Prevention of ischemia/reperfusion-induced cardiac apoptosis and injury by melatonin is independent of glutathione peroxdiase 1

Abstract

Free-radical generation is one of the primary causes of myocardial ischemia/reperfusion (I/R) injury. Melatonin is an efficient free-radical scavenger and induces the expression of antioxidant enzymes. We have previously shown that melatonin can prevent free-radical-induced myocardial injury. To date, the mechanism underlying melatonin's cardioprotective effect is not clear. In this study, we assessed the ability of melatonin to protect against I/R injury in mice deficient in glutathione peroxidase 1 (Gpx1). Mice hearts were subjected to 40 min of global ischemia in vitro followed by 45 min of reperfusion. Myocardial I/R injury (expressed as % of recovery of left ventricular developed pressure x heart rate) was exacerbated in mice deficient in Gpx1 (51 +/- 3% for Gpx1+/+ mice versus 31 +/- 6% for Gpx1(-/-) mice, P < 0.05). Administration of melatonin for 30 min protected against I/R injury in both Gpx1+/+ mice (72 +/- 4.8%) and Gpx1(-/-) mice (63 +/- 4.7%). This protection was accompanied by a significant improvement in left ventricular end-diastolic pressure and a twofold decrease in lactate dehydrogenase (LDH) level released from melatonin-treated hearts. In another set of experiments, mice were subjected to 50 min of ligation of the left descending anterior coronary artery in vivo followed by 4 hr of reperfusion. The infarct sizes, expressed as the percentage of the area at risk, were significantly larger in Gpx1(-/-) mice than in Gpx1+/+ mice (75 +/- 9% versus 54 +/- 6%, P < 0.05) and were reduced significantly in melatonin-treated mice (31 +/- 3.7% Gpx1(-/-) mice and 33 +/- 6.0% Gpx1+/+ mice). In hearts subjected to 30 min of coronary artery occlusion followed by 3 hr of reperfusion, melatonin-treated hearts had significantly fewer in situ oligo ligation-positive myocytes and less protein nitration. Our results demonstrate that the cardioprotective function of melatonin is independent of Gpx1.

Fig. 1

Effect of melatonin on the cardiac function and coronary flow rate of hearts from Gpx1^-/- and wild type mice. Melatonin (150 μg/kg, i.p.) was given 30 min before the hearts were removed for in vitro perfusion. Results are expressed as the percentages of recovery of pre-ischemic values. Values are mean ± SEM of 6 hearts. a = P<0.05, Gpx1^-/- vs. Gpx1^+/+; b = P< 0.05, melatonin-treated group vs. nontreated group;

Fig. 2

Melatonin improves left ventricular end-diastolic pressures of Gpx1^+/+ and Gpx1^-/- mouse hearts. Left ventricular end-diastolic pressure was measured after 40 min of global ischemia and 45 min of reperfusion in isolated hearts from Gpx1^+/+ and Gpx1^-/- mice with or without pretreatment with melatonin (150 μg/kg). Values are mean ± SEM of 6 hearts. a = P<0.05, Gpx1^-/- vs. Gpx1^+/+; b = P< 0.05, melatonin-treated group vs. nontreated group, c = P<0.05, post-ischemic values vs. respective pre-ischemic values.

Fig. 3

Melatonin reduces LDH release in vitro. The release of LDH during 45 min of reperfusion after 40 min of global ischemia was measured. Values were mean ± SEM of 6 hearts. a = P<0.05, Gpx1^-/- vs. Gpx1^+/+; b = P< 0.05, melatonin-treated group vs. nontreated group.

Fig. 4

Protective effect of melatonin on myocardial infarction in Gpx1^+/+ and Gpx1^-/- mouse hearts. Mice were treated with 150 μg/kg melatonin for 30 min. They were subjected to 50 min of LAD ligation followed by 4 h of reperfusion. Values are mean ± SEM of 6 hearts. a = P<0.05, Gpx1^-/- vs. Gpx1^+/+; b = P<0.05, melatonin-treated group vs. nontreated group.

Fig. 5

Melatonin attenuates apoptosis in Gpx1^+/+ and Gpx1^-/- mouse hearts. Mice were subjected to a period of 30 min of LAD ligation and 3 h of reperfusion. Paraffin-embedded sections of hearts were stained by ISOL procedure. Immunolabeled nuclei of myocytes were determined by random counting of 10 fields per section. Each bar represents mean ± SEM of six hearts. a = P<0.05, Gpx1^-/- vs. Gpx1^+/+; b = P< 0.05, melatonin-treated group vs. nontreated group.

Fig. 6

Melatonin reduces nitrotyrosine staining in Gpx1^+/+ and Gpx1^-/- mouse hearts. Mice were subjected to a period of 30 min of LAD ligation and 3 h of reperfusion. Immunohistochemical staining of mouse hearts was performed with anti-nitrotyrosine antibody. Sections from Gpx1^+/+ (A), Gpx1^+/+ + Mel (B), Gpx1^-/- (C), and Gpx1^-/- + Mel mouse hearts (D) were stained with anti-tyrosine antibody. Semi-quantitative analysis was performed using Image Pro Plus. Each bar represents mean ± SEM of 4 hearts. a = P < 0.05, Gpx1^+/+ + Mel vs. Gpx1^+/+; b = P<0.05, Gpx1^-/- + Mel vs. Gpx1^-/-.