myxothiazol and Myocardial-Ischemia

Although a burst of oxidants has been well described with reperfusion, less is known about the oxidants generated by the highly reduced redox state and low O(2) of ischemia. This study aimed to further identify the species and source of these oxidants. Cardiomyocytes were exposed to 1 h of simulated ischemia while oxidant generation was assessed by intracellular dihydroethidine (DHE) oxidation. Ischemia increased DHE oxidation significantly (0.7 +/- 0.1 to 2.3 +/- 0.3) after 1 h. Myxothiazol (mitochondrial site III inhibitor) attenuated oxidation to 1.3 +/- 0.1, as did the site I inhibitors rotenone (1.0 +/- 0.1), amytal (1.1 +/- 0.1), and the flavoprotein oxidase inhibitor diphenyleneiodonium (0.9 +/- 0.1). By contrast, the site IV inhibitor cyanide, as well as inhibitors of xanthine oxidase (allopurinol), nitric oxide synthase (nitro-L-arginine methyl ester), and NADPH oxidase (apocynin), had no effect. Finally, DHE oxidation increased with Cu- and Zn-containing superoxide dismutase (SOD) inhibition using diethyldithiocarbamate (2.7 +/- 0.1) and decreased with exogenous SOD (1.1 +/- 0.1). We conclude that significant superoxide generation occurs during ischemia before reperfusion from the ubisemiquinone site of the mitochondrial electron transport chain.

Topics: Acetophenones; Allopurinol; Animals; Cells, Cultured; Chick Embryo; Cytosol; Enzyme Inhibitors; Heart; Kinetics; Methacrylates; Mitochondria, Heart; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; NG-Nitroarginine Methyl Ester; Oxidation-Reduction; Reactive Oxygen Species; Rotenone; Superoxide Dismutase; Superoxides; Thiazoles

We examined the ability of ACh to mimic ischemic preconditioning in cardiomyocytes and the role of ATP-sensitive potassium (KATP) channels and mitochondrial reactive oxygen species (ROS) in mediating this effect. Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH, PO2, and PCO2 were controlled. Cell viability was quantified with propidium iodide (5 microM), and production of ROS was measured using 2', 7'-dichlorofluorescin diacetate. Data were expressed as means +/- SE. Preconditioning with 10 min of ischemia followed by 10 min of reoxygenation or 10 min of ACh (1 mM) followed by a drug-free period before 1 h of ischemia and 3 h of reoxygenation reduced cell death to the same extent [preconditioning 19 +/- 2% (n = 6, P < 0.05) ACh 21 +/- 5% (n = 6, P < 0.05) vs controls 42 +/- 5% (n = 9)]. Like preconditioning, ACh increased ROS production threefold before ischemia [0.60 +/- 0.16 (n = 7, P < 0.05) vs. controls, 0.16 +/- 0. 03 (n = 6); arbitrary units]. Protection and increased ROS production during ACh preconditioning were abolished with 5-hydroxydecanoate (5-HD, 100 microM), a selective mitochondrial K(ATP) channel antagonist, and the thiol reductant 2-mercaptopropionyl glycine (2-MPG, 1 mM), an antioxidant [cell death: 5-HD+ACh 37 +/- 7% (n = 5), 2-MPG+ACh 47 +/- 6% (n = 6); ROS signals: 5-HD+ACh 0.09 +/- 0.03 (n = 5), 2-MPG+ACh 0.01 +/- 0.04 (n = 4)]. In addition, ACh-induced ROS signaling was blocked by the mitochondrial site III electron transport inhibitor myxothiazol (0.02 +/- 0.07, n = 5). These results demonstrate that activation of mitochondrial K(ATP) channels and increased ROS production from mitochondria are important intracellular signals that participate in ACh-induced preconditioning in cardiomyocytes.

Topics: Acetylcholine; Animals; Anti-Arrhythmia Agents; Cardiotonic Agents; Cells, Cultured; Chick Embryo; Decanoic Acids; Electron Transport; Heart; Heart Ventricles; Hydrogen-Ion Concentration; Hydroxy Acids; Ischemic Preconditioning; Methacrylates; Mitochondria, Heart; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Potassium Channels; Reactive Oxygen Species; Signal Transduction; Thiazoles; Time Factors; Tiopronin