Direct evidence that initial oxidative stress triggered by preconditioning contributes to second window of protection by endogenous antioxidant enzyme in myocytes

Background: We tested the hypothesis that late preconditioning is associated with increased antioxidant enzyme activity induced by initial oxidative stress. Methods and Results: Isolated rat myocytes were preconditioned either with two cycles of 5 minutes of anoxia and 5 minutes of reoxygenation or with exogenous superoxide anion (O₂^-) generated by reaction of xanthine oxidase with xanthine. Myocytes were allowed to recover for 60 minutes or 24 hours, after which they were subjected to 60 minutes of anoxia and 60 minutes of reoxygenation. After 60 minutes or 24 hours, the protection was evidenced by decreased O₂^- production, increased Mn superoxide dismutase (Mn-SOD) activity, increased cell viability, decreased LDH release, reduced malondialdehyde formation, high-energy phosphate preservation, and improved cell morphology in preconditioned and O₂^-- treated myocytes. Immediately after treatment with O₂^- or repetitive, brief anoxia, O₂^- production was increased in myocytes. Longer anoxia resulted in loss of Mn-SOD activity in anoxic controls 24 hours later, whereas it was significantly increased in preconditioned and O₂^--treated myocytes, O₂^- production was inhibited in preconditioned and O₂^--treated myocytes. Myocytes treated with Mn-SOD during short, intermittent anoxia exhibited decreased activity of Mn-SOD and increased O₂^- production 24 hours later. Mn-SOD activity in late preconditioning was considerably higher than that in classic preconditioning. Conclusions: These results suggest that a burst of oxygen free radicals generated during the initial periods of brief, repetitive anoxia increases myocardial antioxidant activity 24 hours later and that it contributes to the late cardioprotective effect of preconditioning.