Diazoxide Needs Mitochondrial Connexin43 to Exert Its Cytoprotective Effect in a Cellular Model of CoCl2-Induced Hypoxia

Hypoxia is the leading cause of death in cardiomyocytes. Cells respond to oxygen deprivation by activating cytoprotective programs, such as mitochondrial connexin43 (mCx43) overexpression and the opening of mitochondrial KATP channels, aimed to reduce mitochondrial dysfunction. In this study we used an in vitro model of CoCl2-induced hypoxia to demonstrate that mCx43 and KATP channels cooperate to induce cytoprotection. CoCl2 administration induces apoptosis in H9c2 cells by increasing mitochondrial ROS production, intracellular and mitochondrial calcium overload and by inducing mitochondrial membrane depolarization. Diazoxide, an opener of KATP channels, reduces all these deleterious effects of CoCl2 only in the presence of mCx43. In fact, our results demonstrate that in the presence of radicicol, an inhibitor of Cx43 translocation to mitochondria, the cytoprotective effects of diazoxide disappear. In conclusion, these data confirm that there exists a close functional link between mCx43 and KATP channels.

Mitochondrial KATP channels stabilize intracellular Ca2+ during hypoxia in retinal horizontal cells of goldfish (Carassius auratus)

Neurons of the retina require oxygen to survive. In hypoxia, neuronal ATP production is impaired, ATP-dependent ion pumping is reduced, transmembrane ion gradients are dysregulated, and [Ca2+]i increases enough to trigger excitotoxic cell death. Central neurons of the common goldfish (Carassius auratus) are hypoxia-tolerant, but little is known about how goldfish retinas withstand hypoxia. To study the cellular mechanisms of hypoxia tolerance, we isolated retinal interneurons (horizontal cells; HCs), and measured intracellular Ca2+ concentration ([Ca2+]i) with Fura-2. Goldfish HCs maintained [Ca2+]i throughout 1 h of hypoxia, whereas [Ca2+]i increased irreversibly in HCs of the hypoxia-sensitive rainbow trout (Oncorhynchus mykiss) with just 20 min of hypoxia. Our results suggest mitochondrial ATP-dependent K+ channels (mKATP) are necessary to stabilize [Ca2+]i throughout hypoxia. In goldfish HCs, [Ca2+]i increased when mKATP was blocked with glibenclamide or 5-HD, whereas an mKATP agonist (diazoxide) prevented [Ca2+]i from increasing in hypoxia in trout HCs. We showed that hypoxia protects goldfish HCs via mKATP channels. Glycolytic inhibition with 2-deoxyglucose increased [Ca2+]i, which was rescued by hypoxia in an mKATP-dependent manner. We found no evidence of plasmalemmal KATP channels in patch-clamp experiments. Instead, we confirmed the involvement of KATP in mitochondria with TMRE imaging, as hypoxia rapidly (<5 min) depolarized mitochondria in an mKATP-sensitive manner. We conclude that mKATP channels initiate a neuroprotective pathway in goldfish HCs to maintain [Ca2+]i and avoid excitotoxicity in hypoxia. This model provides novel insight into the cellular mechanisms of hypoxia tolerance in the retina.

Mitochondrial connexin43 affects arrhythmogenesis with modulation of mitochondrial KATP channels

Introduction Connexin43 (Cx43) forms gap junction channels in ventricular muscle and also exits in the inner mitochondrial membrane (mCx43). We have previously reported that carbenoxolone, a blocker of Cx43, increased the occurrence of arrhythmias with modulation of mitochondrial KATP channels (mKATP). Purpose To examine how mCx43 and mKATP affect arrhythmogenesis, using cardiac-specific Cx43-deficient (Cx43−/−) mice. Methods To generate cardiac-specific Cx43−/− mice, Cx43flox/flox mice were crossed with α-myosin heavy chain (Myh6)-cre+/− mice. The resulting offspring, Cx43flox/flox/ Myh6-cre+/− mice (Cx43−/− mice) and their littermates (Cx43+/+ mice), were used. Trabeculae were dissected from right ventricles of mouse hearts (length ∼0.9 mm, width ∼200 μm, thickness ∼80 μm). Force was measured with a strain gauge, and intracellular Ca2+ (Cai) with fura-2 and a CCD camera (22°C). To assess arrhythmogenesis, the minimal extracellular Ca2+ concentration (Caomin), at which arrhythmias were induced by electrical stimulation (0.3-s stimulus intervals, 100 nM isoproterenol), was determined in mouse trabeculae. Using isolated single ventricular myocytes, mitochondrial membrane potential (ΔΨm) was estimated with tetramethylrhodamine methylester (TMRM) fluorescence, ROS production was estimated with 2',7'-dichlorofluorescein (DCF) fluorescence, and Ca2+ spark frequency was measured with fluo-4 and confocal microscopy. To investigate the roles of mKATP, 0.1 mM diazoxide, a mKATP channel opener, and 0.2 mM 5-hydroxydecanoic acid (5-HD), a mKATP channel inhibitor, were used. Results Most of Cx43−/− mice suddenly died within 8 weeks (p<0.01). Cx43 was present in the inner mitochondrial membrane in Cx43+/+ mice, while it was not present in Cx43−/− mice. The resting and developed force and Cai transients by electrical stimulation (2-s stimulus intervals) showed no difference between Cx43−/− and Cx43+/+ mouse trabeculae (n=7). The Caomin in Cx43−/− mice was lower than that in Cx43+/+ mice (p<0.01), and diazoxide increased the Caomin in Cx43−/− mice (p<0.01), suggesting that arrhythmogenesis is increased in Cx43−/− mice and is decreased by diazoxide. Ca2+ spark frequency and DCF oxidation rate in Cx43−/− mice were higher than those in Cx43+/+ mice (p<0.01). They were decreased by diazoxide and were increased by 5-HD (p<0.01). TMRM fluorescence was decreased after electrical stimulation (1-s stimulus intervals) in Cx43−/− mice and was further decreased by 5-HD (p<0.01), suggesting depolarization of ΔΨm in Cx43−/− mice. Such decrease in TMRM fluorescence in Cx43−/− mice was suppressed by diazoxide and did not occur in Cx43+/+ mice. Conclusions These results suggest that with modulation of mKATP, mCx43 plays important roles in ΔΨm determination, ROS production, and Ca2+ leak from the sarcoplasmic reticulum, thereby affecting arrhythmogenesis. Therefore, it is likely that mCx43 and mKATP could become a therapeutic target for controlling arrythmias. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science

Abstract 287: 5-Hydroxydecanoate, a Mitochondrial Selective KATP Antagonist, Blocks the Rescue Action of Lipid Emulsion in Bupivacaine-Induced Cardiotoxicity

Introduction: Lipid emulsion (intralipid) rescues Bupivacaine-induced cardiac toxicity. We have shown previously that glibenclamide, a non selective ATP potassium channel blocker abolishes the rescue action of intralipid. Here we explored the role of 5-Hydroxydecanoate (5-HD), a mitochondrial selective KATP antagonist in the intralipid-mediated rescue of bupivacaine overdose. Methods: Young male Sprague-Dawley rats (300-350 g) were anesthetized intraperitoneally with a mixture of ketamine (80 mg/kg) and xylazine (8 mg/kg), and ventilated with a ventilator. M-mode echocardiography were acquired throughout the experiment. In protocol-1 (n=5), asystole was induced by a single injection of bupivacaine (10mg/kg over 20 seconds, intravenously) and resuscitation with Intralipid 20% (5ml/kg bolus, and 0.5ml/kg/min maintenance) with chest compression was started immediately. In protocol-2 (n=8) rats were pre-treated with 5-HD (5 mg/kg, IV) 30 minutes prior to inducing asystole with bupivacaine. In both protocols the heart rate (HR), ejection fraction (EF) and fractional shortening (FS) were measured before inducing asystole (baseline), and at 1, 5 and 10 min after lipid treatment. A two way repeated measure analysis of variance (ANOVA) model was used for comparison of means. Values are mean ± SEM, n=5 rats/group, p<0.05 significant. Results: In protocol-1, the baseline HR and EF were 321±21 and 72.3±4.6%. Bupivacaine resulted in asystole and intralipid therapy improved the heart rate and cardiac function within 10 min(HR=86±13beats/min at 1min, 216±10beats/min at 5 min, and 228±14beats/min at 10 min). The left ventricular systolic function fully recovered in all rats within 5 min of intralipid treatment (EF=72±5%, FS=42±4%). In protocol-2, there were no significant differences between HR and EF before (HR=324.6±16.5 beats/min, EF=78.0±2.8%) and 30 min after (HR=340.9±15.6 beats/min, EF=83.3±2.0%) 5-HD adminstration. However, 5-HD (5mg/kg), prevented the intralipid-induced rescue of bupivacaine overdose, as there was no recovery within 10 min of intralipid therapy. Conclusions: Pre-treatment with 5-HD prevents the lipid rescue of bupivacaine-induced cardiotoxicity suggesting that mitochondrial KATP channels are involved.