Biochimica et Biophysica Acta 1830 (2013) 3082–3094

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Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen

Stimulation of σ1-receptor restores abnormal mitochondrial Ca 2 +mobilization and ATP production following cardiac hypertrophy
Hideaki Tagashira a, Chen Zhang b, Ying-mei Lu c, Hideyuki Hasegawa d, Hiroshi Kanai e,
Feng Han f, Kohji Fukunaga a,⁎a

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-Aoba, Aoba-ku, Sendai, Japan
Department of Pharmacy, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 31005, PR China
Department of Neurobiology, Zhejiang University School of Medicine, 388 Yu Hang Tang Road, Hangzhou 310058, PR Chinad
Department of Electrical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6 Aramaki-Aoba, Aoba-ku, Sendai, Japane
Department of Electrical Engineering, Graduate School of Engineering, Tohoku University, 6-6 Aramaki-Aoba, Aoba-ku, Sendai, Japanf
Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, Zhejiang University, Hangzhou, Zhejiang 31005, PR Chinabca r t i c l e

i n f oArticle history:
Received 2 October 2012
Received in revised form 21 December 2012
Accepted 26 December 2012
Available online 6 January 2013
Keywords:
ATP
Calcium signaling
ATP production
Mitochondria
Sigma receptora b s t r a c t
Background: We previously reported that the σ1-receptor (σ1R) is down-regulated following cardiac hypertrophyand dysfunction in transverse aortic constriction (TAC) mice. Here we address how σ1R stimulation with theselective σ1R agonist SA4503 restores hypertrophy-induced cardiac dysfunction through σ1R localized in the sarcoplasmic reticulum (SR).
Methods: We ﬁrst conﬁrmed anti-hypertrophic effects of SA4503 (0.1–1 μM) in cultured cardiomyocytes exposed
to angiotensin II (Ang II). Then, to conﬁrm the ameliorative effects of σ1R stimulation in vivo, we administered
SA4503 (1.0 mg/kg) and the σ1R antagonist NE-100 (1.0 mg/kg) orally to TAC mice for 4 weeks (once daily).
Results: σ1R stimulation with SA4503 signiﬁcantly inhibited Ang II-induced cardiomyocyte hypertrophy. Ang II
exposure for 72 h impaired phenylephrine (PE)-induced Ca2+ mobilization from the SR into both the cytosol
and mitochondria. Treatment of cardiomyocytes with SA4503 largely restored PE-induced Ca2+ mobilization
into mitochondria. Exposure of cardiomyocytes to Ang II for 72 h decreased basal ATP content and PE-induced
ATP production concomitant with reduced mitochondrial size, while SA4503 treatment completely restored
ATP production and mitochondrial size. Pretreatment with NE-100 or siRNA abolished these effects. Chronic
SA4503 administration also signiﬁcantly attenuated myocardial hypertrophy and restored ATP production in
TAC mice. SA4503 administration also decreased hypertrophy-induced impairments in LV contractile function.
Conclusions: σ1R stimulation with the speciﬁc agonist SA4503 ameliorates cardiac hypertrophy and dysfunction
by restoring both mitochondrial Ca2+ mobilization and ATP production via σ1R stimulation.
General signiﬁcance: Our observations suggest that σ1R stimulation represents a new therapeutic strategy to rescue the heart from hypertrophic dysfunction.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Administration of selective serotonin reuptake inhibitors (SSRIs),
which are used to treat depression, to patients who have undergone
myocardial infarction reportedly reduces post-MI morbidity and mortality [1]. Although the mechanism underlying that effect remains
unclear, it is known that SSRIs such as sertraline [2] and ﬂuvoxamine[3], in addition to acting on serotonin transporters, bind with highAbbreviations: SA4503, 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine
dihydrochloride; NE-100, N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy) phenyl]ethylamine monohydrochloride; SR, sarcoplasmic reticulum; TAC, transverse aortic
constriction; IP3R, inositol trisphosphate receptors; ATP, adenosine triphosphate; MAM,
mitochondria-associated ER membrane; TCA cycle, tricarboxylic acid cycle; PE,
phenylephrine⁎ Corresponding author. Tel.: +81 22 795 6837; fax: +81 22 795 6835.
E-mail address: kfukunaga@m.tohoku.ac.jp (K. Fukunaga).
0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbagen.2012.12.029

afﬁnity to the σ1-receptor (σ1R), suggesting that cardioprotective activity is mediated by σ1R stimulation. We recently conﬁrmed that the σ1R
is more highly expressed in neonatal rat cardiomyocytes and rat heart
than it is in brain and other peripheral tissues [4,5]. We also observed
reduction in σ1R levels in the left ventricle accompanying progressionof left ventricular hypertrophy in transverse aortic constriction (TAC)
mice and a signiﬁcant positive correlation between σ1R expression
and impaired LV fractional shortening [6]. σ1R stimulation with
ﬂuvoxamine rescues impaired LV fractional shortening and reduces hypertrophy in the TAC mice [6]. Furthermore treatment with a different
σ1R agonist, the neurosteroid dehydroepiandrosterone (DHEA), signiﬁcantly antagonizes pressure overload (PO)-induced decreases in σ1R
expression in rat heart [7,8] and thoracic arteries [9,10]. On the other
hand, Ito et al. [11] reported that suppression of sympathetic nerve activation by an σ1R agonist in brain improves impaired LV fractional
shortening in a pressure overload- and high-salt diet-induced heart

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failure mouse model. This ﬁnding suggests that ﬂuvoxamine indirectly
blunts impaired LV fractional shortening in this model. We previously
asked whether ﬂuvoxamine directly elicits an anti-hypertrophic effecton cultured cardiomyocytes and found that ﬂuvoxamine reduced
angiotensin II (Ang II)-induced hypertrophy by stimulating the σ1R in
cardiomyocytes [6]. Indeed, σ1R knockdown in cardiomyocytes totallyabolished this effect. However, the mechanism underlying the antihypertrophic effects of ﬂuvoxamine [6] and DHEA [8] and their effecton intracellular Ca2+ regulation remains unclear.
In non-cardiomyocytes, the σ1R localizes to the mitochondriaassociated ER membrane (MAM) and interacts with the IP3 receptor(IP3R), which likely promotes Ca 2 + transport into mitochondria
from the ER [12]. In the IP3R family, IP3R2 is the most prominent
isoform expressed in the heart [13,14]. Transgenic mice expressing
an IP3-chelating protein (IP3 sponge), which binds free IP3 with highafﬁnity and inhibits IP3R function in the heart [15], are resistant toisoproterenol-induced hypertrophy [16]. Taken together, IP3-mediated
Ca2+ release plays an important role in development of cardiac hypertrophy, and σ1R agonists likely modulate IP3-mediated Ca2+ release in
cardiomyocytes. However, it is not known how the σ1R modulates mitochondrial Ca2+ mobilization through IP3R2 in cardiomyocytes orwhether reducing σ1R levels following hypertrophy would alter mitochondrial Ca2+ mobilization.
In this study, to determine whether σ1R stimulation directly affects
IP3R function in cardiomyocytes, we used the speciﬁc, high afﬁnity σ1Ragonist SA4503 (1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride) (Ki = 4.4 nM) to deﬁne the function ofσ1R-mediated IP3R regulation of mitochondrial Ca 2 + transport
into cardiomyocytes in vitro and in vivo. We made the novel
observation that SA4503 ameliorates Ang II-induced impairment of
SR-mitochondrial Ca2+ transport and mitochondrial ATP production
via σ1R stimulation. We found that enhanced ATP production following
SA4503 administration restored cardiac contractile dysfunction in TAC
mice. This study suggests that the σ1R could be a novel therapeutic target to rescue cardiomyocytes from myocardial infarction.
2. Materials and methods
2.1. Materials
Reagents and antibodies were obtained from the following sources:
anti-σ1R antibody (Abcam, Cambridge, UK); anti-IP3 receptor type 2(American Research Products Inc. Waltham, MA); anti-mitofusin 2(Sigma, St. Louis, MO); anti-mitofusin 1 (Abcam, Cambridge, UK);anti-voltage-dependent anion channel (VDAC) (Cell Signaling Technology, Beverly, MA); anti-GRP75 (Cell Signaling Technology, Beverly, MA);
anti-Cytochrome c (BD Biosciences, San Diego, CA); and anti-β-tubulin
antibody (Sigma, St. Louis, MO). The σ1R agonist SA4503 was synthesized in the Laboratory of Medicinal Chemistry, Zhejiang University
according to the method of Fujimura et al. [17]. The speciﬁc σ1Rantagonist NE-100 (N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)
phenyl]-ethylamine monohydrochloride) was generously supplied by
Taisho Pharmaceutical Co. Ltd (Ohmiya, Japan). Other reagents were of
the highest quality available (Wako Pure Chemicals, Osaka, Japan).

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ad libitum. Transverse aortic constriction (TAC) was performed aspreviously described [6] on male ICR mice under anesthesia using a
mixture of ketamine (100 mg/kg, i.p.) (Daiichi Sankyo Pharmaceutical Co. Ltd, Tokyo, Japan) and xylazine (5 mg/kg, i.p.) (Sigma, St.
Louis, MO). Adequate depth of anesthesia was conﬁrmed by a negative toe-pinch reﬂex. If anesthesia was not sufﬁcient, then a top-up
dose of 20% of the initial dose was given.
2.3. Experimental design
ICR mice were randomly separated into six experimentalgroups: 1) sham (n = 5), 2) TAC for 4 weeks (n = 5), 3) TAC plus
SA4503 (0.1 mg/kg, p.o.) treatment (SA 0.1) (n = 5), 4) TAC plus
SA4503 (0.3 mg/kg, p.o.) treatment (SA 0.3) (n = 5), 5) TAC plus
SA4503 (1.0 mg/kg, p.o.) treatment (SA 1.0) (n = 5), 6) TAC plus
SA4503 (1.0 mg/kg, p.o.) plus NE-100 treatment (1.0 mg/kg, p.o.)(SA 1.0 + NE) (n = 5). SA4503 and NE-100 were dissolved in a
0.9% saline solution. Vehicle, SA4503 and NE-100 (1.0 mg/kg)
were administered orally for 4 weeks (once daily) using a metal
gastric zonde formice in a volume of 1 ml/100 g of mouse bodyweight, starting from the onset of aortic banding.
2.4. Echocardiography and measurement of cardiac hypertrophy
Noninvasive echocardiographic measurements were performed as
described [6]. Brieﬂy, noninvasive echocardiographic measurements
were performed in mice anesthetized with pentobarbital-NaCl/EtOH
solution (0.05 g/kg i.p.) using ultrasonic diagnostic equipment(SSD-6500; Aloka, Tokyo, Japan) equipped with a 10-MHz linear arraytransducer (UST-5545; Aloka). The heart was imaged in the twodimensional parasternal short-axis view, and an M-mode echocardiogram of the midventricle was recorded at the level of 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 leading edge to leading edge according
to the American Society of Echocardiography guidelines. The percentage of LV fraction shortening (LV%FS) was calculated as ([LVEDD−
LVESD]/ LVEDD)× 100. After sacriﬁcing by cervical spine fracture dislocation, the thoracic cavity was opened, and hearts were immediately
harvested and weighed.
2.5. Transfection of cultured cardiomyocytes
Neonatal rat ventricular cardiomyocytes were isolated from hearts of
1- to 3-day-old Wistar rats, which had been sacriﬁced by decapitation,and cardiomyocytes were cultured as described [6]. σ1R siRNA (sense,
5′-ACACGTGGATGGTGGAGTA-3′ and anti-sense, 5′-TACTCCACCATC
CACGTGT-3′) was purchased from Exigen Ltd., Tokyo, Japan. Cultured
myocytes were plated on uncoated 90-mm culture dishes on collagencoated cover glasses. Transfections were performed using 100 nM σ1R
siRNAs as described [6].
2.6. Morphological analysis and immunocytochemistry of cultured
cardiomyocytes

2.2. Animals and operations
All procedures for handling animals complied with the Guide forCare 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 to 40 g
were obtained from Nippon SLC (Hamamatsu, Japan). Ten-week-old
males were acclimated to the local environment for 1 week, which
included housing in polypropylene cages at 23 ± 1 °C in a humiditycontrolled environment maintained on a 12-h light/dark schedule(lights on 8:00 AM–8:00 PM). Mice were provided food and water

Morphological analysis was performed as described [6]. The surface
area of control cells was set at 100% and compared with that of treatedcells. For mitochondrial staining, cells were stained for 20 min with
0.02 μM MitoTracker Red CMXRos (Molecular Probes) before ﬁxation.
After permeabilization with 0.1% Triton X-100 in PBS, ﬁxed cells wereincubated with 1% bovine serum albumin in PBS for 30 min. For immunocytochemistry, cells were incubated 24 h at 4 °C with anti-σ1R
antibody (1:500) or anti-Cytochrome c antibody (1:500) in PBS
containing 1% BSA. After washing, cells were incubated 24 h with
Alexa 488 in PBS containing 1% BSA. Immunoﬂuorescent images were3084

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obtained with a confocal laser scanning microscope (TCS SP, Leica
Microsystems).

2.7. Western blot analysis and measurement of ATP content
Dissected LV tissue samples were rapidly frozen in liquid nitrogenand stored at −80 °C before analysis. In the case of cardiomyocytes,
cells were washed with PBS at 4 °C and stored at −80 °C until immunoblotting analyses were performed as described [18]. For assays, each frozen sample was homogenized as described [6,19]. ATP measurement
was performed using an ATP assay kit (Toyo Ink, Tokyo, Japan), according
to the manufacturer's protocol. Brieﬂy, frozen samples were homogenized in homogenate buffer (0.25 M sucrose, 10 mM HEPES-NaOH:
pH 7.4), and lysates were cleared by centrifugation at 1000 g for
10 min at 4 °C. The supernatant was collected, and proteins in the supernatant were solubilized in extraction buffer. After 30 min, luciferin buffer
was added to each sample and oxyluciferin was detected using aluminometer (Gene Light 55, Microtec, Funabashi, Japan).
2.8. Measurement of intracellular Ca 2+
Neonatal ventricular myocytes were cultured on 0.01% poly-L-lysine(Sigma-Aldrich) coated glass-bottom dishes and maintained in
growth medium. After stimulation with 100 nM Ang II for either 48 or
72 h, myocytes were loaded with the Ca2+-sensitive dye Fura-2acetoxymethyl ester (2.5 μM) for 30 min before measuring Ca2+ levels
in a chamber on an inverted microscope stage. Cells were perfused withnormal Tyrode solution containing 150 mM NaCl, 4 mM KCl, 1 mM
MgCl2, 2 mM CaCl2, 5.6 mM glucose, and 5 mM HEPES at 37 °C. When
Ca2+ ﬂuorescence levels reached a steady state, 10 μM phenylephrine(PE) and 10 mM caffeine were applied for 10 s through a small perfusion pipe. The amplitude of the PE-induced Ca2+ transient was used
as an index of IP3R-mediated Ca2+ release. The amplitude of thecaffeine-induced Ca2+ transient served as an index of sarcoplasmic
reticulum Ca2+ content [20,21]. Changes in PE-induced Ca2+ releaseand caffeine-induced Ca2+ release from the sarcoplasmic reticulum
were determined using the ratio of ﬂuorescence emission at 530 nm
in response to excitation at 340 nm and to that at 380 nm.
2.9. Measurement of mitochondrial Ca 2+
Neonatal ventricular myocytes were cultured on 0.01% poly-L-lysine(Sigma-Aldrich) coated glass-bottom dishes and maintained in
growth medium. Transfections were performed with ratiometricpericam targeted to the mitochondrial matrix (ratiometric-pericammt/pcDNA3), which was a kind gift of Dr. Atsushi Miyawaki of
the RIKEN Brain Science Institute (Wako-city, Japan). Brieﬂy, 1 μg/μl
ratiometric-pericam-mt/pcDNA3 in 1 μl was added to 199 μl opti-MEM(Invitrogen), and 1 μl Lipofectamine 2000 (Invitrogen) was added to
9 μl opti-MEM. Both solutions were incubated separately at room temperature for 5 min and then mixed and incubated at room temperaturefor 15 to 20 min. Cells were incubated with 800 μl of opti-MEM towhich 200 μl of the ratiometric-pericam-mt/pcDNA3 solution had been
added. Cells were then incubated at 37 °C in a 5% CO2 atmosphere for
4 h to initiate transfection. Then, 500 μl DMEM supplemented with 5%
FBS was added to each well to maintain cell viability. After 24 h,
100 nM Ang II was added. After stimulation for 48 or 72 h, cells were
perfused with normal Tyrode solution at 37 °C. When Ca 2+ ﬂuorescencelevels reached a steady state, 10 μM PE was applied for 10 s through asmall perfusion pipe. Dual-excitation imaging with ratiometricpericam-mt required two ﬁlters (EX:482/35, DM:506, EM:536/40 and
EX:414/46, DM:510, EM:527/20). Changes in PE-induced Ca2+ transport
from the sarcoplasmic reticulum to mitochondria were determinedusing the Metaﬂuor Imaging system (Molecular Devices, Sunnyvale, CA).

2.10. Statistical analysis
Values are represented as means ± standard error of the mean
(S.E.M.). Results were evaluated for differences using one-way analysis
of variance (ANOVA), followed by multiple comparisons using Scheffe'stest. P b 0.05 was considered statistically signiﬁcant.
3. Results
3.1. SA4503 treatment ameliorates Ang II-induced cardiomyocyte
hypertrophy
We previously documented that ﬂuvoxamine elicits antihypertrophic
effects on Ang II-induced hypertrophy through σ1R stimulation.
We ﬁrst conﬁrmed the antihypertrophic effect of the selective σ1R
agonist SA4503 on Ang II-induced hypertrophy. Neonatal rat cultured
cardiomyocytes were exposed to Ang II for 72 h with or without treatment with SA4503, NE-100 or the selective IP3R inhibitor xestospongin
C for the last 24 h. None of these treatments had an effect on the sizeof untreated (control) cells; however, the size of Ang II-treated cells
signiﬁcantly increased compared to control cells 72 h after Ang II
exposure (Pb 0.01 vs. control) (Fig. 1A and B). SA4503 treatment
inhibited Ang II-induced cell enlargement dose-dependently (Pb 0.01
vs. Ang II) (Fig. 1A and B). Combined treatment with NE-100 reversed
SA4503-mediated inhibition of cardiomyocyte hypertrophy (Pb 0.01 vs.
SA 1.0) (Fig. 1A and B). Interestingly, combined treatment withxestospongin C and SA4503 partially but signiﬁcantly reversed
SA4503-mediated inhibition of cardiomyocyte hypertrophy (Pb 0.01 vs.
SA 1.0) (Fig. 1A and B). These ﬁndings suggest that SA4503 directly
acts on the cardiac σ1R to antagonize Ang II-induced cardiomyocyte
enlargement, an effect partially mediated by IP3R stimulation.
3.2. Effect of SA4503 treatment on PE-induced Ca2+ inﬂux into mitochondria
and the cytosol
To test our hypothesis that σ1R stimulation regulates IP3R-mediated
Ca2+ mobilization in cardiomyocytes, we examined the effects ofSA4503 on PE-induced IP3R-mediated Ca 2+ mobilization into mitochondria and the cytosol in Ang II-treated cells. Treatment with
SA4503 alone for 24 h had no effect on PE-induced mitochondrial
Ca2+ mobilization in control cells. At 72 h after treatment, Ang II exposure severely impaired PE-induced mitochondrial Ca2+ mobilizationcompared to effects seen in untreated control cells (P b 0.01 vs. control)(Fig. 2A, B and C). SA4503 treatment during the last 24 h signiﬁcantly
restored PE-induced Ca 2+ mobilization in mitochondria (P b 0.01 vs.
Ang II) (Fig. 2A, B and C). We also examined the effects of SA4503
on PE-induced IP3R-mediated Ca2+ mobilization into the cytosol.
SA4503 treatment alone for 24 h had no effect on PE-induced Ca2+
mobilization into the cytosol in control cells. Like mitochondrial Ca2+
mobilization, Ang II exposure for 72 h signiﬁcantly suppressed Ca2+
mobilization, and SA4503 treatment partially rescued that effect(P b 0.01 vs. control) (Fig. 2D, E and F). These results suggest thatSA4503 treatment preferentially rescues IP3R2-mediated mitochondrial
Ca2+ mobilization but partly rescues IP3R2-mediated Ca2+ mobilization into the cytosol.
3.3. Effect of SA4503 treatment on σ1R expression, its association withmitochondria, and mitochondrial ATP content
Since SA4503 promotes IP3R-mediated Ca 2+ mobilization, particularly into mitochondria, we asked whether σ1R activity governs mitochondrial morphology and functions in ATP production. MitoTracker
Red CMXRos detects not only mitochondrial morphology but also mitochondrial membrane potential because of intake of mitochondria in amembrane potential-dependent manner [22]. We ﬁrst conﬁrmed an association of the σ1R with mitochondrial membranes in cultured

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A

15µm

Control

Ang II

Ang II +SA 0.1 µM

Ang II +SA 1.0 µM

Ang II +SA 1.0 µM +NE

Ang II +SA 1.0 µM +
xest

**

††
**

B

Cell size (% of control)

250

200

##
**

150

##
**

††
**

100

50

0

Cont

SA

NE

xest

-

SA 0.1

SA 1.0SA 1.0 + SA 1.0 +NE
xest

Ang II
Fig. 1. Effects of SA4503 treatment on Ang II-induced hypertrophy of 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 in each condition for cell size. B, cell size is expressed as apercentage of the relative surface area compared to control cells. Each column represents the mean ±S.E.M. **, P b 0.01 versus the control cells; ##, P b 0.01 versus Ang II-treatedcells; ††, P b 0.01 versus Ang II plus 1 μM SA4503-treated cells. NE: NE-100 (10 μM for 24 h). Xest: xestospongin C (1 μM for 24 h).

cardiomyocytes. In unstimulated control cells, σ1R immunoreactivity
largely merged with that of a MitoTracker Red (Fig. 3A: Cont). SA4503
alone treatment did not alter such co-localization (Fig. 3A: SA 24 h).
After Ang II exposure for 72 h, σ1R immunoreactivity largely
disappeared from the cytosol, and mitochondrial tracer ﬂuorescence
was also slightly reduced (Fig. 3A: Ang II 72 h). Mitochondrial size signiﬁcantly decreased after Ang II exposure, suggesting either mitochondrial degradation or enhanced mitochondrial ﬁssion. Interestingly,
SA4503 treatment for the last 24 h of 72 h Ang II exposure rescued reduced σ1R immunoreactivity and inhibited mitochondrial ﬁssion and/or degradation (Fig. 3A: Ang II 72+ SA 24 h). Finally, NE-100 treatment
antagonized the SA4503-induced protective effect on mitochondrial
degradation, as shown by increased mitochondrial ﬁssion (Fig. 3A:
Ang II 72 + NE + SA 24 h). These data suggest that Ang II treatment ofcells causes mitochondrial dysfunction and that SA4503 treatment

protects cells from Ang II-induced mitochondrial ﬁssion and/or
degradation.
We next examined the effects of Ang II and SA4503 treatment on
ATP content. SA4503 treatment alone slightly but signiﬁcantly enhanced ATP content in control cells, but xestospongin C treatment
alone had no effect. As expected, Ang II exposure for 72 h signiﬁcantly
decreased ATP content (P b 0.01 vs. control), and SA4503 treatment
rescued this effect (P b 0.01 vs. Ang II) (Fig. 3B). Treatment with
NE-100 combined with σ1R knockdown by siRNA totally abolishedthe SA4503 effect (P b 0.01 vs. SA) (Fig. 3B). σ1R siRNA effectively reduced σ1R levels to 30% of control cells, as shown previously [6]. Finally, xestospongin C treatment largely reversed SA4503-mediatedincreases in ATP content (P b 0.01 vs. SA 1.0). Since increased ATP consumption could persist following Ang II exposure, we examined
phenylephrine (PE)-induced ATP production, which peaks within

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A

D
Mitochondria

3

cont

AngII

AngII+SA

AngII+SA

2.5

2

0.35

0.3

1 min

1 min

B

E
Mitochondria

Cytosol

150

100

##
**

50

AUCcyto(% of control)

150

AUCmito(% of control)cont

PE 10µM

AngII

Ratio
(F340/F380) cyto

Ratio
(F480/F410) mito

PE 10µM

Cytosol

0.4

100

#
**

50

**

**
0

Cont

SA

-

0

SA

Cont

SA

-

SA

Ang IIC

Ang IIF
0.1

Mitochondria

0.7

0.08

0.5

##

0.4
0.3
0.2

0

Cont

SA

-

0.06

#
*

0.04

**

0.02

**

0.1

Change in ratio(Rmax -Rmin) cyto

Change in ratio(Rmax -Rmin) mito

0.6

CytosolSA

Ang II

0

Cont

SA

-

SA

Ang II

Fig. 2. Effects of SA4503 treatment on PE-induced Ca2+ inﬂux into mitochondria and the cytosol. A, time courses of PE-induced Ca2+ inﬂux into mitochondria 72 h after 100 nM
Ang II treatment with or without SA4503 treatment during the last 24 h. B, area under the curve (AUC) quantiﬁcation of PE-induced Ca2+ inﬂux into mitochondria. C, peak increases
in [Ca2+]mito induced by 10 μM PE. D, time courses of PE-induced Ca2+ release into the cytosol 72 h after 100 nM Ang II treatment with or without SA4503 treatment for the last
24 h. E, AUC quantiﬁcation of PE-induced Ca2+ release into the cytosol. F, peak increases in [Ca2+]cyto induced by 10 μM PE. Each group consists of more than 10 cells. Data areexpressed as percentages of control cells (mean ± S.E.M.) *, Pb 0.05 and **, P b 0.01 versus control cells; #, P b 0.05 and ##, Pb 0.01 versus Ang II-treated cells.

15 min of treatment. As shown in Fig. 3C, PE stimulated ATP production up to 180% in naive control cells, whereas PE failed to stimulate
ATP production at 72 h after Ang II treatment. When cardiomyocytes

were treated with Ru360, a speciﬁc inhibitor of mitochondrial calcium uptake, PE-induced ATP production was completely inhibited(Fig. 3C), suggesting that mitochondrial Ca 2+ inﬂux functions in

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A

σ1receptor

Merge

MitoTracker

3087

Zoom

Cont

Cont +SA

AngII

AngII +
SA

AngII +
NE + SA

B

150

ATP content(% of control)

*
##

100

††
**

**

†
*

††
**

50

0
Cont

SA

xest

-

SA

SA+NE100 SA+siRNA SA+xest

Ang II

C
200

N.S.
N.S.

ATP content(% of control)

150

N.S.
non

##

100

††
**
50

0
Cont

Ru360

-

SA

Ang II

SA+NE

PE-stimulated

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PE-induced ATP production. Notably, ATP content of Ang II-treatedcells was markedly decreased compared to naive cells 72 h afterAng II treatment (P b 0.01 vs. control) (Fig. 3B and C: white bar), andPE stimulation (Fig. 3C: black bar) failed to increase ATP production.
Treatment with SA4503 for the last 24 h of Ang II exposure signiﬁcantly and completely restored ATP content and PE-induced ATP production. SA4503-mediated rescue of ATP content and PE-induced ATP
production was totally abolished in the presence of NE-100 (Fig. 3C).
Taken together, our observations strongly suggest that SA4503mediated increases in ATP content and PE-induced ATP production
are mediated by σ1R stimulation.
3.4. The effect of sigma-1 receptor stimulation by SA4503 on expressionof σ1R and ER-mitochondria junctional proteins
We further investigated mitochondrial morphology using anothermitochondrial marker protein, Cytochrome c, whose localization is independent of mitochondrial membrane potential. Mitochondrial size
decreased after Ang II exposure, suggesting either mitochondrial degradation or enhanced mitochondrial ﬁssion. Interestingly, SA4503 treatment for the last 24 h of 72 h Ang II exposure inhibited mitochondrial
ﬁssion and/or degradation (Fig. 4A: Ang II 72+ SA 24 h). Finally,
NE-100 treatment antagonized the SA4503-induced protective effecton mitochondrial degradation, as shown by increased mitochondrial
ﬁssion (Fig. 4A: Ang II 72 + NE+ SA 24 h).
To further deﬁne changes in mitochondrial function, we determined
expression levels of proteins associated with the SR and mitochondria.
Consistent with decreased σ1R immunoﬂuorescence observed in Fig. 3A,
σ1R protein expression levels decreased in Ang II-exposured cells(Pb 0.01 vs. control) (Fig. 4B, C). SA4503 treatment signiﬁcantly rescued
reduced σ1R expression (Pb 0.01 vs. Ang II) (Fig. 4B, C). Interestingly,
although NE-100 treatment abolished SA4503-mediated cardioprotective
effects (Fig. 1A), it did not completely abolish SA4503-mediatedupregulation of σ1R expression (Fig. 4B, C).
Recent studies reveal that speciﬁc proteins and chaperones function
as ER-mitochondria linkage proteins. For example, mitofusin-2 (Mfn-2)
not only connects mitochondria but also bridges the ER with mitochondria and functions in IP3-induced mitochondrial Ca 2+ uptake [23].
Moreover, Szabadkai et al. report that glucose-regulated protein(GRP75), a molecular chaperone, links ER and mitochondria byconnecting the IP3R with voltage-dependent anion channels (VDACs)[24]. Thus, we hypothesized that ER-mitochondria linkage proteins
could alter the response of cells to Ang II treatment. Interestingly,
Ang II exposure for 72 h signiﬁcantly decreased GRP75 and Mfn-2 levels(P b 0.01 vs. control), and SA4503 treatment rescued this effect (P b 0.01
vs. Ang II) (Fig. 4B). Treatment with NE-100 combined with SA4503
totally abolished the SA4503 effect (P b 0.01 vs. SA) (Fig. 4B). Unexpectedly, expression of other mitochondria-associated proteins, namely
Mfn-1 and VDAC, was not altered after Ang II exposure for 72 h,
suggesting that mitochondria are not signiﬁcantly degraded afterAng II exposure and that SA4503-induced rescue of ATP production
correlates with alterations in ER-mitochondrial linkage proteins.
3.5. The effect of sigma-1 receptor stimulation by SA4503 on myocardial
hypertrophy and dysfunction
To assess potential SA4503-mediated cardioprotective andantihypertrophic effects in vivo, we administered either SA4503 alone
or SA4503 combined with NE-100 orally to TAC mice for 4 weeks,
starting at TAC onset. One-way ANOVA analysis showed a signiﬁcantgroup effect on LV fractional shortening (%) [F (5, 47)=4.747, Pb 0.01]
and left ventricular end-systolic diameter (LVESD) [F (5, 47)=4.169,
Pb 0.01] in vivo. Post hoc analysis showed that TAC treatment signiﬁcantly decreased LV fractional shortening and increased LVESD (Pb 0.01 vs.
sham) compared with sham-operated mice (Fig. 5A, B and C), consistent
with our previous report [6]. SA4503 treatment restored decreased
LV fractional shortening (P b 0.05 vs. TAC-vehicle for SA 0.3 andP b 0.01 vs. TAC-vehicle for SA 1.0) (Fig. 5A and B) and increased
LVESD dose-dependently (P b 0.05 vs. TAC-vehicle for SA 1.0)
(Fig. 5A and C). Co-administration of NE-100 abolished SA4503mediated amelioration of heart dysfunction, as indicated bydecreased LV fractional shortening (FS%) and increased LVESD(P b 0.01 vs. SA 1.0) (Fig. 5A, B and C). These results suggestthat SA4503 ameliorates cardiac contractile dysfunction throughactivation of the σ1R.
Similarly, one-way ANOVA analysis showed a signiﬁcant group effecton HW-to-BW [F (5, 23)=21.266, Pb 0.01] and LW-to-BW [F (5, 23)=
15.610, Pb 0.01] ratios in vivo. Post hoc analysis showed that SA4503
treatment signiﬁcantly restored the HW-to-BW ratio (Pb 0.01 vs.
TAC-vehicle for SA 0.3 and SA 1.0) (Fig. 5D) and the LW-to-BW ratio(Pb 0.01 vs. TAC-vehicle for SA 0.3 and SA 1.0) dose-dependently(Fig. 5B). Co-administration of NE-100 abolished SA4503 inhibition of
TAC-induced hypertrophy, as indicated by HW-to-BW (Pb 0.05 vs.
sham; Pb 0.01 vs. SA 1.0) (Fig. 5D) and LW-to-BW (P b 0.05 vs. sham;Pb 0.01 vs. SA 1.0) ratios (Fig. 5E). Taken together, we conclude thatthe anti-pathological hypertrophic effect of SA4503 on TAC mice ismediated by σ1R stimulation in vivo.
3.6. Effect of SA4503 treatment on σ1R and IP3R2 expression in TAC mice
We previously documented that the σ1R is down-regulated concomitant with increased contractile dysfunction in TAC mice 4 weeks
after TAC and that ﬂuvoxamine treatment rescued σ1R expression [6].
We conﬁrmed SA4503 effects on σ1R levels. One-way ANOVA analysis
showed a signiﬁcant group effect on σ1R [F (5, 33)= 12.322, P b 0.01]
and IP3R2 [F (5, 32)= 3.033, P b 0.05] expression in vivo. As expected,
SA4503 treatment signiﬁcantly and dose-dependently restored σ1Rlevels (P b 0.05 vs. TAC-vehicle for SA 0.3 and P b 0.01 vs. TAC-vehicle
for SA 1.0) (Fig. 6A, B). By contrast, TAC-induced hypertrophy was
associated with increased IP3R2 expression levels in the LV (P b 0.01
vs. sham). SA4503 administration signiﬁcantly and dose-dependently
decreased hypertrophy (Pb 0.01 vs. TAC-vehicle for SA 1.0) (Fig. 6A, C).
Although co-administration of NE-100 with SA4503 abolished SA4503mediated cardioprotective effects (Fig. 5), co-administration of
NE-100 did not abolish SA4503-mediated increases in σ1R expression(Pb 0.01 vs. TAC-vehicle) in the LV (Fig. 6A, B). On the other hand,co-administration of NE-100 with SA4503 totally abolished SA4503mediated decreases in IP3R2 expression (Fig. 6A, C). These results
suggested that SA4503-induced restoration of imbalance between σ1R
and IP3R2 levels is correlated with its cardioprotective activity in animals subjected to TAC-induced cardiac pathological hypertrophy anddysfunction.
3.7. σ1R stimulation increases ATP content in TAC mice
Our in vitro studies of cardiomyocytes clearly demonstrate that
stimulation of the σ1R and subsequently of the IP3R by SA4503 is criticalfor ATP production (Fig. 3). Thus, we conﬁrmed the effects of SA4503 on
ATP production in the LV of TAC mice. One-way ANOVA analysis
showed a signiﬁcant group effect on ATP content [F (5, 47) = 7.809,

Fig. 3. Intracellular localization of σ1R and ATP content following treatment with Ang II and SA4503. A, cultured cardiomyocytes were processed using immunoﬂuorescence to markthe σ1R (green) and MitoTracker Red CMXRos (red) to mark mitochondria. Scale bar = 8 μm. B, measurement of cellular ATP content 72 h after treatment with 100 nM Ang II withor without SA4503 treatment for the last 24 h. Each group consists of 4 samples. C, measurement of cellular ATP content with or without 10 μM PE stimulation. Each group consistsof 4 samples. Data are expressed as percentages of the control cell value (mean ± S.E.M.) *, P b 0.05 and **, P b 0.01 versus control cells; ##, P b 0.01 versus Ang II-treated cells; †,P b 0.05 and ††, P b 0.01 versus Ang II plus 1 μM SA4503-treated cells; ¶¶, P b 0.01 versus the non-stimulated cells. NE: NE-100 (10 μM for 24 h). Xest: Xestospongin C (1 μM for 24 h).

H. Tagashira et al. / Biochimica et Biophysica Acta 1830 (2013) 3082–3094

A

3089

B

Control

σ1receptor← 27 kDa

GRP75← 75 kDa

Mfn 1← 84 kDa

Mfn 2← 84 kDa

AngII← 31 kDa

VDAC← 60 kDa

β-tubulinCont

AngII + SA-

SA SA+NE
AngII

AngII + SA + NE

C
150

150

150

**
50

0

Cont

-

100

**
50

0

SA SA+NE

Cont

-

AngII

##

100

**

50

0

Cont

-

SA SA+NE

AngII

150

100

100

50

0

SA SA+NE

AngII

VDAC
(% of sham)

Mfn 1(% of sham)

150

Mfn 2
(% of sham)

##

100

GRP 75(% of sham)σ1 receptor(% of sham)

##

Cont

-

SA SA+NE

50

0

Cont

AngII

-

SA SA+NE

AngII

Fig. 4. Effect of SA4503 and NE-100 on expression of σ1R and ER-mitochondria junctional proteins. A, cultured cardiomyocytes were processed using immunoﬂuorescence to monitor mitochondrial morphology using Cytochrome c (red). Scale bar = 15 μm. B, western blot analysis of σ1R, VDAC and ER-mitochondria junctional protein expression in neonatal
rat ventricular myocytes with or without drug treatment. Immunoblotting with anti-β-tubulin antibody indicates equal protein loading in each lane. C, densitometric quantiﬁcationof σ1R and VDAC immunoreactive bands. C, densitometric quantiﬁcation of ER-mitochondria junctional protein immunoreactive bands. Each group consists of 4 samples. Data areexpressed as percentages of control cell values (mean ± S.E.M.) **, P b 0.01 versus control cells; ##, P b 0.01 versus Ang II-treated cells; ††, P b 0.01 versus Ang II plus 1 μM
SA4503-treated cells.

P b 0.01] in vivo. Post hoc analysis showed that ATP content of the TAC
mouse LV was signiﬁcantly decreased compared to sham mice
(P b 0.05 vs. sham) (Fig. 6D). SA4503 treatment (0.3, 1.0 mg/kg) significantly and completely ameliorated reduced ATP content dosedependently (P b 0.05 vs. TAC for SA 0.3 and SA 1.0) (Fig. 6D), and an
effect completely abolished in the presence of NE-100 (Fig. 6D). Theseobservations strongly suggest that SA4503-mediated increases in ATP
content are mediated by the σ1R and that these activities underlie its
cardioprotective effects.

4. Discussion
The present study addresses how σ1R stimulation ameliorates cardiac hypertrophy and dysfunction in vitro and in vivo. We ﬁrst deﬁned effects of SA4503, a selective σ1R agonist, on IP3-mediated
Ca 2 + mobilization into mitochondria and on ATP production. We
also focused on the effects of SA4503 on mitochondrial damage
caused by oxidative stress following Ang II-induced hypertrophy in
cardiomyocytes. Hayashi and Su [12] have proposed an attractive

3090

H. Tagashira et al. / Biochimica et Biophysica Acta 1830 (2013) 3082–3094

A

LVESD

LVEDD

Sham

TAC

TAC + SA 1.0

TAC + SA 1.0 + NE

B

D
##

#
40

FS(%)

**

*

8

†
*

30
20
10
0

sham

vehicleSA 0.1

SA 0.3

HW/BW (mg/g)

50

**
6

##

##

††
*

4
2
0

SA1.0 SA1.0+NE

*

sham

vehicleTAC (4 weeks)

SA 0.1

SA 0.3 SA1.0 SA1.0+NE

TAC (4 weeks)

C

E
4

LVESD

3

††
*

**
#

2
1
0

sham

vehicleSA 0.1

SA 0.3

SA1.0 SA1.0+NE

TAC (4 weeks)**

12

LW/BW (mg/g)

**

†
##

8

##

4

0

sham

vehicleSA 0.1

SA 0.3

SA1.0 SA1.0+NE

TAC (4 weeks)

Fig. 5. Effect of SA4503 and NE-100 on cardiac failure and hypertrophy induced by TAC. A, representative M-mode echocardiograms of mice with or without SA4503 and/or NE-100 treatments. B, changes in percentage of LV fractional shortening (FS%). C, changes of left ventricular end-systolic diameter (LVESD). TAC mice were treated with SA4503 (0.1, 0.5 or 1.0 mg/kg)
or SA4503 (1.0 mg/kg) plus NE-100 (1.0 mg/kg) as indicated. D, TAC-induced cardiac hypertrophy as indicated by the heart weight/body weight (HW/BW) ratio. E, TAC-induced cardiac
hypertrophy as indicated by the lung weight/body weight (LW/BW) ratio. Each group consists of 4–5 mice. Data are expressed as percentages of values of sham-operated animals(mean±S.E.M.) *, Pb 0.05 and **, Pb 0.01 versus the sham group; #, Pb 0.05 and ##, Pb 0.01 versus the TAC/vehicle treated group; †, Pb 0.05 and ††, Pb 0.01 versus the TAC plus
SA4503 (1 mg/kg)-treated group.

model that the σ1R supports cell survival via interaction with theIP3R3, allowing Ca 2+ entry into mitochondria in CHO cells. Our results further strengthen the evidence that the Ca 2+ entry into mitochondria through σ1R/IP3R complex promotes ATP production in
cardiomyocytes.
One of the striking observations shown here is that prolonged (72 h)
exposure to Ang II decreases ATP content and promotes mitochondrial
ﬁssion and/or degradation, suggesting that mitochondrial dysfunction
occurs when cardiac hypertrophy progresses to heart failure. Indeed,
mitochondrial ﬁssion or degeneration was observed in H9c2 and HL-1

cardiac cells in ischemic conditions and, interestingly, pharmacologicalinhibition of mitochondrial ﬁssion reportedly protects cardiomyocytes
from ischemia-induced apoptosis [25,26]. These observations suggestthat mitochondrial ﬁssion or degradation triggers apoptosis or
mitophagy associated with mitochondrial energy depletion [27]. Mitochondrial ﬁssion or degeneration was also observed in mice doublydeﬁcient in mitofusin-1 and -2, mitochondrial dynamin-related proteinsthat promote fusion, and these mice showed LV fractional shortening by
2 weeks of age [28]. Notably, we found that SA4503 treatment during
the last 24 h of 72 h Ang II exposure totally rescued reduced ATP content

H. Tagashira et al. / Biochimica et Biophysica Acta 1830 (2013) 3082–3094

in hypertrophic cardiomyocytes concomitant with reduced hypertrophy.
Since increased ATP consumption could continue after Ang II exposure,
we also deﬁned PE-induced ATP production which was totally abolished

3091

Ru360, a speciﬁc inhibitor of mitochondrial calcium uptake. Prolonged(24 h) SA4503 treatment restored reduced ATP content in vitro and invivo. Moreover, SA4503 rescued impaired PE-induced ATP productionA
σ1 receptor← 25 kDa

IP3R2← 260 kDa

β-tubulin← 50 kDa
shamvehicleSA 0.1

SA 0.3

SA1.0 SA1.0+NE

TAC (4 weeks)

B150

σ1 receptor(% of sham)

##

##

#

100

**

**

50

0

sham

vehicleSA 0.1

SA 0.3

SA1.0 SA1.0+NE

TAC (4 weeks)C200

IP3 receptor type2(% of sham)

**

*

†

150

##

100

50

0

sham

vehicleSA 0.1

SA 0.3

SA1.0 SA1.0+NE

TAC (4 weeks)

D

150

##

ATP content(% of sham)

##
100

††
*

*
**

50

0

sham

vehicleSA 0.1

SA 0.3

SA1.0 SA1.0+NE

TAC (4 weeks)

3092

H. Tagashira et al. / Biochimica et Biophysica Acta 1830 (2013) 3082–3094

SA4503NAAngII
α1 receptor

AT1receptor

Plasmamembrane

L-VDCC
Gαq

Gαq

PLC

Ca2+
RyR

IP3R2

SR
Ca2+
ROS generation
Oxidative stress

Cardiaccontraction

Ca2+

Ca2+

IP3R2

σ 1R

GRP75
VDAC

Mfn2
Mfn1/2

IP3

mitochondrial dysfunction

Ca2+

SERCA2[Ca2+]ihomeostasis
Cardioprotection

Ca2+

ATP

EnergysupplyMitochondria
Fig. 7. Schematic representation of the hypothesis tested and results obtained. Shown is a model of regulation of [Ca2+]i homeostasis and ATP production by SA4503 through σ1Rstimulation, as suggested by ﬁndings presented here. σ1R stimulation with SA4503 promotes mitochondrial Ca2+ inﬂux and mitochondrial Ca2+-dependent ATP production. Increasing ATP production ameliorates sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) activity, preventing SR Ca2+ depletion and increasing the cardiomyocyte energy supply.
σ1R-induced regulation of [Ca2+]i homeostasis and ATP production is crucial for the cardioprotective activity of SA4503 against heart failure. VDAC: voltage-dependent anion
channel.

in cultured cardiomyocytes, an effect closely associated with mitochondrial elongation (Fig. 3A). Importantly, we recently reported that σ1R
over-expression in neuro2A cells promotes mitochondrial elongation
and IP3R-dependent ATP production [29]. Taken together, SA4503 treatments likely promote mitochondrial elongation and stabilization of the
IP3R in SR membranes of cardiomyocytes, enhancing Ca 2+ mobilization
into mitochondria.
We also speculated that σ1R stimulation at the mitochondrial
ER-membrane enhances Ca2+ transport from the ER into mitochondria,
thereby activating the tricarboxylic acid (TCA) cycle. Two hertz electricalstimulation of adult rat cardiomyocytes enhances mitochondrial Ca2+ inﬂux, increasing mitochondrial ATP content [30]. Denton and McCormack
also showed that activities of glycerophosphate dehydrogenase, pyruvate
dehydrogenase, isocitrate dehydrogenase, and oxoglutarate dehydrogenase, which are mitochondrial and mitochondrial matrix dehydrogenase
enzymes functioning in the TCA cycle, are regulated by mitochondrial
Ca2+ concentration [31–34]. We ﬁrst conﬁrmed that SA4503 treatment
rescues PE-induced Ca2+ mobilization into mitochondria (Fig. 2A). Notably, in control cells PE elicited biphasic mitochondrial Ca2+ mobilization,
likely due to IP3R-mediated transient and store- or voltage-operated persistent Ca2+ mobilization in cardiomyocytes. Interestingly, SA4503 had aprominent effect on IP3R-mediated transient Ca2+ mobilization without
altering persistent Ca2+ mobilization, suggesting that SA4503 rescues
IP3R-mediated mitochondrial Ca2+ mobilization. More importantly,
SA4503 slightly but signiﬁcantly enhanced cytosolic Ca2+ mobilization

by PE. Thus, the σ1R is critical to regulate IP3R-mediated mitochondrial
Ca2+ mobilization. Furthermore, mitochondrial elongation observed following SA4503 treatment also likely plays a crucial role in IP3R-mediatedmitochondrial Ca2+ mobilization.
Chronic Ang II exposure caused Ca2+ overload in the cytosol [35]. In
particular, ryanodine receptors (RyRs) are major Ca2+-releasing channels in cardiac myocytes. We examined the role of RyRs on mitchodrial
Ca2+ uptake. Treatment of cultured cardiomyocytes with ryanodine,
which opens the RyR channel, induced Ca2+ release into the cytosol(Supplemental Fig. 1A), but failed to increase mitochondrial Ca 2+ inﬂux(Supplemental Fig. 1B). These data suggested that the RyR does not directly regulate mitochondrial Ca2+ inﬂux.
Recent studies reveal that speciﬁc proteins and chaperones function
as ER-mitochondria linkage proteins. For example, mitofusin-2 not only
connects mitochondria but also bridges the ER and mitochondria andfunctions in IP3-induced mitochondrial Ca2+ uptake [23]. Moreover,
Szabadkai et al. report that glucose-regulated protein (GRP75), a molecular chaperone, links the ER and mitochondria by connecting the IP3R
with voltage-dependent anion channels (VDACs) [24]. Mitofusin-2 islocalized in both ER and mitochondrial membranes, whereas GRP75
and mitofusin-1 are predominantly localized to the mitochindrial membrane. Here, we found, for the ﬁrst time, that σ1R downregulation is associated with reduction in levels of the junctional proteins mitofusin-2
and GRP 75. Notably, levels of mitofusin-1 and VDAC were unchanged
after Ang II-induced mitochondrial fussion, suggesting that Ang II

Fig. 6. Effects of SA4503 and NE-100 on σ1R and IP3R2 expression and ATP content in TAC mice. A western blot analysis of σ1R, IP3R2 and β-tubulin (as a loading control) expression
in the LV of sham and TAC-mice with or without drug treatment. Immunoblotting with anti-β-tubulin antibody indicates equal protein loading in each lane. B, densitometric quantiﬁcation of σ1R immunoreactive bands. C, densitometric quantiﬁcation of IP3R2 immunoreactive bands. D, measurement of cellular ATP content in the LV of TAC mice with or without drug treatment. Each group consists of 4–5 mice. Data are expressed as percentages of values of sham-operated animals (mean ± S.E.M.) *, P b 0.05 and **, P b 0.01 versus thesham group; #, P b 0.05 and ##, Pb 0.01 versus the TAC/vehicle-treated group; †, P b 0.05 and ††, P b 0.01 versus the TAC plus SA4503 (1 mg/kg)-treated group.

H. Tagashira et al. / Biochimica et Biophysica Acta 1830 (2013) 3082–3094

treatment, at least for 72 h, has no apparent effect of mitophagy or mitochondrial breakdown. σ1R agonists likely rescue mitochondrial ﬁssion by stabilizing GRP75 and mitofusin-2, although further studies
are required to identify mechanisms underlying this effect.
Overall, chronic Ang II exposure causes mitochondrial dysfunction via
oxidative stress. Previous studies conducted in various cell lines
suggest that Ang II induces oxidative stress by stimulating NADPH
oxidase-derived superoxide generation, promoting mitochondrial dysfunction [36,37]. Another report suggests that calcineurin dephosphorylates dynamin-related protein (Drp) 1, a key regulator of mitochondrial
ﬁssion, promoting mitochondrial ﬁssion [38]. Further studies are required to identify mechanisms underlying mitochondrial ﬁssion andhow they are regulated by the σ1R.
Ito et al. [11] have reported that pressure overload and high-salt
diet-induced heart failure mice exhibit reduced σ1R levels in thebrain, with concomitant depression-like behavior. Chronic infusion in
those model mice with PRE084, a σ1R agonist, signiﬁcantly reduced
sympathetic nerve activation. These authors conclude that suppressionof sympathetic nerve activation by a σ1R agonist acting in the brain improves heart function in pressure overload mice. We observed that
xestospongin C treatment abolished increased ATP content mediatedby SA4503 and antagonized amelioration of cardiomyocyte hypertrophy by SA4503. This ﬁnding supports our hypothesis that SA4503 directly stimulates the σ1R in cardiomyocytes, rescuing IP3R-mediatedATP production and cardiac dysfunction. We propose that both indirect
and direct mechanisms through the σ1R underlie cardioprotection seen
in TAC mice.
5. Conclusions
For the ﬁrst time, we provide evidence that SA4503, a potent and selective σ1R agonist, ameliorates TAC-induced cardiac hypertrophy anddysfunction by activating the σ1R. Rescue of IP3R2-mediated mitochondrial Ca2+ transport and ATP production following SA4503 treatment
was critical to maintain the energy supply and [Ca 2+]i homeostasis required for proper cardiac contraction and relaxation (Fig. 7). We also report that signaling through the σ1R counteracts Ang II-induced cardiac
myocyte apoptosis by rescuing SR-mitochondria calcium mobilizationand mitochondrial ATP production. Indeed, the effect of SA4503 treatment has recently been tested in humans in a stroke and depression
clinical phase II trial (NCT00639249). Our observations suggest a new
therapeutic strategy to rescue the heart from hypertrophic dysfunction
via σ1R stimulation.
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbagen.2012.12.029.
Conﬂict of interest statement
The authors declare that there are no conﬂicts of interest.
Acknowledgments
This work was supported in part by grants-in-aid for Scientiﬁc Research from the Ministry of Education, Science, Sports and Culture ofJapan (22390109 to K.F.).
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