Effects Of Postconditioning On Cardiomyocytes After Ischemia And Reperfusion In Rabbits

Background and ObjectivesWith increasing application of intracoronary thrombolisis, percutaneous coronary intervention and coronary artery bypass graft surgery in acute myocardial infarction in recent years, myocardial ischemia and reperfusion injury has attracted considerable attention. It is extremely valuable to explore some clinically feasible and effective therapeutic strategies that attenuate ischemia and reperfusion injury. Vinten-Johansen laboratory recently reported a novel cardioprotective strategy—ischemic postconditioning that brief episodes of ischemia occurring in early reperfusion after prolonged coronary artery occlusion reduced myocardial infarct size, decreased endothelial dysfunction, and attenuated neutrophil accumulation. The reduction in infarct size observed with this "ischemic postconditioning" phenomenon was similar in magnitude to that produced by ischemic preconditioning. Sequently a few studies reported that inhalational anaesthetics given just before the onset of reperfusion reduced infarct size. Some studies suggested that ischemic postconditioning involved numerous mechanisms, such as ATP-sensitive potassium channel (K_ATP C) activation, activation of phosphatidylinositol-3-kinase-Akt (PI3K-Akt) pathway and inhibition of the mitochondrial permeability transition pore (mPTP). Cardiomyocytes undergo apoptosis during reperfusion and apoptosis are associated with PI3K-Akt pathway and mPTP. These previous data evoke the hypothesis that the beneficial effects exerted by postconditioning may involve reduction apoptosis. In addition, diazoxide, a mitoK_ATPC opener, had been shown to produce pharmacological preconditioning in variety of animal models. The extent of protection against infarction and apoptosis by these agents was similar to that observed during classic ischemic preconditioning. But whether the administration of diazoxide during early reperfusion after prolonged coronary occlusion can salvage myocardium from reperfusion injury is unknown. So, the objectives of this investigation is to elucidate the protective effects of ischemic postconditioning and diazoxide postconditioning on cardiomyocytes after ischemia and reperfusion.Materials and MethodsRabbits were randomized into 6 groups:①Group IR(n=6): anesthetized rabbits were subjected to a 30 min the left anterior descending coronary artery occlusion followed by 120min reperfusion;②Group IP(n=6): 3 cycles of myocardial ischemia(5 min) and reperfusion(5 min) preceded the ischemia and reperfusion protocol;③Group IPO1(n=6): 4 cycles of 10 s reperfusion and 10 s re-occlusion were applied during the first 80 s of reperfusion;④Group IPO2(n=6): 6 cycles of 10 s reperfusion and 10 s re-occlusion were applied during the first 2min of reperfusion;⑤Group DZO(n=6): intravenous infusion diazoxide 7mg/kg of 3 min before reperfusion for 5 min;⑥Group(IPO1+DZO)(n=6): anesthetized rabbits were exposed to combined 4 cycles of 10 s reperfusion and 10 s re-occlusion and diazoxide (7mg/kg intravenously). Blood sample was taken from for assaied of the concentration of maleic dialdehyde (MDA) with the method of Thibarbituric Acid, the activity of superoxide dismutase (SOD) with xanthine oxidase. Myocardial infarction size was determined by staining with Evans blue and triphenyltetrazolium chloride solution. Cardiomyocyte apoptosis was assessed by in situ TDT-mediated dUTP nick end labeling (TUNEL). The expressions of bcl-2 and bax proteins were measured by immunohistochemical technique in the ischemic area. Results1.The changes of MDA concentration and SOD activity: Compared with group IR, the SOD activity was higher, the MDA concentration were lower in group IPO1,group IPO2,group IP and group(DZO+IPO1). There was significant difference between group IPO1 and group IPO2. But there was no significant difference among group IPO2,group IP and group(DZO+IPOl). There was significant difference between group IPO1 and group IPO2. There was no significant difference between group IR and group DZO.2. Infarct size: Ischemic preconditioning and 6 cycles of ischemic postconditioning produced equivalent effects on infarct size reduction. These beneficial effects were similar in magnitude to that produced by combined 4 cycles of 10 s reperfusion and 10 s re-occlusion and diazoxide together after 30 min ischemia. Diazoxide alone exerted no effect on infarct size.3.Cardiomyocyte apoptosis: Ischemic preconditioning and 6 cycles of ischemic postconditioning produced equivalent effects on apoptosis reduction. These beneficial effects were similar in magnitude to that produced by combined 4 cycles of 10 s reperfusion and 10 s re-occlusion and diazoxide together after 30 min ischemia. Diazoxide alone exerted no effect on apoptosis index.4. Bcl-2 and Bax protein expression: Both ischemic preconditioning and 6 cycles of ischemic postconditioning inhibited equivalently Bax protein and increased the Bcl-2/Bax ratio.Conclusions1. Ischemic postconditioning reduces infarct size and apoptosis, and exertes protective effects that is similar in magnitude to that produced by ischemic preconditioning.2. Ischemic postconditioning attenuates the expression of Bax protein in ischemic myocardium, and this may help increase the ratio of Bcl-2/Bax in preventing the progression of apoptosis 3. Diazoxide postconditioning decreases the protective threshold of ischemicpostconditioning necessary to protect against reperfusion injury.