Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes

We examined the ability of ACh to mimic ischemic preconditioning in cardiomyocytes and the role of ATP-sensitive potassium (K_ATP) 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,$P{O}_2$ , and$P{\text{CO}}_2$ were controlled. Cell viability was quantified with propidium iodide (5 μM), 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 μM), 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_ATPchannels and increased ROS production from mitochondria are important intracellular signals that participate in ACh-induced preconditioning in cardiomyocytes.