Genetic deletion of soluble epoxide hydrolase preserves cardiac function and limits inflammation in acute lipopolysaccharide injury

Abstract

Objective Acute systemic inflammatory syndromes, such as endotoxemia, elicit detrimental multi-organ responses. The heart is particularly susceptible to an acute inflammatory response resulting in myocardial dysfunction which often leads to death. A stress-induced metabolism of N-3 and N-6 polyunsaturated fatty acids (PUFAs) will generate numerous bioactive lipid mediators with beneficial or adverse effects. Cytochrome P450 (CYP) derived epoxylipid mediators may be further hydrolyzed to diol metabolites by soluble epoxide hydrolase (sEH). Emerging evidence suggests epoxylipids can exert cardioprotective effects by modulating the NLRP3 inflammasome pathway. This study investigated whether cardiomyocyte specific sEH knockdown can preserve cardiac function and attenuate inflammation in a model of acute lipopolysaccharide (LPS) injury. Hypothesis Conditional knockdown of cardiomyocyte sEH protects the heart from acute LPS injury via modulation of the NLRP3 inflammasome pathway. Methods Cardiomyocyte targeted sEH knockdown mice were produced by crossing Ephx2 floxed mice with mice expressing Cre recombinase. Male sEHFl/Fl (knockdown) and sEH+/+ (Cre control) mice were treated with tamoxifen (45 mg/kg, 6 i.p injections over 8 days) 5 weeks prior to LPS injection (10mg/kg, i.p.). A comparator group of male wild type (WT) and global sEH null mice were subjected to LPS treatment. Echocardiography was conducted pre-injection and 6 or 24hr post-LPS followed by euthanization. Plasma cytokine and chemokine levels were determined with a multi-plex assay. In vitro studies employed neonatal rat cardiomyocytes treated with LPS (1µg/mL), 19, 20-epoxydocosapentaenoic acid (EDP, 1µM) or sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB, 10µM), for 6hr. NLRP3 and pro-IL-1β expression was assessed using Western immunoblotting. Results Genetic deletion of sEH preserved cardiac function following 24hr LPS exposure indicating an initial early decline at 6hr which was recovered in both sEHFl/Fl and sEH null but not WT mice. The increased plasma levels of pro-inflammatory cytokines and chemokines following LPS treatment was attenuated in mice lacking sEH expression. Cardiomyocytes treated with LPS had increased NLRP3 inflammasome complex formation and pro-IL-1β expression, which was not attenuated by co-treatment with EDP or tAUCB. However, plasma levels of IL-1β were reduced in both sEHFl/Fl and global sEH null mice following LPS exposure. Conclusion Deletion of sEH protects cardiac function and limits pro-inflammatory response following exposure to LPS. Preliminary data suggest epoxylipids do not prevent the formation of the NLRP3 inflammasome complex in cardiomyocytes but may impact downstream effectors. Thus, limiting the NLRP3 inflammasome cascade to reduce LPS-induced cardiac and inflammatory injury.