Journal of Pharmacological Sciences 131 (2016) 172e183

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Haloperidol aggravates transverse aortic constriction-induced heart
failure via mitochondrial dysfunction
Yasuharu Shinoda a, 1, Hideaki Tagashira a, 1, Md. Shenuarin Bhuiyan b,
Hideyuki Hasegawa c, d, Hiroshi Kanai c, d, Kohji Fukunaga a, *a

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-Aoba, Aoba-ku, Sendai, Japan
Department of Pathology and Translational Pathology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71103, USA
Department of Electrical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6 Aramaki-Aoba, Aoba-ku, Sendai, Japand
Department of Electrical Engineering, Graduate School of Engineering, Tohoku University, 6-6 Aramaki-Aoba, Aoba-ku, Sendai, Japanbca r t i c l e i n f o

a b s t r a c t

Article history:
Received 19 April 2016
Received in revised form
25 May 2016
Accepted 27 May 2016
Available online 4 June 2016

Haloperidol is an antipsychotic drug that inhibits the dopamine D2 receptor among others. Haloperidol
also binds the sigma-1 receptor (s1R) and inhibits it irreversibly. A serious outcome of haloperidol
treatment of schizophrenia patients is death due to sudden cardiac failure. Although the cause remains
unclear, we hypothesized that these effects were mediated by chronic haloperidol inhibition of cardiac
s1R. To test this, we treated neonatal rat cardiomyocytes with haloperidol, exposed them to angiotensin
II and assessed hypertrophy, s1R expression, mitochondrial Ca2þ transport and ATP levels. In this context,
haloperidol treatment altered mitochondrial Ca2þ transport resulting in decreased ATP content byinactivating cardiac s1R and/or reducing its expression. We also performed transverse aortic constriction(TAC) and then treated mice with haloperidol. After two weeks, haloperidol-treated mice showedenhanced heart failure marked by deteriorated cardiac function, reduced ATP production and increasingmortality relative to TAC only mice. ATP supplementation via sodium pyruvate rescued phenotypes seenin haloperidol-treated TAC mice. We conclude that s1R inactivation or downregulation in response tohaloperidol treatment impairs mitochondrial Ca2þ mobilization, depleting ATP depletion from cardiomyocytes. These ﬁndings suggest a novel approach to mitigate haloperidol-related adverse effects inschizophrenia patients by ATP supplementation.
© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese PharmacologicalSociety. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

Keywords:
Sigma-1 receptor (s1R)
Haloperidol
Angiotensin II (Ang II)
Myocardial hypertrophy
ATP

1. Introduction
Haloperidol was introduced as a therapeutic over 40 years ago
and remains widely used clinically to treat acute and chronic psychosis, schizophrenia, and delirium. Antipsychotic activity of
haloperidol is thought to be mediated, at least in part, by its
antagonism (Ki ¼ 2.8 nM) of the dopamine D2 receptor(8,30,52,56). In addition, haloperidol has comparable afﬁnity forthe sigma-1 receptor (s1R) and is known to act as a s1R inhibitor(Ki ¼ 3.0 nM) (8,32); however, the consequences of haloperidol* Corresponding author. 6-3 Aramaki-Aoba, Aoba, Sendai 980-8578, Japan.
Tel.: þ81 22 795 6837; fax: þ81 22 795 6835.
E-mail address: kfukunaga@m.tohoku.ac.jp (K. Fukunaga).
Peer review under responsibility of Japanese Pharmacological Society.
1
These authors contributed equally to this work.

binding to s1Rs are less well known. Studies show that haloperidol
administered at therapeutic doses (0.003 mg/kg), as is used inhumans, can inhibit s1 receptors in vivo (38) and that treatment
reportedly decreases the density of brain s1R in patients (38).
Moreover, several studies show that haloperidol is metabolized
in vivo to a reduced form with higher afﬁnity (in the nanomolar
range) for s1Rs (17,32,52); conversely, reduced haloperidol binds
dopamine receptors with 85-fold lower afﬁnity than haloperidol(Ki ¼ 239 nM) (8). Acting as an antagonist, the reduced form canirreversibly inactivate s1Rs in guinea pig brain homogenates and in
SH-SY5Y human neuroblastoma cells (10).
Although haloperidol is generally considered safe, a serious
adverse effect of its use in schizophrenia patients is an abnormal
heart rhythm called torsades de pointes (TdP) and QT prolongation(QTP), which can lead to sudden death from heart failure (14,24,42).
These outcomes have been reported following either oral (18,29) or

http://dx.doi.org/10.1016/j.jphs.2016.05.012
1347-8613/© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Y. Shinoda et al. / Journal of Pharmacological Sciences 131 (2016) 172e183

intravenous (26) normal therapeutic doses of haloperidol or as aresult of overdose (23). The Food and Drug Administration (FDA)
reported a total of 70 cases of intravenous haloperidol
administration-associated fatal events (like QTP and/or TdP) andhas warned patients about these potential outcomes (34). Molecular mechanisms underlying cardiotoxic effects remain unclear. We
proposed that adverse cardiac effects of haloperidol are mediatedby inhibition of cardiac s1Rs, which we conﬁrmed to be abundantly
expressed in mouse heart (7,16). We also reported that s1R activity
is potentially cardioprotective and have shown a direct relationshipbetween reduced s1R expression and impaired heart function in atransverse aortic constriction (TAC) mouse model of left ventricular
hypertrophy (47). Our work has conﬁrmed that s1R stimulation
with selective agonists like ﬂuvoxamine or SA4503 in TAC mice
improved cardiac function and reduced cardiac hypertrophy (50).
We also showed that in rat heart, targeting s1R with neurosteroid
dehydroepiandrosterone (DHEA) or pentazocine can promote cardioprotection in the context of pressure overload (PO)-induced
cardiac dysfunction (44,46).
s1R is localized in the mitochondria-associated endoplasmic
reticulum (ER) membrane (MAM) where it interacts with theinositol 1,4,5-trisphosphate receptor (IP3R) to regulate Ca2þtransport from the ER lumen into mitochondria (22). Cardiomyocytes express all the three types of IP3Rs (approximate
relative abundance: IP3R1, 25%; IP3R2, 48%; and IP3R3, 27%), andthe three subtypes differ in function, expression pattern andsubcellular localization in different cell types (1,53,55). We previously reported that perturbation of IP3R-mediated Ca2þ releaseplays an important role in development of cardiac hypertrophy,
and that s1R agonists modulate IP3R-mediated Ca2þ release in
cardiomyocytes (44,45). However, it is not known whether
haloperidol-induced inactivation and/or reduction in expressionof cardiac s1R would alter mitochondrial Ca2þ mobilization
through IP3R expressed in cardiomyocytes and thereby promote
adverse cardiac effects.
In this study, we asked whether haloperidol treatment induced
cardiotoxicity mediated by s1R inactivation. We discovered that
haloperidol-induced s1R inactivation exacerbates Ang II-induced
impairment of sarcoplasmic reticulum (SR)-mitochondrial Ca2þtransport, leading to decreases in mitochondrial ATP production,
which in turn aggravated cardiac contractile dysfunction in TAC
mice. Interestingly, co-administration of an ATP supplement suchas sodium pyruvate restored cardiac ATP levels and ameliorated
haloperidol-induced cardiac contractile dysfunction in TAC mice.
This study suggests that co-administration of an ATP supplementwith therapeutic doses of haloperidol could be beneﬁcial in preventing adverse effects of haloperidol in schizophrenic patients.

2. Materials and methods
2.1. Materials
Reagents and antibodies were obtained from the following
sources: anti-s1R antibody (Abcam, Cambridge, UK); antivoltage-dependent anion channel (VDAC) antibody (Cell
Signaling Technology, Beverly, MA); anti-p62 antibody (MBL,
Nagoya, Japan); anti-LC3 antibody (MBL); anti-cytochrome cantibody (BD Biosciences, San Diego, CA); anti-b-tubulin antibody (Sigma, St. Louis, MO). Haloperidol was purchased from
Dainippon Sumitomo Pharma Co. Ltd. (Osaka, Japan). Other reagents were of the highest quality available (Wako Pure Chemicals, Osaka, Japan). The s1R agonist SA4503 was synthesized in
the Laboratory of Medicinal Chemistry, Zhejiang University(Hangzhou, China).

173

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
35e40 g were obtained from Nippon SLC (Hamamatsu, Japan). Tenweek-old males were acclimated to the local environment for 1
week, which included housing in polypropylene cages at 23 ± 1  C
in a humidity-controlled environment maintained on a 12-h light/
dark schedule (lights on 8:00 AMe8:00 PM). Mice were provided
food and water ad libitum. Transverse aortic constriction (TAC) was
performed as described (44,50) 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). Adequate depth of anesthesia was conﬁrmed by a negative
toe-pinch reﬂex. If anesthesia was not sufﬁcient, then a top-up doseof 20% of the initial dose was given.

2.3. Experimental design
ICR mice were randomly separated into seven experimentalgroups: 1) Sham (n ¼ 5), 2) Sham plus sodium pyruvate (Pyr)treatment (1.0 mg/kg, p.o.) (n ¼ 4), 3) Sham plus haloperidol (Halo)treatment (0.1 mg/kg, p.o.) (n ¼ 6), 4) TAC plus vehicle treatment(n ¼ 9), 5) TAC plus sodium pyruvate treatment (n ¼ 5), 6) TAC plus
haloperidol treatment (n ¼ 10), and 7) TAC plus haloperidol plus
sodium pyruvate treatment (n ¼ 11). Vehicle, sodium pyruvate andhaloperidol were administered orally for 2 or 4 weeks (once daily)
using a metal gastric zonde for mice in a volume of 1 ml/100 g ofmouse body weight, starting from the onset of aortic banding.

2.4. Echocardiography and measurement of cardiac hypertrophy
Noninvasive echocardiographic measurements were performed
as described (47,50). Brieﬂy, noninvasive echocardiographic measurements were performed in mice anesthetized with a mixture of
ketamine (100 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.) using ultrasonic diagnostic equipment (SSD-6500; Hitachi-Aloka, Tokyo,
Japan) equipped with a 10-MHz linear array transducer (UST-5545;
Hitachi-Aloka). The heart was imaged in the two-dimensional
parasternal short-axis view, and an M-mode echocardiogram ofthe mid-ventricle was recorded at the level of the papillary muscles. Diastolic and systolic LV wall thickness, LV end-diastolic
diameter (LVEDD), LV end-systolic diameter (LVESD) and ejection
fraction (EF) were measured. All measurements were done fromleading edge to leading edge according to the American Society of
Echocardiography guidelines. The percentage of LV fraction shortening (FS) was calculated as ([LVEDD  LVESD]/LVEDD)  100.
Following sacriﬁce by cervical spine fracture dislocation, thethoracic cavity of mice was opened, and hearts were immediately
harvested and weighed.

2.5. Cultured cardiomyocytes
Neonatal rat ventricular cardiomyocytes (NRVMs) were isolated
from hearts of 1 to 3-day-old Wistar rat pups that had been sacriﬁced by decapitation, and cardiomyocytes were cultured as
described (47,50). In the experiments that followed, cells were
stimulated with Ang II (100 nM), haloperidol (10 nM) or SA4503(1 mM) for 1 or 48 h.

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Y. Shinoda et al. / Journal of Pharmacological Sciences 131 (2016) 172e183

2.6. Morphological analysis, immunocytochemistry of cultured
cardiomyocytes and histological analysis of cardiac tissues

forward, 50 - TGTGTCCGTCGTGGATCTGA -30 ; and reverse Gapdh, 50 CACCACCTTCTTGATGTCATCATAC -30 .

Morphological analysis was performed as described (44). The
surface area of control cells was deﬁned as 100% and thencompared with that of treated cells. For mitochondrial staining,cells were stained for 20 min with 0.02 mM Mito Tracker Red
CMXRos (Molecular Probes, Eugene, OR) before ﬁxation (12). After
permeabilization with 0.1% Triton X-100 in PBS, ﬁxed cells wereincubated with 1% bovine serum albumin (BSA) (Sigma) in PBS for
30 min. For immunocytochemistry, cells were incubated 24 h at
4  C with anti-LC3 antibody (1:500), anti-p62 antibody (1:500) or
anti-cytochrome c antibody (1:500) in PBS containing 1% BSA. Afterwashing, cells were incubated 24 h with Alexa 488-conjugatedanti-rabbit IgG and Alexa 488-conjugated phalloidin and/or Alexa
594-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA) in PBS
containing 1% BSA. For nuclear staining, sections were incubatedwith 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI)(Sigma; 1 mg/mL PBS). Immunoﬂuorescent images were analyzed
using a confocal laser scanning microscope (LSM700; Zeiss,
Thornwood, NY). Quantitative analyses of cell size, ﬂuorescenceintensity of Mito Tracker and LC3 granules were undertaken using
ImageJ software. Speciﬁcally, after image capture, lines surrounding
each cell were drawn and signal intensity inside those lines wasdetermined using ImageJ software.
Histological analysis was performed as described (44). Brieﬂy,
ﬁxed and dehydrated murine heart was embedded in O.C.T. compound (Sakura Finetek, Torrance, CA) and sliced. For Masson's trichrome staining, 20-mm slice tissues were stained using a Trichrome
Stain (Masson) Kit (Sigma) according to manufacturer's protocol.

2.9. Measurement of intracellular Ca2þ levels
NRVMs were cultured on 0.01% poly-L-lysine (Sigma)-coatedglass-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 Ca2þ-sensitive dye Fura-2 acetoxymethyl
ester (2.5 mM; Sigma) for 30 min before measuring Ca2þ levels in achamber 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 Ca2þ ﬂuorescence levels reached a steady state, 10 mM
phenylephrine (PE) or 10 mM ATP was applied for 10 s through asmall perfusion pipe. The amplitude of the PE- or ATP-induced Ca2þtransient was used as an index of IP3R-mediated Ca2þ release.
Changes in PE- or ATP-induced Ca2þ release from the SR were
determined using the ratio of ﬂuorescence emission at 530 nm in
response to excitation at 340 nm and to that at 380 nm.
2.10. Measurement of mitochondrial Ca2þ levels

Dissected LV tissue samples were rapidly frozen in liquid nitrogen and stored at 80  C before analysis (47). In the case of
cardiomyocytes, cells were washed with PBS at 4  C and storedat 80  C until immunoblotting was performed as described(46,50). For assays, each frozen sample was homogenized as
described (44,49). ATP measurement was performed using an ATP
assay kit (Toyo Ink, Tokyo, Japan), according to the manufacturer's
protocol (50). Brieﬂy, frozen samples were homogenized in homogenate buffer (0.25M sucrose, 10 mM HEPES-NaOH: pH 7.4), andlysates were cleared by centrifugation at 1000 g for 10 min at 4  C.
The supernatant was collected, and supernatant proteins weresolubilized in extraction buffer. After 30 min, luciferin buffer wasadded to each sample and oxyluciferin was detected using aluminometer (Gene Light 55, Microtec, Funabashi, Japan).

NRVMs were cultured on 0.01% poly-L-lysine (Sigma)-coatedglass-bottom dishes and maintained in growth medium. Transfections were performed with ratiometric-pericam targeted to themitochondrial matrix (ratiometric-pericam-mt/pcDNA3), which
was a kind gift of Dr. Atsushi Miyawaki of the RIKEN Brain Science
Institute (Wako, Japan) (35,40,50). Brieﬂy, 1 mg/mL ratiometricpericam-mt/pcDNA3 in 1 mL was added to 199 mL opti-MEM (Invitrogen), and 1 ml Lipofectamine 2000 (Invitrogen) was added to 9 mL
opti-MEM. Both solutions were incubated separately at room temperature for 5 min and then mixed and incubated at room temperature for 15e20 min. Cells were incubated with 800 mL of optiMEM to which 200 mL 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 ml 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 h, cells were perfused with normal Tyrode solution at 37  C.
When Ca2þ ﬂuorescence levels reached a steady state, 10 mM PE or
10 mM ATP was applied for 10 s through a small perfusion pipe. Dualexcitation imaging with ratiometric-pericam-mt required two ﬁlters(EX:482/35, DM:506, EM:536/40 and EX:414/46, DM:510, EM:527/
20). Changes in PE- or ATP-induced Ca2þ transport from the SR tomitochondria were determined using the Metaﬂuor Imaging system(Molecular Devices, Sunnyvale, CA).

2.8. Real-time PCR

2.11. TUNEL staining

Total RNA from LV tissues was isolated using TRI Reagent(Sigma). Brieﬂy, LV tissues were homogenized in TRI Reagent andpassed through 23-gauge needle ten times on ice. Chloroform wasadded to homogenates, and the mixture was kept on ice for 15 min
and then centrifuged at 15,000 rpm for 5 min at 4  C. The top layerof supernatant was collected and combined with one volume ofisopropanol and then centrifuged at 15,000 rpm for 20 min at 4  C.
Resulting RNA pellets were washed with 75% ethanol and suspended in sterile water containing 0.1% diethyl pyrocarbonate(Sigma) to block RNA degradation. cDNAs were synthesized byreverse transcription as described (40). Real-time PCR was performed with iQ CYBR Green Supermix (Bio-Rad Laboratories, Redmond, WA) following the manufacturer's protocol. The primers
were: mouse forward Anp, 50 - GTCCAACACAGATCTGATGG -30 ;reverse Anp, 50 - GATTTGGCTGTTATCTTCGG -30 ; mouse Gapdh

Double-staining for TUNEL to detect apoptotic cell nuclei andpropidium iodide to detect myocardial cell nuclei was described
previously (40). TUNEL staining was performed using an in situ
apoptosis detection kit (Takara Bio Inc., Shiga, Japan), according to
the manufacturer's protocol. Cardiomyocytes from at least four
randomly selected slides per block were evaluated immunohistochemically to determine the number and percentage of cells
exhibiting staining indicative of apoptosis. The index of apoptosis(number of apoptotic myocytes/the total number of myocytes
counted  100%) was determined.

2.7. Western blot analysis and measurement of ATP content

2.12. Statistical analysis
Values are represented as means ± standard error of the mean
(S.E.M.). Comparison between two experimental groups was made

Y. Shinoda et al. / Journal of Pharmacological Sciences 131 (2016) 172e183

using the unpaired Student's t test to evaluate effects of SA4503 on
cardiomyocyte size. In other experiments, differences were evaluated using analysis of variance (ANOVA) followed by multiple
comparisons using Dunnett's test, except for NRVM morphologicalmeasurement. Results related to NRVM area were evaluated by
Scheffe's test to test the effect of SA4503. Survival probability wascalculated by KaplaneMeier analysis, and statistical signiﬁcancewas analyzed by Log-rank test. P < 0.05 was considered statisticallysigniﬁcant. F and P values are reported in corresponding ﬁgurelegends.
3. Results
3.1. Haloperidol treatment of cultured cardiomyocytes enhances
Ang II-induced hypertrophy and mitochondrial dysfunction
We previously reported that s1R is abundantly expressed inrodent cardiomyocytes and showed that s1R activation with a variety of agonists was cardioprotective in vitro and in vivo

175

(2e4,44e47,50). Thus, we reasoned that haloperidol, acting as anirreversible s1R antagonist, could aggravate Ang II-induced hypertrophy via s1R inactivation (10) and/or by reducing s1R
expression in cardiomyocytes (38). To assess this possibility, we
ﬁrst observed the size of NRVMs exposed to Ang II (100 nM) for 48 hwith or without haloperidol treatment. Treatment with Ang II only
signiﬁcantly increased cell size relative to that of untreated controls(P < 0.05 vs. control) (Fig. 1A and B). Haloperidol treatment (10 nM)
signiﬁcantly enhanced Ang II-induced cell enlargement (P < 0.01 vs.
control; P < 0.05 vs. Ang II) (Fig. 1A and B). Haloperidol treatment
alone also induced cardiomyocyte hypertrophy in mock-treatedcontrols (P < 0.05 vs. control) (Fig. 1A and B). Interestingly, haloperidol treatment of Ang II-exposed cells for 48 h signiﬁcantly
reduced expression of s1R protein (P < 0.05 vs. control) (Fig. 1D),
but treatment with either haloperidol or Ang II did not (Fig. 1D). To
conﬁrm that s1R activity was relevant to these effects, weemployed the selective s1R agonist (SA4503) (33). SA4503 coadministration (1 mM) with haloperidol rescued haloperidolinduced cardiomyocyte hypertrophy (P < 0.01 vs. haloperidol

Fig. 1. Effects of haloperidol treatment on Ang II-induced hypertrophy and mitochondrial structure in cultured cardiomyocytes. A: Cells were stained with Alexa Fluor 488phalloidin (green), Mito Tracker Red CMXRos (red) and DAPI (blue) and assessed by ﬂuorescence microscopy. B: Cell size is expressed as a percentage of surface area relative tountreated control cells. Fifty cells were counted in each experiment. F(7, 408) ¼ 18.680, P < 0.01. C: Mito Tracker Red CMXRos intensity is expressed as a percentage of surface area
relative to untreated control cells. Six to nine randomly selected ﬁelds were counted in each experiment. F(3, 26) ¼ 10.430, P < 0.01. D: Upper, Immunoblot of s1R protein in NRVMs
treated 48 h with or without Ang II or haloperidol (Halo). b-tubulin staining serves as a loading control. Lower, Densitometry analysis of s1R immunoreactive bands. Groups consistof 4 samples. E: TUNEL-positive cells were counted in cultures treated 48 h with or without Ang II or Halo. Eighty cells from 10 randomly selected ﬁelds were counted in eachexperiment. F(3, 19) ¼ 3.086, P < 0.05. Data are expressed as percentages of control values (mean ± S.E.M.).*, P < 0.05 and **, P < 0.01 vs. control cells; #, P < 0.05 and ##, P < 0.01 vs.
Halo-treated cells; y, P < 0.05 and yy, P < 0.01 vs. Ang II-treated cells; ‡, P < 0.05 and ‡‡, P < 0.01 vs. SA4503 () control.

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Y. Shinoda et al. / Journal of Pharmacological Sciences 131 (2016) 172e183

treatment) (Fig. 1B). Both SA4503 and haloperidol block [3H](þ)pentazocine binding to s1R (32,33), suggesting they occupy thesame binding site. Therefore, SA4503 treatment at a relatively highconcentration (1 mM) was sufﬁcient to inhibit haloperidol (10 nM)
binding in s1R-induced function. These ﬁndings suggest that
haloperidol directly acts on the cardiac s1R to enhance Ang IIinduced cardiomyocyte enlargement.
We have also reported that treatment of cardiomyocytes with
s1R agonists has antihypertrophic effects in part by preventing
mitochondrial dysfunction (45,50). We extended that analysis todeﬁne effect of s1R signaling on mitochondrial membrane potential
under stress conditions, as assessed by Mito Tracker Red CMXRosstaining. Staining intensity of cells treated with Ang II or haloperidol signiﬁcantly decreased compared to that of untreated control
cells (44.8% and 51.4% of control, respectively) (P < 0.05 and 0.01 forhaloperidol and Ang II, respectively) (Fig. 1A and C). Treatment withboth haloperidol and Ang II signiﬁcantly reduced mitochondrial
membrane potential (P < 0.01 vs. control) (Fig. 1A and C). We also
investigated a potential effect of haloperidol on Ang II-induced
cardiomyocyte apoptosis in cultured cardiomyocytes. Ang II or

Fig. 2. Effects of haloperidol treatment on phenylephrine (PE) and ATP-induced Ca2þ inﬂux into mitochondria and the cytosol and on ATP content. A: Time course of PEinduced Ca2þ inﬂux into mitochondria treated 48 h with or without Ang II or Halo. B: Peak increases in [Ca2þ]mito induced by 10 mM PE. F(4, 96) ¼ 14.392, P < 0.01. C: Time courses of
PE-induced Ca2þ release into the cytosol in cells treated 48 h with or without Ang II or Halo. D: Peak increases in [Ca2þ]cyto induced by 10 mM PE. F(4, 89) ¼ 16.579, P < 0.01. E: Timecourse of ATP-induced Ca2þ inﬂux into mitochondria in cells treated 48 h with or without Ang II. F: Peak increases in [Ca2þ]mito induced by 10 mM ATP. Each group consists of>10 cells. F(4, 101) ¼ 20.761, P < 0.01. G: Measurement of cellular ATP content with or without Ang II or Halo treatment. Groups consist of 3e4 samples. F(3, 22) ¼ 9.870, P < 0.01.
Data are expressed as percentages of control cell value (mean ± S.E.M.). *, P < 0.05 and **, P < 0.01 vs. control cells; #, P < 0.05 and ##, P < 0.01 vs. Halo-treated cells; yy, P < 0.01 vs.
Ang II-treated cells.

Y. Shinoda et al. / Journal of Pharmacological Sciences 131 (2016) 172e183

haloperidol treatment for 48 h signiﬁcantly increased cardiomyocyte apoptosis, as indicated by a relative increase in thenumber of TUNEL-positive cells (P < 0.05 vs. control for both
treatments), while treatment with both signiﬁcantly enhanced
cardiomyocyte apoptosis (P < 0.01 vs. Ang II) (Fig. 1E).
3.2. Effect of haloperidol treatment on phenylephrine (PE)-induced
Ca2þ mobilization and ATP content in NRVMs
We previously reported that s1R activation by agonists regulates
IP3R-mediated Ca2þ release in cardiomyocytes (45,50). Therefore,
we examined the effects of haloperidol treatment on PE-induced
IP3R-mediated Ca2þ mobilization into mitochondria and cytosolin Ang II-treated cells. Treatment with haloperidol alone for 1 hslightly suppressed PE-induced mitochondrial Ca2þ transport
relative to controls, an effect that was signiﬁcant after 48 h oftreatment (P < 0.01 for Halo) (Fig. 2A and B). Ang II treatment for
48 h severely impaired PE-induced mitochondrial Ca2þ transportcompared to untreated control cells (P < 0.01 vs. control) (Fig. 2A
and B). Treatment with haloperidol plus Ang II for 48 h signiﬁcantly
enhanced the Ang II response (P < 0.01 vs. Ang II) (Fig. 2A and B).
We also examined the effects of haloperidol on PE-induced IP3Rmediated Ca2þ release to the cytosol. Neither haloperidol nor Ang II
treatment alone for 1 h or 48 h had a signiﬁcant effect (Fig. 2C andD); however, combined administration of haloperidol plus Ang II
for 48 h signiﬁcantly increased PE-induced Ca2þ release to thecytosol (P < 0.01 vs. control and vs. Ang II) (Fig. 2C and D). We also
examined the effects of haloperidol on IP3R-mediated mitochondrial Ca2þ mobilization by supplementing Ang II-treated cells with
ATP. Treatment with either haloperidol for 1 or 48 h or Ang II alone
for 48 h had little effect on ATP-induced Ca2þ release to mitochondria (Fig. 2E and F). However, combining haloperidol with Ang

177

II treatment for 48 h signiﬁcantly impaired ATP-induced mitochondrial Ca2þ transport (P < 0.01 vs. Ang II) (Fig. 2E and F). These
results suggest overall that haloperidol enhances Ang II-induced
impairment of Ca2þ mobilization in both mitochondria and cytosol.
We next asked whether s1R inactivation by haloperidol
impaired mitochondrial ATP production in NRVMs. Treatment with
haloperidol alone slightly reduced ATP content relative to untreated control cells (Fig. 2G). Consistent with our previous study(45), Ang II exposure for 48 h increased ATP content (P < 0.01 vs.
control) (Fig. 2G). Interestingly, treatment with haloperidol and
Ang II treatment signiﬁcantly decreased ATP content relative to Ang
II treatment alone (P < 0.01 vs. Ang II) (Fig. 2G). In naive NRVMs,
ATP-induced mitochondrial Ca2þ transport was signiﬁcantly suppressed following acute haloperidol treatment for 1 h (P < 0.05 vs.
control), while co-administration of haloperidol with the s1R
agonist SA4503 eliminated this effect (Fig. 3A and B). By contrast,
ATP-induced Ca2þ release to the cytosol was signiﬁcantly potentiated by haloperidol treatment (P < 0.01 vs. control), which antagonized by SA4503 co-administration (P < 0.01 vs. Halo) (Fig. 3C and
D). These observations suggest that s1R inactivation deregulates
mitochondrial Ca2þ mobilization by decreasing ATP content, an
outcome that may underlie haloperidol-induced cardiotoxicity.
3.3. Effects of haloperidol treatment on autophagy andmitochondrial integrity
Recent reports demonstrate that in cardiomyocytes, multiple
forms of stress, including pressure overload, chronic ischemia, andischemia-reperfusion, stimulate increased autophagy, a maladaptive response that contributes to heart failure (31,36,57). Given that
Ang II and haloperidol co-administration alters mitochondrial Ca2þtransport and ATP production, we hypothesized that these effects

Fig. 3. Effects of acute haloperidol or SA4503 treatment on ATP-induced Ca2þ mobilization into mitochondria and the cytosol. A: Time course of ATP-induced Ca2þ inﬂux into
mitochondria in cardiomyocytes treated with or without Halo or SA4503 (SA) for 1 h. B: Peak increases in [Ca2þ]mito induced by 10 mM ATP. F(3, 71) ¼ 4.281, P < 0.01. C: Time coursesof ATP-induced Ca2þ release to the cytosol. D: Peak increases in [Ca2þ]cyto induced by 10 mM ATP. Groups consist of >10 cells. F(3, 55) ¼ 7.789, P < 0.01. Data are expressed aspercentages of control value (mean ± S.E.M.). *, P < 0.05 and **, P < 0.01 vs. control cells; #, P < 0.05 and ##, P < 0.01 vs. Halo-treated cells.

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may be due to increased autophagic activity. We assessed thispossibility by monitoring the autophagic marker LC3 in cardiomyocytes treated with or without Ang II and/or haloperidol. Ang
II treatment alone for 48 h induced a signiﬁcant increase in theintensity of punctate LC3-positive dots (P < 0.01 vs. control) (Fig. 4A
and B). Moreover, treatment with both haloperidol and Ang II
signiﬁcantly enhanced induction of LC3-positive dots (P < 0.05 vs.
Ang II) (Fig 4A and B), a ﬁnding consistent with earlier studies (57).
Accordingly, we observed induction of expression of p62, adifferent marker of autophagy, in cardiomyocytes treated with Ang
II, haloperidol or both (Fig. 4C). Treatment of cardiomyocytes with a
combination of haloperidol and Ang II also elicited a fragmented
staining pattern of the mitochondrial marker, cytochrome c, which
preferentially distributes in tubular and branching pattern in untreated control cells (Fig. 4A). Fragmented cytochrome c staining
partially co-localized with LC3 granules, suggesting that mitochondria are undergoing mitophagy. We next asked whether
apparent mitochondrial degradation occurred in cardiomyocytes
treated with or without Ang II and/or haloperidol. Combined
treatment with both for 48 h signiﬁcantly decreased expression ofvoltage-dependent anion channel (VDAC), a mitochondrial protein
critical for Ca2þ transport with IP3R (P < 0.05 vs. control andP < 0.01 vs. Ang II) (Fig. 4D and E). Taken together, our ﬁndings
suggest that chronic haloperidol treatment has deleterious effects
on mitochondrial integrity via induction of autophagy in
cardiomyocytes.
3.4. Effects of haloperidol and sodium pyruvate administration on
cardiac hypertrophy and dysfunction in TAC mice
To assess the effect of haloperidol in vivo, we treated both shamoperated and TAC mice with daily oral administration of haloperidol (0.1 mg/kg) beginning two days after surgery. Two weeks later,
treatment groups were analyzed for myocardial hypertrophy basedon assessment of heart weight-to-body weight (HW/BW) ratio,lung weight-to-body weight (LW/BW) and mRNA levels of atrial
natriuretic peptide (ANP), a hypertrophy marker (Fig. 5AeC). TAC
mice showed signiﬁcantly increased HW/BW and ANP mRNA levels
and moderately increased LW/BW compared to sham mice
(P < 0.01 vs. sham-vehicle in HW/BW and ANP mRNA) (Fig. 5AeC).
Furthermore, haloperidol treatment (0.1 mg/kg) of TAC micesigniﬁcantly increased the LW/BW ratio (P < 0.01 vs. sham-vehicle
for TAC-haloperidol), an effect not seen in vehicle-treated TAC
controls (Fig. 5B). Given that haloperidol treatment perturbs
mitochondrial ATP production, we supplemented TAC animals withsodium pyruvate (1.0 mg/kg) to determine whether the haloperidol
effect was due to decreased mitochondrial function. Interestingly,
treatment with both sodium pyruvate (1.0 mg/kg) and haloperidol
prevented TAC and/or haloperidol-induced cardiac hypertrophy(P < 0.01 vs. TAC-haloperidol) (Fig. 5AeC).
To assess a potential effect of haloperidol treatment on cardiac
remodeling, we measured cardiac ﬁbrosis using Masson's trichrome staining. Interestingly, we observed adverse cardiac
remodeling, as indicated by induction of interstitial ﬁbrosis,
following chronic oral administration of haloperidol to TAC mice, anaffect blocked by co-administration of sodium pyruvate (Fig. 5D).
We also assessed cardiac function by echocardiography after
similar treatments (Fig. 5E). Control vehicle-treated TAC mice
showed slightly decreased LC fractional shortening (FS) and ejection fraction (EF) at 2 weeks after surgery, consistent with ourprevious report (47). By contrast, TAC mice treated with haloperidol
showed signiﬁcant cardiac dysfunction, as indicated by decreased
FS and EF, compared to vehicle-treated sham mice (P < 0.01 vs.
sham-vehicle) (Fig. 5F and G). All groups showed comparable
LVESD values [F(6, 44) ¼ 1.961, P ¼ 0.0920] (Fig. 5H), haloperidol

treatment on TAC mice elicited only a slight increase in LVESD.
These results suggest that haloperidol induces some indications ofcardiac dysfunction in TAC mice, such as increased LW/BW ratio(Fig. 5B). Interestingly, co-administration of sodium pyruvate with
haloperidol prevented haloperidol-induced deterioration of cardiac
function and progression of heart failure (P < 0.01 vs. TAChaloperidol in FS and EF; P < 0.05 vs. TAC-haloperidol in LVESD)(Fig. 5EeH). Taken together, we conclude that haloperidol-induced
progression of heart failure is mediated by reduced ATP production
in vivo.
3.5. Effects of haloperidol plus sodium pyruvate treatment on
cardiac s1R expression, ATP content and TAC mouse survival
Earlier studies had reported that haloperidol treatment of human subjects was accompanied by a decrease in the density of s1R
expression in brain (38). Therefore, we asked whether micechronically treated with haloperidol would also exhibit similarchanges in expression pattern in cardiac tissue. Interestingly, after 2
weeks of haloperidol administration, TAC mice showed signiﬁcantly decreased cardiac s1R expression (P < 0.01 vs. sham-vehicle)(Fig. 6A and B), although untreated and haloperidol-treated shammice showed comparable receptor expression (Fig. 6A and B). These
data suggest that cardiac defects seen in TAC mice treated with
haloperidol may be due to decreased s1R expression.
When we examined effects on ATP production we found that
haloperidol treatment of TAC mice signiﬁcantly decreased cardiac
ATP levels relative to sham or TAC mice (P < 0.01 vs. TAC-vehicle)(Fig. 6C). Interestingly, treatment with sodium pyruvate plus
haloperidol signiﬁcantly and restored ATP content in heart (P < 0.05
vs. TAC-haloperidol) but did not rescue decreased s1R expression(P < 0.05 vs. sham-vehicle) (Fig. 6AeC). Furthermore,
KaplaneMeier survival analysis showed that chronic haloperidol
treatment increased mortality of TAC mice, with a survival rate of
50% at 14 days and 33% at 28 days (P < 0.05 vs. TAC-vehicle)
compared to 86% survival at 28 days seen in TAC only mice
(Fig. 6D). Sham mice treated with haloperidol showed 100% survival. Co-administration of sodium pyruvate with haloperidol to
TAC mice prevented haloperidol-induced progression of heart
failure and signiﬁcantly improved mortality (P < 0.05 vs. TAChaloperidol). These correlative observations strongly suggest that
deleterious cardiac effects seen following chronic haloperidol
treatment of TAC mice are mediated by decreased s1R expression
and/or inactivation, which in turn promote decreased mitochondrial ATP production.
4. Discussion
Recently, we have been exploring the potential beneﬁcial effectof s1R agonists on the heart, effects which were heretofore underappreciated. Despite the numerous studies analyzing the effects
of s1R ligands in heart tissues, we still do not fully understand how
s1R function is perturbed in pathological states and whether s1R
exists as active state or resting state without stimulation of s1R
ligands. Here, to understand s1R function in heart, we investigatedtemporal changes in its expression levels in murine models of
cardiac hypertrophy and heart failure. We have reported that alongwith progression of LV hypertrophy, s1R expression was signiﬁcantly reduced in the heart and showed a signiﬁcant positive linear
correlation with heart function (6,47). Our previous studies clearly
demonstrated that s1R activation by selective agonists is cardioprotective in rodent models of cardiac hypertrophy and heart
failure (2e7,44e50) Here, we addressed the deleterious effects ofchronic s1R inactivation and decreased expression in heart broughton by treatment with haloperidol in the progression of heart failure

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179

Fig. 4. Effects of haloperidol treatment on autophagy and mitochondrial structure. A: Cultured cardiomyocytes were assessed using antibodies to LC3 (a marker of autophagy;
green) and Cytochrome c (a mitochondrial marker; Red) and to DAPI (blue) as a nuclear stain. B: Intensity of LC3 staining in granules is expressed as a percentage relative to control
cells. Six to nine randomly selected ﬁelds were counted in each experiment. F(3, 23) ¼ 13.575, P < 0.01. C: Cultured cardiomyocytes were assessed using antibodies to p62 (a marker
of autophagy; green). D: Immunoblot of the mitochondrial protein VDAC in cultured cardiomyocytes treated with or without Ang II or Halo for 48 h. E: Quantiﬁcation bydensitometry of VDAC protein. F(3, 20) ¼ 6.536, P < 0.01. Data are expressed as percentages of control values (mean ± S.E.M.). *, P < 0.05 and **, P < 0.01 vs. control cells; ##, P < 0.01
vs. Halo-treated cells; y, P < 0.05 and yy, P < 0.01 vs. Ang II-treated cells.

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Y. Shinoda et al. / Journal of Pharmacological Sciences 131 (2016) 172e183

Fig. 5. Effects of haloperidol and sodium pyruvate administration on cardiac hypertrophy and function in TAC mice. A: TAC-induced cardiac hypertrophy, as indicated by theheart weight/body weight (HW/BW) ratio. F(6, 17) ¼ 14.409, P < 0.01. B: TAC-induced cardiac hypertrophy, as indicated by the lung weight/body weight (LW/BW) ratio. Halo (0.1 mg/kg) and/or sodium pyruvate (1 mg/kg; Pyr) were administered to sham and TAC mice for 2 weeks after surgery. F(6, 18) ¼ 7.425, P < 0.01. C: Effect of Halo and Pyr treatment on ANPmRNA levels in TAC and control mice. F(6, 55) ¼ 24.810, P < 0.01. D: Effect of Halo and Pyr on scar formation induced by TAC (Masson's trichrome staining). E: Representative Mmode echocardiograms of TAC mice treated with and without Halo and Pyr. F: Changes in percentage of LV fractional shortening (FS). F(6, 43) ¼ 3.804, P < 0.01. G: Changes inpercentage of ejection fraction (EF). F(6, 44) ¼ 3.934, P < 0.01. H: Changes in left ventricular end-systolic diameter (LVESD). F(6, 44) ¼ 1.961, P ¼ 0.0920. Groups consist of 5e9 mice.
Each column represents the mean ± S.E.M. **, P < 0.01 vs. sham/vehicle-treated group; #, P < 0.05 and ##, P < 0.01 vs. TAC/vehicle-treated group; yy, P < 0.01 vs. TAC/Halo-treatedgroup.

and adverse myocardial remodeling. Several lines of evidence
suggest that haloperidol and its more active metabolite irreversibly
inhibit s1R activity and reduce s1R expression levels in vivo
(17,32,52). Although haloperidol treatment was previously reported to have deleterious effects in heart (34), the molecularmechanisms underlying these effects remained obscure. Here we
report that i) chronic haloperidol treatment decreases s1R
expression, ii) decreases myocardial ATP content, and iii) promotes
adverse myocardial remodeling and deterioration of cardiac function, which ultimately led to increased mortality of TAC mice.
Combining s1R agonists with ATP supplementation, we conﬁrmedthat the deleterious effects of chronic haloperidol treatment are
mediated by both s1R inactivation and impaired mitochondrial ATP
production.
Here we address mechanisms underlying deleterious effects ofhaloperidol on cardiac hypertrophy in vitro and in vivo. We reportthat haloperidol treatment impairs ATP production in part byderegulating IP3R-mediated Ca2þ mobilization into mitochondria.
Earlier studies in CHO cells demonstrated that s1R interacts with
IP3R to form a functional complex to regulate Ca2þ entry into
mitochondria, enabling cell survival under stress conditions (22). In
fact, we previously reported that s1R interacts with IP3R in cardiomyocytes and that s1R activation promotes ATP synthesis byenhancing Ca2þ entry into mitochondria (44,50). Here, we conﬁrm
that s1R function is cardioprotective in studies showing that s1R
inhibition by haloperidol decreases mitochondrial ATP content:
haloperidol treatment signiﬁcantly suppressed PE- and ATPinduced mitochondrial Ca2þ transport and potentiated PE- andATP-induced Ca2þ release to the cytosol. Treatment with the selective s1R agonist SA4503 completely reversed haloperidolinduced impairment in Ca2þ mobilization.
We also show that treatment of cardiomyocytes with both
haloperidol and Ang II induces apoptosis, as indicated by thepresence of TUNEL-positive cells, and deregulates autophagy, as

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181

Fig. 6. Effects of combining haloperidol with sodium pyruvate treatment on cardiac s1R expression, ATP content and survival in TAC mice. A: Western blot analysis of s1R
protein in the LV of sham and TAC mice, with or without drug treatment. Anti-b-tubulin staining serves as a loading control. B: Quantiﬁcation by densitometry of s1R immunoreactive bands. F(6, 19) ¼ 6.005, P < 0.01. C: ATP content in left ventricles with or without drug treatment. F(6, 34) ¼ 3.436, P < 0.01. *, P < 0.05 and **, P < 0.01 vs. sham/vehicletreated group; ##, P < 0.01 vs. TAC/vehicle-treated group; y, P < 0.05 vs. TAC/Halo-treated group. D: KaplaneMeier survival analysis following Halo and Pyr treatment of TAC mice.
Halo and/or Pyr or saline along were administered once daily to sham and TAC mice and survival was monitored over 28 days. In the Log-rank test: P < 0.05 for TAC/Halo vs. TAC/
vehicle-treated group; P < 0.05 for TAC/Halo/Pyr-treated group vs. TAC/Halo-treated group.

evidenced by increased accumulation of LC3 puncta and p62
upregulation. Currently it is unclear whether autophagosome
accumulation occurs as a compensatory mechanism to allow
cellular survival under reduced ATP conditions. Interestingly, we
also observed increased mitochondrial fragmentation or degradation, as indicated by decreased Mito Tracker intensity, increased
LC3/cytochrome c co-localization and decreased VDAC expression,suggesting that mitochondrial dysfunction occurs as cardiac hypertrophy progresses to heart failure. These observations suggestthat mitochondrial damage triggers apoptosis or autophagy associated with mitochondrial energy depletion (21). We also foundthat haloperidol treatment remarkably reduced mitochondrial
membrane potential and enhanced similar effects triggered by Ang
II treatment. Ang II exposure generates reactive oxygen species(ROS) via activation of NADPH oxidase in vascular smooth muscle
and mouse heart (9,37), perturbing mitochondrial function (11,20).
Similarly, haloperidol treatment reportedly increases the presenceof ROS in neurons (39). These reports suggest that Ang II treatment
generates mitochondrial ROS via independent mechanisms.
Further studies are required to deﬁne mechanisms underlying
these outcomes in heart.
We also observed signiﬁcantly increased mortality inhaloperidol-treated TAC mice, with a survival rate of 50% at 14 daysand 33% at 28 days as opposed 86% survival at 28 days seen in TAC
only mice. Mitochondria comprise approximately 30% of the volume of cardiac muscle. About 70% of ATP generated in heart serves
to fuel contraction, and the remaining 30% provides energy forvarious ion pumps including the Ca2þ-ATPase in SR (SERCA) (19,43).
Cardiac contraction requires ATP production, and impairment inthis process rapidly induces contractile dysfunction (15). Consistent
with these ﬁndings, we found that haloperidol treatment in TAC
mice decreased ATP production and contractile dysfunction.
However, haloperidol treatment of sham mice had no adverse effecton cardiac morphology and function. This result agrees with aprevious report that a total cumulative dose of intravenous haloperidol of <2 mg is safe for use for patients who lack cardiovascularrisk factors (34).
Earlier studies of schizophrenia patients combining haloperidol
treatment with supplementation with u-3 polyunsaturated fatty
acid, vitamin E and vitamin C support the idea that potential
deleterious effects of haloperidol could be ameliorated by reducing
oxidative stress (41). Here, we suggest a novel mechanism of ATP
supplementation using sodium pyruvate, the end product ofglycolysis and substrate for acetyl-CoA, combined with haloperidol
treatment. Sodium pyruvate is known to be a safe drug (51) andexhibits a cardioprotective effect against ischemia-reperfusion
injury in Langendorff-perfused rat hearts (27). Sodium pyruvatealso possesses a therapeutic potential to treat mitochondrial disease (28). Interestingly, sodium pyruvate combined with haloperidol in TAC mice blocked adverse myocardial remodeling, perturbed
heart function and progression of heart failure and ultimately
increased animal survival by rescuing mitochondrial ATP levels.
Moreover, administration of intracoronary pyruvate at supraphysiological concentrations increases cardiac output and182

Y. Shinoda et al. / Journal of Pharmacological Sciences 131 (2016) 172e183

decreases pulmonary capillary-wedge pressure and heart rate inpatients with dilated cardiomyopathy and heart failure (25).
Indeed, sodium pyruvate treatment reportedly induces mitochondrial biogenesis (54) and can replenish pools of Krebs cycle intermediates, enhancing oxidative phosphorylation (13). These
reports support our result that sodium pyruvate prevents
haloperidol-induced progression of heart failure by protecting
mitochondrial function.
5. Conclusions
We have demonstrated that haloperidol treatment in vitro andin vivo signiﬁcantly decreases expression and activation of cardiac
s1R under stress conditions, which in turn impaired mitochondrial
Ca2þ mobilization and decreased ATP content. These activities
contribute to adverse cardiac remodeling and deterioration of heart
function associated with increased mortality in haloperidol-treated
TAC mice. We also reported for the ﬁrst time that ATP supplementation via sodium pyruvate rescues ATP levels in haloperidoltreated TAC mice, enabling proper cardiac contraction and relaxation. Our study suggests that ATP supplementation could be combined with haloperidol as treatment for serious cases ofschizophrenia, a regime that could mitigate haloperidol's adverse
effects.
Conﬂict of interest statement
The authors declare that they have no competing interests.
Acknowledgments
This work was supported in part by grants-in-aid for ScientiﬁcResearch from the Ministry of Education, Science, Sports and Culture of Japan (Kakenhi 24659024 and 24102505 to K.F.) and the
Smoking Research Foundation (to K.F.)
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