Inhibition of Soluble Epoxide Hydrolase Reduces Inflammation and Myocardial Injury in Arrhythmogenic Cardiomyopathy

Abstract

We analyzed the role of pro- and anti-inflammatory eicosanoids in the pathogenesis of arrhythmogenic cardiomyopathy (ACM). Lipidomics revealed reduced levels of anti-inflammatory oxylipins in plasma and increased levels of pro-inflammatory eicosanoids in hearts of Dsg2^mut/mut mice, a preclinical model of ACM. Disease features were reversed in vitro in rat ventricular myocytes expressing mutant JUP by the anti-inflammatory epoxyeicosatrienoic acid 14-15-EET, whereas 14,15-EEZE, which antagonizes the 14,15-EET receptor, intensified nuclear accumulation of the desmosomal protein plakoglobin. Inhibition of soluble epoxide hydrolase (sEH), an enzyme that converts anti-inflammatory EETs into polar, less active diols, prevented progression of myocardial injury in Dsg2^mut/mut mice and promoted recovery of contractile function. This was associated with reduced myocardial expression of genes involved in innate immune signaling and fewer injurious macrophages expressing CCR2. These results suggest that pro-inflammatory eicosanoids contribute to the pathogenesis of ACM. Inhibition of sEH may be an effective, mechanism-based therapy for ACM patients.

Keywords: lipidomics; oxylipins; pro-inflammatory eicosanoids.

Graphical abstract

Figure 1

Lipidomics Assays and Expression of Endoplasmic Reticulum Stress Genes in Dsg2^mut/mut Mice (A) Free oxlipin levels in plasma and hearts from wild-type (WT) and Dsg2^mut/mut mice at ages 8 or 16 weeks quantified by liquid chromatography/tandem mass spectrometry; n = 5-9, ∗P < 0.05 vs WT. Heart oxylipins are displayed in a volcano plot of Log₂ fold relative concentration. Oxylipins above the dashed line represent significant differences between WT and Dsg2^mut/mut mice. (B) quantitative polymerase chain reaction showing increased expression of the endoplasmic reticulum chaperone gene BiP and the protein folding protein disulfide isomerase gene Pdi, both markers of endoplasmic reticulum stress. Gene expression values in WT samples were normalized to 1; values in Dsg2^mut/mut samples are shown as relative levels; ∗P < 0.05 vs WT by t-test.

Figure 2

Effects of 14,15-EET and its Antagonist on ACM Features In Vitro Effects of 14,15-eicosatrienoic acid (14,15-EET) and 14,15-eicosa-5(Z)-enoic acid (14,15-EEZE) on the distribution of immunoreactive signals for plakoglobin, connexin43 (Cx43), and RelA/p65 in primary cultures of wild-type (WT) neonatal rat ventricular myocytes and myocytes transfected to express the arrhythmogenic cardiomyopathy disease allele JUP2157del2.

Figure 3

Uniform Manifold Approximation and Projection Plots Showing Populations of Cells Isolated From Hearts of 16-Week-Old Dsg2^mut/mut Mice and Relative Density of Ephx2 Expression in Each Population sEH (Ephx2) is expressed mainly in cardiac myocytes in the heart with lower levels of expression in endothelial cells.

Figure 4

Effects of sEH Inhibition on ACM Features In Vitro Effects of 1μM TPPU (1-trifluoro-methoxy-phenyl-3-(1-propionylpiperidin-4-yl) urea) and 500 nM PTUPB (4-(5-phenyl-3-{3-[3-(4-trifluoromethylphenyl)-ureido]-propyl}-pyrazol-1-yl)-benzenesulfonamide) on the distribution of immunoreactive signals for plakoglobin, connexin43 (Cx43), and RelA/p65 in primary cultures of wild-type (WT) neonatal rat ventricular myocytes and myocytes transfected to express the arrhythmogenic cardiomyopathy disease allele JUP2157del2.

Figure 5

Effects of sEH Inhibition on Contractile Function in Dsg2^mut/mut Mice Effects of TPPU (1-trifluoro-methoxy-phenyl-3-(1-propionylpiperidin-4-yl) urea) on left ventricular ejection fraction (A) and fractional shortening (B) in wild-type (WT) and Dsg2^mut/mut mice. (C) Representative echocardiograms at the follow-up time point after treatment. Baseline echocardiography was performed in 9-week-old mice and then repeated after treatment for 4 weeks with TPPU or vehicle (Veh). Data are shown for each group (left) and each individual animal (right). The Shapiro-Wilk test was used to assess normality within each of the 4 groups. No significant deviations from normality were found, indicating an approximate normal distribution. Then, Tukey’s 1-way analysis of variance was used. ∗∗∗∗P < 0.0001 Dsg2^mut/mut + Vehicle follow-up vs Dsg2^mut/mut + TPPU follow-up. Data are shown as mean with SEM.

Figure 6

Effects of sEH Inhibition on Fibrosis and CCR2+ Cells in Heart of Dsg2^mut/mut Mice Effects of TPPU (1-trifluoro-methoxy-phenyl-3-(1-propionylpiperidin-4-yl) urea) on the amount of myocardial fibrosis (A) and the number of cells expressing CCR2 (B) in wild-type (WT) and Dsg2^mut/mut mice (positive cells identified by arrows in representative immunostained tissue sections). Hearts were excised from animals after treatment for 4 weeks with TPPU or vehicle (Veh) and analyzed by histology in trichrome stained sections or by immunohistochemistry in sections stained with an anti-CCR2 antibody. The Shapiro-Wilk test was used to assess normality within each of the 4 groups. No significant deviations from normality were found, indicating an approximate normal distribution. Then, Tukey’s 1-way analysis of variance was used. ∗P < 0.05 WT vs vehicle-treated Dsg2^mut/mut; ∗P < 0.05 vehicle-treated Dsg2^mut/mut vehicle-treated vs 1770-treated Dsg2^mut/mut mice. Data are shown as mean with SEM.

Figure 7

Effects of sEH Inhibition on Expression of Innate Immune Signaling Genes Effects of TPPU (1-trifluoro-methoxy-phenyl-3-(1-propionylpiperidin-4-yl) urea) on expression of Tnfα, Tlr4, and Ccr2 in hearts of wildtype (WT) and Dsg2^mut/mut mice treated with TPPU or vehicle (Veh). Gene expression values in wildtype samples treated with vehicle were normalized to 1; values in all other groups are shown as relative levels. The Shapiro-Wilk test was used to assess normality within each of the 4 groups. No significant deviations from normality were found, indicating an approximate normal distribution. All tests were 1-way analysis of variance. ∗P < 0.05 Dsg2^mut/mut vs WT Veh; ∗P < 0.05 Dsg2^mut/mut vs Dsg2^mut/mut + TPPU. Data are shown as mean with SEM.