS production, MitoSox Red fluorescence was measured before (Fl) and after the addition of 10 mM H2O2 (FlH2O2), and the ratio of Fl to FlH2O2 was calculated. To estimate arrhythmia susceptibility, the minimal extracellular Ca2+ concentration (Cao), at which arrhythmias were induced by electrical stimulation, was determined (Caomin). Arrhythmias were induced by electrical stimulation with 0.3-s stimulus intervals for 30 s. Results Most of Cx43-/- mice died within 8 weeks (p<0.01). Body and heart weights and tibial length of Cx43-/- mice were similar to those of Cx43+/+ mice. Cx43 was present in the inner mitochondrial membrane in Cx43+/+ mice but not in Cx43-/- mice. Developed force of Cx43-/- mice was similar to that of Cx43+/+ mice at Cao ranging from 0.2 to 6 mM. Rhod-2 fluorescence in Cx43-/- mice was higher than that of Cx43+/+ mice, especially at higher Cao such as 4 and 6 mM (n=7, p<0.01), and the Caomin in Cx43-/- mice was lower than that in Cx43+/+ mice (n=5, p<0.01), suggesting that Cx43-/- mice shows higher arrhythmia susceptibility with higher mitochondrial Ca2+. In Cx43-/- mice, Ru360 (5 μM), a mitochondrial calcium uniporter inhibitor, increased the Caomin, that is, decreased arrhythmia susceptibility with decreases in rhod-2 and MitoSox Red fluorescence (n=6, p<0.05). Additionally, antioxidant peptide SS-31 (50 µM) and N-acetyl-L-cysteine (1 mM) increased the Caomin in Cx43-/- mice (n=5, p<0.05), and the ratio of Fl to FlH2O2 in Cx43-/- mice was larger that that in Cx43+/+ mice, suggesting that an increase in ROS production plays important roles in the occurrence of arrhythmias in Cx43-/- mice. Conclusions Deficiency of Cx43 increases arrhythmia susceptibility with increases in mitochondrial Ca2+ and ROS, providing an attractive therapeutic target for improving arrhythmias in diseased hearts.