CYP2J2-derived epoxyeicosatrienoic acids suppress endoplasmic reticulum stress in heart failure

Abstract

Prolonged endoplasmic reticulum (ER) stress causes apoptosis and is associated with heart failure. Whether CYP2J2 and its arachidonic acid metabolites [epoxyeicosatrienoic acids (EETs)] have a protective influence on ER stress and heart failure has not been studied. Assays of myocardial samples from patients with end-stage heart failure showed evidence of ER stress. Chronic infusion of isoproterenol (ISO) or angiotensin II (AngII) by osmotic mini-pump induced cardiac hypertrophy and heart failure in mice as evaluated by hemodynamic measurements and echocardiography. Interestingly, transgenic (Tr) mice with cardiomyocyte-specific CYP2J2 expression were protected against heart failure compared with wild-type mice. ISO or AngII administration induced ER stress and apoptosis, and increased levels of intracellular Ca(2+). These phenotypes were abolished by CYP2J2 overexpression in vivo or exogenous EETs treatment of cardiomyocytes in vitro. ISO or AngII reduced sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a) expression in hearts or isolated cardiomyocytes; however, loss of SERCA2a expression was prevented in CYP2J2 Tr hearts in vivo or in cardiomyocytes treated with EETs in vitro. The reduction of SERCA2a activity was concomitant with increased oxidation of SERCA2a. EETs reversed SERCA2a oxidation through increased expression of antioxidant enzymes and reduced reactive oxygen species levels. Tempol, a membrane-permeable radical scavenger, similarly decreased oxidized SERCA2a levels, restored SERCA2a activity, and markedly reduced ER stress response in the mice treated with ISO. In conclusion, CYP2J2-derived EETs suppress ER stress response in the heart and protect against cardiac failure by maintaining intracellular Ca(2+) homeostasis and SERCA2a expression and activity.

Fig. 1.

SERCA2a expression and activity were reduced in failing human hearts. (A) The expression of SERCA2a in normal (N1 and N2) and failing (P1–P6) human hearts is shown. Corresponding clinical characteristics of the six patients with heart failure are shown in Table 1. (B) SERCA2a activity was decreased in failing human hearts. Proteins were normalized to β-actin (normal human hearts, n = 4; failing human hearts, n = 6). *P < 0.05 versus normal hearts.

Fig. 2.

Attenuation of cardiac hypertrophy and dysfunction induced by ISO or AngII in CYP2J2 Tr mice. (A) CYP2J2 protein level was increased in CYP2J2 Tr mice compared with WT mice. (B) Urinary 14,15-DHET levels were increased in CYP2J2 Tr mice compared with WT mice (n = 10 per group). *P < 0.05 versus WT mice. (C and E) Representative gross appearance of hearts (left) from CYP2J2 Tr and WT mice treated with ISO or AngII, respectively. (D and F) The ratio of heart weight/body weight (HW/BW) of CYP2J2 Tr and WT mice treated with ISO or AngII, respectively (n = 5). *P < 0.05 versus WT mice treated with saline; ^#P<0.05 versus WT mice treated with ISO or AngII. Scale bar, 5 mm.

Fig. 3.

ER stress signaling was suppressed in CYP2J2 Tr mice. (A) ISO-induced ER stress and apoptosis was alleviated in CYP2J2 Tr mice. (B) Densitometric analysis of ER stress markers was shown (n = 5 per group). (C) AngII-induced ER stress and apoptosis were also inhibited in CYP2J2 Tr mice. (D) Densitometric analysis of ER stress markers was shown (n = 5 per group). (E) Representative images of TUNEL staining showing cardiac myocytes apoptosis and quantitative analysis of TUNEL-positive myocardial cells in mice. Nuclei of normal cells are blue, and nuclei of apoptotic cells (TUNEL-positive cells) from the same fields are identified by green fluorescence (n = 5). *P < 0.05 versus WT mice treated with saline; ^#P<0.05 versus WT mice treated with ISO or AngII. Scale bar, 50 μm. Original magnification, 400×.

Fig. 4.

CYP2J2 overexpression restored CaMKII and SERCA2a expression and activity in mice failing hearts. (A) Elevated CaMKII activation induced by ISO or AngII was inhibited in CYP2J2 Tr mice (n = 5 per group). (B) Changes in b-IAM–SERCA2a and expression of SERCA2a were attenuated in CYP2J2 Tr mice exposed to ISO or AngII (n = 5 per group). (C) The activity of SERCA2a was restored in CYP2J2 Tr mice exposed to ISO or AngII (n = 5). (D) The detection of ROS using dihydroethidium (DHE) staining in mouse hearts. Quantification of ROS from three random fields per mouse (n = 5 per group). *P < 0.05 versus WT mice treated with saline; ^#P<0.05 versus WT mice treated with ISO or AngII. (E–G) The effect of Tempol on SERCA2a expression and activity, b-IAM–SERCA2a levels, and expression of ER stress markers (n = 5). *P < 0.05 versus control; ^#P<0.05 versus ISO. (H) Antioxidant enzyme expression in CYP2J2 Tr mice (n = 5 per group). *P < 0.05 versus WT mice treated with saline; ^#P<0.05 versus WT mice treated with ISO or AngII. Scale bar, 50 μm. Original magnification, 400×.

Fig. 5.

14,15-EET reduced ER stress signaling and apoptosis in cultured cardiomyocytes. (A–D) Effects of 14,15-EET and/or 14,15-EEZE on ER stress signaling induced by TG (A), TM (B), ISO (C), and AngII (D). (E–H) Hoechst staining and Annexin V–fluorescein isothiocyanate (FITC) binding with flow cytometry analysis showed that 14,15-EET inhibited apoptosis induced by ISO or AngII, respectively (n = 3 for each experiment). *P < 0.05 versus control; ^#P<0.05 versus ISO or AngII; ^P < 0.05 versus ISO + EET or AngII + EET. DMSO, dimethyl sulfoxide.

Fig. 6.

14,15-EET attenuates the ER stress–induced increase of intracellular Ca²⁺ in cardiomyocytes. (A and B) 14,15-EET attenuated the rise in intracellular Ca²⁺ levels and overexpression of p-CaMKII in H9C2 cells exposed to ISO or AngII, respectively (n = 3 for each experiment). *P < 0.05 versus control; ^#P<0.05 versus ISO or AngII. (C) BAPTA attenuated the ER stress signaling in H9C2 cells exposed with ISO (n = 3 for each experiment). *P < 0.05 versus control; ^#P<0.05 versus ISO.

Fig. 7.

14,15-EET preserved the expression and activity of SERCA2a in cardiomyocytes after ER stress. (A) 14,15-EET attenuated the reduction of SERCA2a expression and prevented its oxidation in H9C2 cells treated with ISO or AngII, respectively. (B) 14,15-EET restored the activity of SERCA2a (n = 3 for each experiment). (C) 14,15-EET attenuated ISO- or AngII-induced ROS production in cardiomyocytes (n = 3 for each experiment). (D) 14,15-EET cotreatment attenuated the loss of ZnCu-SOD, Mn-SOD, and catalase protein levels observed in ISO- or AngII-treated cells (n = 3 for each experiment). *P < 0.05 versus control; ^#P<0.05 versus AngII.