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

Abstract

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.

Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation

“…For example, more severe and prolonged hypoxia treatments may be necessary to induce mitochondrial depolarization in cardiomyocytes (45). Notwithstanding, our results with dopamine-containing PC12 cells confirm the conclusions of previous studies in other systems that hypoxia depolarizes ⌬⌿ m , as demonstrated by using different voltage-sensitive dyes (44,45), and that hypoxia increases the overall cellular ROS level before the onset of reperfusion (6,31,46).…”

“…Oxidative stress is implicated in reperfusion after an ischemic episode (30), and hypoxia may enhance production of ROS before reoxygenation (5,6,31). We used two groups of PC12 cells, one group treated with control adenovirus particles containing no insert and the other treated with MSRA virus particles, and challenged them with normoxia, hyperoxia, and hypoxia (Fig.…”

Reversible redox-dependent modulation of mitochondrial aconitase and proteolytic activity during in vivo cardiac ischemia/reperfusion

“…Preconditioning, a series of brief periods of ischemia and reperfusion, protects the myocardium from deleterious events associated with extended durations of ischemia and reperfusion (7)(8)(9). Cardioprotection afforded by preconditioning appears mediated, in part, by the production of oxygen radicals (35)(36)(37)(38)(39)(40) and is associated with reductions in Ca 2ϩ overload (7,(41)(42)(43) and free radical production (44) during extended periods of ischemia͞ reperfusion.…”

Reactive species mechanisms of cellular hypoxia-reoxygenation injury

“…Pulmonary artery smooth muscle cells, cardiomyocytes, and several other cell types produce increased ROS in hypoxia that are usually detected as oxidation of the fluorescent probe DCF (54,133). Hypoxic pulmonary artery smooth muscle cells significantly increased DCF fluorescence fivefold above that of normoxic cells (55), and simulated ischemia (hypoxia and low glucose) of embryonic ventricular myocytes increased DCF fluorescence threefold over that of normoxic cells (133). Simulated in vitro ischemia of cardiomyocytes also increased dihydroethidine (DHE) oxidation due to ROS, so these observations are not simply an artifact related to the use of oxidizable DCF.…”