Activation of GPR30 attenuates diastolic dysfunction and left ventricle remodelling in oophorectomized mRen2.Lewis rats.
ABSTRACT GPR30 is a novel oestrogen receptor expressed in various tissues, including the heart. We determined the role of GPR30 in the maintenance of left ventricular (LV) structure and diastolic function after the surgical loss of ovarian hormones in the female mRen2.Lewis rat, a model emulating the cardiac phenotype of the post-menopausal woman.
Bilateral oophorectomy (OVX) or sham surgery was performed in study rats; the selective GPR30 agonist, G-1 (50 µg/kg/day), or vehicle was given subcutaneously to OVX rats from 13-15 weeks of age. Similar to the cardiac phenotype of sham rats, G-1 preserved diastolic function and structure relative to vehicle-treated OVX littermates independent of changes in blood pressure. G-1 limited the OVX-induced increase in LV filling pressure, LV mass, wall thickness, interstitial collagen deposition, atrial natriuretic factor and brain natriuretic peptide mRNA levels, and cardiac NAD(P)H oxidase 4 (NOX4) expression. In vitro studies showed that G-1 inhibited angiotensin II-induced hypertrophy in H9c2 cardiomyocytes, evidenced by reductions in cell size, protein content per cell, and atrial natriuretic factor mRNA levels. The GPR30 antagonist, G15, inhibited the protective effects of both oestradiol and G-1 on this hypertrophy.
These data show that the GPR30 agonist G-1 mitigates the adverse effects of oestrogen loss on LV remodelling and the development of diastolic dysfunction in the study rats. This expands our knowledge of the sex-specific mechanisms underlying diastolic dysfunction and provides a potential therapeutic target for reducing the progression of this cardiovascular disease process in post-menopausal women.
- SourceAvailable from: circres.ahajournals.org[show abstract] [hide abstract]
ABSTRACT: Abnormalities of diastolic function are common to virtually all forms of cardiac failure. However, their underlying mechanisms, precise role in the generation and phenotypic expression of heart failure, and value as specific therapeutic targets remain poorly understood. A growing proportion of heart failure patients, particularly among the elderly, have apparently preserved systolic function, and this is fueling interest for better understanding and treating diastolic abnormalities. Much of the attention in clinical and experimental studies has focused on relaxation and filling abnormalities of the heart, whereas chamber stiffness has been less well studied, particularly in humans. Nonetheless, new insights from basic and clinical research are helping define the regulators of diastolic dysfunction and illuminate novel targets for treatment. This review puts these developments into perspective with the major aim of highlighting current knowledge gaps and controversies.Circulation Research 07/2004; 94(12):1533-42. · 11.86 Impact Factor
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ABSTRACT: We investigated the effects of transdermal 17beta-estradiol, combined with standard antihypertensive therapy, on the modification of the cardiovascular risk profile in hypertensive postmenopausal women. In a randomized, double-blind, placebo-controlled study, we enrolled 200 postmenopausal women with mild to moderate hypertension. Patients received 17beta-estradiol (50 microg/day, transdermal) and norethisterone acetate (2.5 mg/ day, orally) or placebo. At baseline serum total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, glucose, and fibrinogen plasma levels were measured and all subjects underwent complete M-mode and 2-D echocardiograms, which were repeated after 6, 12, and 18 months of hormonal replacement therapy. Compared with placebo, all values decreased significantly except for HDL cholesterol. In both groups, no modifications were observed in echocardiographic parameters, except for left ventricular mean diastolic and systolic wall thickness and left ventricular mass index, which showed a significant decrease in both groups. The reduction was greater in the treated group; the percentage of patients with left ventricular hypertrophy was 46% before randomization and 17.2% after 18 months of treatment (P < .0001), whereas in group II the percentage was 48% at baseline and 31.5% after 18 months (P < .05). In conclusion, transdermal 17beta-estradiol, associated with antihypertensive therapy, may contribute to the reduction of cardiovascular risk profile in hypertensive postmenopausal women.American Journal of Hypertension 10/1999; 12(10 Pt 1):1000-8. · 3.67 Impact Factor
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ABSTRACT: In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.Endocrinology 11/2008; 150(2):687-98. · 4.72 Impact Factor
(50 mg/kg/day), or vehicle was given subcutaneously to OVX rats from 13–15 weeks of age. Similar to the cardiac
phenotype of sham rats, G-1 preserved diastolic function and structure relative to vehicle-treated OVX littermates
independent of changes in blood pressure. G-1 limited the OVX-induced increase in LV filling pressure, LV mass, wall
thickness, interstitial collagen deposition, atrial natriuretic factor and brain natriuretic peptide mRNA levels, and
cardiac NAD(P)H oxidase 4 (NOX4) expression. In vitro studies showed that G-1 inhibited angiotensin II-induced
hypertrophy in H9c2 cardiomyocytes, evidenced by reductions in cell size, protein content per cell, and atrial natri-
uretic factor mRNA levels. The GPR30 antagonist, G15, inhibited the protective effects of both oestradiol and G-1 on
These data show that the GPR30 agonist G-1 mitigates the adverse effects of oestrogen loss on LV remodelling and
the development of diastolic dysfunction in the study rats. This expands our knowledge of the sex-specific mechan-
isms underlying diastolic dysfunction and provides a potential therapeutic target for reducing the progression of this
cardiovascular disease process in post-menopausal women.
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Cardiac hypertrophy † Diastolic dysfunction † GPR30 † mRen2.Lewis rat † Post-menopausal women
Activation of GPR30 attenuates diastolic
dysfunction and left ventricle remodelling
in oophorectomized mRen2.Lewis rats
Hao Wang1, Jewell A. Jessup1, Marina S. Lin1, Clarissa Chagas1, Sarah H. Lindsey2,
and Leanne Groban1,3*
1Department of Anesthesiology, Wake Forest School of Medicine, Winston-Salem, Medical Center Boulevard, Winston-Salem, NC 27157-1009, USA;2Hypertension and Vascular Research
Center, Wake Forest School of Medicine, Winston-Salem, NC, USA; and3Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston-Salem, NC, USA
Received 23 August 2011; revised 1 February 2012; accepted 7 February 2012; online publish-ahead-of-print 10 February 2012
Time for primary review: 23 days
GPR30 is a novel oestrogen receptor expressed in various tissues, including the heart. We determined the role of
GPR30 in the maintenance of left ventricular (LV) structure and diastolic function after the surgical loss of ovarian
hormones in the female mRen2.Lewis rat, a model emulating the cardiac phenotype of the post-menopausal woman.
Bilateral oophorectomy (OVX) or sham surgery was performed in study rats; the selective GPR30 agonist, G-1
The prevalence of diastolic dysfunction dramatically increases in post-
menopausal women.1Unlike men of equivalent age, post-menopausal
women predominantly exhibit impairments in diastolic function with
concentric left ventricular (LV) remodelling and eventual diastolic
heart failure rather than systolic dysfunction, eccentric remodelling,
and systolic heart failure.2Diastolic heart failure, also known as
heart failure with preserved ejection fraction, accounts for 40–60%
of chronic heart failure cases in industrialized nations,3and over
75% of these cases are in older women.4Hospital admissions for dia-
stolic heart failure have increased steadily over the past two decades,
with female patients having the highest annual percentage increase in
hospitalization rates when compared with males with heart failure.3
This may be due in part to delayed diagnoses of heart failure in
women compared with men.5,6In addition, the prevalence and prog-
nostic impact of its precursor, diastolic dysfunction, has only recently
been recognized and the mechanisms are not yet clear.7
Evidence suggests that the loss of oestrogens contributes to the de-
velopment of hypertension and cardiac hypertrophy in post-
menopausal women;8–10and these diseases are risk factors for
diastolic dysfunction.11,12While the prevalence of hypertension rises
with age for both genders, the near doubling in the prevalence of
hypertension in women aged 70–79 compared with those aged 50–
* Corresponding author. Tel: +1 336 716 4498; fax: +1 336 716 8190, Email: email@example.com
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2012. For permissions please email: firstname.lastname@example.org.
Cardiovascular Research (2012) 94, 96–104
59 may be attributed, in part, to the decline in oestrogen during meno-
pause.13,14Moreover, the evolution of LV hypertrophy differs between
pre- and post-menopausal women, with a greater induction of hyper-
trophy when circulating oestrogen levels are reduced.15Experimental
studies in adult female rats show that ovariectomy eliminates female-
specific protection against volume-induced cardiac hypertrophy and re-
modelling,16while oestradiol administration attenuates hypertrophy
associated with pressure overload17and ageing.18Although activation
of the classical oestrogen receptors (ER)-a and ER-b located on myo-
cytes, fibroblasts, and the extracellular matrix might be involved in
oestrogen-mediated cardioprotection,19the exact mechanisms for
the beneficial effects of oestrogen on cardiac hypertrophy, fibrosis,
and diastolic dysfunction remain unclear.
With the recent discovery of the G protein-coupled oestrogen
receptor (GPR30) in the heart,12,20the potential mechanisms for
oestrogen-mediated cardioprotection are expanded. GPR30, also
known as GPER, is located on the cell membrane and endoplasmic re-
ticulum,21,22and is widely distributed, independent of species and sex,
among a variety of mammalian tissues including the heart, lung, liver,
adrenal gland, intestine, ovary, and brain.23,24GPR30 activation by its
specific agonist, G-1, improves contractile function and reduces infarct
size in isolated rat and mouse hearts subjected to ischaemia/reperfu-
sion injury.20,25–27We found increased cardiac GPR30 mRNA levels
in the female mRen2.Lewis rat following chronic consumption of a
high-salt diet12or surgically induced oestrogen loss (see Supplemen-
tary material online). Further studies from our laboratory show that in
vivo activation of GPR30, with G-1, attenuates the adverse effects of
high salt on diastolic function and cardiac remodelling.12Indeed, en-
dogenous oestrogens could have contributed to the anti-remodelling
and lusitropic benefits following high salt consumption,28since oestra-
diol binds GPR30 at an affinity similar to G-1.29Nonetheless, these
data support the notion that cardiac GPR30 is increased during
periods of physiological and pathophysiological stress, and that its ac-
tivation could provide valuable cardioprotection.
Therefore, using the oophorectomized (OVX) mRen2.Lewis rat, an
established rodent model that mimics the cardiac phenotype of
women following surgical or natural cessation of ovarian hormone
production,28,30–34we hypothesized that GPR30 activation by G-1
preserves diastolic function and mitigates cardiac remodelling follow-
ing the loss of oestrogens.
Female mRen2.Lewis rats were obtained from the Hypertension and Vas-
cular Research Center Congenic Colony at Wake Forest School of Medi-
cine and all studies were approved by the institution’s Animal Care and
Use Committee. All animal procedures conformed to the Guide to the
Care and Use of Laboratory Animals published by the US National Institutes
of Health. Rats were individually housed in an Association for Assessment
and Accreditation of Laboratory Animal Care-approved, temperature
(22+28C) and light (12 h light/dark cycle) controlled facility with ad
libitum access food and water.
2.2 Experimental protocol
At 4 weeks of age, rats were randomly assigned to undergo either OVX
(n ¼ 16) or sham operation (Sham; n ¼ 10) performed under 2% isoflur-
ane anaesthesia, as previously described.30,31The adequacy of anaesthesia
was monitored by observation of slow breathing, loss of muscular tone,
and no response to surgical manipulation. The success of OVX and the
subsequent depletion of circulating oestrogens were confirmed using a
serum oestradiol assay (5 pg/mL detection limit; Polymedco, Cortlandt
Manor, NY, USA) at the end of the experiment (data not shown).
Once the rats reached 13 weeks of age, the OVX group was further ran-
domly divided to receive either the GPR30 agonist, G-1, (OVX-G1; n ¼ 7;
Cayman Chemical Company, Ann Arbor, MI, USA) diluted in a DMSO/
EtOH12mixture for a targeted dose of 50 mcg/kg/day or the DMSO/
EtOH vehicle (OVX-V; n ¼ 9) and were administered subcutaneously
via osmotic mini-pumps (DURECT Corporation, Cupertino, CA, USA).
This dose of G-1 was determined by an initial pilot study conducted in
our laboratory that demonstrated lusitropic effectiveness without signifi-
cant blood pressure reductions (Figure 1). Weekly body weight and systol-
ic blood pressures by tail-cuff plethysmography (NIBP-LE5001, Panlab,
Barcelona, Spain) were monitored throughout the study. Rats were eutha-
nized via exsanguination by cardiac puncture while under ketamine/xyla-
zine anaesthesia (ketamine HCL 60 mg/kg and xylazine HCL 5 mg/kg) at
15 weeks of age, following echocardiographic evaluation. Whole hearts
were isolated and further dissected to isolate the left ventricle, right ven-
tricle, and atria. Tissue weights and tibial lengths were measured with an
analytical scale and a micrometer, respectively. The left ventricle was cut
into pieces for RNA, western blot, and histological analyses.
2.3 Echocardiographic evaluation
LV function and dimensions were assessed prior to the protocol termin-
ation in anaesthetized (ketamine/xylazine: 80/12 mg/kg) animals using a
Philips 5500 echocardiograph (Philips Medical Systems, Andover, MA,
USA) and a 12 MHz phased array probe as previously described.12,28,30,31
2.4 Determination of collagen deposition
in the heart
LV specimens were fixed in 4% paraformaldehyde and embedded into
paraffin blocks; 4 mm sections were stained with picrosirius red, as previ-
ously described.30,31Images were captured using an Axiovert 200 micro-
scope (Thornwood, NY, USA). The ratio of collagen-stained pixels to
unstained pixels was quantified using NIH ImageJ software (http://
2.5 Cardiomyocyte size measurement
embedded LV sections. Alexa Flour 488-conjugated wheat germ agglutinin
(WGA, 10 mg/mL, Invitrogen, Carlsbad, CA, USA) was employed to delin-
eate cellular membranes. TO-PRO-3 (1/1000 dilution, Invitrogen, Carls-
bad, CA, USA) was used for nuclear staining. Images were captured
areas were measured in paraffin-
sham-operated and ovariectomized female mRen2.Lewis rats
treated with G-1 or vehicle for 2 weeks. Values are means+SEM;
OVX, ovariectomized; V, vehicle. *P , 0.05 vs. sham. n ¼ 7–10/
1 Tail-cuffsystolicblood pressure in conscious,
GPR30 attenuates diastolic dysfunction
using a Zeiss LSM 510 confocal microscope (Thornwood, NY, USA) and
analysed with Zeiss LSM Image Browser (version 3,2,0,70). For each
section, at least 300 round cells with centrally located nuclei were mea-
sured, and the average value was used as the final cardiomyocyte size.
2.6 Immunofluorescence staining and
Immunofluorescent staining of heart sections and immunocytochemistry
in cultured cells were performed using standard procedures. Images
were captured using a Zeiss LSM 510 confocal microscope. For detailed
methodology, please refer to the Supplementary material online.
2.7 Cell culture and treatment
The H9c2 myoblast cell line, derived from embryonic rat heart, was
obtained from American Type Culture Collection (ATCC; Manassas,
VA, USA). Cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM, Gibco) with 10% heat-inactivated foetal bovine serum (FBS),
penicillin (100 U/mL), streptomycin (100 mg/mL), at 378C in 5% CO2
and 95% air, at a relative humidity of 95% and split 1:4 at subconfluence
(80%). Before each experiment, cells were seeded in six-well plates or
chamber slides (Lab-TekTMChamber Slides: Thermo Scientific NUNC
177437) at the density of 5 × 104cells/cm2and starved for 18 h in
DMEM containing 0.1% FBS. Cells were subsequently treated with angio-
tensin II (Ang II, 1027M, Bachem), oestradiol (E2) (1027M, Sigma), G-1
(1027M, Cayman Chemical Company), G15 (1026M, Tocris Bioscience).
After an additional 24-h incubation, cells were fixed with cold ethanol
for immunocytochemical staining, lysed with TRIzol (Invitrogen, Carlsbad,
CA, USA) for real-time (RT)–PCR analysis, or trypsinized for protein ana-
lysis. Protein content was determined using a Biorad protein assay kit
(Hercules, CA, USA).
2.8 Analysis of gene expression by quantitative
RT–PCR was used to detect gene mRNA levels in cardiac tissue or H9c2
cells. For detailed methodology, please refer to the Supplementary mater-
2.9 Western blot analysis
LV tissue homogenates were separated by SDS–PAGE and transferred
onto membranes as described previously.12,31
probed using antibodies for NAD(P)H 4 (NOX4) (2 mg/mL; Abcam, Cam-
bridge, MA, USA) collagen I (1:200; Santa Cruz Biotechnology, Santa Cruz,
CA, USA), collagen III (1:200; Santa Cruz Biotechnology), transforming
growth factor beta-1 (TGF-b1) (1:500; Santa Cruz Biotechnology), sarco-
plasmic endoplasmic reticulum ATPase (SERCA2;1:1000; Abcam), and
phospholamban (PLB) (1:5000; Abcam). The PLB-to-SERCA2 ratio was
used as a measure of SERCA2 inhibition. Glyceraldehyde-3-phosphate de-
hydrogenase (GAPDH; 1:5,000; Cell Signaling, Danvers, MA, USA) was
used as a loading control. The bands were digitized using MCID image
analysis software (Imaging Research, Inc., Ontario, Canada). Each band
was expressed in arbitrary units and normalized to its own GAPDH.
2.10 Statistical analyses
All results were expressed as mean+SEM. For all endpoints, one-way
ANOVA evaluated significant effects among the groups. Significant inter-
actions between the groups were further characterized using Student–
Newman–Keuls post hoc analyses. Analyses were performed using Graph-
Pad Prism version 5 (GraphPad, San Diego, CA, USA). Differences were
considered significant at P , 0.05.
3.1 Blood pressure changes during the
Consistent with our previous studies,28,30,31loss of ovarian hormones
exacerbated hypertension in the female mRen2.Lewis rat. At 13
weeks of age, systolic blood pressure was 173+6 mmHg in OVX
rats vs. 147+3 mmHg in intact littermates (P , 0.001). Since
chronic G-1 treatment, at the dose of 400 mg/kg/day, has significant
blood pressure lowering effects in this strain,34and since afterload
is an important factor that affects diastolic function and myocardial re-
laxation,35we conducted a pilot study to determine the ideal lusitro-
pic dose of G-1, as determined by tissue Doppler mitral annular
descent (e′) that did not alter blood pressure (Figure 1). Accordingly,
we chose to chronically administer G-1 at 50 mg/kg/day, as ,100 mg/
kg/day of G-1 had no effect on blood pressure (data not shown).
Serial systolic blood pressures during the 2-week treatment period
were not different between OVX-vehicle and OVX-G-1 treated
rats, but both OVX-groups had significantly higher blood pressures
compared with their oestrogen-intact littermates at each time-point
3.2 G-1 attenuated the adverse effects
of oestrogen loss on diastolic function
In the present study, we found that cardiac GPR30 mRNA levels
were increased by 60% in OVX rats when compared with
sham-operated littermates (see Supplementary material online,
Figure S1). We evaluated the cardioprotective potential of GPR30
activation by its agonist, G-1, in OVX-mRen2.Lewis rats using
both conventional and tissue Doppler echocardiography. While
overt differences in the early-to-late (E/A) transmitral Doppler
filling ratios were not observed among groups (P ¼ 0.08; see Sup-
plementary material online, Table S1), the 2-week subcutaneous
infusion of G-1 attenuated the decrease in myocardial relaxation
(e′), increase in Doppler-derived left ventricle filling pressures
(E/e′), and increase in cardiac gene expression of brain natriuretic
peptide (BNP) and atrial natriuretic factor (ANF) that occurred
following the surgical loss of oestrogens in this strain (Figure 2). Sys-
tolic function, as determined by per cent fractional shortening and
velocity of circumferential fibre shortening (Vcf), was not affected
by the loss of oestrogens or the administration of G-1 (Figure 2B
and Table 1).
3.3 G-1 attenuated the effect of oestrogen
loss on hypertrophic and extracellular LV
The early surgical loss of oestrogen (at 4 weeks of age) led to
significant increases in heart weight and left ventricle weight by
15 weeks of age (Table 1 and Figure 3). The whole heart weight
increased by 20% and the LV weight increased by 14% in the
OVX- compared with sham-operated rats (P , 0.05). Interestingly,
2 weeks of G-1 attenuated the effect of ovarian hormone loss on
heart weight. (Table 1, Figure 3C). These findings are consistent
with echocardiographic-derived morphometric observations. In the
hearts of OVX rats, the posterior and anterior wall thicknesses,
relative wall thickness, and LV mass were significantly increased
when compared with measures from the sham-operated group.
H. Wang et al.
These increases were also mitigated by 2 weeks of G-1 treatment
(Table 1, Figure 3A and B). Moreover, cardiomyocyte size was signifi-
cantly increased in OVX- vs. oestrogen-intact rats, and this effect
was inhibited by G-1 (Figure 3D and E).
Consistent withour previous
OVX-mRen2.Lewis rats when compared with sham-operated litter-
mates (OVX: 5.7+0.4% vs. Sham: 4.3+0.4%, P , 0.05). This in-
crease in interstitial fibrosis was attenuated by G-1 (OVX-G-1:
5.0+0.4% vs. OVX-V: 5.7+0.4%, P , 0.05) (Figure 4A). In
hearts from OVX rats, collagen III protein expression was
increased and collagen I was decreased when compared with
hearts from sham-operated rats. These changes were attenuated
in hearts from G-1 treated rats (Figure 4B). In addition, cardiac
TGF-b1 mRNA was significantly increased by OVX and attenuated
by G-1 (see Supplementary material online, Figure S2B). Likewise,
TGF-b1 protein expression was increased in hearts from OVX
rats and attenuated by G-1 (see Supplementary material online,
Figure 2 (A) Representative transmitral and tissue Doppler images of sham and OVX mRen2.Lewis female rats receiving either vehicle or G-1 treat-
ment for 2 weeks. (B) Per cent fractional shortening. (C) Early mitral annular velocity (e′). (D) The ratio of transmitral early filling-to-early mitral
annular descent (E/e′), or index of filling pressures. (E and F) Cardiac brain natriuretic peptide (BNP) and atrial natriuretic factor (ANF) mRNA
levels. Values are mean+SEM; *P , 0.05 vs. sham, #P , 0.05 vs. OVX-vehicle. n ¼ 7–10/group.
Heart rate (b.p.m.) 246+8
Body weight (g) 232+17
LVEDd (cm) 0.68+0.02
Table 1 Heart weight, heart rate, and echocardiography
HW/TL, heart weight/tibial length; LVESd, left ventricular end-systolic dimension;
LVEDd, left ventricular end-diastolic dimension; RWT, relative wall thickness; LV mass,
left ventricular mass; Vcf, velocityof circumferentialfibreshortening. Dataaremean+
*P , 0.05 vs. sham.
**P , 0.05 vs. OXV-V.
GPR30 attenuates diastolic dysfunction
3.4 G-1 inhibited the increase in cardiac
NOX4 associated with oestrogen loss
In the hypertrophied hearts from OVX-mRen2.Lewis rats, we found
that NOX4 mRNA was increased by 80% when compared with
sham-operated rats. Importantly, this increase in cardiac NOX4
mRNA was inhibited by G-1 (Figure 5A). Consistent with these
changes in gene levels, cardiac NOX4 protein was also increased
with OVX and attenuated by G-1 (Figure 5B and C).
RT–PCR did not reveal differences in the cardiac mRNA levels of
NOX2, p22phox, and p47phox (see Supplementary material online,
3.5 Calcium-related gene expression
in the heart
Cardiac mRNA levels of sarcoendoplasmic reticulum calcium ATPase
(SERCA) 2, PLB, sodium-calcium exchanger 1 (NCX1), NCX3, calse-
questrin 1 (CASQ1), CASQ2, triadin, cardiac ryanodine receptor 2
(RyR2), calreticulin, and calmodulin-1 were similar across all groups
(see Supplementary material online, Figure S3). Likewise, the
PLB-to-SERCA2 protein ratio, an index of SERCA2 inhibition, was
not significantly influenced by ovarian hormonal withdrawal or G-1
treatment (see Supplementary material online, Figure S4).
Figure 3 (A and B) Echocardiographic-derived posterior (left) and anterior (right) LV wall thicknesses. (C) Heart weight of rats. (D) Quantification
of cardiomyocyte size. (E) Representative confocal imagines showing wheat germ agglutinin (WGA) membrane and TO-PRO-3 nuclear staining in the
left ventricular cardiomyocytes from sham, OVX-V, and OVX-G-1 rats. Values are mean+SEM; * P , 0.05 vs. sham, #P , 0.05 vs. OVX-vehicle.
n ¼ 7–10/group.
Figure 4 (A) Upper panel: representative LV images, in each
group, of collagen deposition (×200 magnification); lower panel:
quantification of collagen deposition. Values are mean+SEM;
*P , 0.05 vs. sham, #P , 0.05 vs. OVX-vehicle. n ¼ 5–6/group.
(B) Pictures from western blot showing cardiac collagen I and colla-
gen III protein expression.
H. Wang et al.
3.6 Protective effects of GPR30 on
angiotensin II-induced hypertrophy in H9c2
To better understand the hypertrophic limiting capacity of GPR30, we
used H9c2 cardiomyocytes treated with angiotensin II (Ang II)
(Figure 6). Consistent with previous reports,36Ang II (1027M) treat-
ment for 24 h induced hypertrophy in H9c2 cardiomyocytes, demon-
strated by increases in cell size (Figure 6A), protein content per cell
(Figure 6C), and ANF mRNA levels (Figure 6B). These indicators of
cell hypertrophy were significantly inhibited following treatment
with either E2 or G-1 (1027M). This anti-remodelling effect of E2
was partially prevented by co-incubation with the GPR30 antagonist
G15 (1026M), while the protective effect of G-1 on cell size was com-
pletely blocked by G15 (Figure 6).
The most important findings of this study are that chronic GPR30 ac-
tivation by its agonist G-1 for 2 weeks attenuated the adverse effects
of ovariectomy on diastolic function and LV remodelling in the
female mRen2.Lewis rat. Importantly, this GPR30-mediated lusitropic
and anti-remodelling effect occurred in the absence of a significant re-
duction in systolic blood pressure. Together with our previous data
from the oestrogen-intact-mRen2.Lewis rat fed a high-salt diet,12
these observations provide evidence that an increase in GPR30 recep-
tor expression in the heart may contribute to cardiac functional and
structural stability during periods of physiological (e.g. oestrogen loss)
and pathophysiological stress (e.g. high salt intake). Given the
cardioprotective reports of G-1 in other animal models and
strains,20,25–27,37our findings are unlikely exclusive to the female
The adult mRen2.Lewis female rat, a congenic rodent model of
angiotensin II- and oestrogen-dependent hypertension,32consistently
manifest elevations in systolic blood pressure, LV hypertrophy, and dia-
stolic functional impairments when ovariectomy occurs between 4 and
5 weeks of age.26,30–32Our approach of initiating treatment after the
establishment of the exacerbated hypertension is practical as it emu-
lates the cardiovascular phenotype of both pre- and post-menopausal
women after the surgical or natural cessation of ovarian hormone pro-
duction.15,38While others have shown that non-selective ER stimula-
hypertrophy and fibrosis,19our study using the selective agonist
G-129strongly suggests that the cardiac GPR30 receptor is involved
in the maintenance of LV structure and function, particularly in females.
LV hypertrophy commonly occurs in women after the cessation of
ovarian oestrogen production39and is an independent risk factor for
the development of ventricular stiffness and impaired myocardial re-
laxation.40In rodent studies, cardioprotective and anti-hypertrophic
oestrogenic effects have been well described. In adult rats, ovariec-
tomy inhibits female-specific protection against pressure-induced
hypertrophic remodelling16and attenuates the effect of age on ven-
tricular remodelling,18while oestradiol replacement reverses these
Consistent with these studies and our own,30–32
OVX-mRen2.Lewis rats exhibited increased myocyte cell size, ven-
tricular wall thickness, and LV weight compared with oestrogen-intact
littermates. One striking and novel finding from our in vivo study was
that administration of G-1, the selective agonist for the G protein-
coupled membrane ER GPR30, attenuated hypertrophic remodelling
Figure 5 (A) Cardiac NOX4 mRNA levels. (B) NOX4 protein expression. Upper panel: representative pictures from western blot; lower panel:
quantification with densitometry and corrected by internal control GAPDH expression. (C) Representative confocal images of double-staining for
desmin (red) and NOX4 (green) in left ventricle of rats in sham, OVX-V, and OVX-G-1 groups. The nuclei were counterstained with DAPI
(blue) (×200 magnification). Values are mean+SEM; *P , 0.05 vs. sham; #P , 0.05 vs. OVX-V.
GPR30 attenuates diastolic dysfunction
without affecting blood pressure. These favourable effects of GPR30
activation were further corroborated in H9c2 cultured cardiomyo-
cytes where G-1 reversed Ang-II mediated increases in cell size,
ANF expression, and cell protein content. While additional studies
are needed to determine the signalling pathways associated with
GPR30-mediated reductions in cardiomyocyte size, our data strongly
suggest that GPR30 activation with G-1 preserves diastolic function
after oestrogen loss, in part by attenuating hypertrophic remodelling.
Cardiac collagen deposition not only contributes to hypertrophic
remodelling, it directly influences LV compliance and, subsequently,
diastolic function.41In this study and our previous studies,28,30,31oes-
trogen loss by early ovariectomy in the mRen2.Lewis strain led to
interstitial and perivascular fibrosis with associated increases in
Doppler-derived filling pressures (E/e′). In the absence of overt valvu-
lar heart disease or atrial arrhythmias, increases in LV filling pressure,
or E/e′, are indicative of increased LV chamber stiffness, advanced dia-
stolic dysfunction, and worsening clinical outcomes.42Different lines
of evidence indicate that oestrogens influence the extracellular
matrix in the heart, specifically the collagens. In vitro studies show
that oestradiol suppresses rat cardiac fibroblast proliferation along
with their capacity to produce collagen.43In aged rats, ovariectomy
leads to increased collagen I, reduced collagen III, and metalloprotei-
nase 2 (which degrades collagen), all of which are reversed by oestro-
Surprisingly,in mRen2.Lewis rats,early
ovariectomy reduced cardiac collagen I and increased collagen III sug-
gesting that the changes in extracellular matrix remodelling following
oestrogen loss might be age- and strain-dependent. In 8-week-old
female Sprague-Dawley rats subjected to volume overload, collagen
I is reduced while collagen III tended to be elevated.44In this same ex-
perimental model of volume overload, ovariectomy had no effect on
either type of cardiac collagen.44Our present data suggest that
9 weeks following ovariectomy, a chronic 2-week subcutaneous infu-
sion of G-1 inhibited interstitial myocardial fibrosis and improved
Doppler-derived filling pressures in OVX-G-1 rats when compared
with ovary-intact littermates. Although we do not yet know the mech-
anism by which GPR30 influences cardiac collagen deposition and
cardiac stiffness, further investigations in cultured cardiac fibroblasts
will help clarify the GPR30-related signalling pathways involved in miti-
gating fibroblast proliferation and collagen production.
Changes in the content and isoforms of proteins involved intracel-
lular Ca2+handling could also contribute to impairments in myocar-
dial relaxation, and result in diastolic dysfunction.45SERCA2 and PLB
are two key proteins involved in calcium re-uptake into the sarcoplas-
mic reticulum, and subsequent maintenance of lusitropic function.45
Unlike our findings in the hypertensive male mRen2.Lewis rat,46we
have not observed overt differences in SERCA2 or PLB protein ex-
pression following ovariectomy of the mRen2 female, consistent
with other observations.47,48Likewise, in the current study, we did
not find differences in either SERCA2 or PLB mRNA levels among
OVX-vehicle, OVX-G-1, and sham-operated mRen2.Lewis rats. Al-
though oestrogen has been shown to alter the L-type Ca2+channel
and the Na/Ca exchanger,49,50cardiac mRNA levels of these and
ryanodine-2, calreticulin, and calmodulin-1, were not affected by oes-
trogen loss or G-1 administration. Indeed, other mechanisms that
modulate LV relaxation, such as cardiac bioenergetics, conformation
of the cytoskeleton, or titin isoform expression could be involved in
the improved lusitropic function induced by G-1.51,52
The downstream signalling mechanisms of GPR30 in the heart are
still unclear. Acute ex vivo studies show that G-1 inhibits Akt and Erk
pathways which become activated by ischaemia and reperfusion
The present study shows that G-1 attenuated the
up-regulation of NOX4 in OVX-rat hearts. NOX4 expression is
increased by hypertrophic stimuli, including angiotensin II, and med-
iates cardiac hypertrophy and heart failure induced by pressure over-
load and chronic angiotensin II treatment.53Therefore, NOX4 may
also play an important role in LV remodelling in the female mRen2.-
Lewis rat, an established model of oestrogen and angiotensin
II-sensitive hypertension.32Since NOX4 is known to be a significant
source of reactive oxygen species (ROS) in the failing heart,54
future studies evaluating cardiac ROS production will be required to
determine whether the loss of endogenous activation of cardiac
GPR30 or the interaction between angiotensin II and cardiac
GPR30 signalling leads to an increase in oxidative stress, and subse-
quent NOX4-related hypertrophic remodelling.
4.1 Limitations of this study
Subcutaneous administration of a selective GPR30 agonist was used to
determine the role of this receptor in the maintenance of LV struc-
ture and diastolic function. Although our dose of G-1 had no overt
effect on blood pressure, we cannot exclude the possibility that
G-1 has some other systemic action that could indirectly alter vascular
haemodynamics and subsequently improve cardiac structure and
Figure 6 Activation of GPR30 inhibits angiotensin II-induced
hypertrophy in H9c2 cardiomyocytes. Cell hypertrophy was deter-
mined by (A) cell size, (B) ANF mRNA levels, and (C) protein
content per cell. Data represent mean+SEM. *P , 0.05 vs.
vehicle; #P , 0.05 vs. angiotensin II. n ¼ 4/group.
H. Wang et al.
diastolic function.46The presence and content of G-1 in the heart
after subcutaneous administration was not determined in this study.
However, evidence strongly suggests that the cardiac effects of G-1
occur mainly through its activation of GPR30 in the heart: (i) data
show the presence of GPR30 in the heart (see Supplementary mater-
ial online, Figure S1 and Ref.12) and the high affinity and specificity of
G-1 to GPR30;29and (ii) exogenous administration of G-1 is com-
monly used to study the roles of cardiac GPR30 in both ex vivo
studies20,25–27and in vivo studies,12without leading to confounding
factors as reported in the GPR30 knockout model.55Although no
specific myocardial phenotypic changes were reported in the global
GPR30 knockout mouse,55studies using conditional cardiomyocyte-
specific GPR30 knockout animals will be needed to determine the
exact physiological functions of GPR30 in the female heart. A
second limitation is that early ovariectomy in the mRen2.Lewis
female does not address the age-related changes that also influence
the cardiac diastolic dysfunction phenotype of older women.
Indeed, a more hormonally relevant model mimicking human meno-
pause might be useful in future studies.56Thirdly, although tissue
Doppler echocardiography is a well-accepted methodology for the
clinical assessment of lusitropic function, other load-independent
approaches, such as cardiac catheterization with pressure–volume
analyses, could provide more quantitative information of diastolic
function in the experimental setting.
4.2 Clinical implications
Preclinical studies support the potential benefits of oestrogen therapy
in reducing the risk of cardiovascular diseases.16,32,57,58Oestrogen re-
placement therapy in hypertensive post-menopausal women has also
been shown to diminish cardiac fibrosis and hypertrophy.10,59
However, oestrogen replacement therapy remains controversial
given that data from the Women’s Health Initiative (WHI)60and the
Heart and Estrogen/Progestin Replacement Study (HERS)61did not
show protective cardiovascular benefits in older women who
received conjugated equine oestrogen (Premarinw Pfizer Pharmaceu-
ticals, Inc., New York, NY, USA) with or without synthetic progestins
(medroxyprogesterone acetate) well after the onset of menopause.
Prior to halting the hormone replacement arms of the WHI study,
there was an increased risk for myocardial events and strokes
among the oestrogen-replete post-menopausal women.60Similarly,
the HERS study found an increased risk for thromboembolic events
in a large group of hormone-replaced post-menopausal women
with established coronary disease.62,63Fortunately, it does not
appear that the increased cardiovascular risks, including myocardial in-
farction, stroke, pulmonary embolism, and venous thrombosis risks
persist once hormone replacement is discontinued.64,65Preclinical
data also suggest that late-in-life oestrogen may adversely influence
cardiovascular risk. Specifically, ovariectomy in mid-aged mRen2.Lewis
female rats conveyed protection against salt-induced renal damage
when compared with oestrogen-intact littermates.66Certainly, in
order to ascertain the appropriate composition of oestrogens,
source, dosage, and the therapeutic window for perimenopausal
women, more studies focused on the dynamic relationship between
oestrogens and the cardiovascular system are needed.
Despite a disease continuum of great clinical importance and
urgency, diastolic dysfunction in post-menopausal women is not
well understood and its medical management remains largely empirical.
Herein, using a reverse translational rodent model that emulates the
cardiovascular phenotype of women after the surgical or natural
cessation of ovarian hormonal production,67,68we conclude that the
loss of endogenous GPR30 activation in the heart could provoke
enhanced deposition of interstitial collagen and cardiomyocyte hyper-
trophy, eventually leading to diastolic dysfunction. The improved myo-
cardial relaxation, reduced filling pressures, and anti-remodelling that
occurred with the administration of a highly specific GPR30 agonist,
late after ovariectomy, further reveals the importance of the GPR30 re-
ceptor in the maintenance of female-sex-specific cardiac function and
Supplementary material is available at Cardiovascular Research online.
This work was supported by grants from the National Institutes of Health
R01-AG033727-03 (to L.G.).
Conflict of interest: none declared.
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