Effects of Ischemic Preconditioning on Hypoxia-Reoxygenation Injury of Cardiac Myocyte in Culture

Sung, Hoon Ki; Park, Jeong Hyun; Lee, Yung Chang

Abstract

Short period of ischemia and reperfusion protect heart against subsequent prolonged ischemia-reperfusion injury. This phenomenon was first described by Murry et al in 1986, who demonstrated that four 5-minute coronary artery occlusions followed by equal period of reflow at each time before a subsequent prolonged occlusion resulted in a reduction of infarct size in dog. Although the precise mechanism of preconditioning remains unknown, this phenome-non is present among different species of mammals, including dogs, rats, pigs, rabbits, and human. The objects of present study was to investigate effect of ischemic preconditioning on cell viability, structural changes and apoptosis during 60 min hypoxia and 60 min reoxygenation of the cell. In present study we investigated through cell culture system using myocyte of three days old neonatal rat cultured for three days. During hypoxia and reoxygenation, differences between preconditioned and nonpreconditioned of beating counts, morphological and structural changes are investigated through inverted phase contrast microscope and transmis-sion electron microscope. To detection of apoptotic cell, TUNEL (TdT-mediated dUTP-biotin nick end labeling) stain was accomplished, and through which we invesigate the effects of preconditioning on apoptosis. Viabiliy of each cell and it's mitochondria were measured quantitatively by MTT assay. After 60 min of hypoxia and 60 min of reoxygenation, beating rate decreased remarkably. But at the time of 60 min of reoxygenation, there was marked increase in beating count in pre-conditioned cell. Swollen mitochondria with amorphous granules in inner membrane, destroyed mitochondrial cristae, indented nuclear envelope, chromatin condensation, contracture of myofibril, fragmentation of myofilaments, cytoplasmic shrinkage were observed in both preconditioned cell and nonpreconditioned cell. But it is much less in pre-conditioned cell than in nonpreconditioned cell. MTT activity decreased in both experimental groups in compared with normal group, but in preconditioned group, MTT activity increased markedly in compared with nonpreconditioned group. And apoptosis is decreased by precontitioning in TUNEL staining. These results suggest that cardioprotective effects of ischemic preconditioning is mediated by attenuating structural destroy, increasing cell viability, decreasing apoptosis.