Reperfusion of cardiac tissue results in declines in mitochondrial respiration, the severity of which increases with age. Critical to the energy status and function of the heart, cardiac mitochondria exhibit reperfusion-induced increases in free radical production. A direct link between free radicals and mitochondrial dysfunction has yet to be established. 4- Hydroxy-2-nonenal (HNE), a major product of lipid peroxidation readily reacts with and inactivates enzymes. Work from the Principal Investigator's laboratory had established that reperfusion results in modification of specific proteins by HNE. The level of HNE modification increases with age and parallels declines in mitochondrial respiration. Treatment of intact cardiac mitochondria with concentrations of HNE expected during reperfusion causes rapid declines in NADH-dependent respiration similar to that observed during reperfusion. The Principal Investigator thus proposes that: Reperfusion-induced declines in mitochondrial respiration are due, in part, to modification of specific mitochondrial proteins(s) by HNE and that these processes contribute to age-related increases in myocardial reperfusion injury. To test this hypothesis, hearts isolated from rats of different ages will be subjected to varying durations of ischemia and reperfusion. Mitochondria will then be isolated to determine: 1. Specific respiratory enzymes inactivated during ischemia and reperfusion. 2. The identities of mitochondrial protein(s) modified by HNE and the level of HNE modification. 3. Changes in mitochondrial susceptibility to HNE damage and superoxide anion generation. Chemical, immunochemical, and mass spectroscopic techniques will be utilized to detect and purify HNE-modified protein. Relationships between the level and identity of mitochondrial proteins modified by HNE (Aim 2) and those exhibiting reperfusion-induced declines in activity (Aim 1) will define mechanisms responsible for loss in mitochondrial function during reperfusion. Determinants of HNE-mediated damage to mitochondria will be established by evaluating changes in mitochondrial properties which occur during aging and ischemia (Aim 1) and result in elevated rates of HNE formation and/or increased susceptibility of specific respiratory enzymes to modification (Aim 3). Identification of specific mechanisms of myocardial reperfusion injury and conditions under which they occur is necessary if intervention is to be achieved.
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