| While restoration of blood flow is the sole method for preventing death, reperfusion is associated with additional loss in cardiac function, the severity of which increases with age. Cardiac reperfusion is associated with increased rates of mitochondrial free radical production. Mitochondria are therefore a likely site of reperfusion-induced oxidative damage. 4-Hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, increases in concentration upon reperfusion of ischemic cardiac tissue, can react with and inactivate enzymes, and has been shown to inhibit mitochondrial respiration in vitro. HNE modification of mitochondrial protein(s) might, therefore, be expected to occur during reperfusion and result in loss in mitochondrial function. In addition, this process may be more prevalent in aged animals. To begin to test this hypothesis, hearts from adult and senescent rats were subjected to periods of ischemia and reperfusion. Mitochondria were then isolated and enzyme activities were measured. In addition, the level, relative distribution, and identity of HNE-modified proteins were assessed. We provide evidence that cardiac reperfusion results in modification of specific myocardial proteins by HNE. Modification occurs exclusively during reperfusion, increases dramatically with age, and parallels declines in mitochondrial NADH-linked respiration. In addition, we report the selective age-dependent inactivation of Complex I during ischemia, and α-ketoglutarate dehydrogenase (KGDH) and Complex IV during reperfusion. In contrast, other electron transport complexes and dehydrogenases examined were not significantly affected. Complex I and IV were determined not to be rate limiting for respiration and were not responsible for declines in mitochondrial respiratory rates during ischemia and reperfusion. It has been reported that inhibition of Complex I results in increased rates of free radical production. The age-related decline in Complex I activity during ischemia may therefore predispose senescent animals to more severe oxidative damage during reperfusion. Lipoic acid (LA), a component of KGDH, is highly susceptible to HNE inactivation. Mitochondrial LA levels are reported to declines in an age-dependent fashion only during reperfusion. Thus, our results suggest a likely mechanism by which free radical production mediates reperfusion-induced declines in mitochondrial and cardiac function. |
