Role of Epoxyeicosatrienoic Acids in Protecting Against Ischemic Cardiomyopathy

Abstract

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid (AA) epoxidation with important cardioprotective and signaling properties. AA is a twenty carbon ω-6 polyunsaturated fatty acid containing four cis-double bonds. Like other PUFAs, both free and membrane-bound AA undergo autoxidation in the presence of initiators such as air, light, heat, and transition metal ions. While autoxidation yields both cis- and trans-EETs with preference to the later, cytochrome P450 (CYP) epoxygenases, especially CYP2J2, exclusively catalyze the formation of all regioisomer of cis-EETs. The overall goals of this dissertation were to elucidate the formation of enzymatic and non-enzymatic EETs during P450 incubations and in biological systems and determine the protective role EETs play during ischemic cardiomyopathy. To achieve these goals, we used in vitro systems, a mouse model that overexpresses human CYP2J2 in the heart cardiomyocytes, and tissue from diseased and control human subjects. In in vitro work, formation of EETs in free radical initiated reactions of AA in benzene and in liposomes exhibited time- and AA concentration-dependence and favored the formation of trans-EETs over cis-EETs. Experimental conditions were optimized to minimize non-enzymatic EET formation during P450 reactions by adding pyruvate, a hydrogen peroxide scavenger, and an iron chelating agent. In the in vivo mouse model, stressors such as age and disease altered EET levels in transgenic (Tr) mice. EET levels in erythrocyte membranes increased with age while alterations appeared to be regioisomer-specific for cardiac EETs. Effects of acute ischemic cardiac events were evaluated in both a mouse model and human sudden cardiac arrest (SCA) subjects. Acute myocardial infarction in the mouse model increased both erythrocyte membrane and cardiac tissue cis- and trans-EETs in Tr mice, while human SCA cases had significantly lower EET levels in their erythrocyte membrane compared to the control group. In ventricular cardiac tissue obtained from patients with cardiovascular disease and controls, EET levels were significantly higher in control tissue compared to diseased. Lower EET levels in diseased cardiac tissue were associated with lower protein expression of CYP2J2, NADPH-Cytochrome P450 oxidoreductase and soluble epoxide hydrolase. Finally, specific activity of CYP2J2 in hydroxylating the probe substrate, terfenadine was shown to be significantly decreased with lower levels of CYP2J2, the main cardiac epoxygenase, in diseased cardiac tissue. These results suggest that higher levels of EETs, specifically cis-EETs are cardioprotective, and that erythrocyte membrane of cis-EETs could potentially serve as surrogate markers to report on cis-EET levels in cardiac tissue.