Beneficial effects of soluble epoxide hydrolase inhibitors in myocardial infarction model: Insight gained using metabolomic approaches

Abstract

Myocardial infarction (MI) leading to myocardial cell loss represents one of the common causes leading to cardiac failure. We have previously demonstrated the beneficial effects of several potent soluble epoxide hydrolase (sEH) inhibitors in cardiac hypertrophy. sEH catalizes the conversion of epoxyeicosatrienoic acids (EETs) to form the corresponding dihydroxyeicosatrienoic acids (DHETs). EETs are products of cytochrome P450 epoxygenases that have vasodilatory properties. Additionally, EETs inhibit the activation of nuclear factor (NF)-kappaB-mediated gene transcription. Motivated by the potential to uncover a new class of therapeutic agents for cardiovascular diseases which can be effectively used in clinical setting, we directly tested the biological effects of sEH inhibitors (sEHIs) on the progression of cardiac remodeling using a clinically relevant murine model of MI. We demonstrated that sEHIs were highly effective in the prevention of progressive cardiac remodeling post MI. Using metabolomic profiling of the inflammatory lipid mediators, we documented a significant decrease in EETs/DHETs ratio in MI model predicting a heightened inflammatory state. Treatment with sEHIs resulted in a change in the pattern of lipid mediators from one of inflammation towards resolution. Moreover, the oxylipin profiling showed a striking parallel to the changes in inflammatory cytokines in this model. Our study provides evidence for a possible new therapeutic strategy to improve cardiac function post MI.

Figures

Fig. 1

Beneficial effects of sEHIs (AEPU and t-AUCB) in a mouse MI model. a, Structure of the two sEHIs used in our studies: 1-adamantan-1-yl-3-{5-[2-(2-ethoxy-ethoxy)-ethoxy]-pentyl}-urea (AEPU, 100 mg/L)[5] and trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB). b, Examples of whole hearts from MI mice treated with AEPU or t-AUCB compared to MI alone or sham-operated hearts. The mice were sacrificed after three weeks of follow up. Scale, 1 cm. c, Summary data for heart/tibial length ratios (mg/mm) from MI mice compared to untreated MI mice. Error bars are ± s.e.m. (n = 12). *p<0.05. Similar results were obtained with AEPU.

Fig. 2

a, Examples of two-dimensional and M-mode echocardiography in mouse models with sham operation, MI and MI treated with AEPU or t-AUCB after three weeks of treatment showing evidence of cardiac failure with chamber dilatation in MI mice. AEPU and t-AUCB prevented the development of chamber dilatation in MI mice. b, Histologic sections (Masson’s trichrome stain) of sham-operated and MI mouse hearts, showing infarct area with scarring and gross cardiac dilatation at 3 weeks in the MI mouse. Treatment of MI mice with t-AUCB in drinking water prevented the development of cardiac remodeling. All histologic cross sections are presented with right ventricles to the right. Scale bar, 100 μm. c, Summary data for % fractional shortening (FS). Data shown are mean ± s.e.m., n = 12 for each group, *p<0.05 comparing MI and sham animals, #p<0.05 comparing treated vs. untreated MI animals.

Fig. 3. Quantification of LV remodeling after MI using histologic analysis and whole-cell capacitance

a, Histologic sections (Masson’s trichrome stain) of LV anterior wall of sham-operated and MI mouse hearts, showing connective tissues in blue. b, Representative photomicrographs of bright-field images of single isolated LV cardiomyocyte remote from infracted area. c, d, Summary data for % infracted area and cell capacitance, respectively. Data shown are mean ± s.e.m., n = 12 mice for each group, *p<0.05. Similar data were obtained with AEPU.

Fig. 4. Prevention of cardiac arrhythmia inducibility and electrical remodeling in MI mice by sEH inhibitors

a,In-vivo EPS in sham and treated compared to untreated MI animals. Sham and treated MI animals are shown in the upper panels. Lower panels are from untreated MI animals showing evidence of inducible atrial fibrillation (lower left panel) and ventricular tachycardia (lower right panel). Upper four tracing are surface ECG (Lead I, II and aVF). Lower two tracings are intracardiac electrogram showing atrial, and ventricular electrograms. Summary data are presented in Supplemental Table 1. b, Examples of AP recordings from LV free wall myocytes isolated from sham animals compared to treated and untreated MI animals at three weeks of follow up. APs recorded from MI mice were significantly prolonged compared to sham animals. Treatment with t-AUCB resulted in the normalization of the AP prolongation. Summary data for APD at 50 and 90% repolarization (APD50 and APD90 in ms) are shown in Panel c. (*p<0.05 comparing sham and treated MI animals to MI alone, n=12-15 cells for each group). d, Examples of Ca2+-independent outward K+ current traces elicited from a holding potential of −80 mV using test potentials of 2.5 seconds in duration from −60 to +60 mV in 10-mV increments. e, Summary data for the density of the peak outward components (*p<0.05 comparing sham and treated MI animals to MI alone). n=11-13 cells for each group.

Fig. 5. Plasma Levels of Selected Oxylipin and Cytokines

a, b, Oxylipin profiling from sham, MI and MI treated with t-AUCB at 3 weeks of follow up (*p<0.05 comparing sham or treated MI groups to MI alone). c, Serum concentrations (in pg/ml) of IL-12p70, TNF-α, IFN-γ, MCP-1, IL-10 and IL-6 from sham, MI and MI treated with t-AUCB at 3 weeks of follow up (*p<0.05 comparing sham or treated MI groups to MI alone). See supplementary materials for full oxylipin profile.

Fig. 6. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) and Western blot analysis

a,In situ cell death detection kit was used for the detection of apoptotic cells. Three independent experiments were performed. TUNEL-positive cells were visualized using a confocal microscope. b, An example of a TUNEL-positive myocyte (red) was shown with cardiac myocyte membranes (green) and nuclei (blue). The fraction of apoptotic cells was determined by dividing the number of TUNEL-positive cells (red) by the total number of DAPI-positive cardiac myocyte nuclei (blue). c, Immunoblots showing sEH protein from LV free wall in four different groups of animals; treated and untreated sham and MI. GAPDH protein was used as an internal loading control. d, Summary data showing the densitometry of sEH protein level normalized to GAPDH level in the four groups of animals.