Nitroglycerin Aggravates Myocardial Ischemia Reperfusion Injury

BackgroundClinical use of GTN continues to be a treatment of choice to improve cardiac output through its peripheral dilatory effects, decreasing preload and afterload on the heart. However, more recently, several large-scale clinical trials showed that nitrate use failed to exert any protective effect to ischemic heart, even one of them reported that long-term use of nitrate may aggravate myocardial ischemia reperfusion (MI/R) injury. In addition, several animal experiments demonstrated nitroglycerin tolerance, a "final state" of nitroglycerin use, may induce the production of superoxide (O2") from the vascular endothelial cells . It had been well known that O₂- is a cytotoxic factor, which increases cardiomyocyte death during reperfusion. In addition, the earlier studies suggested that an interaction between nitroglycerin and sulfhydryl (-SH)-containing cellular receptors was necessary for vascular smooth muscle relaxation to occur and that repeated administration of nitroglycerin caused sulfhydryl depletion (via oxidation).AimThe purposes of the first part of this study was 1) to determine whether GTN is deleterious to postischemic cardiomyocyte, and if so, 2) to elucidate the role of ONOO" in these GTN -induced myocardial injury, and (3) to investigate whether treatment with exogenous GSH may attenuate the increased cardiomyocyte injury induced by GTN. We adopted isolated heart perfusion model because nitroglycerin may make coronary flow increase, which may compromise the "potential" deleterious effect of nitroglycerin.The purposes of the second one was: 1) to determine whether nitrate tolerance is deleterious to postischemic cardiomyocyte, and if, 2) to elucidate the role of ONOO" in these nitrate tolerance-induced myocardial injury, and (3) to investigate whether treatment with exogenous GSH may attenuate the increased cardiomyocyte injury induced by nitrate tolerance.The purposes of the third one was examined the effects of nitroglycerin tolerance on isolated arterial function and tissue injury following simulative ischemia (90 min) and reperfusion (120 min).Methods1. Isolated rat hearts were subjected to 30 minutes of regional ischemia followed by 2 h of reperfusion. During reperfusion, the isolated hearts received randomly one of the following treatments, control (KH buffer), low-dose GTN (0.75 ug/h , LN), medium-dose GTN (3.75 ug/h, MN), high-dose GTN (15 ug/h, HN), HN plus GSH (1 mmol/L GSH). Functional parameters (LVSP, LVEDP and HR) were obtained from five groups throughout the experiment. Creatine kinase (CK) and lactate dehydrogenase (LDH) activity in the coronary effluent was determined. The apoptosis and necrosis of cardiomyocyte were detected with TUNEL way and TTC stain. The peroxynitrite(ONOO") formation was analyzed with immunohistochemical stain of nitrotyrosine.2. Male Sprague-Dawley rats were randomized to receive nitroglycerin (60 ug/kg/h) or saline for 12 hours followed by 40 min of MI and 4 hours of reperfusion. Myocardial apoptosis, infarct size, nitrotyrosine formation, plasma CK and LDH activity, and cardiac function were determined.3. Male Sprague-Dawley rats were infused with nitroglycerin or saline for 12 h and ascendens aorta was rapidly isolated. The isolated aorta was subjected to one of the following treatments: simulative ischemia (90 min)/reperfusion (120 min), simulative ischemia /reperfusion (I/R) plus glutathione (GSH, O.lmmol/L) during reperfusion, or control treatment (Kreb's solution for 3.5 h). Contractile function and vasorelaxation responses of ascendens aorta was measured, NO production and CK, LDH activity in reperfusion solution were analysed and nitrotyrisine formation was evaluated with immunohistochemical stain.Results1. HN significantly exacerbated cardiac reperfusion injury as evidenced by increased coronary effluent CK, LDH activity, cardiomyocyte apoptosis and necrosis, decreased cardiac function recovery after reperfusion. In addition, HN/ MN increased the formation of nitrotyrosine compared with control group. GSH decreased the formation of nitrotyrosine, coronary effluent CK, LDH activity, cardiomyocyte apoptosis and necrosis in HN group.2. MI/R resulted in significant apoptotic cell death, which was further increased in animals with nitrate tolerance. In addition, nitrate tolerance further increased plasma CK and LDH activity, enlarged infarct size, and impaired cardiac functional recovery after ischemia.