Premenopausal females have a lower incidence of cardiovascular disease than their male counterparts, but the mechanism is unclear. Estrogen has been thought to signal through two nuclear receptors: estrogen receptor-α or estrogen receptor-β; however, a third, membrane-bound receptor G protein-coupled estrogen receptor (GPER), has been identified and shown to bind estrogen with high affinity. To date, there is little information on GPER in the heart and no study has looked at the effect of GPER activation during myocardial ischemia-reperfusion (I/R). Therefore, the goal of this study was to determine whether activation of GPER is cardioprotective in rats. A highly specific GPER agonist, G-1, was administered to Sprague-Dawley (200–350 g) rat hearts 10 min before 20 min of ischemic followed by 120 min of reperfusion using a Langendorff model. Similar levels of GPER were found in both male and female rat hearts. With administration of 110 nM of G-1, postischemic contractile dysfunction was significantly reduced compared with untreated controls (43.8 ± 4.3% vs. 26.9 ± 2.1% of preischemic rate pressure product; P < 0.05). Additionally, infarct size was reduced in the G-1-treated animals when compared with control (18.8 ± 2.7% vs. 32.4 ± 2.1%; P < 0.05). These observations were demonstrated in both male and intact female rat hearts. Through Western blot analysis, it was demonstrated that G-1 induces the activation of both Akt and ERK1/2. Furthermore, the protection afforded by G-1 was blocked by coadministration of a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin, 100 nM). Taken together, the data show that G-1 activation of GPER improves functional recovery and reduces infarct size in isolated rat hearts following I/R through a PI3K-dependent, gender-independent mechanism.
- G protein-coupled receptor 30
- G protein-coupled estrogen receptor
epidemiological studies have demonstrated that premenopausal women have a reduced risk of cardiovascular disease compared with their male counterparts (3). However, postmenopause the risk reaches or even exceeds the rates for men (3). These studies, therefore, suggest that estrogen (E2) may be a cardioprotective agent. However, two clinical trials designed to test the effects of E2 replacement in postmenopausal women, the Women's Health Initiative (WHI) and the Heart and Estrogen/Progestin Replacement Study (HERS), found that E2 did not reduce adverse cardiovascular events and, in fact, increased the number of cardiovascular events (1, 17). For that reason, understanding the mechanisms by which E2 exerts its protective effects in animal studies is important.
Historically, the effects of estrogen have been attributed to binding to one of its two nuclear hormone receptors: estrogen receptor-α (ER-α) or estrogen receptor-β (ER-β). These receptors then translocate to the nucleus and induce or repress transcription of certain genes (i.e., genomic changes). However, it has recently been shown that E2 can also induce rapid changes and rapid signaling events (24, 33). The relative importance of rapid signaling versus genomic changes is unclear. It has been suggested that the classical nuclear hormone receptors can be tethered to the plasma membrane and/or other intracellular signaling molecules to induce rapid effects via activation of phosphatidylinositol 3-kinase (PI3K) (21, 32). To complicate matters further, a G protein-coupled receptor (GPCR), GPR30, was shown to bind E2 and also induce rapid signaling events (29). Filardo and colleagues (9) identified E2 as a GPR30 ligand by stimulating SKBR3 breast cancer cells, which do not express ER-α or ER-β, with estrogen and demonstrating rapid activation of the mitogen-activated protein kinases ERK-1 and ERK-2. Additionally, the authors overexpressed GPR30 in GPR30-negative MDA-MB-231 cells and demonstrated activation of ERK with E2, which they did not observe without GPR30 overexpression (9). Since the discovery of estrogen as a ligand for GPR30, the nomenclature for this receptor changed to G protein-coupled estrogen receptor (GPER) (26). To further characterize the function of this novel receptor, Bologa et al. (5) used virtual and biomolecular screening methods to identify a highly specific agonist for GPER. This agonist, G-1, demonstrated high specificity to GPER with little to no binding to either ER-α or ER-β (5). Most studies find that acute treatment of animals or perfused hearts with E2 results in reduced ischemia-reperfusion (I/R) injury (7, 12, 13, 27).
In the present study, we examined the acute effects of the GPER agonist G-1 in a Langendorff-perfused model of I/R in the rat. We sought to determine whether activation of this receptor was protective and, if so, by what mechanism. This is the first study to examine the role of this receptor in cardioprotection. We report that G-1 reduced postischemic dysfunction and reduced infarct size after I/R and that this protection is blocked by the addition of the PI3K inhibitor, wortmannin.
MATERIALS AND METHODS
Male and female Sprague-Dawley rats (200–350 g) were used in this study. All animals were treated and cared for in accordance with the Guide for the Care and Use of Laboratory Animals [National Institutes of Health (NIH), Revised 1996], and protocols were approved by the Institutional Animal Care and Use Committee.
G-1 was purchased from Cayman Chemical (Ann Arbor, MI) and was dissolved in DMSO and diluted to the final concentration (110 nM) in perfusate immediately before use. Wortmannin (Calbiochem, San Diego, CA) was made up as a 4 mM stock solution in DMSO and diluted in perfusate to a concentration of 100 nM before use. Rabbit polyclonal anti-phospho-ERK (Thr-202/Thr-204), anti-total ERK, anti-phospho-Akt (Ser473), and anti-total Akt were purchased from Cell Signaling Technologies (Beverly, MA) and were used at a concentration of 1:1,000. GPER antibody was purchased from Affinity Bioreagents (Golden, CO) and used at a concentration of 1:8,000. PD-98059 (Calbiochem, San Diego, CA) was dissolved in DMSO and diluted in perfusate to a concentration of 10 μM before use.
Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (0.25 cc/100 g of body weight). A transverse incision was made, the abdominal cavity was exposed, and 0.1 cc of 1,000 U iv heparin sodium was administered intravenously. The heart was then quickly excised, placed in ice-cold Krebs-Henseleit buffer containing 120 nM NaCl, 4.6 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.25 mM CaCl2, 25 mM NaHCO3
The design of the experimental protocol is illustrated in Fig. 1. After connection to the cannula and insertion of the balloon, hearts were stabilized for 20 min with Krebs-Henseleit buffer gassed with 95% O2-5% CO2 followed by a 10-min treatment period with or without G-1. This was subsequently followed with 20 min of global, no-flow ischemia. Hearts were then reperfused for 120 min. The heart remained at 37°C throughout the entire protocol. DMSO controls were tested and revealed no difference in recovery from control animals similar to previous studies (15, 37).
Infarct size measurement.
The cannula with the heart attached was removed at the end of the protocol and immediately connected to a syringe containing 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in Krebs-Henseleit buffer to a concentration of 1%. This solution was perfused through the heart at a rate of 2.5 ml/min and incubated in TTC for an additional 15 min at 37°C. The hearts were subsequently fixed in Formalin, and 5-6 cross-sectional slices were taken. These slices were imaged on a Leica Stereoscope, and the percentage of infarct (white area) and viable tissue (red area) was analyzed using NIH ImageJ software. Infarct area is expressed as a percentage of total ventricular area.
Samples for immunoblotting were obtained after treatment, immediately before ischemia. Myocardial extracts containing 10 μg total protein were separated electrophoretically on a 4–12% Bis-Tris gel and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline-Tween (TBS-T) followed by a 2-h incubation with primary antibody in 2.5% nonfat milk in TBS-T. Membranes were subsequently incubated with anti-rabbit horseradish peroxidase conjugated secondary antibody (1:5,000; Cell Signaling Technologies) for 1 h at room temperature. Proteins were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL) and visualized by autoradiography. Optical densities for each band were obtained using ImageJ. Ratios of phosphorylated to total protein levels were measured.
All data are presented as means ± SE. Hemodynamic measurements, functional recovery, infarct size, and immunoblot analysis were first analyzed using a one-way ANOVA followed by Fisher's least-significant difference post-hoc test if significant. A P value of ≤0.05 was considered statistically significant.
GPER is present in heart.
We were interested in testing the hypothesis that the acute protective effects of E2 were mediated by GPER. We first examined whether GPER is present in cardiomyocytes. Adult cardiac myocytes were isolated by collagenase perfusion as previously described (18), and an extract was examined by Western blotting to determine whether GPER was present. As shown in Fig. 2A, GPER is present in adult rat cardiomyocytes. In addition, we wanted to determine whether GPER is present in similar amounts in male and female rats. Total heart homogenate was examined by Western blot analysis. As shown in Fig. 2B, there are similar amounts of GPER in males and females when normalized to total protein levels (Ponceau).
Dose determination studies.
We chose the concentration of G-1 based on preliminary studies and previously published data (5, 6). We tested concentrations from 11 nM to 110 nM and found that a concentration of 110 nM (10 × Ki value) produced the greatest increase in functional recovery and that this increase was dose dependent (Fig. 3). Previous studies have used a similar concentration of 100 nM to elicit cell-specific responses (5, 6). In addition, Bologa et al. (5) demonstrated that G-1 had no substantial binding to either ER-α or ER-β at concentrations up to 1 μM.
To determine whether GPER activation is involved in acute protection mediated by estrogen, we treated both male and female rat hearts with a specific activator of GPER: G-1. Table 1 shows hemodynamic parameters measured in male and female hearts in the control, G-1-treated, and G-1/wortmannin-treated groups. There were no differences in any of the hemodynamic parameters between males or females; however, we did find that there was less postischemic contractile dysfunction in the G-1-treated males compared with male control and G-1-treated females compared with female controls. Likewise, treatment with wortmannin was able to block the G-1 protection in both genders. Because of the similarity of protection by G-1 treatment observed in males and females, all hemodynamic values were combined (Table 2).
Activation of GPER is cardioprotective.
To test the hypothesis that G-1 pretreatment is cardioprotective, we treated hearts with 110 nM for 10 min before 20 min of ischemia and 120 min of reperfusion (see Fig. 1). As shown in Fig. 4A, untreated hearts recovered 26.9 ± 2.1% of their preischemic rate pressure product (RPP). In contrast, G-1-treated hearts had reduced postischemic contractive dysfunction (43.8 ± 4.3% of RPP; Fig. 4A). To evaluate whether G-1 treatment reduced cell death (infarct size), we measured TTC staining after 2 h of reperfusion. Consistent with the improved functional recovery, we saw reduced infarct sizes in G-1-treated hearts compared with untreated (18.8 ± 2.7% vs. 32.4 ± 2.1%; P < 0.05; Fig. 4B).
Mechanism of G-1-mediated protection.
We were interested in determining the mechanism involved in the G-1-mediated cardioprotection. Activation of GPER has been shown to activate the PI3K pathway (30), and the PI3K pathway has been shown to be protective in a number of I/R model systems (16, 34). We therefore tested whether PI3K was involved in G-1-mediated protection by infusing wortmannin along with G-1 10 min before ischemia. We found that the G-1-mediated improvement in recovery of RPP was abrogated by coadministration with wortmannin (Fig. 4A). Likewise, we saw that the reduction in infarct size provided by G-1 was eliminated with wortmannin (Fig. 4B). These data suggest that the protection afforded by G-1 is mediated by activation of PI3K.
Stimulation of GPER results in activation of survival pathways.
If G-1-mediated cardioprotection is occurring via activation of PI3K, one would expect that G-1 would lead to an increase in phosphorylation of downstream kinases such as protein kinase B (Akt). To test this hypothesis control, G-1 and G-1/wortmannin-treated hearts were snap frozen at the end of the treatment period as shown in Fig. 1 (before ischemia). As demonstrated in Fig. 5, administration of G-1 for 10 min caused a modest but significant increase in the ratio of phosphorylated Akt to total levels of Akt. We further tested whether ERK activation occurred with G-1 treatment. As shown in Fig. 5, the ratio of phosphorylated ERK1/2 to total ERK1/2 was increased in G-1-treated hearts. With the addition of wortmannin, the ratio of phosphorylated Akt and ERK1/2 to their total levels was significantly reduced below basal levels (Fig. 5).
Inhibition of MEK signaling does not block G-1-mediated protection.
Because we saw a significant increase in ERK phosphorylation with G-1 treatment, we treated isolated hearts with an inhibitor of MEK-1, PD-98059 (10 μM), to determine whether ERK signaling was involved in mediating this protection. As shown in Fig. 6, blocking ERK activation did not block protection afforded by G-1 and did not reduce infarct sizes compared with G-1-treated hearts. PD-98059 treatment did block ERK phosphorylation as shown in Fig. 6C, demonstrating that this dose of PD-98059 was effective. These data suggest that although ERK is activated by G-1, its activation is not required for acute protection.
Traditionally, the effects of estrogen have been attributed to estrogen binding to the nuclear receptors ER-α and ER-β. These nuclear receptors act as ligand gated transcription factors to alter gene expression. However, recent studies have shown that estrogen can acutely activate signaling pathways (14). It has been reported that ER-α can localize to the plasma membrane and that estrogen binding results in activation of PI3K (22, 32). Recently, GPER has been proposed to bind estrogen and activate rapid signaling pathways such as PI3K (36). There have been no studies examining the role of GPER in cardioprotection. Therefore, in this study, we tested whether activation of the novel estrogen G protein-coupled receptor GPER is protective with I/R. We show here that acute activation of GPER with G-1 is cardioprotective in both male and intact female rat hearts using the Langendorff model of I/R. Using the PI3K inhibitor wortmannin, we further demonstrated that G-1 exerts its protective effects through the PI3K/Akt pathway, which has been extensively shown to be involved in cardioprotection.
Studies in tissues other than heart have shown that activation of GPER results in increased phosphorylation of Akt and ERK (9, 36). Consistent with this, we found that G-1 increased phosphorylation of both Akt and ERK. However, somewhat surprisingly, we found that wortmannin blocked activation of both ERK and Akt. The activation of the PI3K pathway by GPER has been reported to occur via GPER-mediated transactivation of the EGF receptor (9). This transactivation leads not only to activation of PI3K but also to activation of ERK. Consisitent with our findings, ERK activation has been shown to be dependent on PI3K under conditions of low activation of EGF receptor as might be expected in the heart (4, 28). Multiple studies have demonstrated that transactivation of the EGF receptor due to a physiological or nonsaturating levels of agonist can induce the activation of ERK and that this activation can be blocked with inhibitors of PI3K (8, 31). These observations are consistent with our data.
Although we demonstrated an increase in ERK activation with G-1 treatment, blocking MEK activation, which is upstream of ERK, did not inhibit G-1-induced protection. These data suggest that activation of ERK is not required in the acute cardioprotective effects we observed with G-1 treatment. This study is consistent with results published by Quesada et al. (29a). These investigators demonstrated that neuroprotection elicited by E2 treatment resulted in the activation of both Akt and ERK, but only inhibiting PI3K (LY-294002) and not MEK (PD-98059) blocked protection (29a). It is quite possible that although G-1 activates ERK, this activation is not important in the acute window of protection. It perhaps plays a role in the nonacute window of protection through gene expression changes. A study by Maggiolini et al. (23) demonstrated that after a 5-min exposure of estrogen to ER-negative, GPER-positive SKBR3 breast cancer cells, ERK1/2 phosphorylation was significantly increased, and this activation induced a genomic response via c-fos upregulation.
It is interesting that acute addition of G-1 to females, who are presumably already exposed to E2, should mediate cardioprotection. We found that G-1 mediated protection in our model in both male and female rats. This protection was blocked by wortmannin in both genders as well. A study by Haas et al. (11) deleted GPER and demonstrated an increase in body weight in both male and female knockout mice, suggesting that this receptor plays a role in both genders.
Interestingly, GPER has been demonstrated to be activated by tamoxifen, a selective estrogen receptor modulator, and ICI 182,780 (ICI), a putative pure estrogen receptor antagonist (10). A study by Mercier et al. (25) treated isolated, neonatal cardiomyocytes and fibroblasts with tamoxifen and ICI and demonstrated a rapid activation of ERK1/2 and that this ERK activation led to changes in DNA synthesis. The authors suggested that this occurred by an estrogen receptor-independent mechanism (25). In another study, Ullrich et al. (35) looked at the effects of estrogen on the L-type channel calcium current in isolated ventricular myocytes of control and ER-α knockout and ER-β knockout animals. Estrogen has previously been shown to be a negative inotropic agent (19). Interestingly and in contrast with a study by Johnson et al. (20), they demonstrated that the inhibition of calcium current via estrogen does not depend on either ER-α or ER-β (35). Regulation of calcium by GPER was also shown in a paper by Haas and colleagues. They studied isolated human aortic smooth muscle cells and demonstrated that external application of G-1 was able to increase intracellular calcium concentration, and this increase was blocked with small-interfering RNA to GPER (11). Taken together, these data suggest that GPER can regulate intracellular calcium, although the precise regulation may differ depending on cell type. Regardless, these studies suggest that GPER activation modulates intracellular calcium and kinase activation.
In summary, these data suggest that G-1 can mediate protection by activation of acute signaling pathways. We demonstrated in this study that activation of the GPER pathway by a 10-min infusion of G-1 resulted in less postischemic dysfunction and reduced infarct size. Blocking PI3K activation resulted in reduced phosphorylation of Akt and resulted in reduced recovery and larger infarct sizes compared with G-1-treated hearts. These results demonstrate that the acute activation of the newly identified estrogen receptor, GPER, is protective in the Langendorff model of I/R. Thus the role of estrogen in activating this GPCR and activation of acute signaling pathways needs to be considered in studying the role of estrogen in the heart.
These studies were supported by the National Heart, Lung, and Blood Institute intramural program.
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