Melatonin protects against ischemia-reperfusion injury and inhibits apoptosis in isolated working rat heart.

INTRODUCTION: Melatonin (MEL), a pineal hormone, is well known as a potent antioxidant in a variety of ischemia-reperfusion models. Recent studies have assumed a pivotal role of reactive oxygen species (ROS) in the development of apoptosis. There are few pieces of information concerning a possible protective role of MEL against apoptosis in ischemia-reperfusion injury of myocardium. METHODS: We conducted an in vitro experiment: (1) to study the effect of MEL in the model of isolated and perfused working rat heart; (2) to evaluate the antioxidant capacity of MEL by a simple fluorescence test; and (3) to analyze the extent of apoptosis inhibition by MEL. Four groups of male Wistar rat were used: (a) group 'MEL 50 muM' (n=8); (b) group 'ischemia 30 min' (n=8); (c) group 'controls' (n=8); and (d) group 'controls+MEL 50 muM' (n=8). The perfusion medium was an oxygenated Krebs-Henseleit buffer (KHB). Hearts in groups (a) and (b) underwent 30 min of global normothermic ischemia and 45 min of reperfusion; 3 min before ischemia the hearts of group (a) received KHB with MEL 50 muM (and MEL 50 muM was also present in KHB solution during reperfusion). Hearts of group (c) were only perfused by KHB, and hearts of group (d) perfused by KHB+MEL 50 muM throughout the experiment. Registered were basic hemodynamic parameters: coronary, aortic, cardiac output and heart rate. At the end of each experiment, a left ventricle samples were taken for in situ detection of apoptosis using a TUNEL in-situ detection kit (POD) and quantitative analysis was performed. Malonedialdehyde concentrations were evaluated from heart homogenate to determine the severity of oxidative damage. To study the antioxidant capacity of MEL, a fluorescence test with allophycocyanin as an indicator was performed. A peroxyl radical generator, 2,2'-azobis(2-amidinopropan)-4-hydrochloride (AAPH) was used, and the antioxidant effect of MEL was expressed in oxygen-radical absorbing capacity (ORAC) units. RESULTS: Treatment by MEL resulted in a significant improvement of hemodynamic parameters and reduction of postischemic arrhythmias during reperfusion. All hearts in group 'ischemia 30 min' developed fatal ventricular fibrillations. MEL significantly reduced the incidence of apoptotic cells (14+/-4.3%; **P<0.01) vs. group 'ischemia 30 min' (58+/-2.1%). No apoptotic cells were detected in both control groups (c) and (d). In the fluorescence test, MEL exhibited a significant dose-dependent protective effect against peroxyl radical; MEL also reduced significantly the level of lipoperoxidation (MDA; *P<0.05). Analysis of hemodynamic parameters in both control groups (c) and (d) did not show any significant differences; the presence of MEL 50 muM in KHB solution did not have any important influence on cardiac performance in this type of experiment. CONCLUSION: We confirmed the previously reported beneficial effects of MEL against ischemia-reperfusion injury, presumably via its antioxidant properties. A significant suppression of apoptosis and the peroxyl radical scavenging properties of MEL in our study could contribute to the hypothesis of a close link between oxidative stress and apoptosis promotion.