Nitrotyrosine and 8-isoprostane formation indicate free radical-mediated injury in hearts of patients subjected to cardioplegia

Abstract

Objective: Myocardial ischemia and reperfusion induced by cardioplegic arrest subjects the heart to free radical-mediated stress. The purpose of our study was to investigate the effect of cardioplegia-induced ischemia and reperfusion on myocardial formation and distribution of (1) nitrotyrosine as an indicator for peroxynitrite-mediated tissue injury resulting from increased nitric oxide release and (2) 8-isoprostane as an indicator for oxygen-derived free radical-mediated lipid peroxidation. Methods: In 10 patients undergoing coronary artery operations (64 ± 6 [mean ± SD] years, 3 women and 7 men) subjected to cardiopulmonary bypass and intermittent cold blood cardioplegia, we collected transmural left ventricular biopsy specimens before and at the end of cardiopulmonary bypass. Specimens were cut at 10 μm and subjected to immunocytochemical staining against the nitric oxide-producing enzyme constitutive nitric oxide synthase, cyclic guanosine monophosphate (intracellular second messenger of nitric oxide), nitrotyrosine, and 8-isoprostane by using polyclonal antibodies. For global left ventricular function determination, we measured the fractional area of contraction using transesophageal echocardiography. Results: Nitric oxide synthase activity in cardiac myocytes increased from 34 ± 10 gray units before cardiopulmonary bypass to 47 ± 12 gray units at the end of bypass (P = .015), and all hearts showed increased cyclic guanosine monophosphate content in both myocytes and endothelial cells at the end of bypass. The number of nitrotyrosine-positive capillaries increased from 36 ± 29/mm2 before bypass to 82 ± 47/mm2 at the end of bypass (P = .012), and 8-isoprostane-positive capillaries increased from 92 ± 72/mm2 before bypass to 209 ± 108/mm2 at the end of bypass (P = .005). The fractional area of contraction was 53% ± 12% before bypass and 56% ± 12% after bypass (P = .47) but was slightly decreased to 45% ± 14% at 4 hours after bypass (P = .121). Conclusions: Our data show that cardioplegia-induced myocardial ischemia and reperfusion is associated with nitrotyrosine and 8-isoprostane formation mainly in the coronary endothelium, indicating injury mediated by both peroxynitrite and oxygen-derived free radicals. Because nitric oxide synthase activation was accompanied with increased cyclic guanosine monophosphate, these data suggest that direct effects of nitric oxide on cardiac myocytes, as well as nitric oxide-mediated coronary endothelial injury, might contribute to injury associated with cardioplegia and cardiopulmonary bypass.