A Study on the Mechanism of Myocardial Ischemic-Reperfusion Injury: The Role of Oxygen-free Radicals In Hypoxic-Reoxygenation Injury in Isolated Cardiomyocyte of Rat

Abstract

The possible involvement of intracellulary-producing oxygen-free radiclas in the genesis of the reperfusion injury of the heart was investigated in preparations of isolated cardiac myocyte. Calcium-tolerant cardiomyocytes were isolated from an adult rat heart by treatment with collagenase. The freshly isolated myocyte preparations contained more than 86% of viable cells by trypan blue exclusion criterion, with 84% or more of the cells showing the elongated rod-shaped morphology of the typical heart cell. The ATP content was 4.45 p moles/ g wet wt., which was comparable to that of the intact rat heart tissue. Incubation of the myocyte preparation in hypoxic medium with limited energy substrate and low calcium produced round-shaped cells associated with a decrease in number of the typical rod-shaped cells and total viable cells. Reoxygenation of the medium following a period of hyposix incubation induced a further decrease in viable cell numbers with a concomitant increase in the release of intracellular enzymes (CPK, LDH) from the myocytes. The ATP content of the isolated cardiomyocyte rapidly decreased by hyposic incubation, but this decrease was prevented considerably by reoxygenation. Digitonized cardiomyocyte preparations incubated with succinate produced a superoxide anion under aerobic conditions as well as under reoxygenated conditions following hypoxia. The capacity of superoxide anion production was much more highly maintained during the reoxygenation period than in the continuous aerobic condition. The cellular activites of defensive enzyme systems against oxygen free radicals, superoxide dismutase and glutathione peroxidase, were significantly lowered with hypoxic incubation. It is suggested from this study that durinq reoxygenation cardiomyocyte, and these radicals may contribute to the development of reoxygenation or reperfusion injury of the myocardium.