Soluble epoxide hydrolase plays an essential role in angiotensin II-induced cardiac hypertrophy

Abstract

Pathophysiological cardiac hypertrophy is one of the most common causes of heart failure. Epoxyeicosatrienoic acids, hydrolyzed and degraded by soluble epoxide hydrolase (sEH), can function as endothelium-derived hyperpolarizing factors to induce dilation of coronary arteries and thus are cardioprotective. In this study, we investigated the role of sEH in two rodent models of angiotensin II (Ang II)-induced cardiac hypertrophy. The protein level of sEH was elevated in the heart of both spontaneously hypertensive rats and Ang II-infused Wistar rats. Blocking the Ang II type 1 receptor with losartan could abolish this induction. Administration of a potent sEH inhibitor (sEHI) prevented the pathogenesis of the Ang II-induced hypertrophy, as demonstrated by decreased left-ventricular hypertrophy assessed by echocardiography, reduced cardiomyocyte size, and attenuated expression of hypertrophy markers, including atrial natriuretic factor and beta-myosin heavy chain. Because sEH elevation was not observed in exercise- or norepinephrine-induced hypertrophy, the sEH induction was closely associated with Ang II-induced hypertrophy. In vitro, Ang II upregulated sEH and hypertrophy markers in neonatal cardiomyocytes isolated from rat and mouse. Expression of these marker genes was elevated with adenovirus-mediated sEH overexpression but decreased with sEHI treatment. These results were supported by studies in neonatal cardiomyocytes from sEH(-/-) mice. Our results suggest that sEH is specifically upregulated by Ang II, which directly mediates Ang II-induced cardiac hypertrophy. Thus, pharmacological inhibition of sEH would be a useful approach to prevent and treat Ang II-induced cardiac hypertrophy.

Fig. 1.

sEH protein level is elevated in the heart of SHR rats and Ang II-infused Wistar rats. (A–D) Wistar rats and SHR rats (180–280 g, male) had free access to tap water supplemented with 2% NaCl for 14 days. (E–G) Sprague–Dawley rats received a minipump implantation in the dorsal region to deliver Ang II at 450 ng/kg/min (A2) or PBS for 14 days. Western blot analysis was performed with GAPDH level as a control. The ratio of whole heart weight to body weight (HW/BW) and plasma Ang II (A2) levels were measured after the animals were killed. The cross-sections of the rat left ventricle underwent immunohistochemical staining with anti-sEH antibody in C. The sections were counterstained with hematoxylin and the average cell area of myocytes was measured on confocal microscopy in F. The results are presented as mean ± SD from 6 rats in each group (*, P < 0.05; **, P < 0.01).

Fig. 2.

Expression of sEH protein in the heart in three rat models. (A) Wistar rats (180–280 g, male) received losartan (Los, 25 mg/kg/d) or PBS (Ctrl) by oral gavage for 6 days. A minipump was then implanted in the dorsal region to deliver Ang II (A2) at 450 ng/kg/min (A2) or PBS for 3 days. (B) Rats were infused with norepinephrine (NE, 4.8 mg/kg/d) or vehicle (0.01% wt/vol Vit C) for 7 days. (C) Rats were trained with swimming (150 min/day, 5 days/week) for 8 weeks. n = 6 in each group. The ratios of whole heart weight to body weight (HW/BW) and/or left ventricular weight to body weight (LVW/BW) were measured after the animals were killed. Western blot and real-time PCR analysis was performed and the results shown are representative of 6 rats in each group.

Fig. 3.

sEH inhibitor attenuates Ang II-induced cardiac hypertrophy in Sprague–Dawley rats. Sprague–Dawley rats(180–280 g, male) received TUPS (0.65 mg/kg/day) or PEG400 by oral gavage for 21 days. Seven days after beginning TUPS administration, a minipump was implanted in the dorsal region to deliver Ang II (A2) for 14 days. PBS treatment was used as a control (Ctrl). (A) Systolic blood pressure (BP) was measured every 2 days after implantation. (B) Heart weight to body weight (HW/BW) was measured after rats were killed. (C) Cross-sections of rat left ventricles histochemically stained with picric-sirius red for fibrosis. (D) Real-time RT–PCR analysis of ANF and β-MHC mRNA level. Data are means ± SD of the relative mRNA normalized to that of 18S from 6 rats in each group. (E) Western blot analysis of sEH and GAPDH proteins was performed. The results are presented as mean ± SD from 6 rats in each group (*, P < 0.05; **, P < 0.01).

Fig. 4.

Role of sEH inhibitor in Ang II-induced cardiomyocyte hypertrophy: rat (A and C) or mouse (B). NCMs were treated with 100 nM Ang II (A2) with or without TUPS (1 μM) for 24 h. (A) Western blot analysis was performed with GAPDH level as a control. (B) The DHETs and EETs in cytosolic extracts were separated on reverse-phase HPLC and then quantified by use of a 4000 QTRAP tandem mass spectrometer. For cell area, cells were stained with α-actin, and the cell area of myocytes was measured on confocal microscopy. Data are means ± SD of 100 cell measurements from 3 independent experiments. (C) Quantitative RT–PCR analysis of mRNA level of ANF and β-MHC, the relative mRNA normalized to that of 18S (*, P < 0.05).

Fig. 5.

Role of sEH overexpression in cultured NCMs. (A and B) Rat NCMs were infected with Ad-sEH or Ad-GFP for 24 or 48 h. Western blot analysis of sEH protein level in A and quantitative RT–PCR analysis of mRNA level of ANF and β-MHC in B were measured. (C and D) Murine NCMs were infected with Ad-sEH or Ad-GFP for 24 h and sEH activity and the size of the myocytes were determined as described in Fig. 4. Data are means ± SD from 3 independent experiments (*, P < 0.05).

Fig. 6.

Role of sEH knockout on Ang II-induced hypertrophy in cultured mouse NCMs. Murine NCMs were isolated from sEH^−/− mice or their wild-type littermates (WT). Cell area and mRNA expression of β-MHC and ANF were determined on treatment with or without 100 nM Ang II (A2). Data are means ± SD from 3 independent experiments (*, P < 0.05).