Inhibition of soluble epoxide hydrolase preserves cardiomyocytes: role of STAT3 signaling

Abstract

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs), primarily 14,15-EET. EETs are derived from arachidonic acid via P-450 epoxygenases and are cardioprotective. We tested the hypothesis that sEH deficiency and pharmacological inhibition elicit tolerance to ischemia via EET-mediated STAT3 signaling in vitro and in vivo. In addition, the relevance of single nucleotide polymorphisms (SNPs) of EPHX2 (the gene encoding sEH) on tolerance to oxygen and glucose deprivation and reoxygenation and glucose repletion (OGD/RGR) was assessed in male C57BL\6J (WT) or sEH knockout (sEHKO) cardiomyocytes by using transactivator of transcription (TAT)-mediated transduction with sEH mutant proteins. Cell death and hydrolase activity was lower in Arg287Gln EPHX2 mutants vs. nontransduced controls. Excess 14,15-EET and SEH inhibition did not improve cell survival in Arg287Gln mutants. In WT cells, the putative EET receptor antagonist, 14,15-EEZE, abolished the effect of 14,15-EET and sEH inhibition. Cotreatment with 14,15-EET and SEH inhibition did not provide increased protection. In vitro, STAT3 inhibition blocked 14,15-EET cytoprotection, but not the effect of SEH inhibition. However, STAT3 small interfering RNA (siRNA) abolished cytoprotection by 14,15-EET and sEH inhibition, but cells pretreated with JAK2 siRNA remained protected. In vivo, STAT3 inhibition abolished 14,15-EET-mediated infarct size reduction. In summary, the Arg287Gln mutation is associated with improved tolerance against ischemia in vitro, and inhibition of sEH preserves cardiomyocyte viability following OGD/RGR via an EET-dependent mechanism. In vivo and in vitro, 14,15-EET-mediated protection is mediated in part by STAT3.

Fig. 1.

Experimental timeline and treatments. A: cardiomyocytes were subjected to 90 min oxygen and glucose deprivation (OGD) followed by 180 min reoxygenation and glucose repletion (RGR). Viability was assessed at baseline, the end of OGD, and at 1, 2, and 3 h of reperfusion. TAT-soluble epoxide hydrolase (sEH) fusion proteins were administered 24 h before OGD. Small interfering RNAs (siRNAs) were added to the culture medium 48 h before OGD. 14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-epoxyeiosa-5(Z)-enoic acid (14,15-EEZE), and sEH inhibitors were given for 1 h prior to OGD. The STAT3 inhibitor compound Stattic or STAT3 inhibitor VI was added 5 min before EET or N-adamantanyl-N′-dodecanoic acid urea (AUDA) treatment. B: adult male C57BL\6J mice underwent temporal left coronary artery (LCA) occlusion followed by 2 h of reperfusion. Infarct size in relation to ischemic area at risk was assessed. STAT3 inhibitor compound was given intravenously 20 min and 14,15-EET was given 15 min before occlusion. 4-PCO, 4-phenylchalcone oxide.

Fig. 2.

sEH polymorphism. A: time course of transduction with human wild-type (WT) sEH, as assessed by Western blot for human recombinant (hr) sEH over 24 h. The right-hand lane is nontransduced naive cardiomyocytes. B: representative image of cardiomyocytes successfully transfected with human WT sEH (left side) by costaining with anti-His-tag antibody and 4,6-diamidino-2-phenylindole. Right: naive, nontransfected control (n = 3). C: Arg287Gln mutations reduced cell death compared with nontransduced controls (45 ± 1 vs. 58 ± 1%, P < 0.001). The Arg287Gln mutation was previously shown to exhibit reduced hydrolase activity. As shown in C, excess 14,15-EET (1 μM) improved cell survival in all polymorphisms tested except for Arg287Gln (45 ± 1% TAT-hr-sEH-Arg287Gln vs. 45 ± 1% TAT-hr-sEH-Arg287Gln + 14,15-EET). Similarly, D shows that pharmacological blockade of sEH with 4-PCO improved cell survival in all mutants with the exception of Arg287Gln (34 ± 1% TAT-hr-sEH-Arg287Gln vs. 33 ± 1% TAT-hr-sEH-Arg287Gln + 4-PCO, respectively). E: sEH activity of different sEH single nucleotide polymorphism of the EPHX2 gene as indexed by 14,15-dihydroxyeicosatrienic acid (DHET) production. Production of 14,15-DHET was lower in TAT-hr-sEH-Arg287Gln mutants compared with TAT-hr-sEH-WT. All samples were spiked with 1 μM 14,15-EET to ensure adequate substrate. F: transduction with human WT sEH increased cell death in cardiomyocytes from sEH knockout (sEHKO), whereas the human Arg287Gln polymorphism had no effect on viability (n = 3 replicas). Data are presented as means ± SE.

Fig. 3.

sEH inhibition and 14,15-EET administration provide tolerance to OGD. A: to determine whether the tolerance to OGD that is induced by 14,15-EET (1 μM) is sustained or merely a delay in the expression of cell death, cardiomyocytes were subjected to OGD and then followed for 6 h RGR. As can be seen in the figure, the 14,15-EET-induced survival advantage was maintained for the entire duration of the 6 h of assessment period, suggesting that the salutary effect is long lasting (n = 3 replicates). B: to examine whether sEH inhibition and 14,15-EET (1 μM) show additive cytoprotection, cardiomyocytes were treated with 14,15-EET and then subjected to OGD followed by RGR in the presence or absence of the sEH inhibitor 4-PCO (2 μM). The main substrate of sEH, 14,15-EET, provided tolerance to OGD that was comparable to that elicited by sEH inhibition with 4-PCO. However, coadministration of 14,15-EET and 4-PCO did not provide additive protection (n = 5 replicates). C: to examine whether the cytoprotection of exogenous administration of 14,15-EET, or sEH inhibition with 4-PCO (2 μM) or AUDA (2 μM), is sensitive to blockade with the EET antagonist EEZE (1 μM), cells were subjected to 1.5-h OGD followed by 3-h RGR. Cardiomyocytes were incubated with 14,15-EET or sEH inhibitors for 1 h prior to initiation of OGD. The EET antagonist EEZE was given concomitantly with 14,15-EET, 4-PCO, or AUDA. Exogenous 14,15-EET produced a marked increase in cell survival following OGD. Inhibition of sEH with 4-PCO or AUDA elicited a comparable protective effect. Tolerance to OGD was abolished by administration of 14,15-EEZE, showing that the salutary effect observed with EET treatment or sEH inhibition was specifically due to EETs (n = 3 replicates). D: to determine whether sEH inhibition or gene deletion mediates a comparable cytoprotective effect, cardiomyocytes from male WT and sEHKO mice were subjected to OGD, and cell death was assessed at 180 min of RGR. The effect was compared with exogenous 14,15-EET (n = 4–5 replicas). AUDA resulted in improved survival following OGD in WT, which was less than the survival benefit of 14,15-EET. However, in cardiomyocytes from sEHKO mice, AUDA produced an increase in cell death whereas 14,15-EET improved survival. Of note, cell death was lower in untreated sEHKO cardiomyocytes compared with WT (solid bars). No additive effect on cell viability was detected in cardiomyocytes treated simultaneously with the sEH inhibitor AUDA (2 μM) and 3 increasing doses of 14,15-EET (0.1, 0.3, and 1 μM) (E; n = 3 replicates). Data are presented as means ± SE.

Fig. 4.

sEH inhibition and 14,15-EET rapidly increase phosphorylation of STAT3. STAT3 phosphorylation is seen at Tyr⁷⁰⁵ following incubation in AUDA (2 μM) (A) or 14,15-EET (1 μM) (B) but not at Ser⁷²⁷ following 5 min of 14,15-EET (1 μM) incubation (C). After 5 min of incubation, cardiomyocytes showed a significantly increased degree of phosphorylation (at tyrosine 705) compared with untreated control (n = 5–6 replicates). D: individual Western blots. STAT3 p/t, ratio of phosphorylated (pStat3) to total STAT3 (tStat3) protein. Data are shown as means ± SE.

Fig. 5.

STAT3 inhibition partially abolishes cytoprotective effect of sEH inhibition and 14,15-EET. To determine the downstream mechanism of sEH inhibition, we used 2 different STAT3 inhibitors and assessed cardiomyocyte viability following OGD and RGR. Both STAT3 inhibitors partially abolished the cytoprotective effect of AUDA (2 μM) and 14,15-EET (1 μM). However, cells still remained partially protected against OGD-induced cell death compared with vehicle control (n = 3–5 replicas). A: cell death as a function of time. B: same data plotted as cell death at 180 min RGR. C: data from B are normalized as a percentage of control (because of a slight increase in cell death with Stattic or STAT3 inhibitor VI (S31-201) alone: AUDA and 14,15-EET are normalized to vehicle control, and inhibitor+AUDA and inhibitor+14,15-EET are normalized to inhibitor alone). NS, not significant. Data are presented as means ± SE.

Fig. 6.

STAT3 siRNAs abolish 14,15-EET- and AUDA-mediated cytoprotection, but not JAK2 siRNA. To determine the role of JAK2 and STAT3 in 14,15-EET-mediated cytoprotection and cytoprotection via sEH inhibition (AUDA) following OGD and RGR, isolated cardiomyocytes were pretreated with siRNA for STAT3 and JAK2 for 48 h and cell death was assessed. Shown is the effect on cardiomyocyte viability in the presence or absence of 14,15-EET (1 μM; n = 5 replicas) pretreatment (A) and pretreatment with AUDA (2 μM; n = 4 replicas; B). STAT3 siRNA completely abolished the cytoprotective effect of 14,15-EET and AUDA, whereas cells pretreated with JAK2 siRNA still showed protection. C: relative reduction in protein levels (STAT3 and JAK2) compared with negative siRNA after 48 h incubation with siRNA. D: 2 representative Western blots for JAK2 and STAT3 following 48 h of incubation with siRNA. STAT3 siRNA, small interfering RNA against STAT3; JAK2 siRNA, small interfering RNA against JAK2; siRNA control, silencer select negative control no. 1 small interfering RNA. Data are presented as means ± SE.

Fig. 7.

STAT3 inhibition abolishes 14,15-EET-induced reduction in infarct size in vivo. To determine the role of STAT3 in 14,15-EET-mediated cardioprotection in vivo, adult male mice were subjected to 40 min of temporary occlusion of the LCA followed by 2 h of reperfusion. A: 14,15-EET (2.5 μg/g iv) significantly reduced the infarct size, and STAT3 inhibition by STAT3 inhibitor VI (6 μg/g iv) completely abolished this cardioprotective effect (n = 8 per group). B: shows that the ischemic area at risk was similar in all 4 groups tested. EtOH, ethanol; AAR, area at risk; LV, left ventricle. Data are shown as means ± SE.