Molecular aspects of myocardial ischemia/reperfusion injury and the protective effects of allopurinol

A growing body of evidence has now accumulated supporting the involvement of oxygen-derived free radicals in the development of myocardial ischemia/reperfusion (I/R) injury. We have, therefore, undertaken the present study to examine (1) I/R-related alterations in myocardial antioxidant capacity in pentobarbital anesthetized open-chest rabbits subjected to left circumflex coronary artery ligation followed by reperfusion; (2) the protective effects of pretreatrnent with allopurinol or the 21-aminosteroid U74006F; (3) alternative mechanisms to xanthine oxidase inhibition for allopurinol protection against I/R injury; and (4) the effect of allopurinol treatment on the antioxidant capacity of erythrocytes in pigs used in a heart-lung transplantation study. In the rabbit myocardium, a marked impairment in myocardial antioxidant capacity developed in association with the onset of irreversible injury, as reflected in the enhancement in glutathione (GSH) depletion and formation of thiobarbituric acid-reactive substances (TBARS) following in vitro incubation of tissue homogenate with tert-butylhydroperoxide (TBHP). During the course of post-ischemic reperfusion, the protracted time-course of alterations in antioxidant capacity dissociated them from the early burst of radical formation known to occur at the onset of post-ischemic reperfusion of the myocardium. When the time-dependent changes in functional indices of antioxidant status (TBHP-induced GSH depletion and formation of TBARS) were analysed in relation to activities of antioxidant enzymes, evidence suggestive of functionally relevant impairments in Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and glutathione reductase (GRD) activities was found. These results and our demonstration of significant decreases in the activity of GSH-dependent antioxidant enzymes under acidotic conditions suggest that a transient impairment in the functioning of antioxidant enzymes may be involved in triggering irreversible myocardial I/R injury. Repetitive brief episodes of I/R produced a progressive decrease in myocardial ATP levels, which was not associated with any detectable changes in myocardial antioxidant capacity. Ischemic preconditioning produced by brief episodes of I/R did not affect the severity of subsequently induced I/R injury. These results suggest that brief episodes of myocardial ischemia do not produce oxidative tissue damage and the ischemia-induced depletion in myocardial ATP level is at least partially dissociable from the I/R-related impairment in tissue antioxidant capacity. Isolated Langendorff-perfused rabbit hearts subjected to I/R did not show any changes in antioxidant capacity. However, when intact hearts were subjected to ischemia in vivo and a subsequent reperfusion in vitro, an impairment in myocardial antioxidant capacity became apparent. These results suggest that blood elements, possibly activated neutrophils, may be a crucial factor involved in the development of I/R-induced oxidant injury. Chronic allopurinol pretreatment (1 mg/ml in drinking water or approximately 75 mg/kg/day) for 7 days prior to ischemia provided significant protection against I/R-induced alterations in myocardial antioxidant capacity, but not the decrease in tissue ATP levels. This chronic allopurinol regimen was found to enhance myocardial GRD activity in nonischemic tissue. In addition, both allopurinol and oxypurinol inhibited the transition metal ion-catalysed ascorbate oxidation and lipid peroxidation in vitro, likely as a consequence of their metal chelating properties. Similarly, myoglobin-TBHP-catalysed oxidation of uric acid and lipid peroxidation were also suppressed by allopurinol. All these suggest that allopurinol may favorably alter myocardial antioxidant capacity directly by virtue of its transition metal chelating properties and its antioxidant actions in myoglobin-mediated oxidative processes. The acute administration of 21-aminosteroid U74006F (3 mg/kg, i.v) under conditions comparable to those known to protect against trauma-induced damage in the central nervous system failed to reduce manifestations of oxidative injury in rabbit hearts subjected to ischemia and reperfusion. Although reactive oxy-radicals have been implicated in both types of tissue damage, the observed difference in susceptibility to protection by this steroidal antioxidant suggests that the molecular mechanisms involved are not identical. In the heart-lung transplantation study, erythrocytes from allopurinol-treated pigs (given repeatedly at an oral dose of 50 mg/kg) showed a time/dose-dependent increase in antioxidant capacity as reflected in the decrease in malondialdehyde production following in vitro oxidative challenge. The extent of red cell protection in both donor and recipient animals correlated significantly with the functional viability of the transplanted lung tissue, as assessed by tissue water content. These results suggest that the measurement of erythrocyte antioxidant capacity may provide an useful assessment of generalized alterations in tissue antioxidant status produced by pharmacological interventions.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat