σ1-Receptor stimulation with fluvoxamine ameliorates
transverse aortic constriction-induced myocardial
hypertrophy and dysfunction in mice
Hideaki Tagashira, Shenuarin Bhuiyan, Norifumi Shioda, Hideyuki Hasegawa,
Hiroshi Kanai and Kohji Fukunaga
Am J Physiol Heart Circ Physiol 299:H1535-H1545, 2010. First published 27 August 2010;
doi:10.1152/ajpheart.00198.2010
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Am J Physiol Heart Circ Physiol 299: H1535–H1545, 2010.
First published August 27, 2010; doi:10.1152/ajpheart.00198.2010.

␴1-Receptor stimulation with fluvoxamine ameliorates transverse aortic
constriction-induced myocardial hypertrophy and dysfunction in mice
Hideaki Tagashira,1 Shenuarin Bhuiyan,1 Norifumi Shioda,1 Hideyuki Hasegawa,2 Hiroshi Kanai,3and Kohji Fukunaga1
1

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, and 2Graduate School of Biomedical
Engineering and 3Graduate School of Engineering, Tohoku University, Sendai, Japan
Submitted 26 February 2010; accepted in final form 21 August 2010

protein kinase B; Akt; endothelial nitric oxide synthase
ACCUMULATING CLINICAL EVIDENCE suggests that depression after
myocardial infarction (MI) is associated with higher morbidity
and mortality and that selective serotonin reuptake inhibitors(SSRIs) are safe for use in patients with cardiovascular diseases and may even reduce post-MI morbidity and mortality(47). However, the benefit of SSRIs to patients with cardiovascular disease has not been established, because the molecular mechanism underlying SSRI-induced cardioprotection islargely unknown. Notably, SSRIs such as sertraline (38) andfluvoxamine (24) are potent agonists of the ␴1-receptor (␴1R)
as well as serotonin uptake inhibitors. The order of affinity ofSSRIs for ␴1R is as follows: fluvoxamine (Ki: 36 nM) ⬎Address for reprint requests and other correspondence: K. Fukunaga, Dept.
of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku Univ.,
Aramaki-Aoba, Aoba-ku, Sendai 980-8578, Japan (e-mail: fukunaga@mail.
pharm.tohoku.ac.jp).
http://www.ajpheart.org

sertraline (Ki ⫽ 57 nM) ⬎ fluoxetine (Ki ⫽ 120 nM) ⬎citalopram (Ki ⫽ 292 nM) ⬎ paroxetine (Ki ⫽ 1,893 nM) (30).
Thus, ␴1R is a potential physiological target for SSRIs not onlyin the brain but also in the heart.
␴-Receptors, which are unique in having two transmembrane segments that show no homology with any mammalian
G protein-coupled receptor, are widely expressed in heart
muscle. In cardiac tissue, modulation of contractility by ␴-receptor ligands was first reported in rat neonatal cultured cardiomyocytes (11). Later, ␴-receptors were found in the membranes of adult rat ventricular cardiomyocytes (32). Approximately 80% of ␴-receptors in the rat ventricular myocardium
are the ␴1-subtype and 20% are the ␴2-subtype, based on an
analysis of membrane binding of specific ␴-ligands in isolated
rat cardiomyocyte preparations (32). Several studies (12, 33)
have indicated the effects of ␴-ligands in isolated rat hearts andreported desensitization of ␴-receptors in heart muscle byrepeated treatment with ␴-ligands. However, the physiological
function of cardiac ␴-receptor agonists in the normal heart andtheir therapeutic potential in cardiac disease remain unclear.
Despite information available relevant to the modulation ofcardiomyocyte function by ␴-receptor ligands, no study has
described the expression level of ␴-receptors, nor have theirdownstream targets in cardiomyocytes been identified. Most
studies of ␴-receptors have been carried out using isolatedheart preparations (11, 12, 13, 31, 32) and indirect interpretations using nonspecific ␴-ligands {such as haloperidol, 1,3di-o-tolylguanidine, and (⫹)-3-3-(3-hydroxyphenyl)-N-(1-propyl)
piperidine [(⫹)3-PPP]} (22, 27). Moreover, the expression of
␴1R in the heart is upregulated by strong stress stimuli, such as
immobilization and hypoxia, and is not affected by aging (31).
To assess both the normal biological function and pathophysiological role of ␴1Rs, we recently analyzed ␴1R expression in
the heart (2), kidney (3), and thoracic aorta (5). We found thatpressure overload-induced hypertrophy significantly decreased
the expression of ␴1Rs in the left ventricle (LV) of ovariectomized rats and observed a significant correlation between heart
dysfunction and decreased ␴1R expression in the LV (2).
Since SSRIs, including fluvoxamine, are used to treat depressive patients with or without cardiac disease, our goal wasto define the molecular mechanism underlying fluvoxamineinduced cardioprotection through ␴1Rs and evaluate the pathophysiological relevance of ␴1Rs in cardiac hypertrophy andheart failure. In the present study, we found that fluvoxamine’s
potent ␴1R stimulatory effect largely blunts the development ofpathological LV hypertrophic remodeling and promotes cardiac functional recovery. In contrast, paroxetine, which has a
low affinity for ␴1Rs, had no antihypertrophic effect. We also0363-6135/10 Copyright © 2010 the American Physiological Society

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Tagashira H, Bhuiyan S, Shioda N, Hasegawa H, Kanai H, Fukunaga K. ␴1-Receptor stimulation with fluvoxamine ameliorates transverse
aortic constriction-induced myocardial hypertrophy and dysfunction in mice.
Am J Physiol Heart Circ Physiol 299: H1535–H1545, 2010. First published
August 27, 2010; doi:10.1152/ajpheart.00198.2010.—Selective serotonin
reuptake inhibitors (SSRIs) are known to reduce post-myocardial
infarction-induced morbidity and mortality. However, the molecularmechanism underlying SSRI-induced cardioprotection remains unclear. Here, we investigated the role of ␴1-receptor (␴1R) stimulation
with fluvoxamine on myocardial hypertrophy and cardiac functional
recovery. Male ICR mice were subjected to transverse aortic constriction (TAC) in the cardiac aortic arch. To confirm the cardioprotectiverole of fluvoxamine by ␴1R stimulation, we treated mice with fluvoxamine (0.5 or 1 mg/kg) orally once per day for 4 wk after the onset of
aortic banding. Interestingly, in untreated mice, ␴1R expression in theleft ventricle (LV) decreased significantly over the 4 wk as TACinduced hypertrophy increased. In contrast, fluvoxamine administration significantly attenuated TAC-induced myocardial hypertrophy
concomitant with recovery of ␴1R expression in the LV. Fluvoxamine
also attenuated hypertrophy-induced impaired LV fractional shortening. The fluvoxamine cardioprotective effect was nullified by treatment with a ␴1R antagonist [NE-100 (1 mg/kg)]. Importantly, another
SSRI with very low affinity for ␴1Rs, paroxetine, did not elicit
antihypertrophic effects in TAC mice and cultured cardiomyocytes.
Fluvoxamine treatment significantly restored TAC-induced impaired
Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in
the LV. Our findings suggest that fluvoxamine protects against TACinduced cardiac dysfunction via upregulated ␴1R expression andstimulation of ␴1R-mediated Akt-eNOS signaling in mice. This is thefirst report of a potential role for ␴1R stimulation by fluvoxamine inattenuating cardiac hypertrophy and restoring contractility in TAC
mice.

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␴1-RECEPTOR STIMULATION AMELIORATES MYOCARDIAL HYPERTROPHY

defined a fluvoxamine-induced cardioprotective mechanism
through Akt signaling in LV hypertrophic remodeling.
MATERIALS AND METHODS

AJP-Heart Circ Physiol • VOL 299 • NOVEMBER 2010 • www.ajpheart.org

Downloaded from ajpheart.physiology.org on February 21, 2011Materials. Reagents and antibodies were obtained from the following sources: anti-␴1R antibody (Abcam, Cambridge, UK); anti-endothelial nitric oxide synthase (eNOS) antibody and anti-␤-tubulin
antibody (Sigma, St. Louis, MO); anti-phospho-eNOS antibody, antiphospho-p44/42 MAPK (ERK1/2) antibody (Thr202/Tyr204), total
p44/42 MAPK (ERK1/2), anti-phospho-Akt antibody (Ser437), andtotal Akt antibody (Cell Signaling Technology, Beverly, MA);
anti-phospho-Akt antibody (Thr308), anti-phospho-PKC-␣ antibody(Ser657), total PKC-␣ antibody, and anti-heat shock protein 90(HSP90) antibody (Upstate Biotechnology, Lake Placid, NY), anticaveolin-3 antibody (BD Biosciences, San Jose, CA); and anti-rabbit
antibody (Amersham Biosciences, Piscataway, NJ). The ␴1R agonist
fluvoxamine maleate was supplied by Meiji Seika Kaisha (Tokyo,
Japan), and the specific ␴1R antagonist N,N-dipropyl-2-[4-methoxy3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100)
was generously supplied by Taisho Pharmaceutical (Tokyo, Japan).
Other reagents were of the highest quality available (Wako Pure
Chemicals, Osaka, Japan).
Animals. All procedures for handling animals complied with the
Guide for Care and Use of Laboratory Animals and were approved by
the Animal Experimentation Committee of Tohoku University Graduate School of Pharmaceutical Sciences. Adult male ICR mice weighing 35– 40 g were obtained from Nippon SLC (Hamamatsu, Japan).
Ten-week-old male mice were acclimated to the local environment for
1 wk, which included housing in polypropylene cages at 23 ⫾ 1°C in
a humidity-controlled environment maintained on a 12:12-h light-darkschedule (lights on 8:00 AM to 8:00 PM). Mice were provided foodand water ad libitum.
Primary cardiomyocyte cultures from neonatal rats. Neonatal ventricular myocytes were isolated from the hearts of 1- to 3-day-old
Wistar rats by collagenase digestion and cultured according to themethods of Lu et al. (25). Briefly, neonatal rats were decapitated, andtheir hearts immediately removed. Ventricles were separated from theheart and washed in HBSS (137.0 mM NaCl, 5.4 mM KCl, 0.4 mM
KH2PO4, 0.3 mM Na2HPO4·12H2O, 6.12 mM glucose, and 4.2 mM
NaHCO3; pH 7.4), and myocytes were dissociated from the ventricles
by serial digestion with 0.1% trypsin and 0.05% DNase I in HBSS.
After digestion, dissociated cardiomyocytes were collected and suspended in DMEM with 10% FBS and 0.02% trypsin inhibitor to
inhibit further digestion. Cells were collected by centrifugation (4°C,
1,000 g for 10 min). After the supernatant was discarded, DMEM
containing 10% FBS was added. Cells were gently agitated and thenplated on uncoated 90-mm culture dishes. Plates were allowed tostand for 90 min at 37°C in a CO2 incubator to remove nonmyocytes,
which attached to the plates. Unattached myocytes were collected andplated at 1–2 ⫻ 106 cells/5-mm dish and incubated with DMEM and
10% FBS in a humidified incubator with DMEM and 5% CO2 at 37°C
for 48 h. Cells were cultured in serum-free DMEM for 24 h before
treatment with ANG II (100 nM).
Morphological analysis of cultured cardiomyocytes. Cultured myocytes were plated on collagen-coated coverglasses at a density of
1–2 ⫻ 106 cells/coverslip of 12-mm diameter. After an incubation in
the presence or absence of ANG II for 48 h, cultured cells were
washed three times in PBS (pH 7.4) and fixed in PBS plus 4%
formaldehyde. After permeabilization with 0.1% Triton X-100 in
PBS, fixed cells were incubated with 1% BSA in PBS for 30 min. For
cell size measurement, cells were incubated 3 h at room temperature
with rhodamine-conjugated phalloidin (1:300; Molecular Probes, Eugene, OR) in PBS containing 1% BSA. After cell images wereacquired using an Olympus fluorescence microscope, the surface areaof the cells was quantitated using the Image J program. All cells fromrandomly selected fields were examined in each condition (at least

100 myocytes/group). The surface area of control cells was expressed
as 100% and compared with that of treated cells.
Small interfering RNA transfection in cultured cardiomyocytes.
␴1R small interfering (si)RNA (sense: 5=-ACACGTGGATGGTGGAGTA-3= and antisense: 5=-TACTCCACCATCCACGTGT-3=) was
purchased from Exigen (Tokyo, Japan). Cultured myocytes wereplated on uncoated 90-mm culture dishes on collagen-coated coverglasses. Transfections were performed with 100 nM ␴1R siRNAs
according to the methods of Wang et al. (49). Briefly, 20 ␮M ␴1R
siRNA in 5 ␮l was added to 180 ␮l opti-MEM (Invitrogen), and 5 ␮l
Lipofectamine 2000 (Invitrogen) was added to 10 ␮l opti-MEM. Bothsolutions were incubated separately at room temperature for 5 min,mixed, and then incubated at room temperature for 15–20 min.
Meanwhile, medium containing serum was removed from the cells
and replaced with 1 ml opti-MEM. This solution was then removed andreplaced with 800 ␮l fresh opti-MEM to which 200 ␮l of the siRNA
solution was added. Cells were then incubated at 35°C in a 5% CO2
atmosphere for 4 h to initiate transfection. DMEM (500 ␮l) supplemented
with 5% FBS was then added to each well, and the transfection was
continued for up to 72 h. Cultured cells were washed with cold PBS andstored at ⫺80°C until Western blot analysis was performed.
Surgical procedures. Transverse aortic constriction (TAC) was
performed on male ICR mice as previously described (35). After
acclimatization for 7 days, animals were anesthetized with tribromoethanol (0.25– 0.3 g/kg ip). The animal was placed supine, and an
endotracheal intubation was rapidly performed. The cannula wasconnected to a volume-cycled rodent ventilator with a tidal volume of
0.5 ml of room air and respiratory rate of 110 breaths/min. The chest
cavity was opened using scissors to make a small incision at the levelof the second intercostal space at the left upper sternal border. Afterthe aortic arch was isolated, the transverse aorta was isolated between
the carotid arteries and constricted by a 7-0 silk suture ligature tiedfirmly against a 27-gauge needle. The needle was promptly removed
to produce an aortic constriction of 0.4 mm in diameter. The chest
cavity was then closed with a 6-0 nylon suture, and mice were allowed
to recover from anesthesia while their body temperature was kept at
37°C. Sham-operated animals, which underwent a similar surgicalprocedure without aortic constriction, served as controls.
Experimental design. Initially, we observed a downregulated expression of ␴1Rs in TAC-induced hypertrophy. ICR mice were randomly separated into four groups: 1) sham-operated animals (sham;n ⫽ 9), 2) TAC for 2 wk (n ⫽ 6), 3) TAC for 4 wk (n ⫽ 6), and 4) TAC
for 6 wk (n ⫽ 7). We then observed the cardioprotective role of␴1R-mediated signaling and randomly separated ICR mice into 10
treatment groups: 1) sham (n ⫽ 15), 2) sham plus fluvoxamine (1mg/kg, n ⫽ 5), 3) sham plus NE-100 (1 mg/kg, n ⫽ 5), 4) TAC plus
vehicle (TAC-vehicle; n ⫽ 12), 5) TAC plus 0.5 mg/kg fluvoxamine(F 0.5; n ⫽ 7), 6) TAC plus 1.0 mg/kg fluvoxamine (F 1.0; n ⫽ 7), 7)
TAC plus 1.0 mg/kg fluvoxamine plus NE-100 (F 1.0-NE; n ⫽ 7), 8)
sham plus 0.4 mg/kg paroxetine (n ⫽ 5), 9) TAC plus 0.2 mg/kg
paroxetine (n ⫽ 4), and 10) TAC plus 0.4 mg/kg paroxetine (n ⫽ 4).
Drug administration. Fluvoxamine and paroxetine maleate weredissolved in 0.5% calboxymetylcellulose, and NE-100 was dissolved
in 0.9% saline water. Vehicle, fluvoxamine (0.5 and 1.0 mg/kg),
paroxetine (0.2 and 0.4 mg/kg), and NE-100 (1.0 mg/kg) wereadministered orally for 4 wk (once daily) in a volume of 1 ml/100 gmouse body wt (BW), starting from the onset of aortic banding.
Measurement of cardiac hypertrophy. After 4 wk of drug or vehicle
administration, animals were subjected to terminal surgery. Mice were
weighed and anesthetized with a mixture of ketamine (100 mg/kg ip)
and xylazine (5 mg/kg ip) (34). The thoracic cavity was opened, andhearts were immediately harvested and weighed. Cardiac indexes,
expressed as the heart weight (HW)-to-BW ratio (HW/BW; in mg/g),
were used to estimate the degree of cardiac hypertrophy.
Echocardiography. Noninvasive echocardiographic measurements
were performed in mice anesthetized with 2.5% avertin (8 ␮l/g) (50)
using an echocardiograph (SSD-6500, Aloka, Tokyo, Japan) equipped␴1-RECEPTOR STIMULATION AMELIORATES MYOCARDIAL HYPERTROPHY

detection system (Amersham Biosciences) and visualized on X-ray
film (Fuji Film). Autoradiographic films were scanned by densitometry (Lasergraphics, Irvine, CA) and quantitated using Imagegauseversion 3.41 (Fuji Film). Relative amounts of proteins were expressedas percent increases over sham values.
Statistical analysis. Values are represented as means ⫾ SE. Morphometric changes and SDS-PAGE results were evaluated for differences by one-way ANOVA combined with Dunnett’s post hoc test. P
values of ⬍0.05 were considered statistically significant.
RESULTSTime course analysis of cardiac hypertrophy and ␴1Rexpression. To define the role of ␴1R expression in cardiac
hypertrophy and heart failure, we evaluated the time course of
LV function, cardiac hypertrophy, and ␴1R expression after
TAC. Echocardiographic data for the time course experiment isprovided in the Supplemental Material (Supplemental Table 1)1 andFig. 1. We found a time-dependent impairment of LV function,
as indicated by decreased FS 4 and 6 wk after TAC in mice1
Supplemental Material for this article is available online at the American
Journal of Physiology-Heart and Circulatory Physiology website.

Fig. 1. Temporal changes in cardiac hypertrophy and failure induced by transverse aortic constriction (TAC) in mice. A: representative M-mode echocardiograms of mice before and after 2– 6 wk of TAC. LVEDD, left
ventricular (LV) end-diastolic diameter; LVESD,
LV end-systolic diameter. B: changes in thepercentage of LV fraction shortening (FS). C
and D: TAC-induced myocardial hypertrophy as indicated by the heart weight-to-bodyweight ratio (HW/BW; C) and lung weightto-body weight ratio (LW/BW; D). E: Western blot analysis (top) and densitometry
quantification (bottom) of ␴1-receptor (␴1R)
expression in the LV. Each group consistedof 5–10 mice. Immunoblot analysis with
anti-␤-tubulin antibody showed equal protein loading in each lane. Data are expressedas percentages of values in sham-operated
animals (sham group; means ⫾ SE). *P ⬍0.05 and **P ⬍ 0.01 vs. the sham group.

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with a 10-Hz linear transducer (UST-5545, Aloka). The heart was
imaged in the two-dimensional parasternal short-axis view, and an
M-mode echocardiogram of the midventricle was recorded at the levelof the papillary muscles. Diastolic and systolic LV wall thickness, LV
end-diastolic diameter (LVEDD), and LV end-systolic diameter(LVESD) were measured. All measurements were done from leadingedge to leading edge according to American Society of Echocardiography guidelines (26). The percentage of LV fraction shortening (FS) was
calculated as follows: FS ⫽ [(LVEDD ⫺ LVESD)/LVEDD] ⫻ 100.
Western blot analysis. Four weeks after aortic banding, mice wereanesthetized, and hearts were excised and quickly perfused with PBS
to wash out blood from coronary vessels. Heart tissue was sliced at2-mm thickness using a slicer (RBS-2, Zivic-Miller Laboratories,
Zelienople, PA). LV tissue samples were then rapidly frozen in liquid
nitrogen and stored at ⫺80°C before use. For assays, each frozen
sample was homogenized by methods we have previously described(4, 6). An equal amount of protein for each sample (25 ␮g total
protein) was separated on 7.5–15% SDS-polyacrylamide gels andtransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). After being blocked with 5% lowfat milk in Tris-bufferedsaline plus Tween 20, membranes were incubated with specificprimary antibodies overnight at 4°C. This procedure was followed byan incubation with donkey anti-rabbit IgG coupled to horseradish
peroxidase, and blots were developed using an ECL immunoblotting

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␴1-RECEPTOR STIMULATION AMELIORATES MYOCARDIAL HYPERTROPHY

found time-dependent increases in the LW-to-BW ratio (LW/
BW; Fig. 1D) from 1 to 6 wk after TAC in mice. Interestingly,␴1R expression in the LV markedly decreased time dependently with significantly decreased expression observed 4 and6 wk after TAC in mice (P ⬍ 0.01 vs. the sham group; Fig.
1E). Thus, our study revealed a significant negative linear
correlation of heart dysfunction with reduced ␴1R expression
in the LV.
Effect of ␴1R stimulation on myocardial hypertrophy anddysfunction. To confirm the cardioprotective role of fluvoxamine and paroxetine, which are typical SSRIs, we treated mice

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Fig. 2. Effect of fluvoxamine (Flux) and NE-100 (NE) on cardiac hypertrophyand failure induced by TAC in mice. A: representative M-mode echocardiograms of mice with and without Flux and/or NE treatment. B: changes in thepercentage of LV FS. TAC mice were treated with Flux [0.5 mg/kg (F 0.5
group) or 1.0 mg/kg (F 1.0 group)] or Flux (1.0 mg/kg) plus NE (1.0 mg/kg;
F 1.0 ⫹ NE group) as shown. C and D: TAC-induced myocardial hypertrophy
as indicated by HW/BW (C) and LW/BW (D). Each group consisted of 5–10
mice. Each bar represents the mean ⫾ SE. *P ⬍ 0.05 and **P ⬍ 0.01 vs. thesham group; †P ⬍ 0.05 and ††P ⬍ 0.01 vs. the TAC-vehicle group; ##P ⬍0.01 vs. the TAC ⫹ F 1.0 group.

(P ⬍ 0.01 vs. the sham group; Fig. 1, A and B). Time course
experimetns to assess HWs and lung weights (LWs) indicatedthat HW/BW (Fig. 1C) increased time dependently from 1 to 6
wk after TAC in mice (P ⬍ 0.01 vs. the sham group). We also

Fig. 3. Effect of paroxetine (Parox) on cardiac hypertrophy and failure inducedby TAC in mice. A: Representative M-mode echocardiograms of mice with andwithout drug (Parox) treatments. B: changes in the percentage of LV FS. TAC
or sham mice were treated with Parox at 0.2 mg/kg (P 0.2 group) or 0.4 mg/gk(P 0.4 group) as shown. C and D: TAC-induced myocardial hypertrophy as
indicated by HW/BW (C) and LW/BW (D). Each group consisted of 4 –5 mice.
Each bar represents the mean ⫾ SE. **P ⬍ 0.01 vs. the sham group.

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␴1-RECEPTOR STIMULATION AMELIORATES MYOCARDIAL HYPERTROPHY

with either drug or with the ␴1R antagonist NE-100 for 4 wk,
starting at the onset of TAC. Echocardiographic data are shownin Supplemental Table S3. Oral administration of fluvoxamine(1 mg/kg) or NE-100 (1 mg/kg) alone to mice that had notundergone TAC for 4 wk had no effect on echocardiographic
parameters (Supplemental Table S2) and LV FS (Supplemental
Fig. S1B). Consistent with temporal changes in heart function,
as shown in Fig. 1B, TAC treatment significantly decreased LV
FS (P ⬍ 0.01) compared with the sham group (Figs. 2, A andB, and 3, A and B). Fluvoxamine treatment restored decreased
LV FS dose dependently (P ⬍ 0.01 vs. the TAC-vehicle groupfor both the F 0.5 and F 1.0 groups; Fig. 2B). Paroxetine
treatment, however, failed to restore FS (Fig. 3B). Coadministration of NE-100 with fluvoxamine nullified the fluvoxamine-mediated amelioration of heart dysfunction, as indicated by
the decreased LV FS (P ⬍ 0.01 vs. the F 1.0 group; Fig. 2B).
Similarly, fluvoxamine treatment significantly restored boththe elevated HW/BW (P ⬍ 0.01 vs. the TAC-vehicle groupfor the F 1.0 group; Fig. 2C) and elevated LW/BW (P ⬍ 0.05
vs. the TAC-vehicle group for the F 1.0 group; Fig. 2D).
Paroxetine administration failed to inhibit increases in both
HW/BW (Fig. 3C) and LW/BW (Fig. 3D). Coadministration of
NE-100 with fluvoxamine nullified the latter’s inhibition of
TAC-induced hypertrophy, as indicated by HW/BW (P ⬍ 0.05
vs. the sham group and P ⬍ 0.01 vs. the F 1.0 group; Fig. 2C)
and LW/BW (P ⬍ 0.05 vs. the sham group and P ⬍ 0.01 vs.
the F 1.0 group; Fig. 2D). Moreover, oral administration of
fluvoxamine (1 mg/kg) and NE-100 (1 mg/kg) alone to micethat had not undergone TAC for 4 wk had no effect on
HW/BW (Supplemental Fig. S1C) and LW/BW (Supplemental
Fig. S1D) ratio in the sham group. Taken together, we conclude that the antihypertrophic effect of fluvoxamine on TAC
mice is mediated by ␴1R stimulation.
Effect of fluvoxamine treatment on ␴1R expression. Since
␴1R stimulation by fluvoxamine ameliorated TAC-induced LV

hypertrophy in mice, we asked whether treatment with fluvoxamine and NE-100 had any effect on ␴1R expression in the LV.
TAC-induced hypertrophy significantly decreased ␴1R expression in the LV (P ⬍ 0.01 vs. the sham group), whereas
fluvoxamine administration significantly and dose dependently
increased it (P ⬍ 0.01 vs. the TAC-vehicle group for the F 1.0
group; Fig. 4). Interestingly, although NE-100 treatment nullified fluvoxamine-mediated cardioprotective effects (Fig. 2),
coadministration of NE-100 with fluvoxamine did not nullify
fluvoxamine-dependent increases in ␴1R expression (P ⬍ 0.01
vs. the TAC-vehicle group) in the LV (Fig. 4). Moreover, oral
administration of fluvoxamine (1 mg/kg) and NE-100 (1 mg/kg) for 4 wk in sham mice had no effect on ␴1R expression in
the LV (Supplemental Fig. S1E). Thus, fluvoxamine-induced
restoration of ␴1R levels is correlated with its cardioprotective
action in the context of TAC-induced hypertrophy; however,that effect is likely not through stimulation of ␴1Rs.
␴1R activation induces Akt phosphorylation. Our previous
studies (4, 6) showed that LV Akt phosphorylation levels
markedly decrease after pressure overload-induced hypertrophy in ovariectomized rats. We speculated that fluvoxamineFig. 5. Effects of Flux and NE on Akt phosphorylation. TAC mice were treatedwith Flux (0.5 or 1.0 mg/kg) or Flux (1.0 mg/kg) ⫹ NE (1.0 mg/kg) as shown.
A–C: Western blot analysis (A) and densitometric quantification of phosphorylated (p-)Akt (Ser473; B) and p-Akt (Thr308; C) in the LVs of TAC mice withor without drug treatment. Data are expressed as percentages of the value ofsham mice. Densitometric quantification of p-Akt (Ser473; B) and p-Akt(Thr308; C) was relative to total Akt in the LV. Each group consisted of 5–10
mice. Each bar represents the mean ⫾ SE. *P ⬍ 0.05 vs. the sham group;††P ⬍ 0.01 vs. the TAC-vehicle group; #P ⬍ 0.05 vs. the TAC ⫹ F 1.0 group.

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Fig. 4. Effects of Flux and NE-100 on ␴1R expression. A: Western blot analysisof ␴1R expression and ␤-tubulin as a loading control in the LVs of sham and
TAC mice with or without drug treatment. Immunoblot analysis with anti-␤tubulin antibody showed equal protein loading in each lane. B: densitometricquantification of ␴1R-immunoreactive bands. Data are expressed as percentages of values of sham mice (means ⫾ SE). Each group consisted of 5–10
mice. **P ⬍ 0.01 vs. the sham group; ††P ⬍ 0.01 vs. the TAC-vehicle group.

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administration may act to maintain Akt in a phosphorylated
state, which is required for its activation. Consistent with our
hypothesis, fluvoxamine administration (1.0 mg/kg) significantly increased Akt phosphorylation at Ser473 (P ⬍ 0.01 vs.
the TAC-vehicle group for the F 1.0 group) and Thr308 (P ⬍0.01 vs. the TAC-vehicle group for the F 1.0 group; Fig. 5,
A–C) in LV tissues. Interestingly, coadministration of NE-100
with fluvoxamine nullified the fluvoxamine-induced rescue of
Akt phosphorylation at Ser473 (P ⬍ 0.05 vs. the sham group
and P ⬍ 0.05 vs. the F 1.0 group) and Thr308 (P ⬍ 0.05 vs. thesham group and P ⬍ 0.05 vs. the F 1.0 group; Fig. 5, A–C).
These findings suggest that fluvoxamine-mediated cardioprotection is closely associated with the maintenance of Akt
phosphorylation/activity in mice.
␴1R activation induces eNOS expression and phosphorylation.
Since eNOS is a physiological substrate for Akt in humanvascular endothelial cells (21, 52), we asked whether fluvoxamine-induced Akt activation results in increased eNOS phosphorylation in hypertrophied cardiomyocytes of ovariectomized rats (4, 6). Consistent with our previous observation inthis model (4, 6), we found that TAC-induced myocardial

hypertrophy was associated with significantly decreased eNOS
expression (P ⬍ 0.05 vs. the sham group; Fig. 6, A and B) as
well as markedly reduced Akt-mediated eNOS phosphorylation at Ser1177 (P ⬍ 0.05 vs. the sham group; Fig. 6, A and C).
Notably, fluvoxamine administration increased both eNOS
levels (P ⬍ 0.01 vs. the TAC-vehicle group for the F 1.0 group)and Akt-mediated eNOS phosphorylation (P ⬍ 0.05 vs. theTAC-vehicle group for the F 0.5 group and P ⬍ 0.01 vs. theTAC-vehicle group for the F 1.0 group; Fig. 6, A–C). NE-100
treatment antagonized fluvoxamine-mediated eNOS upregulation(P ⬍ 0.05 vs. the F 1.0 group), confirming that fluvoxaminemediated increased eNOS expression in the LV is mediated by
␴1Rs. Likewise, coadministration of NE-100 with fluvoxamine
blocked fluvoxamine-mediated increased eNOS phosphorylation (P ⬍ 0.05 vs. the sham group and P ⬍ 0.01 vs. the F 1.0
group; Fig. 6, A and C). These results suggest that fluvoxamine-mediated increased expression of ␴1Rs promotes increased
eNOS phosphorylation by Akt, as indicated by the increased
ratio of eNOS phosphorylation to total eNOS expression (Fig.
6D). Enhanced Akt activity and concomitant eNOS phosphorylation are likely mediated by ␴1R stimulation.

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Fig. 6. Effects of Flux and NE on the levelsof endothelial nitric oxide synthase (eNOS),eNOS phosphorylation, heat shock protein
90 (HSP90), and caveolin-3. TAC mice weretreated with Flux (0.5 or 1.0 mg/kg) or Flux
(1.0 mg/kg) plus NE (1.0 mg/kg) as shown.
A–F: Western blot analysis (A) and densitometric quantification of eNOS (B), p-eNOS(Ser1179; C), ratio of p-eNOS to eNOS (D),HSP90 (E), and caveolin-3 (F) in the LVs of
TAC mice with or without drug treatment.
Data are expressed as percentages of thevalue of sham mice. Each group consisted of
5–10 mice. Each bar represents the mean ⫾SE. *P ⬍ 0.05 vs. the sham group; †P ⬍
0.05, ††P ⬍ 0.01, and †††P ⬍ 0.001 vs. theTAC-vehicle group; #P ⬍ 0.05 and ##P ⬍0.01 vs. the TAC ⫹ F 1.0 group.

␴1-RECEPTOR STIMULATION AMELIORATES MYOCARDIAL HYPERTROPHY

Fig. 7. Effects of Flux and NE on ERK1/2 and PKC-␣ phosphorylation. A and
B: Western blot analysis (top) and densitometric quantification (bottom) of
ERK1/2 phosphorylation (Thr202/Tyr204; A) and PKC-␣ phosphorylation(Ser657; B) relative to respective total proteins in the LVs of TAC mice with orwithout drug treatment. Data are expressed as percentages of the value of shammice. Each group consisted of 5–10 mice. Each bar represents the mean ⫾ SE.

together, our observations strongly suggest that the antihypertrophic effect of fluvoxamine on ANG II-induced cardiomyocyte hypertrophy is mediated by ␴1R stimulation.
DISCUSSION

␴-Receptors have been suggested to regulate the cardiovascular system, as evidenced by the fact that several ␴-receptor
ligands influence cardiovascular function and that cardiomyocytes exhibit binding sites for ␴-receptor ligands (10, 11).
␴-Receptor ligands such as (⫹)-3-PPP, (⫹)-pentazocine, andhaloperidol alter contractility, Ca2⫹ influx, and rhythmic activity in cultured cardiac myocytes, but these actions arecomplex, and some are controversial (11, 12, 28, 32). In
addition, mechanisms underlying the potential reduction in MI
by SSRIs administered to humans are largely unknown.
We first hypothesized that SSRIs with a high affinity for␴1Rs have direct cardioprotective effects via ␴1Rs expressedon cardiomyocytes. We confirmed the cardioprotective role ofone such high-affinity reagent, fluvoxamine (Ki: 36 nM), for␴1Rs in pathophysiological conditions mediated by TAC andcompared those with the effects of the low- affinity agonist
paroxetine (Ki: 1,893 nM). The most significant observations in

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In cardiomyocytes, the localization and activity of eNOS are
regulated by a complex formation between it and scaffold
proteins, such as HSP90 and caveolin-3, especially in caveolae(16). Since eNOS protein levels are reduced by TAC, we
determined whether fluvoxamine administration modulates
HSP90 and caveolin-3 levels after TAC in mice. We observed
no changes in the expression of HSP90 (Fig. 6E) and caveolin-3 (Fig. 6F) after TAC. Moreover, treatment of TAC micewith fluvoxamine and NE-100 had no effect on the expressionof HSP90 (Fig. 6E) and caveolin-3 (Fig. 6F). Taken together,fluvoxamine treatment increased the expression and Akt-mediated phosphorylation of eNOS without affecting eNOS scaffold proteins, including HSP90 and caveolin-3, in cardiomyocyte caveolae.
Effect of fluvoxamine treatment on ERK1/2 and PKC-␣
phosphorylation. To determine the involvement of other protein kinase signaling in fluvoxamine effects, we examined therole of ERK1/2 phosphorylation in the LV after TAC-induced
cardiac hypertrophy and after fluvoxamine treatment. We did
not observe significant changes in ERK1/2 phosphorylation in
the LV when expressed as the ratio of phosphorylated to total
ERK1/2 after aortic banding and after treatment of mice withfluvoxamine and/or NE-100 (Fig. 7A).
PKC-␣, a major PKC isoform expressed in endothelial cells,
regulates eNOS phosphorylation at Ser1177 in endothelial cells
in vitro and in vivo (7). Moreover, in rat pheochromocytoma
PC12 cell lines, a ␴1-agonist induces PKC-␣ activation andunderlies cell survival in serum deprivation-induced apoptotic
conditions (8). Thus, we evaluated PKC-␣ phosphorylation
associated with activation after TAC-induced hypertrophy andfluvoxamine treatment. We did not observe significant changes
in PKC-␣ phosphorylation in the LV when expressed as theratio of phosphorylated to total PKC-␣ after TAC and after
treatment with fluvoxamine and/or NE-100 in mice (Fig. 7B).
Role of ␴1R stimulation on ANG II-induced cardiomyocyte
hypertrophy in vitro. To confirm the antihypertrophic effect of
fluvoxamine through ␴1Rs on ANG II-induced hypertrophy,
we treated neonatal rat cultured cardiomyocytes with ANG II
in the presence or absence of various combinations of fluvoxamine, paroxetine, and NE-100. The size of ANG II-treatedcells was significantly increased compared with untreated (control) cells 48 h after treatment, as previously described (25)(P ⬍ 0.001 vs. control; Figs. 8, A and B, and 9, A and B).
Fluvoxamine treatment dose dependently inhibited this effect(P ⬍ 0.001 vs. ANG II for both the F 1.0 and F 5.0 groups; Fig.
8, A and B). Combined NE-100 and fluvoxamine treatment
reversed the fluvoxamine-mediated inhibition of cardiomyocyte hypertrophy (P ⬍ 0.001 vs. the F 5.0 group; Fig. 8, A and
B). Consistent with our in vivo findings, paroxetine treatment
did not inhibit hypertrophy (Fig. 8, A and B).
To obtain direct evidence that ␴1R stimulation mediates the
fluvoxamine-induced inhibition of cardiomyocyte hypertrophy,
we treated cardiomyocytes with ␴1R siRNA to downregulate
␴1Rs. As reported by Wang et al. (49), ␴1Rs were downregulated ⬃70% by ␴1R siRNA treatment (P ⬍ 0.01 vs. control;
Fig. 9A). ␴1R siRNA treatment alone resulted in a slightly
enlarged cell size of cardiomyocytes in the absence or presenceof ANG II, but these changes were not statistically significant.
Fluvoxamine-mediated inhibition of cardiomyocyte hypertrophy was largely abolished by pretreatment with ␴1R siRNA(P ⬍ 0.001 vs. the F 5.0 group; Fig. 9, B and C). Taken

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␴1-RECEPTOR STIMULATION AMELIORATES MYOCARDIAL HYPERTROPHY

the present study are that chronic fluvoxamine but not paroxetine treatment totally inhibited LV hypertrophy and allowed
functional recovery and that fluvoxamine administration may
rescue the downregulation of ␴1Rs in the LV associated with
contractile impairment after TAC. We also report, for the firsttime, that restored Akt activity through ␴1R stimulation by
fluvoxamine ameliorates the impairment of eNOS expression
and phosphorylation in TAC cardiomyocytes.
Currently, we do not know how chronic fluvoxamine treatment upregulates ␴1R expression. Whether ␴1R expression isregulated by agonists and antagonists is not clear from theliterature. Zamanillo et al. (51) reported that chronic treatmentusing E-5842 as a ␴1R agonist increased ␴1R mRNA levels in
the rat brain. Shirayama et al. (42) reported a decrease in␴1-binding sites in the rat brain after chronic treatment withimipramine, a weak ␴1R agonist (Ki: 343 nM). Chronic haloperidol (a ␴1R antagonist) treatment reportedly promoted a
reduction in ␴1R-binding sites (23). These apparent discrepancies may be due to different methodologies or cell types used,
including in vivo versus in vitro analysis and binding assays
versus immunodetection. Our study indicates that treatment
with the ␴1R antagonist NE-100 did not alter ␴1R expression insham mice. Similarly, the combination of NE-100 with fluvoxamine had no effect on fluvoxamine-induced upregulation of
␴1Rs in the LV but nullified fluvoxamine-mediated antihypertrophic effects. These data suggest that antihypertrophic effects
and the upregulation of Akt signaling are mediated by ␴1Rstimulation and that stabilization or upregulation of ␴1Rs by
fluvoxamine is not mediated by ␴1R-stimulated signaling. In
another words, the stabilization or upregulation of ␴1Rs is not
mediated by Akt signaling. Further studies are required to
identify the mechanisms underlying the stabilization or upregulation of ␴1Rs by fluvoxamine administration.
Our novel findings indicate that fluvoxamine prevents thedevelopment of not only TAC-induced LV hypertrophy in vivo
but also ANG II-induced cardiomyocyte hypertrophy in vitro
and that Akt and eNOS signaling via ␴1Rs likely mediates
antihypertrophic effects. Indeed, fluvoxamine treatment stimulated Akt phosphorylation in vitro in PC12 cells (29). Our
hypothesis regarding fluvoxamine-mediated Akt activation is
supported by the fact that ␴1R antagonists such as rimcazole(BW 234U) promote calcium-independent inhibition of phosphatidylinositol 3=-kinase (PI3K) signaling pathways, inhibiting Akt phosphorylation in tumor cell lines (45). Moreover,␴1R knockdown via siRNA reportedly inhibited thrombinstimulated Akt phosphorylation, increasing cell death in lens
cells (49). Taken together, although the ␴1R is not a G
protein-coupled transmembrane receptor, ␴1R stimulation activates PI3K/Akt signaling in diverse cell lines.
Akt directly phosphorylates recombinant human eNOS oreNOS in situ at Ser1177 (bovine residue 1179) (16), enhancing
eNOS activity. Interestingly, continuous administration of fluvoxamine for 4 wk not only increased Akt-mediated eNOS
phosphorylation on Ser1177 but also enhanced eNOS protein

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Fig. 8. Effect of Flux and Parox treatments on ANG
II-induced hypertrophy in cultured cardiomyocytes. A: cells
were fixed with 4% paraformaldehyde, stained with rhodamine-conjugated phalloidin, and processed for fluorescence microscopy. One hundred cells from randomly selected fields were evaluated for cell size in each condition.
B: cell size expressed as a percentage of the relative surface
area in each condition compared with control cells. Eachbar represents the mean ⫾ SE. ***P ⬍ 0.001 vs. the control
group; †††P ⬍ 0.001 vs. the ANG II-treated group; ###P ⬍0.001 vs. the ANG II ⫹ 5 ␮M Flux-treated (F 5.0) group.

␴1-RECEPTOR STIMULATION AMELIORATES MYOCARDIAL HYPERTROPHY

expression in the LV. Treatment with the ␴1R antagonist
NE-100 significantly nullified fluvoxamine-mediated eNOS
upregulation and Akt-mediated eNOS phosphorylation, confirming th ␴1R-mediated modulation of eNOS activity in theheart. ␴1R ligands, such as dehydroepiandrosterone, rapidly

activate eNOS through a nontranscriptional mechanism requiring ERK1/2 activity but independent of the PI3K/Akt-mediatedpathway (44). However, we did not observe changes in
ERK1/2 phosphorylation in the LV after TAC as well as after
continuous treatment with ␴-ligands. PKC-␣, a major PKC
isoform expressed in endothelial cells, also regulates eNOS
activity in endothelial cells in vitro and in vivo by increasing
the phosphorylation of eNOS at Ser1177 (7). We did notobserve significant changes in PKC-␣ phosphorylation in the
LV after aortic banding or after treatment with fluvoxamine.
Therefore, the fluvoxamine-mediated cardioprotective effect ispartly mediated by the increased expression of ␴1Rs, and ␴1Rs
stimulate both increased eNOS expression and increased Aktmediated eNOS phosphorylation at Ser1177 in the heart.
Here, we used therapeutic doses of fluvoxamine and paroxetine, which have ␴1R activities with a Ki of 36 nM (potent)
and 1,893 nM (weak), respectively (20). A positron emission
tomography study in humans demonstrated that fluvoxamine(50 –200 mg/BW) binds to ␴1Rs but that a paroxetine dose of
20 mg/BW has no effect in the intact human brain (20),suggesting that ␴1Rs function in the pharmacological actionsof fluvoxamine (20). Notably, SSRIs such as fluvoxamine
appear to be generally safe and effective in treating depression
in patients with cardiovascular disease. SSRIs generally do notslow cardiac conduction, cause orthostatic hypotension, decrease heart rate variability, or alter QT variability measures (9,14). In regard to treatment, SSRIs appear to be safe for use in
cardiac patients and can improve both depressive symptoms
and quality of life (17, 36, 46).
SSRIs have several physiological effects, as evidenced bystudies indicating that in non-depressive individuals, SSRIs
can decrease sympathetic nervous system activity at rest (asindicated by reduced plasma norepinephrine concentrations)and during mental stress tasks (as measured by lowered heartrate, blood pressure, and plasma catecholamine concentrations)(18, 19, 43). Moreover, SSRIs reportedly decreased plateletactivation in patients treated for depression and in healthy
volunteers (1, 34, 40, 41). Sauer et al. (37) reported a significant reduction of the risk of MI in SSRI-treated smokers,
whereas in the Sertraline Antidepressant Heart Attack Randomized Trial (17), the incidence of severe cardiac events was
only 14.5% in the sertraline (Ki ⫽ 57 nM)-treated groupcompared with 22.4% in the placebo group. Likewise, when
457 fatal and nonfatal cardiovascular events were followed up
for 29 mo, the risk of death or recurrent MI was significantly
lower in patients taking SSRIs (47). However, the mechanisms
underlying reversal of cardiac dysfunction by SSRIs are largely
unknown. Our study is the first to define the SSRI-induced
cardioprotection through ␴1Rs. Current American College of
Cardiology/American Heart Association guidelines for coronary artery bypass graft surgery, acute MI, and chronic angina
all recommend an evaluation of the symptoms of depression
and consideration of treatment of depression (36). Future
large-scale followup studies should define SSRI cardioprotective mechanisms and reveal the links between depression andcardiovascular disease. SSRIs increase brain monoaminergic
levels and reverse many physiological derangements associated with depression. In addition, SSRI therapy normalizes
urinary cortisol excretion and improves heart rate variability,
reduces platelet activation, and antagonizes the expression of
inflammatory markers (15, 17, 36, 39, 48). Our observations

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Fig. 9. Effect of ␴1R small interfering (si)RNA on Flux-induced antihypertorphic effects in cultured cardiomyocytes. A: Western blot analysis (top) anddensitometric quantification (bottom) of ␴1R expression with or without(control) ␴1R siRNA treatment. Immunoblot analysis with anti-␤-tubulin
antibody showed equal protein loading in each lane. Data are expressed aspercentages of the control value. B: cells were fixed with 4% paraformaldehyde, stained with rhodamine-conjugated phalloidin, and processed for fluorescence microscopy. One hundred cells from randomly selected fields were
examined in each condition. C: cell size results expressed as surface area
relative to control cells. Each bar represents the mean ⫾ SE. **P ⬍ 0.01 and***P ⬍ 0.001 vs. the control group; †††P ⬍ 0.001 vs. the ANG II-treatedgroup; ###P ⬍ 0.001 vs. the ANG II ⫹ F 5.0 group.

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␴1-RECEPTOR STIMULATION AMELIORATES MYOCARDIAL HYPERTROPHYGRANTSThis work was supported in part by Ministry of Education, Science, Sports
and Culture of Japan Grant-In-Aid for Scientific Research 22390109 (to K.
Fukunaga).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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Downloaded from ajpheart.physiology.org on February 21, 2011strongly suggest that SSRIs that stimulate ␴1Rs can reduce therisk of MI not only in depressive patients but in a wide rangeof cardiovascular disease patients.
In conclusion, we provide, for the first time, evidence for the
potential role of ␴1R expression in the heart to attenuate
TAC-induced hypertrophy in mice. With regard to the particular function of ␴1Rs in the heart, our data confirmed thatfluvoxamine is a ␴1R agonist in the heart, because its effectwas not only abrogated by NE-100 treatment but also by
siRNA knockdown of the receptor protein. In addition, fluvoxamine treatment protects the heart from TAC-induced hypertrophy and tissue injury via an upregulation of ␴1Rs andstimulation of ␴1R-mediated Akt-eNOS signaling in mice. We
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