Mitochondrial connexin43 and mitochondrial KATP 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 KATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43-/-) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/- mice. The resulting offspring, Cx43flox/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 KATP. 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 KATP channels in mouse hearts.

Effect of transient elevation of glucose on contractile properties in non-diabetic rat cardiac muscle.

In non-diabetic patients with severe disease, such as acute myocardial infarction or acute heart failure, admission blood glucose level is associated with their short-term and long-term mortality. We examined whether transient elevation of glucose affects contractile properties in non-diabetic hearts. Force, intracellular Ca2+ ([Ca2+]i), and sarcomere length were measured in trabeculae from rat hearts. To assess contractile properties, maximum velocity of contraction (Max dF/dt) and minimum velocity of relaxation (Min dF/dt) were calculated. The ratio of phosphorylated troponin I (P-TnI) to troponin I (TnI) was measured. One hour after elevation of glucose from 150 to 400 mg/dL, developed force, Max dF/dt, and Min dF/dt were reduced without changes in [Ca2+]i transients at 2.5 Hz stimulation and 2.0 mM [Ca2+]o, while developed force and [Ca2+]i transients showed no changes at 0.5 Hz stimulation and 0.7 mM [Ca2+]o. In the presence of 1 µM KN-93, a Ca2+/calmodulin-dependent protein kinaseII (CaMKII) inhibitor, or 50 µM diazo-5-oxonorleucine, a L-glutamine-D-fructose-6-phosphate amidotransferase inhibitor, the reduction of contractile properties after elevation of glucose was suppressed. Furthermore, 1 h after elevation of glucose to 400 mg/dL at 2.0 mM [Ca2+]o, the ratio of P-TnI to TnI was increased. These results suggest that in non-diabetic hearts under higher Ca2+-load, transient elevation of glucose for 1 h reduces contractile properties probably by activating CaMKII through O-GlcNAcylation. Thus, in the patients with severe disease, transient elevation of blood glucose, such as due to stress, may worsen cardiac function and thereby affect their mortality without known diabetes.

Transient Elevation of Glucose Increases Arrhythmia Susceptibility in Non-Diabetic Rat Trabeculae With Non-Uniform Contraction.

BACKGROUND: In non-diabetic patients with acute coronary syndrome, stress hyperglycemia occasionally occurs and is related to their mortality. Whether transient elevation of glucose affects arrhythmia susceptibility in non-diabetic hearts with non-uniform contraction was examined.Methods and Results:Force, intracellular Ca2+([Ca2+]i), and membrane potential were measured in trabeculae from rat hearts. Non-uniform contraction was produced by a jet of paralyzing solution. Ca2+waves and arrhythmias were induced by electrical stimulation (2.0 mmol/L [Ca2+]o). The activity of Ca2+/calmodulin-dependent protein kinaseII (CaMKII) was measured. An elevation of glucose from 150 to 400 mg/dL increased the velocity of Ca2+waves and the number of spontaneous action potentials triggered by electrical stimulation. Besides, the elevation of glucose increased the CaMKII activity. In the presence of 1 µmol/L KN-93, the elevation of glucose did not increase the velocity of Ca2+waves and the number of triggered action potentials. In addition, in the presence of 1 µmol/L autocamtide-2 related inhibitory peptide or 50 µmol/L diazo-5-oxonorleucine, the elevation of glucose did not increase the number of triggered action potentials. Furthermore, the elevation of glucose by adding L-glucose did not increase their number. CONCLUSIONS: In non-diabetic hearts with non-uniform contraction, transient elevation of glucose increases the velocity of Ca2+waves by activating CaMKII,probably through glycosylation with O-linked ß-N-acetylglucosamine, thereby increasing arrhythmia susceptibility.

Regional increase in ROS within stretched region exacerbates arrhythmias in rat trabeculae with nonuniform contraction.

In diseased hearts, impaired muscle within the hearts is passively stretched by contractions of the more viable neighboring muscle during the contraction phase. We investigated whether in the myocardium with nonuniform contraction such passive stretch regionally generates ROS within the stretched region and exacerbates arrhythmias. In trabeculae from rat hearts, force, intracellular Ca2+, and membrane potential were measured. To assess regional ROS generation, the slope of the change in the 2',7'-dichlorofluorescein fluorescence (DCFslope) was calculated at the each pixel position along the long axis of trabeculae using DCF fluorescence images. Ca2+ waves and arrhythmias were induced by electrical stimulation. A H2O2 (1 mmol/L) jet regionally increased the DCFslope within the jet-exposed region. A blebbistatin (10 µmol/L) jet caused passive stretch of the muscle within the jet-exposed region during the contraction phase and increased the DCFslope within the stretched region, the velocity of Ca2+ waves, and the number of beats after electrical stimulation (0.2 µmol/L isoproterenol), while 3 µmol/L diphenyleneiodonium (DPI), NADPH oxidase inhibitor, decreased them. A jet of a solution containing 0.2 mmol/L H2O2 in addition to 10 µmol/L blebbistatin also increased them. A H2O2 jet within the region where Ca2+ waves propagated increased their velocity. In the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally increases ROS within the stretched region, and the regional ROS exacerbates arrhythmias by activating the propagation of Ca2+ waves.