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. 2008 Mar 27;149(7):3361–3369. doi: 10.1210/en.2008-0133

Estrogen Inhibits Cardiac Hypertrophy: Role of Estrogen Receptor-β to Inhibit Calcineurin

Ali Pedram 1, Mahnaz Razandi 1, Dennis Lubahn 1, Jinghua Liu 1, Mani Vannan 1, Ellis R Levin 1
PMCID: PMC2453079  PMID: 18372323

Abstract

Estrogen has been reported to prevent development of cardiac hypertrophy in female rodent models and in humans. However, the mechanisms of sex steroid action are incompletely understood. We determined the cellular effects by which 17β-estradiol (E2) inhibits angiotensin II (AngII)-induced cardiac hypertrophy in vivo. Two weeks of angiotensin infusion in female mice resulted in marked hypertrophy of the left ventricle, exacerbated by the loss of ovarian steroid hormones from oophorectomy. Hypertrophy was 51% reversed by the administration of E2 (insertion of 0.1 mg/21-d-release tablets). The effects of E2 were mainly mediated by the estrogen receptor (ER) β-isoform, because E2 had little effect in ERβ-null mice but comparably inhibited AngII-induced hypertrophy in wild-type or ERα-null mice. AngII induced a switch of myosin heavy chain production from α to β, but this was inhibited by E2 via ERβ. AngII-induced ERK activation was also inhibited by E2 through the β-receptor. E2 stimulated brain natriuretic peptide protein expression and substantially prevented ventricular interstitial cardiac fibrosis (collagen deposition) as induced by AngII. Importantly, E2 inhibited calcineurin activity that was stimulated by AngII, related to E2 stimulating the modulatory calcineurin-interacting protein (MCIP) 1 gene and protein expression. E2 acting mainly through ERβ mitigates the important signaling by AngII that produces cardiac hypertrophy and fibrosis in female mice.

FROM ANIMAL AND human studies, estrogen has been shown to modulate vascular tone and arterial resistance, arterial dilation, and blood flow; lower blood pressure; mitigate the damage of arteries and the heart to various forms of injury; decrease vascular inflammation and atherosclerosis; and prevent cardiac hypertrophy (reviewed in Ref. 1).

As a sequel to myocardial infarction or poorly controlled arterial hypertension, cardiac hypertrophy often results and is an independent risk factor for the development of ischemia, arrhythmia, and sudden death (2,3). Many factors contribute to myocardial hypertrophy, but signaling by vascular hormones such as angiotensin II (AngII) and endothelin-1 (ET-1) is frequently involved (4). Downstream of the AngII or ET-1 receptors, Gαq/11 activation (5) and increased calcium flux (6) stimulate kinases (e.g. ERK MAPK) and phosphatases that induce the expression of the hypertrophic gene program in ventricular myocytes (7). AngII signaling through the AT1 receptor stimulates calcium-related activation of protein phosphatase 2B (calcineurin) that dephosphorylates several members of the nuclear factor of activated T cells (NFAT) family of transcription factors. As a result, the NFATs translocate to the nucleus of the cardiomyocyte where they induce genes such as myosin heavy chain (MHC)-β that result in cardiomyocyte hypertrophy. The protein products result in remodeling of the muscle sarcomere and neurofilaments and increase protein synthesis, which leads to increased cell size.

Another prominent feature of the hypertrophied myocardium is interstitial fibrosis (8). Fibrosis interferes with coordinated excitation-contraction coupling of cardiomyocytes in systole and diastole and induces diastolic stiffness, impairing cardiac output (9). Increased expression of collagen genes is typically seen in hypertrophied myocardium, resulting in fibrosis.

Myocardial hypertrophy frequently develops in older humans. With aging, a sexual dimorphism appears, and the incidence of hypertrophy in postmenopausal women exceeds that of age-matched men (10). The latter can be reversed in postmenopausal women by hormone replacement therapy (11), suggesting that estrogen may oppose the developmental events in the cardiomyocyte and stroma that produce hypertrophy. Various animal studies also support the anti-hypertrophic action of estrogen in the heart. As an example, estrogen supplementation of ovariectomized female mice causes a 30% reduction in pressure overload-induced hypertrophy (12).

The mechanisms by which estrogen inhibits cardiac hypertrophy are not fully understood. Here we examined the role of estrogen receptor (ER) isoforms to mediate the anti-hypertrophic effects of 17β-estradiol (E2). Upon infusing AngII to create in vivo cardiac hypertrophy, E2 acts mainly through ERβ to prevent critical cellular and molecular events that characterize the pathological condition.

Materials and Methods

Mice models

All studies were approved by the Animal Use and Research and Development Committees at the Long Beach Veteran’s Affairs Medical Center and the University of California, Irvine. Thirty 10-wk-old female C57/BJ6 mice, intact or ovariectomized, were obtained from Harlan/Sprague Dawley and were housed in 12-h light, 12-h dark lighting and fed rodent chow devoid of soy or most plant products. Osmotic mini-pumps (Alzet, Durect Corp., Cupertino, CA) filed with AngII (1.1 mg/kg·d) in saline or saline alone provided a 14-d infusion after sc insertion under inhaled chlorofluorane anesthesia. In some mice, an E2 pellet (0.1 mg, 21-d-release pellets; Innovative Research of America, Sarasota, FL) or placebo pellet was also inserted under the skin. This pellet is well documented to produce physiological levels of E2 in the serum of mice (13). At inception and after 14 d, the mice were weighed. At 14 d, the hearts were arrested in diastole by injection of CdCl2 (0.1 mol/liter iv), followed by cervical dislocation. The hearts were removed and weighed, and the ratio of heart to total body weight was determined. Estrogen loss or administration did not significantly affect body weight over the 2-wk period of the study, but heart weight was normalized to initial body weight. Some mice also underwent echocardiographic assessment (see below). For comparison, ovariectomized female wild-type (WT) and ERα or ERβ gene-deleted mice were obtained from Dennis Lubahn (University of Missouri) (14). The ERβ mice were originally created by Drs. Smithies, Gustafsson, and colleagues (15), and control mice were WT littermates. These mice were subjected to the same conditions of AngII and E2, and all mice were identically housed and fed the same chow at the Veteran’s Affairs animal research facility.

Cardiac hypertrophy

In vivo assessment of various parameters was accomplished by transthoracic echocardiography. Mice were anesthetized with 2% isoflurane, and echocardiograms were performed (Sequoia; Siemens, Mountain View, CA) using a 14-MHz linear probe (15L8; Acuson). An advanced high-frame-rate imaging technique (Paragon; Siemens) was adopted to increase temporal resolution at a frame rate of 120 frames/sec. B-mode images of left ventricular (LV) parasternal long-axis, parasternal short-axis, and apical views were digitally acquired at two to three cardiac cycle lengths. Images of LV short axis were standardized at three levels, base, mid, and apex. M-mode imaging was unified according to American Society of Echocardiography guidelines for measurements of wall thickness, chamber dimensions, and functional parameters. LV wall thicknesses and cavity dimensions were measured on LV M-mode spectra. LV ejection fraction, fractional shortening, and wall thickness ratio were calculated. Vector velocity imaging was employed for quantifications of apical rotation (degrees), circumferential strain (percent), and radial strain (percent).

After 2 wk of hormone infusion, the mice were euthanized, and the hearts were removed and sectioned and stained with hematoxylin and eosin or Masson stain (for collagen). Eight sections per heart were created for analysis, and the data represent six to eight mice per condition. Ventricular protein and RNA was also extracted (16) for additional studies. Heart and body weights were also quantified for comparison among the different mice. Insufficient knockout (KO) mice were available to meaningfully carry out the echocardiographic studies that were done at a later date.

Gene expression and protein detection

RT-PCR was accomplished using the following primers, as we have previously described (16): MCIP1, 5′-GACTGGAGCTTCATTGACTGCGAGA and AAGGAACCTACAGCCTCTTGGAAAG; GAPDH, 5′-AGCCACATCGCTCAGAACAC and GAGGCATTGCTGATGATCTTG. GAPDH expression served as control gene for all studies.

For relative protein detection, immunoblots were carried out on protein extracted from the left ventricle of mice from all conditions, after separation by SDS-PAGE and transfer to nitrocellulose, as we described (17,18). Cardiac MHC and modulatory calcineurin-interacting protein (MCIP) antibodies (Abcam, Cambridge, MA; and Santa Cruz Biotechnology Inc., Santa Cruz, CA), and brain natriuretic peptide (BNP) antibodies (Peninsula Labs, Mountain View, CA) were used.

Phosphatase and kinase activity

Total phosphatase activity was determined using a BIOMOL kit (Plymouth Meeting, PA) as per the manufacturer’s instructions from proteins isolated from the left ventricle of the various mice at 2 wk of treatment (17). Calcineurin activity was specifically determined by running duplicates of the samples in the presence of EGTA and subtracting this value from the total phosphatase activity. ERK activity was determined as previously described (17) by immunoprecipitating ERK protein from the ventricles of mice subjected to the various experimental conditions. ERK activity was determined in vitro against myelin basic protein substrate.

Fibrosis

Left ventricular tissues were fixed in 4% paraformaldehyde solution. Paraffin-embedded tissue sections (5 μm) were stained with Masson’s trichrome for the presence of interstitial collagen fiber accumulation indicative of cardiac fibrosis. The ratio of interstitial fibrosis to the total LV area was calculated from 10 randomly selected microscopic fields from each of five sections per heart using National Institutes of Health ImageJ analysis software (n = 5 mice per condition).

Further quantification of collagen deposition was made by ventricular content of hydroxyproline, a breakdown product of collagen, determined by a modified method of Bergman and Loxley (19). The ventricular tissues were homogenized and hydrolyzed in 6 N HCl at 110 C for 24 h in a sealed reaction vial. The sample was dried and the residue resuspended in sterile water. Then, 0.5 ml chloramine T was added for 5 min, and Ehrlich’s reagent (3 ml) was added and the mixture left for 18 h at room temperature. The intensity of the red coloration that developed was measured by a spectrophotometer at 558 nm.

Statistics

Data were compared by two-way ANOVA plus Scheffe’s test for significant differences between conditions. Statistical significance of difference was at the 0.05 level.

Results

E2 inhibits cardiac hypertrophy via ERβ

Female mice receiving AngII infusion over 2 wk demonstrated significant cardiac hypertrophy, especially of the left ventricle (Fig. 1A). AngII-induced hypertrophy was most significant in ovariectomized mice not receiving concomitant E2 replacement (about a 70% increase in heart/body weight, 5.2 ± 0.2 to 8.7 ± 0.3 mg/g). E2 replacement significantly reversed by 51% the ventricular enlargement caused by the hypertrophic agent (Fig. 1A). Functionally, AngII stimulated significantly increased LV wall thickness, apical rotation, and circumferential and radial strain as determined by echocardiography of anesthetized mice, and these parameters were significantly reversed by E2 (Table 1). The strain measurements indicated that AngII induced a hypertrophic muscle phenotype, whereas E2 prevented both the muscle shortening and the enhanced contraction seen. AngII infusion over this 2-wk period did not impair the LV ejection fraction, indicating functional compensation for the induced hypertrophy.

Figure 1.

Figure 1

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E2 prevents cardiac hypertrophy induced by AngII. A, Whole hearts were removed and weighed for calculating the ratio of heart weight to body weight. The bar graph is the mean ± sem from six to eight mice per condition. *, P < 0.05 for WT vs. same receiving AngII or ovariectomized (Ovx) WT vs. same plus AngII. +, P < 0.05 for Ovx WT plus AngII vs. same plus E2. Mice receiving E2 alone were similar to control mice (data not shown). Bar, 2 mm. B, Transverse sections of the ventricles stained with hematoxylin and eosin. a–c, WT mice; d and e, ERαKO mice; f and g, ERβKO mice; a is saline-infused control; b, d, and f are AngII infusion; and c, e, and g are AngII plus E2 replacement. All mice were ovariectomized. The bar graph represents six to eight mice per condition. *, P < 0.05 for WT (saline control) vs. other condition; +, P < 0.05 for AngII vs. same plus E2 for WT or ERαKO mouse. Bar, 2 mm.

Table 1.

Effect of AngII and estrogen on cardiac parameters

Ovx WT (saline) Ovx WT (E2) Ovx WT (Ang II) Ovx WT (Ang II + E2)
LVWT (mm) 0.64 ± 0.05 0.63 ± 0.04 0.79 ± 0.05a 0.70 ± 0.02b
LVEF (%) 69 ± 3.1 69 ± 1.6 75 ± 5 76 ± 1.1
AR (degrees) 2.0 ± 0.1 1.6 ± 0.1 2.5 ± 0.2a 2.1 ± 0.2b
CS (%) −6.7 ± 0.6 −6.5 ± 0.5 −9.3 ± 1.0a −6.2 ± 0.7b
RS (%) 6.6 ± 0.6 6.0 ± 0.6 7.9 ± 0.8a 5.6 ± 0.6a,b
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Echocardiogram data are from five mice per group and are mean ± sd. AR, Apical rotation; CS, circumferential strain; LVEF, LV ejection fraction; LVWT, LV wall thickness; Ovx, ovariectomized; RS, radial strain. 

a

P < 0.05 for condition vs. saline. 

b

P < 0.05 for Ang II vs. Ang II plus E2 by ANOVA plus Scheffé’s test. 

We then determined which of the two ER isoforms, α or β (14,15), mediates the ability of E2 to counteract the effects of AngII. E2 replacement prevented the hypertrophy induced by AngII in both ovariectomized WT and ERαKO mice (Fig. 1B). In contrast, ERβKO mice did not show reversal of AngII-induced hypertrophy upon administration of the sex steroid. Both ERα and ERβ are produced in isolated adult and neonatal rodent cardiomyocytes (17). We conclude that the anti-hypertrophic effects of E2 are mediated mainly via ERβ.

E2 inhibits fibrosis due to collagen deposition

An important contributor to the deterioration of cardiac function during prolonged hypertrophy is the development of interstitial fibrosis due to collagen deposition (9). In our model, AngII significantly increased collagen deposition in the interstitium of the left ventricle (Fig. 2A). As a novel finding, estrogen replacement inhibited as much as 90% of the fibrosis (Fig. 2, A and B) and reduced by about 70% the hydroxyproline content (collagen breakdown product) in the ventricle (Fig. 2C). This was seen in both ovariectomized WT and ERαKO mice. In contrast, E2 had no significant effects in the ERβKO mice. Thus, inhibition of cardiac fibrosis by E2 occurs via ERβ.

Figure 2.

Figure 2

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E2 prevents AngII-induced cardiac fibrosis. A, Representative Masson trichrome staining of collagen deposition in the left ventricle of ovariectomized female mice exposed to the indicated conditions is shown (n = 5 mice per condition). Arrows indicate fibrosis. Bar, 0.1 mm. B, The percent area of fibrosis is quantified as described in Materials and Methods, and the bar graph data are the means ± sem (n = 5 mice per condition). C, Hydroxyproline content of the ventricle was measured by spectrophotometry, and means ± sem were calculated. *, P < 0.05 for control vs. AngII or vs. AngII plus E2 in the ERβKO mice; +, P < 0.05 for AngII vs. AngII plus E2.

E2 modulates important markers of cardiac hypertrophy

In response to agents such as AngII, the hypertrophic gene and protein program is induced. In the normal heart, myosin light chain α is dominantly expressed with little MHCβ produced, but this is reversed in the hypertrophic heart (20). Here, we find strong expression of MHCα protein in the ventricle of saline-infused, ovariectomized WT mice (control) and weak expression of MHCβ (Fig. 3A). The relative expression of MHC protein isoforms is reversed from 2 wk of AngII treatment, and the AngII effect is prevented significantly by E2 coadministration. Maintenance of normal MHC isoform expression by E2 is evident in the WT and ERαKO mice but is not seen in ERβKO mice (Fig. 3A). This is consistent with ERβ mediating the estrogen effects to prevent hypertrophy.

Figure 3.

Figure 3

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E2 modulates important markers of cardiac hypertrophy. A, MHCα and -β proteins were determined by immunoblot from the pooled ventricles of six mice per condition. GAPDH is shown as a loading control. B, BNP expression in the left ventricle of mice. Individual mouse ventricles were processed for protein extraction to determine BNP expression by immunoblot (n = 6 per condition). *, P < 0.05 for control vs. AngII or E2 or both together. C, AngII activation of ERK is inhibited by E2. Kinase activity in the left ventricle was determined at 2 wk of treatment, from equal amounts of ERK2 protein immunoprecipitated from the three mouse models. Total ERK2 protein is shown as loading control. MBP is myelin basic protein substrate. Bar graph is the mean ± sem of three experiments combined. *, P < 0.05 for control vs. AngII or AngII plus E2 in ERβKO mice; +, P < 0.05 for AngII vs. AngII plus E2.

We also determined the protein expression of BNP in the ventricles of the various mice. In WT mice, both AngII and E2 separately stimulated BNP production; the former probably occurs as compensation for increased cardiac hypertrophy (21) (Fig. 3B). Similarly, AngII and E2 each stimulated BNP in ERαKO mice (compared with saline-infused mice, control). However, only AngII caused this in ERβKO mice. Interestingly, the effect of E2 in ERαKO mice was approximately 50% reduced from the effects of the sex steroid in WT female mice. This suggests that the stimulation of BNP by E2 also occurs through ERα, perhaps as a heterodimer with ERβ. The latter idea is suggested by our finding that E2 has no significant effect in the ERβKO mice, where ERα homodimers are present. We previously showed that both atrial natriuretic peptide (ANP) and BNP production is stimulated by E2 in neonatal rat cardiomyocytes (17). We now report that in vivo, ERα and ERβ mediate chronic production of this important anti-hypertrophic peptide.

G protein-coupled receptors such as β-adrenergic and ET-1 receptors signal to cardiomyocyte hypertrophy in part through activating ERK (22). Here, we find that 2 wk of AngII administration induced a strong activation of ERK in the ventricles of all mouse models (Fig. 3C). E2 significantly inhibited ERK activation by AngII in both WT and ERαKO mice but failed to do so in ERβKO mice. These findings support our previous short-term observations in isolated cardiomyocytes (17) but additionally implicate ERβ as mediating the chronic inhibition of this hypertrophic signal in vivo.

Calcineurin activity is inhibited and MCIP1 gene expression is induced by E2

An important pathway for the induction of cardiac hypertrophy in response to several relevant stimuli depends upon the activation of calcineurin (protein phosphatase 2B) (23,24). We now report that AngII strongly stimulates calcineurin activity in WT and either ERKO isoform mice (Fig. 4A). E2 significantly inhibits AngII-stimulated calcineurin by 60–75% in both WT and ERαKO mice, but has little effect in the absence of ERβ.

Figure 4.

Figure 4

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Inhibition of calcineurin and stimulation of MCIP1 by E2. A, Calcineurin activity was determined in individual samples from six mouse ventricles per condition in WT or KO mice. *, P < 0.05 for control vs. AngII; +, P < 0.05 for AngII vs. AngII plus E2. B, MCIP gene and protein expression were determined by PCR (top panel) from the pooled ventricular samples of mice (n = 6 per condition) and by Western blot (bottom panel), respectively. Actin is shown as loading control.

An important protein that clamps calcineurin activity is MCIP1 (24,25,26). To understand whether the effect of E2 could be mediated through this protein, we determined the MCIP1 gene and protein expression in the left ventricle. MCIP expression was strongly stimulated by E2 in WT and ERαKO mice but much less so in ERβKO mice (Fig. 4B). Interestingly, AngII had no effect on this action of E2. We recently reported that E2 signaling through phosphoinositide-3 (PI3) kinase up-regulates MCIP1 gene expression in cultured cardiomyocytes. Furthermore, knockdown of MCIP1 with small interfering RNA in these cells reversed the ability of E2 to inhibit both calcineurin activity and cell hypertrophy in vitro (17). Our data here indicate that ERβ is particularly important for E2 to up-regulate MCIP1 expression in vivo. This is consistent with ERβ mediating E2 inhibition of calcineurin activity in the setting of induced hypertrophy (Fig. 4A). Based upon our in vitro and in vivo observations, we suggest this is an important mechanism by which E2 prevents the induction of the hypertrophic gene response to AngII (17).

Discussion

Estrogen has been previously proposed to significantly prevent cardiac hypertrophy. In ovariectomized mice, E2 administration prevents ventricular remodeling after myocardial infarction (27). Compared with postmenopausal women not taking sex hormone replacement (HR), sex steroid use results in a significant decrease in LV mass (28). HR also decreases significantly the left ventricular mass index in hypertensive women (11,29). Because blood pressure was not significantly affected in these studies, a direct action of sex steroids on the heart may have occurred. After myocardial infarction, heart failure and death occur less frequently in women taking HR (30). In all these studies, the mechanism of estrogen action was not determined.

In the studies here, we find that E2 strongly inhibits the hypertrophic response to AngII infusion in mice. This includes E2 inhibiting the increased interstitial fibrosis/collagen deposition that is induced by AngII and that eventually impairs cardiac function (8,9). AngII acting through the type I receptor induces TGF-β1 and SMAD3, responsible for induction of cardiac fibrosis (32,33). This may be in part related to perivascular fibrosis, resulting from altered extracellular matrix composition and from the transition of cardiac fibroblasts to myofibroblasts (33). At a cellular level, these changes may be related to the activation of c-Jun N-terminal kinase and activator protein I, stemming from reactive oxygen species (ROS) production that stimulates p38 and c-Jun N-terminal kinase (34,35). ROS formation has increasingly been implicated in myocardial remodeling and hypertrophy (36,37). We previously showed that E2/ER inhibits hypoxia/reoxygenation-induced ROS formation and resulting p38α activation, accounting for cell survival in cultured cardiomyocytes (18). We propose that similar mechanisms may underlie E2/ER prevention of cardiac hypertrophy and will serve as a basis for future investigation.

AngII activates several pathways to produce hypertrophy, but a prominent signal for many hypertrophic stimuli is calcium signaling to the up-regulation of calcineurin (protein phosphatase 2B) activity (23,24,25). Much data support a central role for calcineurin, and mice that are null for the catalytic subunit of this phosphatase show impaired hypertrophic responses to AngII, aortic constriction, or isoproterenol (38). When calcineurin is induced, it dephosphorylates and promotes the translocation of the transcription factor NFATc3 to the nucleus. In the nucleus, NFATc3 cooperates with GATA-4 and myocyte-enhancing factor transcription factors to stimulate hypertrophic gene expression (38,39,40). An important protein that prevents calcineurin activity is the anti-hypertrophic protein MCIP1. Functionally, overexpression of MCIP1 reduces cardiac hypertrophy after aortic banding (24,25). Here we report that E2 inhibits calcineurin activity that is stimulated by AngII and up-regulates MCIP1 expression in the ventricle. We recently found that small interfering RNA directed to MCIP1 substantially reversed the inhibitory effects of E2 on calcineurin activity, new protein synthesis, and cardiomyocyte hypertrophy. This mechanism accounted for the modulation of NFAT protein translocation to the nucleus and related transcriptional activity, stimulated by AngII and inhibited by E2 (17).

There is also evidence that AngII administration in vivo causes hypertension (41), potentially inducing cardiac hypertrophy indirectly. Estrogen administration might decrease the resulting cardiac phenotype conceptually by lowering blood pressure. This probably occurs through ERβ stimulating nitric oxide synthase activity (42). Very recent studies from Jazbutyte et al. (43) show that administration of an ERβ-specific agonist, 8β-VE2 (Schering Pharmaceutical), to ovariectomized spontaneously hypertensive rats lowers systolic blood pressure and peripheral arterial resistance and attenuates cardiac hypertrophy. It is known that nitric oxide is an anti-hypertrophic factor (44), generated directly in the heart or in endothelial and vascular smooth muscle cells. In cultured rodent cardiomyocytes, we previously showed direct hypertrophic effects of AngII, mitigated by E2. This includes E2 stimulation of nitric oxide production from these cells (17). Thus, the interplay of these two hormones modulates cardiac hypertrophy through several in vivo mechanisms. Consistent with this, activation of ERK is a known stimulus for cardiomyocyte hypertrophy (22), and we find that AngII stimulation of this pathway occurs directly in these cells (17) and now in our in vivo models, both blocked by E2.

From our investigations, a potential mechanism for E2 action can be postulated to understand a model of hypertrophy in genetically altered mice. Targeted deletion of the FKBP12.6 gene from the cardiomyocyte leads to disordered calcium sparking through the ryanodine receptor (45). In these mice, only the postnatal males developed cardiac hypertrophy. However, female mice developed severe hypertrophy when administered the ER antagonist tamoxifen. It is unknown how estrogen modulates the dysregulation of intracellular calcium and downstream events to prevent this hypertrophic phenotype. We suggest that deleted FKBP12.6 enhances calcium-dependent calcineurin activity and subsequent cardiac hypertrophy. Estrogen would mitigate the loss of the FKBP12.6 gene by increasing MCIP1 to down-regulate calcium-induced calcineurin activity, preventing hypertrophy. Mutation and functional loss of FKBP12.6 also predisposes mice or humans to fatal cardiac arrhythmias (46), but it is unknown whether sexual dichotomy also exists in these respects.

As another reported mechanism of E2 action, the sex steroid up-regulates the ANP gene, and this induces a decrease in phenylephrine-induced cardiomyocyte hypertrophy (47). Genetic deletion of the guanylate cyclase A protein, the functional receptor for ANP and BNP, results in pronounced hypertrophy as induced by several stimuli (48). Here we found the BNP protein is stimulated by either AngII or E2. Natriuretic peptides inhibit ET-1 and AngII signaling to cardiomyocyte hypertrophy (49), and we showed that E2 stimulates ANP and BNP production in cultured cardiomyocytes (17).

We also determined that the ERβ mediates the anti-hypertrophic effects of E2. This finding is consistent with previous studies (50,51), and we report the novel observations that inhibition of ERK and calcineurin activities, stimulation of MCIP1 and BNP, and inhibition of interstitial fibrosis are all mediated through ERβ. This receptor is present mainly in the mitochondria of cardiomyocytes (52), but we have also demonstrated the presence of both ERα and ERβ at the plasma membrane of adult and neonatal heart muscle cells (17). The membrane-localized ER population (and not the nuclear ER pool) is the one responsible for the modulation of rapid signal transduction in many organs (53). The ability of E2 to signal through PI3 kinase protects rats from ischemia-reperfusion injury in muscle (54), and it has recently been shown that this sex steroid modulates a variety of kinases in the in vivo heart and cultured cardiomyocytes (55). We previously reported that membrane ER/E2 induces PI3 kinase activity that up-regulates MCIP1 transcription (17) and now propose that ERβ mediates these in vivo effects of E2.

In summary, E2 and ERβ significantly inhibit AngII-induced cardiac hypertrophy through multiple novel mechanisms. Direct effects on the myocardium are summarized in Fig. 5. AngII-stimulated cardiac hypertrophy is a leading cause of this disorder in humans. If similar actions of E2 occur in women, E2 or ERβ agonist administration may pose a preventative strategy in some individuals. Selective ERβ agonists are particularly attractive for this purpose because they lack the breast and uterine proliferative effects of E2 or selective ER modulators that act at ERα and thus support the malignant transformation of these tissues (31,56).

Figure 5.

Figure 5

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E2 and ERβ inhibit AngII-induced cardiac hypertrophy. The illustration represents data from in vitro (17) and in vivo studies here. AngII binds the AT1 receptor that activates Gqα and Gβγ signaling to up-regulate calcium. This signaling induces the activity of protein phosphatase 2B (calcineurin), dephosphorylating NFAT transcription factors in the cytosol. As a result, NFAT proteins (especially NFATc3) move to the nucleus where they collaborate with GATA-4 and MEF-2 transcription factors to stimulate the hypertrophic gene program. AngII also stimulates ERK MAPK signaling to effect gene up-regulation. E2 acting through ERβ stimulates the MCIP1 gene via PI3 kinase (PI3K) signaling, the protein product of which binds/clamps the catalytic activity of calcineurin. This prevents NFAT translocation to the nucleus and inhibits the hypertrophic genes required for cardiomyocyte increase in size (hypertrophy). E2 activating both ERα and ERβ stimulates the ANP and BNP genes, whose secreted protein products bind the guanylate cyclase A receptor and inhibit AngII-induced ERK activation. NCX, Sodium/calcium exchanger.

Footnotes

This work was supported by grants from the Research Service of the Department of Veteran’s Affairs and National Institutes of Health CA-100366 (to E.R.L.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 27, 2008

Abbreviations: AngII, Angiotensin II; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; E2, 17β-estradiol; ER, estrogen receptor; ET-1, endothelin-1; HR, hormone replacement; KO, knockout; LV, left ventricular; MCIP, modulatory calcineurin-interacting protein; MHC, myosin heavy chain; NFAT, nuclear factor of activated T cells; ROX, reactive oxygen species; WT, wild type.

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