In this proposal, we present new data implicating cytochrome P450 monooxygenases (CYP) in myocardial ischemia/reperfusion (I/R) injury. We have shown that inhibition of CYP with compounds such as chloramphenicol or sulfaphenazole can reduce tissue injury following myocardial ischemia and reperfusion. Importantly, infarct size is reduced by up to 70 percent by pretreatment with chloramphenicol or sulfaphenazole. In the isolated perfused heart model, these drugs are beneficial even when administered after ischemia.
In Aim 1 we will extend the existing preliminary studies with the following objectives: to establish the minimum effective dose in the isolated perfused heart model; to establish how long a delay before drug administration is still protective; and to assess efficacy of sulfaphenazole (the most potent lead compound identified thus far) in a model of regional ischemia and reperfusion in the anesthetized rabbit. Since several of the CYP inhibitors we have shown to be cardioprotective are already safely used in humans for other purposes, it is hoped that these preclinical studies will lay the groundwork for a subsequent clinical trial. We will also establish whether upregulation of CYPs in the heart increases I/R injury.
In Aim 2, we seek to understand the mechanism of cardioprotection by these CYP inhibitors, as this will yield insights into the role of CYPs in causing myocardial damage after ischemia/reperfusion. To this end, we will identify the CYP isoforms in heart that are inhibited by sulfaphenazole and chloramphenicol, and will consider the two likely mechanisms of injury-production of excessive reactive oxygen species, and the dysregulated metabolism of arachidonic acid to vasoactive and cardiotoxic eicosanoids.
In Aim 3 we consider the potential effect of CYPs on the SR Ca+2 ATPase and the mitochondrial KATP channel. We address the potential role of CYPs in apoptosis, based on the finding that the anti-apoptotic proteins ARC (apoptosis repressor with CARD domain) and Bcl-xL inhibit CYP activity, while pro-apoptotic tBid stimulates CYP activity in rat liver microsomes. This proposal identifies cytochrome P450 monooxygenases as a previously underestimated factor in myocardial reperfusion injury, and establishes the basis for a novel therapeutic approach ? ?
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