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 Ca2+. 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 Ca2+ homeostasis and SERCA2a expression and activity.
Introduction
The endoplasmic reticulum (ER) is a central organelle of eukaryotic cells that participates in lipid synthesis, protein folding and maturation, and calcium storage (Lin et al., 2008). Various cellular stresses, such as ischemia, hypoxia, oxidative stress, reactive oxygen species (ROS), Ca2+ depletion of ER stores, and excessive accumulation of unfolded protein can lead to impairment of ER function (Xu C et al., 2005; Marciniak and Ron, 2006). The accumulation of unfolded protein causes activation of transmembrane sensors/transducers, including inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), RNA-dependent protein kinase–like ER kinase (PERK), and activation of transcription factor 6 (ATF6) (Kaufman, 2002). Activation of these sensors induces a marked upregulation of ER chaperones such as glucose-regulated proteins 78 and 94 kDa (GRP78 and GRP94, respectively). When ER stress is excessive and/or prolonged, initiation of the apoptotic pathways is triggered by induction of C/EBP homologous protein (CHOP), activation of c-Jun NH2-terminal kinase (JNK), or cleavage of caspase-12 (Mori, 2000; Kaufman, 2002). Increasing evidence suggests that ER stress–mediated apoptosis is involved in numerous diseases including heart failure (Wencker et al., 2003; Ozcan et al., 2004; Wellen and Hotamisligil, 2005; Lindholm et al., 2006).
Ca2+ storage and signaling, as well as the folding, modifying, and packaging of newly synthesized proteins, are among the main functions of the ER in mammalian cells (Orrenius et al., 2003). It has long been known that Ca2+ signaling governs vital cell functions that are necessary for cell survival. More recently, it has become clear that cellular Ca2+ overload, perturbation of intracellular Ca2+ compartmentalization, or depletion of the ER Ca2+ pool can result in ER stress and ER stress–mediated apoptosis (Orrenius et al., 2003). The cardiac isoform of the sarcoplasmic/ER calcium ATPase (SERCA2a), which transfers calcium ions from the cytosol of the cardiomyocyte to the lumen of the endoplasmic reticulum during muscle relaxation, is a Ca2+ pump powered by ATP hydrolysis (Kawase and Hajjar, 2008). The pivotal role of SERCA2a in cardiomyocyte Ca2+ homeostasis has been confirmed. Several reports indicate that decreased SERCA2a protein levels and activity are a hallmark of heart failure in both human and experimental animal models (Meyer et al., 1995; Minamisawa et al., 1999). Restoration of SERCA2a expression by gene transfer effectively improves cardiac function in animal models (Adachi et al., 2004), as well as in patients with heart failure (Jessup et al., 2011). Previous studies demonstrated that the activity of SERCA2a is decreased by ROS, which is increased in heart failure (Kaneko et al., 1989a,b; Liu et al., 2010). Indeed, several thiol residues in SERCA2a are potential targets for oxidation (Adachi et al., 2004; Gutiérrez-Martin et al., 2004; Cohen and Adachi, 2006; Dremina et al., 2007). Cys674, the most important thiol residue required for SERCA2a activity, is known to be oxidized during oxidative stress (Adachi et al., 2004).
CYP2J2 is abundantly expressed in the human myocardium and metabolizes arachidonic acid (AA) to four biologically active epoxyeicosatrienoic acid (EET) regioisomers (5,6-, 8,9-, 11,12-, and 14,15-EET) (Zhao et al., 2012). Over the past 2 decades, it has become increasingly evident that CYP2J2-derived EETs exert many protective effects on the cardiovascular system. These effects include inhibition of cytokine-induced vascular cell adhesion molecule expression and leukocyte adhesion to the vascular wall (Node et al., 1999), upregulation of endothelial nitric oxide synthase in endothelial cells (Jiang et al., 2007), protection of endothelial cells from apoptosis (Yang et al., 2007), and promotion of endothelial cell proliferation and angiogenesis via activation of mitogen-activated protein kinases and phosphatidylinositide 3-kinase (PI3K)/Akt signaling (Jiang et al., 2007). Importantly, 11,12-EET can induce antioxidant enzymes such as superoxide dismutase (SOD) and catalase, which can reduce intracellular ROS levels and attenuate several major apoptotic signaling events (Liu et al., 2011). Xu et al. (2013) recently reported that CYP2J3 overexpression and increased EETs prevented high-fat diet–induced ER stress in adipose tissue of rats. However, the role of EETs in ER stress and heart failure remains unknown. The goal of the present study was to establish whether EETs function as physiologic inhibitors of aberrant ER stress and heart failure via their antioxidant effects.
Materials and Methods
Antibodies and Reagents.
Thapsigargin (TG), tunicamycin (TM), isoproterenol (ISO), angiotensin II (AngII), N-ethylmaleimide, diethylenetriaminepentaacetic acid, and 4-morpholineethanesulfonic acid were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for p-PERK, ATF6, GRP78, p-JNK, CHOP, phosphorylated calmodulin kinase II (p-CaMK II), and β-actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody for p–inositol-requiring transmembrane kinase and endonuclease 1 was from Thermo Scientific Pierce Antibodies (Rockford, IL). Antibody for GRP94 was from Cell Signaling Technology (Beverly, MA). Antibody for caspase-12 was from Chemicon (Millipore, Billerica, MA). Dihydroethidium was from Invitrogen (Carlsbad, CA). The DeadEnd fluorometric terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) system was from Promega (Madison, WI). 8,9-EET, 11,12-EET, and 14,15-EET were from Cayman Chemical Company (Ann Arbor, MI). pCMV6-SERCA2a was from Origene, Inc. (Rockville, MD). All other reagents were purchased from commercial suppliers unless otherwise indicated.
Preparation of Human Heart Samples.
Samples of human cardiac tissue were obtained under a protocol approved by the Institutional Review Board of Tongji Hospital and Tongji Medical College. The investigation conforms to the principles outlined in the Declaration of Helsinki. Subjects recruited to the study provided written informed consent. Human heart failure left ventricular (LV) specimens were obtained at the time of heart transplantation. Normal human heart tissue was obtained from traffic accident victims. Tissue samples were frozen in liquid nitrogen and then stored at −80°C until use.
Animals.
Cardiac-specific CYP2J2 transgenic (Tr) mice on a pure C57BL/6 genetic background with transgene expression driven by the α major histocompatibility complex (αMHC) promoter were obtained from Dr. Darryl C. Zeldin at the National Institutes of Health (NIH) National Institute of Environmental Health Sciences (Research Triangle Park, NC), and were bred in the Experimental Animal Center of Tongji Medical College (Wuhan, China). All animal experimental protocols complied with the NIH Guide for the Care and Use of Laboratory Animals. The study was approved by the Institutional Animal Research Committee of Tongji Medical College. Mice were housed at the animal care facility of Tongji Medical College at 25°C with 12-hour light/dark cycles and were allowed free access to normal mice chow and water throughout the study period. Mice were randomly assigned to different treatment groups.
Animal Treatment.
C57BL/6 (wild-type) (n = 15) and αMHC-2J2 Tr (n = 15) mice were implanted with mini-osmotic pumps (Alzet model 1007D; DURECT Corp., Cupertino, CA) as described previously (Son et al., 2010). Pumps were filled with ISO dissolved in 0.002% ascorbic acid or AngII dissolved in saline to deliver at rates of 30 μg/g per day or 1.5 μg/kg per minute, respectively, for 14 days. Control mice were implanted with pumps that delivered 0.002% ascorbic acid or saline. In separate experiments, C57BL/6 mice were implanted with mini-osmotic pumps with vehicle or ISO (30 μg/g per day) for 14 days. Half of the mice from each group were treated for 14 days with normal drinking water or water containing Tempol (Sigma-Aldrich), a membrane-permeable radical scavenger, at a concentration of 400 mg/l.
Hemodynamic Measurements.
Mice were anesthetized with pentobarbital (100 mg/kg i.p.) and placed on heating pads to maintain a core temperature of 37°C. A microtip pressure-volume catheter (SPR-839; Millar Instruments, Houston, TX) was inserted into the right carotid artery and advanced into the left ventricle under pressure control as described (Pacher et al., 2003). After stabilization for 20 minutes, the signals were recorded continuously at a sampling rate of 1000/s using an ARIA pressure-volume conductance system (Millar Instruments) coupled to a Powerlab/4SP analog-to-digital converter (ADinstruments, Mountain View, CA) and then stored and displayed on a personal computer. Heart rate, left ventricular end diastolic pressure, left ventricular end systolic pressure, arterial elastance, tau Weiss, maximal slope of systolic pressure increment, and diastolic pressure decrement were analyzed and corrected according to in vitro and in vivo volume calibrations with a cardiac pressure-volume analysis program (PVAN3.6; Millar Instruments).
Analysis of Cardiac Function by Echocardiography.
Cardiac function was assessed by echocardiography, using a Visual sonic Vevo 2100 ultrasound machine equipped with a 30-MHz transducer applied to the chest wall. Ventricular dimensions and ejection fraction were assessed using short axis view in two-dimensional and three-dimensional modes using the onboard Visual Sonics cardiac analysis package (Oikonomopoulos et al., 2011; He et al., 2012).
Cell Culture.
H9c2 cells, a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue, were obtained from American Type Culture Collection (CRL-1446; American Type Culture Collection, Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 IU/ml) in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
Western Blot Analysis.
Proteins were analyzed by SDS-PAGE and blotted using standard protocols (Duan et al., 2012). Expression was quantified by densitometry and normalized to β-actin expression. All groups were then normalized to their respective controls.
Hoechst Staining.
Cultured cells were suspended at approximately 1–2×106/ml in buffered media (pH 7.2) and incubated with 10 mM Hoechst33258 dye at 37°C for 30 minutes. The cells were then observed under a fluorescence microscope. Hoechst33258-stained cells were illuminated with an argon laser tuned for ultraviolet (346–352 nm) and the resulting fluorescence was detected at 460 nm.
Annexin V–Fluorescein Isothiocyanate Apoptosis Assay.
Cultured cells were harvested with trypsin and resuspended in binding buffer. Five microliters Annexin V–fluorescein isothiocyanate and 5 μl propidium iodide (50 mg/ml) were added according to the manufacturer’s protocol. Cells were then analyzed with a FACStar-Plus flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
TUNEL Assay.
Apoptosis in hearts was assayed with the DeadEnd Fluorometric TUNEL System (No. G3250; Promega), following the manufacturer’s instructions.
Measurement of Intracellular Ca2+.
H9C2 cells were cultured and pretreated with 14,15-EET 1 hour before treatment with ISO (10 μM) or AngII (100 nM). Twenty-four hours later, the intracellular Ca2+ concentration was measured using a Fluo-3 AM kit from Invitrogen according to the supplier’s instructions, as previously described (Gao et al., 2011). Relative intracellular Ca2+ was expressed as a percentage of the control.
Microsome Preparation and SERCA2a Activity Measurements.
Preparation of microsomes from H9C2 cells and heart tissues from mice or humans was performed as described previously (Dong et al., 2010). SERCA2a activity was measured using a Ca2+-ATPase assay kit (Jiancheng Bio-Engineering Institute, Nanjing, China) according to the manufacturer’s instructions (Zhang et al., 2010). SERCA2a activity was normalized to protein concentration.
Biotinylated-Iodoacetamide Labeling of SERCA2a Cys674.
Oxidation of Cys674 in SERCA2a in H9C2 cells and heart tissues of mice was assessed using the biotinylated-iodoacetamide (b-IAM) labeling technique. b-IAM labeling of SERCA2a Cys674 was performed as previously described (Tong et al., 2008). Proteins were separated by SDS-PAGE and b-IAM–labeled Cys674-SERCA2a was detected using SERCA2a specific antibody.
Detection of ROS.
DCFH-DA (dichloro-dihydro-fluorescein diacetate) was used as an ROS detection reagent using previously described methods (Liu et al., 2011). Cells were incubated with 10 µM DCFH-DA at 37°C for 20 minutes. Fluorescence was detected by flow cytometry (Liu et al., 2011). To detect superoxide, frozen heart tissue sections were incubated with dihydroethidium (D23107; Invitrogen) according to the manufacturer’s instructions (Son et al., 2010).
Evaluation of Urine 14,15-DHET by Enzyme-Linked Immunosorbent Assay.
The stable 14,15-EET metabolite, 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), was detected in the urine of mice using an enzyme-linked immunosorbent assay kit (Detroit R&D, Detroit, MI) according to the manufacturer’s instructions as previously described (Jiang et al., 2005).
Statistical Analysis.
Continuous data are expressed as the mean ± S.E.M. Group differences of continuous variables were compared by one-way analysis of variance or t test as appropriate. Relationships between variables were determined by the Pearson correlation coefficient. P < 0.05 was accepted as statistically significant.
Results
Induction of ER Stress in Failing Human Hearts.
Similar to previous reports (Okada et al., 2004; Fu et al., 2010; Ni et al., 2011), ER stress and its associated apoptosis signaling pathways were a common occurrence in failing human hearts. Importantly, the expression of SERCA2a protein was significantly decreased in failing human hearts, which is consistent with previous studies (Meyer et al., 1995; Zarain-Herzberg et al., 1996; Minamisawa et al., 1999). We collected heart samples from 4 recipients of heart transplantation who suffered from dilated cardiomyopathy with end-stage heart failure (Table 1). The decrease in SERCA2a protein levels in failing hearts was accompanied by a reduction in SERCA2a activity (Fig. 1). pCMV6-SERCA2a was transfected into human embryonic kidney 293 cells as a positive control (Supplemental Fig. 1).
Attenuation of Cardiac Hypertrophy and Dysfunction Induced by ISO or AngII in αMHC-2J2 Tr Mice.
CYP2J2 protein levels were determined in the heart tissues of wild-type (WT) and αMHC-2J2Tr mice. As showed in Fig. 2A, CYP2J2 protein was abundant in αMHC-2J2 Tr mice. Some cross-reactivity of the antibody with endogenous murine CYP2J proteins can be observed in WT mouse hearts. Here, cardiomyocytes were transfected with rAAV-CYP2J2 or rAAV-GFP, and the expression of CYP2J2 was determined as a positive control (Supplemental Fig. 2). CYP2J2 functionality was demonstrated by the nearly 4-fold increase in urinary 14,15-DHET levels in CYP2J2 Tr mice compared with WT mice (Fig. 2B).
To investigate the effect of cardiomyocyte-specific CYP2J2 expression on ER stress signaling of failing hearts in vivo, we induced heart failure by continuous infusion of ISO for 2 weeks in WT and CYP2J2 Tr mice. There were no significant differences in baseline body weight and hemodynamic parameters between WT and CYP2J2 Tr mice (data not shown). On gross examination, CYP2J2 Tr mice showed less enlargement of the heart compared with WT mice after ISO treatment of 2 weeks (Fig. 2C; Supplemental Fig. 3A). Consistent with this observation, the ratio of heart weight to body weight at 2 weeks after ISO treatment was smaller in CYP2J2 Tr mice than in WT mice (Fig. 2D).
Heart function was examined by echocardiography and invasive pressure-volume analysis. As shown in Table 2, cardiac function in WT mice treated with ISO was decreased compared with that of CYP2J2 Tr mice. Thus, both LVIDd and IVSd were increased in WT mice treated with ISO compared with CYP2J2 Tr mice. WT mice also showed markedly reduced LV ejection fraction, LV fractional shortening, maximal slope of systolic pressure increment, and diastolic pressure decrement, and increased left ventricular end diastolic pressure compared with CYP2J2 Tr mice (Table 2).
A second animal model of heart failure compared WT and CYP2J2 Tr mice after continuous infusion of AngII for 2 weeks. Results were very similar to those observed with ISO. CYP2J2 Tr mice showed reduced heart failure compared with WT mice (Fig. 2, E and F; Supplemental Fig. 3B; Table 2). Taken together, these data demonstrate that CYP2J2 Tr mice are protected against cardiac hypertrophy and decline of cardiac function in models of heart failure.
ER Stress Signaling in Failing Hearts of WT and CYP2J2 Tr Mice.
We investigated ER stress signaling in failing hearts of WT and CYP2J2 Tr mice. Western blots showed that expression levels of ER stress molecules, GRP78, CHOP, and cleaved ATF6 (c-ATF6) were markedly elevated, and PERK (p-PERK) and IRE1 (p-IRE1) as well as p-JNK were markedly activated in failing hearts of WT mice after ISO or AngII treatment (Fig. 3, A–D). Interestingly, induction of these ER stress–initiated signaling markers was suppressed in hearts from CYP2J2 Tr mice (Fig. 3, A–D). These data provide compelling evidence that the activation of ER stress–mediated apoptotic signaling pathways in the failing hearts is attenuated by CYP2J2 expression. We also employed TUNEL staining to quantify the number of cardiomyocytes undergoing apoptosis in the two groups. Compared with vehicle-treated controls, ISO- or AngII-treated hearts from WT mice exhibited an increase in the percentage of cardiomyocyte-shaped, TUNEL-positive cells. In comparison, CYP2J2 Tr mice displayed fewer TUNEL-positive cardiomyocytes after ISO or AngII treatment (Fig. 3E). These data suggest that ER stress and ER stress–mediated apoptosis are inhibited in CYP2J2 Tr mice.
Restoration of SERCA2a Expression and Activity in CYP2J2 Tr Mice.
Elevation of intracellular Ca2+ is a common mechanism for aberrant ER stress and ER stress–mediated apoptosis (Orrenius et al., 2003; Biagioli et al., 2008; Deniaud et al., 2008). Therefore, we assessed the activation of calmodulin kinase II (CaMKII), a Ca2+-dependent kinase that can elevate intracellular Ca2+ levels, in the failing hearts of mice (Dzhura et al., 2000). CaMKII was significantly activated in the failing hearts of WT mice, as determined by an increase in phosphorylated CaMKII (p-CaMKII), relative to normal hearts (Fig. 4A). CaMKII phosphorylation was attenuated in CYP2J2 Tr mice treated with ISO or AngII compared with WT mice (Fig. 4A). These results suggest that CYP2J2 decreases activation of CaMKII, which could decrease intracellular Ca2+ and suppress ER stress.
SERCA2a plays an important role in maintaining intracellular Ca2+ homeostasis through its ability to pump cytosolic Ca2+ into the sarcoplasmic or endoplasmic reticulum stores (Cohen et al., 1999; Tong et al., 2008). Because the intracellular Ca2+ levels were elevated in failing mouse hearts, we reasoned that SERCA2a activity may have been inhibited. Indeed, the expression of SERCA2a was downregulated in WT mice exposed to ISO or AngII. Interestingly, this effect was reversed by expression of CYP2J2 in transgenic mice (Fig. 4B). We further examined SERCA2a activity by measuring the inorganic phosphate liberated from ATP hydrolysis. Both ISO and AngII markedly reduced SERCA2a activity in WT mice compared with CYP2J2 Tr mice (Fig. 4C). Thus, expression and activity of SERCA2a were decreased in failing mouse hearts, whereas CYP2J2 expression attenuated this decline.
Oxidation of Cys674 in SERCA2a inhibits SERCA2a activity (Adachi et al., 2004). Therefore, we determined whether the oxidation of SERCA2a at Cys674 was increased in failing hearts using the b-IAM labeling technique (Tong et al., 2008). b-IAM labeling suggests nonoxidized Cys674, which is conducive to normal SERCA2a function. Failing hearts in WT mice exhibited decreased levels of b-IAM–SERCA2a compared with normal hearts, which indicates both increased Cys674 oxidation and decreased SERCA2A function. CYP2J2 expression in transgenic mice significantly attenuated the decline in b-IAM–SERCA2a in both ISO and AngII models of heart failure (Fig. 4B). Thus, CYP2J2 at least partially prevented SERCA2a oxidation, which is normally increased in failing WT mouse hearts.
A growing body of evidence suggests that ROS is increased and plays an important role in heart failure (Seddon et al., 2007; Liu et al., 2010), and that ROS depresses the activity of SERCA2a (Kaneko et al., 1989a,b). In addition, CYP2J2 overexpression has been shown to suppress ROS levels (Liu et al., 2011). Therefore, we sought to determine whether CYP2J2 Tr hearts restored SERCA2a activation through reduction of ROS levels. As shown in Fig. 4D, CYP2J2 expression significantly decreased the cellular levels of ROS compared with WT mice treated with ISO or AngII. Tempol, a potent antioxidant, also significantly increased SERCA2a activity and the amount of b-IAM–SERCA2a in ISO-infused mice. Tempol did not affect SERCA2a protein levels but reduced the expression of ER stress markers (Fig. 4, E–G). Taken together, these data suggest that the reduced oxidation of SERCA2a and protection against heart failure in CYP2J2 Tr hearts may occur at least partly through suppression of ROS levels.
The reduction in ROS levels in CYP2J2 Tr mice appears to occur through upregulation of antioxidant enzymes. As seen in Fig. 4H, expression of ZnCu-SOD, Mn-SOD, and catalase are suppressed in WT mice treated with ISO or AngII. CYP2J2 expression protected against the decline in each of these enzymes. Therefore, attenuation in the reduction of ZnCu-SOD, Mn-SOD, and catalase protein levels may be involved in the maintenance of SERCA2a activity in CYP2J2 Tr hearts during models of heart failure.
14,15-EET Reduced ER Stress Signaling and Apoptosis in Cultured Cardiomyocytes.
EETs are the major metabolites of CYP2J2. To investigate the role of EETs in ER stress signaling in vitro, H9C2 cells were cultured with 8,9-, 11,12-, or 14,15-EET (1 μM each) for 1 hour before treatment with TM for 24 hours. Cell lysates were assayed by Western blotting. Each of the EETs decreased ER stress, and 14,15-EET exhibited the greatest effect (Supplemental Fig. 4A). We also treated H9C2 cells with 14,15-EET at different concentrations (50–1000 nM). The results showed that 14,15-EET significantly decreased GRP78 expression in a dose-dependent manner (Supplemental Fig. 4B).
We investigated the inhibition of 14,15-EET on ER stress signaling in cultured cells. H9C2 cells were treated with TG or TM (as positive controls for ER stress), ISO or AngII, and 14,15-EET. Similarly to the results in in vivo study in CYP2J2 Tr mice (Fig. 3, A–D), the addition of exogenous 14,15-EET in vitro significantly decreased activation of ER stress pathway and ER stress–mediated apoptosis induced by pharmacological agents TG, TM, ISO, and AngII (Fig. 5, A–D, respectively). To confirm the importance of EETs in our models, we used 14,15-EEZE [14,15-epoxyeicosa-5(Z)-enoic acid], a selective EET signaling antagonist (Gauthier et al., 2004; Seubert et al., 2006). 14,15-EEZE abolished the benefits of 14,15-EET on ER stress signaling (Fig. 5, A–D). ER stress–mediated apoptosis, Hoechst staining, and Annexin V–fluorescein isothiocyanate binding showed similar results (Fig. 5, E–H). In addition, results showed that the inhibitory effects of CYP-2J2 overexpression on ER stress were attenuated by 14,15-EEZE (Supplemental Fig. 2). Thus, EETs inhibit in vitro ER stress signaling and apoptosis in cardiomyocytes induced by a variety of stressors.
14,15-EET Upregulated the Expression and Activity of SERCA2a in Cardiomyocytes.
Loss of Ca2+ homeostasis is a hallmark of cardiac failure. We examined the effect of EETs to restore Ca2+ levels in ER-stressed cardiomyocytes. Indeed, 14,15-EET reduced intracellular Ca2+ levels in H9C2 cells exposed to ISO or AngII (Fig. 6A). 14,15-EET also attenuated ISO- or AngII-induced overexpression of p-CaMKII (Fig. 6B). These data suggest that 14,15-EET suppresses the elevation of intracellular [Ca2+] induced by pharmacological stress agents in vitro. We next determined whether the upregulation of intracellular Ca2+ levels triggered an aberrant ER stress response in H9C2 cells. We assessed whether BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid] (20 μM), an intracellular Ca2+ chelator (Harrison and Bers, 1987), inhibited the ER stress response in H9C2 cells. The results showed that the addition of BAPTA attenuated the ER stress response and ER stress–mediated apoptosis in H9C2 cells exposed to ISO (Fig. 6C).
The effect of EETs on the expression and activation of SERCA2a was assessed in H9C2 cells exposed to ISO or AngII. 14,15-EET significantly alleviated the changes in expression and activity of SERCA2a in H9C2 cells treated with ISO or AngII (Fig. 7, A and B). A growing body of evidence suggested that ROS can depress the activity of SERCA2a (Kaneko et al., 1989a,b). Treatment of H9C2 cells with ISO or AngII for 24 hours led to a significant increase in ROS production. Interestingly, preincubation with 14,15-EET significantly attenuated ISO- or AngII-induced ROS production in cardiomyocytes measured by flow cytometry (Fig. 7C).
We further determined whether oxidation of SERCA2aCys674 was reduced in H9C2 cells treated with 14,15-EET. The data showed that 14,15-EET markedly reduced the oxidation of SERCA2a at Cys674 induced by ISO or AngII in these cells using the b-IAM labeling technique (Fig. 7A) (Tong et al., 2008). We also determined the expression of antioxidant enzymes in H9C2 cells. Similar to that observed in CYP2J2 transgenic hearts, 14,15-EET increased ZnCu-SOD, Mn-SOD, and catalase protein levels (Fig. 7D).
These data suggest that intracellular Ca2+ overload triggers ER stress. CYP2J2 expression or EET treatment markedly attenuates the loss of antioxidant enzymes, intracellular Ca2+ overload, and ER stress via maintenance of SERCA2a expression and function.
Discussion
This study provides evidence for CYP2J2-derived EETs as important regulators of intracellular Ca2+ levels and ER homeostasis in vivo and in vitro. These actions are mediated through maintenance of SERCA2a expression and suppression of SERCA2a oxidation. We found that the levels of ER stress and associated apoptosis were significantly increased in hearts of mice treated with ISO or AngII, as well as in cardiac tissue from patients with heart failure. Consistent with previous studies, our data suggest that ER stress–initiated apoptosis plays an important role in cardiomyocyte apoptosis in failing hearts (Okada et al., 2004; Fu et al., 2010; Ni et al., 2011). CYP2J2 expression significantly reduced ER stress and associated apoptosis and attenuated the development of heart failure in vivo. Interestingly, administration of tauroursodeoxycholic acid, a chemical chaperone with clinical potential (Ozcan et al., 2006), significantly reduced the expression of ER stress markers and suppressed the development of heart failure in a manner similar to EETs (data not published). 14,15-EEZE, as selective EET antagonist, abolished these benefits. These results suggested that CYP2J2-derived EETs might confer their protective effects against heart failure by inhibiting the ER stress–initiated apoptosis in vivo and in vitro.
Elevation of intracellular Ca2+ is a common mechanism for aberrant ER stress and ER stress–mediated apoptosis (Orrenius et al., 2003; Biagioli et al., 2008; Deniaud et al., 2008). Intracellular Ca2+ chelator BAPTA inhibited aberrant ER stress–induced apoptosis, which is consistent with other reports (Minamisawa et al., 1999; Hajjar et al., 2008). The pivotal role of SERCA2a in Ca2+ homeostasis has been described. Reports consistently show decreased levels of SERCA2a expression and activity in failing human hearts (Meyer et al., 1995; Zarain-Herzberg et al., 1996; Minamisawa et al., 1999). Accordingly, restoration of SERCA2a expression has proved to be effective in improving cardiac function in patients with heart failure (Jessup et al., 2011) and in animal models of heart failure (Hajjar et al., 2008). In this study, we showed that SERCA2a protein levels and activity were reduced, levels of intracellular Ca2+ were increased, and severe contractile dysfunction was present. CYP2J2 expression in vivo improved both systolic and diastolic function. In vitro, exogenous EET supplementation attenuated the rise in intracellular Ca2+ and increased SERCA2a protein levels and activity. Thus, CYP2J2-derived EETs could maintain Ca2+ homeostasis and SERCA2a protein expression and activity.
A growing body of evidence suggests that ROS is increased and plays an important role in heart failure (Seddon et al., 2007; Liu et al., 2010), and ROS could depress the activity of SERCA2a (Kaneko et al., 1989a,b). Indeed, we observed increased ROS generation in cardiac tissue or cardiomyocytes exposed to ISO or AngII. Both CYP2J2 expression and exogenous EETs supplementation increased the expression of antioxidant enzymes (ZnCu-SOD, Mn-SOD, and catalase) and attenuated the rise in ROS levels after ISO or AngII. This may be, at least in part, the underlying mechanism by which EETs reduces ROS levels. However, there are also other possible mechanisms through which EETs may induce ROS scavenging. For example, EETs are reported to upregulate heme oxygenase-1 in endothelial cells, which results in decreased oxidative stress and a lower rate of apoptosis (Jozkowicz et al., 2007; Sacerdoti et al., 2007). The direct relationship between EETs and ROS has not been reported, but we could not exclude the possibility. CYP2J2-derived EETs thus limited oxidation of SERCA2a Cys674 in vivo and in vitro. Taken together, we conclude that CYP2J2-derived EET mediated inhibition of ER stress response is likely to occur through inhibition of oxidant-mediated SERCA2a.
SERCA2a activity can be affected by several processes such as oxidation of the enzyme, a lower level of enzyme expression, reduced PKA activity, or reduced phosphorylation of phospholamban (Muller and Simonides, 2005). In our study, we found that the restoration of SERCA2a activity was mediated by inhibition of oxidant-mediated SERCA2a or upregulation of SERCA2a expression via CYP2J2-derived EETs; however, the relative contributions of these two mechanisms were not examined in this study. Many reports have demonstrated that EETs can exert effects through PI3K-Akt signaling (Node et al., 1999; Jiang et al., 2007). Kim et al. (2008) reported that upregulation of the SERCA2a protein can be mediated by the PI3K-Akt-SERCA2a signaling cascade. Yu et al. (2006) also reported enhancement of SERCA2a activity involved the PI3K-Akt-endothelial nitric oxide synthase pathway. Exactly how CYP2J2-derived EETs influence SERCA2a expression and activity warrants further investigation.
In summary, the present study provides insight on the protective role of CYP2J2-derived EETs in heart failure. The effects of CYP2J2 expression and EETs on cardiac protection are likely mediated through upregulation of antioxidant enzymes, suppression of ROS, and inhibition of ER stress. These effects maintain SERCA2a activity and intracellular Ca2+ homeostasis. Our results suggest that CYP2J2-derived EETs may be a logical target for the development of drugs to prevent cardiac hypertrophy and cardiomyocyte apoptosis in failing hearts.
Acknowledgments
The authors thank Xiaoyong Tong for technical assistance.
Authorship Contributions
Participated in research design: X. Wang, Ni, Chen, D. W. Wang.
Conducted experiments: X. Wang, Yang.
Contributed new reagents or analytic tools: X. Wang, Duan.
Performed data analysis: X. Wang.
Wrote or contributed to the writing of the manuscript: X. Wang, Edin, Zeldin, D. W. Wang.
Footnotes
- Received May 13, 2013.
- Accepted October 18, 2013.
X.W. and L.N. contributed equally to this work.
This work was supported by the National 973 Program [Grant 2012CB518004] and the National Natural Science Foundation of China [Grants 31130031, 30930039, 81000097, and 30770882]. This work was also supported, in part, by the Intramural Research Program of the National Institutes of Health [National Institute of Environmental Health Sciences] [Grant Z01-ES025034].
↵This article has supplemental material available at molpharm.aspetjournals.org.
Abbreviations
- 14,15-DHET
- 14,15-dihydroxyeicosatrienoic acid
- AA
- arachidonic acid
- ATF6
- activation of transcription factor 6
- AngII
- angiotensin II
- BAPTA
- 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- b-IAM
- biotinylated-Iodoacetamide
- CaMKII
- calmodulin kinase II
- CHOP
- C/EBP homologous protein
- DCFH-DA
- dichloro-dihydro-fluorescein diacetate
- EET
- epoxyeicosatrienoic acid
- ER
- endoplasmic reticulum
- 14,15-EEZE
- 14,15-epoxyeicosa-5(Z)-enoic acid
- FITC
- fluorescein isothiocyanate
- GRP78
- glucose-regulated protein 78 kDa
- GRP94
- glucose-regulated protein 94 kDa
- IRE1
- inositol-requiring transmembrane kinase and endonuclease 1
- ISO
- isoproterenol
- JNK
- c-Jun NH2-terminal kinase
- LV
- left ventricular
- αMHC
- α major histocompatibility complex
- PERK
- RNA-dependent protein kinase–like ER kinase
- PI3K
- phosphatidylinositide 3-kinase
- ROS
- reactive oxygen species
- SERCA2a
- sarcoplasmic/endoplasmic reticulum calcium ATPase
- SOD
- superoxide dismutase
- TG
- thapsigargin
- TM
- tunicamycin
- Tr
- transgenic
- TUNEL
- terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling
- WT
- wild-type
- U.S. Government work not protected by U.S. copyright