Deletion of soluble epoxide hydrolase enhances coronary reactive hyperemia in isolated mouse heart: role of oxylipins and PPARγ

Abstract

The relationship between soluble epoxide hydrolase (sEH) and coronary reactive hyperemia (CRH) response to a brief ischemic insult is not known. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects in ischemia/reperfusion injury. sEH converts EETs into dihydroxyeicosatrienoic-acids (DHETs). Therefore, we hypothesized that knocking out sEH enhances CRH through modulation of oxylipin profiles, including an increase in EET/DHET ratio. Compared with sEH^+/+, sEH^-/- mice showed enhanced CRH, including greater repayment volume (RV; 28% higher, P < 0.001) and repayment/debt ratio (32% higher, P < 0.001). Oxylipins from the heart perfusates were analyzed by LC-MS/MS. The 14,15-EET/14,15-DHET ratio was 3.7-fold higher at baseline (P < 0.001) and 5.6-fold higher post-ischemia (P < 0.001) in sEH^-/- compared with sEH^+/+ mice. Likewise, the baseline 9,10- and 12,13-EpOME/DiHOME ratios were 3.2-fold (P < 0.01) and 3.7-fold (P < 0.001) higher, respectively in sEH^-/- compared with sEH^+/+ mice. 13-HODE was also significantly increased at baseline by 71% (P < 0.01) in sEH^-/- vs. sEH^+/+ mice. Levels of 5-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids were not significantly different between the two strains (P > 0.05), but were decreased postischemia in both groups (P = 0.02, P = 0.04, P = 0.05, P = 0.03, respectively). Modulation of CRH by peroxisome proliferator-activated receptor gamma (PPARγ) was demonstrated using a PPARγ-antagonist (T0070907), which reduced repayment volume by 25% in sEH^+/+ (P < 0.001) and 33% in sEH^-/- mice (P < 0.01), and a PPARγ-agonist (rosiglitazone), which increased repayment volume by 37% in both sEH^+/+ (P = 0.04) and sEH^-/- mice (P = 0.04). l-NAME attenuated CRH in both sEH^-/- and sEH^+/+ These data demonstrate that genetic deletion of sEH resulted in an altered oxylipin profile, which may have led to an enhanced CRH response.

Keywords: coronary reactive hyperemia; dihydroxyeicosatrienoic acids; epoxyeicosatrienoic acids; isolated perfused heart; oxylipins; soluble epoxide hydrolase.

Fig. 1.

Comparison of repayment volume (RV) between female and male wild-type soluble epoxide hydrolase (sEH^+/+) and sEH^−/− (sEH-null) mice. Repayment volume was not significantly different between female (F-sEH^+/+) and male (M-sEH^+/+) WT mice (P > 0.05) and between female (F-sEH^−/−) and male (M-sEH^−/−) sEH-null mice (P > 0.05). However, M-sEH^−/− had more enhanced (P = 0.04) repayment volume compared with M-sEH^+/+. Also, F-sEH^−/− had more enhanced (P = 0.01) repayment volume compared with F-sEH^+/+. *P ≤ 0.05 vs. baseline F-sEH^+/+. #P ≤ 0.05 vs. M-sEH^+/+. n = 6 per group.

Fig. 2.

Comparison of coronary reactive hyperemia (CRH) between wild type (sEH^+/+) and sEH^−/− (sEH-null) mice. A: tracing depicting coronary flow changes at baseline (before CRH) and after CRH was induced by 15-s no-flow ischemia in both sEH^+/+ (dash line) and sEH^−/− (continuous line) mice. Repayment volume (B; P < 0.001) and repayment/debt ratio (C; P = 0.0004) were enhanced in sEH^−/− vs. sEH^+/+ mice. Repayment duration (D) was increased in sEH^−/− vs. sEH^+/+, but did not reach statistical significance (P = 0.31). Peak hyperemic flow, baseline coronary flow, left-ventricular developed pressure (LVDP) and heart rate (HR) were not significantly different between the two groups (data not shown). *P ≤ 0.05 sEH^+/+. n = 12 per group.

Fig. 3.

LC-MS/MS analysis for 14, 15-EETs and 14,15-DHETs in sEH^+/+ and sEH^−/− mice heart perfusate at baseline (preischemia) and directly after 15-s ischemia (post-ischemia). A: at baseline and post-ischemia, 14,15-EET was increased 2.5 fold in sEH^−/− vs. sEH^+/+ mice (P < 0.001), whereas 14,15-DHET was decreased (P < 0.001). There was no difference in either metabolite preischemia and postischemia within the same strain. B: ratio between 14,15-EET and 14,15-DHET was increased at baseline and postischemia in sEH^−/− vs. sEH^+/+ mice (P < 0.001). *P ≤ 0.05 vs. baseline sEH^+/+. #P ≤ 0.05 vs. postischemia sEH^+/+. n = 7 sEH^+/+, n = 10 sEH^−/−.

Fig. 4.

LC-MS/MS analysis of HETEs in sEH^+/+ and sEH^−/− heart perfusate at baseline (preischemia) and directly after 15-s ischemia (post-ischemia). All detected HETEs (5-, 11-, 12-, and 15-HETE), were decreased postischemia (after perfusion was reinstated) compared with baseline in both sEH^+/+ and sEH^−/− mice (P = 0.02, P = 0.04, P = 0.05, and P = 0.03 respectively; A–D). Only 12-HETE in sEH^+/+ mice did not reach statistically significant level (P = 0.09). There was no difference in any of the measured HETEs between sEH^+/+ and sEH^−/− mice (P > 0.05). *P ≤ 0.05 vs. baseline sEH^+/+. #P ≤ 0.05 vs. baseline sEH^−/−. n = 7 sEH^+/+, n = 10 sEH^−/−.

Fig. 5.

LC-MS/MS analysis of 9,–10-epoxyoctadecaenoic acids (EpOMEs) and dihydroxyoctadecaenoic acids (DiHOMEs) in sEH^+/+ and sEH^−/− mouse heart perfusate samples at baseline (preischemia) and directly after 15-s ischemia (postischemia). A: 9,10-EpOME was increased at baseline (P = 0.03) and postischemia, but was not statistically significant (P = 0.21), whereas 12,13-EpOME was increased both at baseline and postischemia (P = 0.03) in sEH^−/− vs. sEH^+/+ mice. B: 9,10- and 12,13-DiHOMEs were decreased at baseline (P = 0.001), and post-ischemia (P < 0.001) in sEH^−/− vs. sEH^+/+ mice. A decreasing trend in EpOMEs postischemia compared with baseline was observed in sEH^−/− mice, but was not statistically significant. C: 9,10- and 12,13-EpOME/DiHOME ratios were increased at baseline (P = 0.002) and postischemia (P = 0.0008). *P ≤ 0.05 vs. baseline sEH^+/+. #P ≤ 0.05 vs. postischemia sEH^+/+. n = 7 sEH^+/+, n = 10 sEH^−/−.

Fig. 6.

LC-MS/MS analysis of hydroxyoctadecadienoic acid (HODEs) in sEH^+/+ and sEH^−/− mouse heart perfusate samples at baseline (preischemia) and directly after 15-s ischemia (postischemia). Baseline and postischemia analysis 9- and 13-HODEs in sEH^−/− vs. sEH^+/+. 13-HODE, but not 9-HODE, was increased in sEH^−/− vs. sEH^+/+ mice at baseline and postischemia (P = 0.006). There was no change in either HODEs before and after ischemia in both groups (P > 0.05). *P ≤ 0.05 vs. baseline sEH^+/+. #P ≤ 0.05 vs. postischemia sEH^+/+. n = 7 sEH^+/+, n = 10 sEH^−/−.

Fig. 7.

Effect of PPARγ-antagonist (T0070907, 10 μM) on CRH in sEH^+/+ and sEH^−/− mice. Infusion of T0070907 decreased CRH in both sEH^+/+and sEH^−/− mice. In sEH^+/+ mice, T0070907 decreased repayment volume (RV) by 25% (A; P < 0.001), repayment/debt (R/D) ratio by 14% (B; P = 0.004), and repayment duration (RD) by 54% (C; P = 0.02). In sEH^−/−, T0070907 decreased RV by 33% (A; P = 0.0039), repayment/debt ratio by 32% (B; P = 0.01), and RD by 54% (C; P = 0.003). Comparing sEH^−/− and sEH^+/+ mice: repayment volume and repayment/debt ratio were increased more in sEH^−/− vs. sEH^+/+ mice (A: P = 0.048 and B: P = 0.04, respectively). In both sEH^+/+ and sEH^−/− mice, baseline CF (D), PHF (E), HR (F), and LVDP (data not shown) were not changed by T0070907. *P ≤ 0.05 vs. sEH^+/+. #P ≤ 0.05 vs. sEH^−/−. n = 6 per group.

Fig. 8.

Comparison CRH for wild-type (sEH^+/+) mice before and after infusion of rosiglitazone (PPARγ-agonist, 10 μM); administration of rosiglitazone increased CRH in both sEH^+/+and sEH^−/− mice. In sEH^+/+ mice, repayment volume (RV) was increased by 37% (A; P = 0.04), repayment/debt ratio (R/D) by 38% (B; P = 0.03), repayment duration (RD) by 38% (C; P = 0.05), and baseline coronary flow (CF) (D; P = 0.0280). In sEH^−/− mice, rosiglitazone increased RV by 37% (A; P = 0.04), repayment/debt ratio (R/D) by 23% (B; P = 0.03), RD by 50% (C; P = 0.01), and baseline coronary flow (CF) (D; P = 0.04). Comparing sEH^−/− and sEH^+/+ mice: RV and R/D were increased more in sEH^−/− vs. sEH^+/+ mice (P = 0.02 and P = 0.05, respectively). In both sEH^+/+ and sEH^−/− mice, PHF (E), HR (F), and LVDP (data not shown) were not significantly changed by rosiglitazone. *P ≤ 0.05 vs. sEH^+/+. #P ≤ 0.05 vs. sEH^−/−. n = 6 sEH^+/+, n = 7 sEH^−/−.

Fig. 9.

Comparison of the effect of N^ω-nitro-l-arginine methyl ester hydrochloride (l-NAME) (nitric oxide synthase inhibitor) on CRH in sEH^−/− and sEH^+/+ mice before and after infusion of l-NAME (100 μM). l-NAME decreased CRH in both sEH^+/+ and sEH^−/− mice, and CRH responses between the two groups were not statistically significant (P = 0.36). In sEH^+/+, l-NAME decreased RV by 43% (A; P = 0.002), RD by 47% (C; P = 0.002), baseline coronary flow (CF) by 36% (D; P < 0.001), peak hyperemic flow (PHF) (E; P = 0.001), and heart rate (HR) (F; P = 0.01). Repayment/debt (R/D) ratio (B) and LVDP (not shown) were not different between the two groups. In sEH^−/− mice, l-NAME decreased RV by 54% (A; P = 0.005), R/D ratio by 26% (B; P = 0.03), RD by 61% (C; P < 0.001), and baseline CF by 36% (D; P < 0.001), PHF (E), HR (F) and LVPD (not shown) were not significantly changed by l-NAME. RV and RD were increased more in untreated sEH^−/− vs. untreated sEH^+/+ mice (A, P = 0.01 and C, P = 0.01, respectively). n = 8 per group. *P < 0.05 vs. sEH^+/+. #P < 0.05 vs. sEH^−/−.

Fig. 10.

A schematic diagram comparing the changes observed for selected oxylipins in response to short-lived ischemia and their possible impact on CRH between sEH^−/− and sEH^+/+ mice. sEH deletion enhanced CRH possibly through increased 14,15-EET/DHET ratio, increased 13-HODE, and increased EpOME/DiHOME ratio. Role of NO, PPARγ, and midchain HETEs was comparable in both sEH^−/− and sEH^+/+ mice.