Ultrastructural Injury and Its Mechanism of Cultured Cardiac Myocytes under Anoxia-Reoxygenation

Park, Jeong Hyun; Kim, Joo Young; Jeong, See Ryun; Lee, Yung Chang

Abstract

Abrupt reoxygenation (or reperfusion) after anoxia (or ischemia)-induced injury resulted in the loss of contractile property, destruction of cell organelles, and ultimately, cell death in cardiac myocytes. This phenomenon has been called 'oxygen paradox' or 'reperfusion injury'. The purpose of this study was to investigate the changes of fine structures and enzyme activities associated with oxygen paradox during 60 min. of anoxia, followed by a 30 min. of reoxygenation. Cardiac myocytes were dissociated from neonatal rat ventricles and cultured for three days. While they were exposed to anoxia and reoxygenation, the cardiac myocytes were investigated through beating counts, enzyme cytochemistry, immunofluorescence, electron microscopy for morphological study. Activity staining and Western blot for Cu, Zn-SOD, NADPH-diaphorase stain and nitrite concentration mesurement for nitric oxide synthase, and catalase activity measurement were performed. After 60 min. of anoxia, the beating rate increased remarkably. Swollen mitochondria with amorphous dense clumps, mild contracture of myofibrils and retraction of cytoplasmic processes were observed in cardiac myocytes. Under confocal microscope, weak reaction of Mn-SOD and myosin were observed, whereas reaction of Cu, Zn-SOD was enhanced in perinuclear region. Cu, Zn-SOD and catalase activity in cardiac myocytes increased markedly. Nitric oxide synthase activity increased gradually with time. After 30 min. of reoxygenation following 60 min anoxia, structural changes of myocardial cells was more pronounced than in the cells of anoxic group. Beating rate was variable but decreased gradually. Myocardial cells showed evidence of severe structural alterations, including marginal clumping of chromatids, varying-sized bleb formation, many vacuoles, mitochondrial matrix exposed to cytoplasm and fragmen-tation of cristae, myofibrillar hypercontracture. Decline of immunocytochemical reaction of Mn-SOD, myosin and Cu, Zn-SOD were observed under confocal microscope. The declines of activity and quantity of Cu, Zn-SOD were severe compared to control. In contrast, nitric oxide synthase activity significantly increased. Catalase activity was lower than in anoxic group, but still higher than in control activity. These results suggested that there were two possible mechanisms for the drastic morphological changes induced by anoxia-reoxygenation; 1) direct effect of oxygen free radicals, and 2) reaction of nitric oxide with superoxide radicals, which resulted in generation of toxic metabolites of nitric oxide, exacerbated myocardial cellular damages.