Cardiac reperfusion injury: aging, lipid peroxidation, and mitochondrial dysfunction

Abstract

Cardiac reperfusion and aging are associated with increased rates of mitochondrial free radical production. Mitochondria are therefore a likely site of reperfusion-induced oxidative damage, the severity of which may increase with age. 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 inhibits 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 8- and 24-month-old rats were perfused in Langendorff fashion and subjected to periods of ischemia and/or reperfusion. The rate of state 3 respiration of mitochondria isolated from hearts exposed to ischemia (25 min) was approximately 25% less than that of controls, independent of age. Reperfusion (40 min) caused a further decline in the rate of state 3 respiration in hearts isolated from 24- but not 8-month-old rats. Furthermore, HNE modification of mitochondrial protein (approximately 30 and 44 kDa) occurred only during reperfusion of hearts from 24-month-old rats. Thus, HNE-modified protein was present in only those mitochondria exhibiting reperfusion-induced declines in function. These studies therefore identify mitochondria as a subcellular target of reperfusion damage and a site of age-related increases in susceptibility to injury.

Figure 1

Age-related differences in the effect of ischemia and reperfusion on ADP-dependent mitochondrial respiration. After perfusion (P), ischemia (I), or reperfusion (R) of hearts from 8- and 24-month-old rats, mitochondria were isolated and respiratory activities were assessed by using a Clark-style oxygen electrode. Mitochondria at a concentration of 0.5 mg/ml were incubated with 15 mM glutamate for 2.0 min at 25°C. State 3 respiration was then initiated by addition of 0.5 mM ADP.

Figure 2

SDS/gel electrophoresis and Western blot analysis of protein from cardiac mitochondria. Hearts obtained from 8- and 24-month-old rats were exposed to perfusion (P), ischemia (I), or reperfusion (R). Mitochondria were then isolated and proteins were resolved by SDS/gel electrophoresis using duplicate 10% gels (12.5 μg of total protein per lane). One gel of the series was transferred to a nitrocellulose membrane for Western blot analysis with anti-HNE antibody (A). The second gel was stained with Coomassie blue for analysis of the molecular weight distribution of mitochondrial protein (B).