RESEARCH ARTICLE

Corticosteroids Mediate Heart FailureInduced Depression through Reduced σ1Receptor Expression
Yasuharu Shinoda1☯, Hideaki Tagashira1☯, Md. Shenuarin Bhuiyan2,
Hideyuki Hasegawa3, Hiroshi Kanai3, Chen Zhang4, Feng Han4, Kohji Fukunaga1*

a11111

1 Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3
Aramaki-Aoba, Aoba-ku, Sendai, Japan, 2 Division of Molecular Cardiovascular Biology, Department ofPediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, United States of America,
3 Department of Electrical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6
Aramaki-Aoba, Aoba-ku, Sendai, Japan, 4 Department of Pharmacy, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou, Zhejiang, 31005, P. R. China☯ These authors contributed equally to this work.
* kfukunaga@m.tohoku.ac.jp

Abstract
OPEN ACCESS
Citation: Shinoda Y, Tagashira H, Bhuiyan M.S,
Hasegawa H, Kanai H, Zhang C, et al. (2016)
Corticosteroids Mediate Heart Failure-Induced
Depression through Reduced σ1-Receptor
Expression. PLoS ONE 11(10): e0163992.
doi:10.1371/journal.pone.0163992
Editor: Kenji Hashimoto, Chiba Daigaku, JAPANReceived: April 19, 2016
Accepted: September 19, 2016
Published: October 14, 2016
Copyright: © 2016 Shinoda et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.
Funding: This work was supported in part by
grants-in-aid for Scientific Research from the
Ministry of Education, Science, Sports and Cultureof Japan (KAKENHI 25293124 and 26102704 to K.
F.), the Smoking Research Foundation (K.F.), andfrom a Research Fellowship from the Japan
Society for the Promotion of Science (265570 o Y.
S.).

Cardiovascular diseases are risk factors for depression in humans. We recently proposedthat σ1 receptor (σ1R) stimulation rescued cardiac hypertrophy and heart failure induced bytransverse aortic constriction (TAC) in mice. Importantly, σ1R stimulation reportedly ameliorates depression-like behaviors in rodents. Thus, we hypothesized that impaired σ1R activity in brain triggers depression-like behaviors in animals with cardiovascular disease.
Indeed, here we found that cardiac hypertrophy and heart failure induced by TAC wereassociated with depression-like behaviors concomitant with downregulation of σ1R expression in brain 6 weeks after surgery. σ1R levels significantly decreased in astrocytes in boththe hippocampal CA1 region and dentate gyrus. Oral administration of the specific σ1R agonist SA4503 (0.3–1.0mg/kg) significantly improved TAC-induced depression-like behaviorsconcomitant with rescued astrocytic σ1R expression in CA1 and the dentate gyrus. Plasma
corticosterone levels significantly increased 6 weeks after TAC, and chronic treatmentof mice with corticosterone for 3 weeks elicited depression-like behaviors concomitantwith reduced astrocytic σ1R expression in hippocampus. Furthermore, the glucocorticoid
receptor antagonist mifepristone antagonized depressive-like behaviors and ameliorated
decreased hippocampal σ1R expression in TAC mice. We conclude that elevated corticosterone levels trigger hippocampal σ1R downregulation and that σ1R stimulation with
SA4503 is an attractive therapy to improve not only cardiac dysfunction but depression-likebehaviors associated with heart failure.

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Competing Interests: The authors have declaredthat no competing interests exist.
Abbreviations: SA4503, 1-(3,4dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine
dihydrochloride; NE-100, N,N-dipropyl-2-[4methoxy-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.

Introduction
The prevalence of depression in heart failure (HF) patients has drawn attention because it isassociated with poorer clinical outcomes [1, 2]. Indeed, depression following myocardial
infarction (MI) is associated with higher morbidity and mortality [3]. Current American College of Cardiology/American Heart Association guidelines for coronary artery bypass graft(CABG) surgery, acute MI, and chronic angina all recommend evaluation of depression andsubsequent therapy [4]. Notably, administration of selective serotonin reuptake inhibitors(SSRIs) to MI patients reduces post-MI morbidity and mortality [3]. However, it remains
unclear whether MI causes depression, because the relationship of the heart to emotional
responses is largely unknown.
Since selective serotonin reuptake inhibitors (SSRIs) such as fluvoxamine and sertraline arepotent σ1R ligands, we recently focused on cardioprotective mechanisms of SSRI through theσ1R in heart failure models using transverse aortic constriction (TAC) mice and ovariectomised rats [5–7]. We found that fluvoxamine, which exhibits a high σ1R affinity (Ki = 36 nM),
but not paroxetine, which binds with lower affinity (Ki = 1893 nM), markedly rescued heart
failure and cardiac dysfunction in both TAC models of mice and rats [5, 7]. In addition, we
previously confirmed that the selective σ1R agonist SA4503 (Ki = 4.4 nM) antagonizes cardiac
dysfunction associated with heart failure in mice [8]. Interestingly, σ1R is highly expressed inneonatal rat cardiomyocytes and in brain [8, 9]. σ1R protein levels in left ventricle markedly
decreased accompanied by progression of left ventricular hypertrophy in TAC mice and σ1Rprotein expression was significantly and positively correlated with impaired LV fractional
shortening [7].
σ1R protein is predominantly localized to the mitochondria-associated ER membrane(MAM) where it functions as molecular chaperone of the IP3 receptor (IP3R), thereby regulating Ca2+ transport into mitochondria from the ER [10]. In cultured cardiomyocytes, σ1R stimulation inhibits angiotensin II-induced hypertrophy and cardiomyocyte injury by promoting
mitochondrial Ca2+ transport and ATP production [8]. σ1R knockdown by siRNA totally eliminates σ1R-mediated cardioprotection in cultured cardiomyocytes, suggesting that SSRI or
SA4503 directly acts on σ1R expressed in cardiomyocytes.
Recently, Ito et al. reported that pressure overload- and high salt diet-induced heart failure
elicits depression-like behaviors in mouse with concomitant reduction of σ1R levels in brain,
and that sympathetic nervous system suppression by administration of a σ1R agonist in the
CNS antagonized both σ1R downregulation and impaired LV fractional shortening [11]. However, how mechanistically σ1R is downregulated and in what cells remains unclear.
Here, we report that TAC surgery induces depression-like behaviors evident 6 weeks laterand that σ1R stimulation with the high affinity σ1R agonist SA4503 ameliorates those behaviors. We also show that σ1R is preferentially downregulated following TAC in hippocampal
astrocytes. Depressive-like behaviors and decreased σ1R levels in TAC mice were ameliorated
by treatment with the glucocorticoid receptor inhibitor mifepristone. Finally, we demonstrated
that SA4503 treatment ameliorates TAC-induced depression-like behaviors by rescuing σ1R
proein expression in brain and heart. We conclude that σ1R agonists such as fluvoxamine andSA4503 are potential therapeutics to treat depression following cardiovascular disease.

Material and Methods
Materials
Reagents and antibodies were obtained from the following sources: anti-σ1R antibody (Abcam,
Cambridge, UK); anti-glial fibrillary acidic protein (GFAP) (Sigma, St. Louis, MO); anti-β-

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tubulin antibody (Sigma, St. Louis, MO). SA4503 was synthesized in Laboratory of Medicinal
Chemistry, Zhejiang University according to the method of Fujimura et al. [12]. NE-100 (N,Ndipropyl-2-[4-methoxy-3-(2-phenylethoxy) phenyl]-ethylamine monohydrochloride) was
generously supplied by Taisho Pharmaceutical Co. Ltd (Ohmiya, Japan). Paroxetine andmifepristone was purchased from Sigma and Cayman Chemical (Ann Arbor, MI), respectively.
Other reagents were of the highest quality available (Wako Pure Chemicals, Osaka, Japan).

Animals and Operations
All procedures for handling animals complied with the Guide for Care and Use of Laboratory
Animals and were approved by the Animal Experimentation Committee of Tohoku University
Graduate School of Pharmaceutical Sciences. Adult male ICR mice weighing 35 to 40 g wereobtained 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 ahumidity-controlled environment maintained on a 12-h light/dark schedule (lights on 8:00
AM–8:00 PM). Mice were provided food and water ad libitum. Transverse aortic constriction(TAC) was performed as described [7] 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 anegative toe-pinch reflex. If anesthesia was not sufficient, then a top-up dose of 20% of the initial dose was given.

Experimental Design
ICR mice were randomly separated into six experimental groups: 1) sham (n = 7), 2) TAC for 6
weeks (n = 8), 3) TAC plus SA4503 (0.1 mg/kg, p.o.) treatment (SA 0.1) (n = 7), 4) TAC plus
SA4503 (0.3 mg/kg, p.o.) treatment (SA 0.3) (n = 6), 5) TAC plus SA4503 (1.0 mg/kg, p.o.)treatment (SA 1.0) (n = 8), 6) TAC plus SA4503 (1.0 mg/kg, p.o.) plus NE-100 treatment (1.0mg/kg, p.o.) (SA 1.0+NE) (n = 8), 7) TAC plus paroxetine (0.4 mg/kg, p.o.) (n = 7). SA4503
and NE-100 were dissolved in a 0.9% saline solution. Paroxetine was dissolved in 0.5%
carboxymethylcellulose. Vehicle, SA4503 and NE-100 (1.0 mg/kg) were administered orally(once daily) over the last 4 weeks of TAC using a metal gastric zonde for mice in a volume of
1ml/100g body weight, starting from the onset of aortic banding. To define the effect of corticosterone (CORT) on σ1R expression, ICR mice were randomly separated into three experimental groups: 1) vehicle (n = 8), 2) CORT for 1 week (n = 11), and 3) CORT for 3 weeks (n = 8).
CORT (20 mg/kg, s.c.) was was dissolved in 0.5% carboxymethyl cellulose solution and injected
for 1 or 3 weeks (once daily) in a volume of 1ml/100g of body weight as described [13]. To
investigate mifepristone effects, ICR mice were separated into four groups: 1) sham plus vehicle(n = 6), 2) sham plus mifepristone (50 mg/kg s.c.) (n = 7), 3) TAC plus vehicle (n = 4), and 4)
TAC plus mifepristone (50 mg/kg, s.c.) (n = 4). Mifepristone was dissolved in PEG400 vehicle(Nacalai Tesque, Inc., Kyoto, Japan). Vehicle and mifepristone were injected subcutaneously
daily for 4 weeks starting 2 weeks after TAC surgery.

Tail Suspension Test
The tail suspension test is widely used in mice to test anti-depressant activity. The test is basedon the fact that animals subjected to the short-term, inescapable stress of being suspended by
the tail will develop an immobile posture. The depressed mice show increased immobility time.
Immobility duration following tail suspension was scored according to the method of Steru
et al. [14]. In brief, acoustically and visually isolated mice were suspended 50 cm above thefloor by adhesive tape placed approximately 1 cm from the tip of the tail. Immobility time was

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recorded during a 5-min period. Mice were considered immobile only when they hung passively and remained completely motionless.

Forced Swim Test
The forced swim test is also widely used to assess antidepressant-like activity. Mice were placed
individually in glass cylinders (height: 25 cm, diameter: 16 cm) filled 20 cm of water at 24°C,
and the duration of immobility time in a 5-min test was scored as described by Porsolt et al.
[15]. Mice were judged ‘‘immobile” when they ceased struggling and remained floating
motionless in the water, making only movements necessary to keep their head above water.

Sucrose Preference Test
Mice were randomly placed into cages with two bottles containing drinking water and 1%
sucrose solution. Bottle’s weights were measured once in each day and location was switched in
the next day to prevent bias from preference for location. Tests were conducted for five concecutive days, and sucrose preference (%) was calculated as ratio of 1% sucrose consumption tototal consumption.

Echocardiography and Measurement of Cardiac Hypertrophy
Noninvasive echocardiographic measurements were performed as described [7, 8]. Briefly, procedures 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 witha 10-MHz linear array transducer (UST-5545; Aloka). The heart was imaged in a two-dimensional parasternal short-axis view, and an M-mode echocardiogram of the midventricle wasrecorded at the level of the papillary muscles. Diastolic and systolic LV wall thickness, LV enddiastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) were measured, all fromleading edge to leading edge according to American Society of Echocardiography guidelines.
The percentage of LV fraction shortening (LV%FS) was calculated as ([LVEDD—LVESD]/LVEDD) x 100. After animals were sacrificed by cervical spine fracture dislocation, the thoracic
cavity was opened, and hearts were immediately harvested and weighed. Blood pressure wasmeasured using the tail-cuff method (BP-98A-L, Softron, Tokyo, Japan), according to the manufacturer’s protocol.

Western Blot Analysis and Measurement of ATP Content
Frozen samples were homogenized as described [8]. Western blot analysis was performed aspreviously described [7, 16]. Blots were subjected to the ECL immunoblotting detection system(Amersham Biosciences, U.K.), and emitted light was captured by a Luminescent Image Analyzer (LAS-4000 mini, Fuji Film, Tokyo, Japan) attached to a CCD camera. Densitometricquantification of blot signals was performed with Image Gauge Ver3.0 (Science Lab, Fuji Film,
Tokyo, Japan). ATP measurement was performed using an ATP assay kit (Toyo Ink, Tokyo,
Japan), according to the manufacturer’s protocol as described [17, 18]. Briefly, frozen samples
were homogenized in homogenate buffer (0.25M sucrose, 10mM HEPES-NaOH, pH 7.4),
and lysates were cleared by centrifugation at 1000xg for 10 min at 4°C. Supernatants werecollected, and supernatant proteins were solubilized in extraction buffer. After a 30 min incubation, luciferin buffer was added to each sample and oxyluciferin was detected using a luminometer (Gene Light 55, Microtec, Funabashi, Japan).

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Measurement of Corticosterone Levels in Plasma and Brain Tissue
Corticosterone measurement was performed using a corticosterone EIA kit (Enzo Life Sciences, Farmingdale, NY), according to the manufacturer’s protocol. Plasma samples were collected as previously reported [19]. Briefly, the mice were decapiyated and then blood wascollected into tubes that contained EDTA. After centrifugation at 4°C, 1500×g for 10 min,plasma was aliquoted and stored at -80°C until CORT analysis.

Immunohistochemistry
Immunohistochemistry was carried out as described [20, 21]. Briefly, 50 μm sliced mouse brain
tissues were incubated 3 days at 4°C with anti-σ1R antibody (1:200) and anti-GFAP antibody(1:250) in 0.5% Blocking Reagent (TSA Fluorescence system, PerkinElmer, Inc. Waltham,MA). After washing, sections were incubated for 24hr with a species-specific secondary antibody conjugated with Alexa 594 and Alexa 488 in 0.5% blocking reagent. For nuclear staining,
sections were incubated with DAPI (Vector Laboratories, Burlingame, CA). Sections of rat
heart tissue embedded in VECTASHIELD (Vector Laboratories, Burlingame, CA) were examined using a fluorescence microscope. Immunofluorescent images were obtained with confocal
laser scanning microscope (LSM700, Carl Zeiss Microscopy).

Statistical Analysis
Values are represented as means ± standard error of the mean (S.E.M.). Comparison between
two experimental groups was made using the unpaired Student’s t test. Other results were evaluated for differences using two- or one-way analysis of variance (ANOVA), followed by multiple comparisons using Dunnett’s test. P<0.05 was considered statistically significant.

Results
Time Course Analysis of Depression-Like Behaviors and σ1RExpression
To define σ1R function in TAC-induced depression-like behaviors, we first evaluated temporalchanges in both depression-like behaviors and brain σ1R expression in mice after TAC or shamsurgery. In TAC animals, behaviors indicative of depression were observed following analysis
of forced swimming and tail suspension tasks 6 weeks after TAC (P<0.01 vs. sham in forced
swimming task and P<0.05 vs. sham in tail suspension task) (Fig 1A and 1B). Importantly, σ1R
expression in the hippocampal CA1 region and dentate gyrus (DG) but not in medial prefrontal cortex (mPFC) region markedly decreased both at both 4- and 6-week times points after
TAC (P<0.05 vs. sham in CA1 at 4 and 6 weeks after TACs, and P<0.01 vs. sham in DG atboth 4 and 6 weeks) (Fig 1C and 1D). Temporal changes in σ1R levels were comparable tothose previously reported in the heart [7].
Since σ1R stimulation with the novel σ1R agonist SA4503 reportedly improves depressionlike behaviors in olfactory bulbectomized rats [22], we next tested potential anti-depression
effects of SA4503 in TAC mice. As expected, SA4503 treatments starting 2 weeks after TAC
and continuing for 4 weeks dose-dependently antagonized increased immobility times, which
are indicative of depression, seen in the forced swimming task (P<0.01 vs. TAC-vehicle for SA
0.3 and SA 1.0) (Fig 1E). In tail suspension task, SA4503 treatment (1.0 mg/kg) reduced immobility times to identical levels to sham mice (Fig 1F). However, mice treated with both SA4503
and NE100 showed similar increase in immobility times with sham mice (P<0.01 vs. sham forSA 1.0+NE in forced swimming task, P<0.05 vs. sham for SA 1.0+NE in tail suspension task)(Fig 1E and 1F). Treatment with paroxetine (0.4 mg/kg), a conventional SSRI which has a

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Fig 1. Time course of depressive-like behaviors and σ1R expression, and effect of SA4503 treatment
on depressive-like behaviors in TAC mice. A: Immobility time in a forced swimming test was measured 2,
4 and 6 weeks after TAC surgery. B: Immobility time in a tail suspension test was similarly measured. Groups
consisted of 6–8 mice. C: Western blot analysis of σ1R and β-tubulin (as a loading control) proteins in the
CA1 region, dentate gyrus (DG) and medial prefrontal cortex (mPFC) of sham and TAC mice.
Immunoblotting with anti-β-tubulin antibody indicates equal protein loading. D: Densitometric quantificationof σ1R immunoreactivity (n = 6). Columns represent means ± S.E.M. F(3, 20) = 4.875, P<0.05 (CA1); F(3,20) = 6.182, P<0.01 (DG); F(3, 20) = 0.698, P = 0.564 (mPFC). *, P<0.05 and **, P<0.01 versus shamoperated mice. E: Immobility time in a forced swimming test was measured 6 weeks after TAC surgery withor without drug treatment for the last 4 weeks. F: Immobility time in a tail suspension test was similarly
measured. 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 (n = 6). F(5, 37) = 20.740, P<0.01 (forced swimming); F(5, 38) = 2.692, P<0.05 (tailsuspension). G: Consumption of normal water and 1% sucrose solution was measured in continuous 5 days.
Sucrose preference (%) was calculated as ratio of 1% sucrose solution consumption to total consumption.
TAC mice were treated with SA4503 (1.0 mg/kg) or SA4503 (1.0 mg/kg) plus NE-100 (1.0 mg/kg), as
indicated (n = 6). Columns represent means ± S.E.M. *, P<0.05 and **, P<0.01 versus the sham group; ##,P<0.01 versus the TAC/vehicle treated group; ††, P<0.01 versus the TAC plus SA4503 (1 mg/kg)-treatedgroup.
doi:10.1371/journal.pone.0163992.g001

relatively low affinity of σ1R [23], partially but significantly reduced immobility times of TAC
mice in forced swimming task (63.4 ± 5.9 sec) (P<0.05 vs. TAC-vehicle), but not in tail suspension task (102.3 ± 10.8 sec)(S1 Fig). In sucrose preference test, sucrose preference was significantly decreased in TAC mice (P<0.01 vs. sham) (Fig 1G). SA4503 treatment recovered
sucrose preference in TAC mice (P<0.05 vs. TAC veh) and the effect was blocked by coadministration of NE100 (Fig 1G). Overall, these findings indicate that SA4503 treatment ameliorates TAC-induced depression-like behaviors in TAC mice.

Effect of SA4503 Treatment on Hippocampal σ1R Expression
Since σ1R stimulation by SA4503 ameliorated TAC-induced depression-like behaviors, we next
asked whether treatment of mice with SA4503 restored brain σ1R expression using the same
experimental design employed for behavioral tests. SA4503 administration at 1 mg/kg significantly restored σ1R levels in both CA1 (P<0.05 vs. TAC-vehicle for SA 1.0) and DG regions(P<0.01 vs. TAC-vehicle for SA 1.0) (Fig 2A and 2B). Co-administration of NE-100 failed to

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Fig 2. Effect of SA4503 treatment on hippocampal σ1R expression and ATP content. A: Western blot
analysis of σ1R and β-tubulin (as a loading control) proteins in the CA1 region and dentate gyrus (DG) of
sham and TAC-mice. Immunoblotting with anti-β-tubulin antibody indicates equal protein loading. B:
Densitometric quantification of σ1R immunoreactivity. F(5, 32) = 7.128, P<0.01 (CA1); F(5, 26) = 9.868,
P<0.01 (DG). C: Western analysis of σ1R and β-tubulin (as a loading control) proteins in the LV of sham andTAC-mice with or without drug treatment. Immunoblotting with anti-β-tubulin antibody indicates equal protein
loading. D: Densitometric quantification of σ1R immunoreactivity. F(5, 47) = 20.484, P<0.01. In B and D, **,P<0.01 versus the sham group; #, P<0.05 and ##, P<0.01 versus the TAC/vehicle-treated group E:
Measurement of cellular ATP content in hippocampal tissue of TAC mice 6 weeks after surgery with orwithout drug treatment during the last 4 weeks (n = 6). F(5, 47) = 6.284, P<0.01. F: Measurement of cellular
ATP content in the LV of TAC mice with or without drug treatment (n = 6). F(5, 54) = 15.089, P<0.01 (CA1).
Data are expressed as percentages of values of sham-operated animals (mean ± S.E.M.) In E amd F, **,P<0.01 versus the sham group; #, P<0.05 and ##, P<0.01 versus the TAC/vehicle-treated group; †, P<0.05
and ††, P<0.01 versus the TAC plus SA4503 (1 mg/kg)-treated group.
doi:10.1371/journal.pone.0163992.g002

abolish SA4503-induced increases in σ1R expression (Fig 2A and 2B). We also analyzed expression levels of another ER chaperone protein, 78 kDa glucose-regulated protein (GRP78, Bip),
which makes a complex with σ1R [10]. Bip expression levels in CA1 and DG regions were notsignificantly changed among four groups (sham, TAC-vehicle, SA1.0 and SA1.0+NE) [CA1: F(3,8) = 1.707, P = 0.242, DG: F(3, 8) = 0.033, P = 0.991] (n = 3)(S2 Fig).
As previously reported by Tagashira et al. [8], SA4503 administration at 0.3 and 1 mg/kg
significantly rescued σ1R downregulation seen in heart following TAC (P<0.01 vs. TAC-vehicle for SA 0.3 and SA 1.0) (Fig 2C and 2D). In that study we also found that SA4503 treatment
immediately after surgery increased heart ATP production concomitant with improved heart
function [8]. In the present study, we found that SA4503-mediated restoration of ATP production also occurred in hippocampus (P<0.05 vs. TAC-vehicle for SA 1.0) (Fig 2E), and coadministration of NE-100 abolished this effect (P<0.01 vs. SA 1.0) (Fig 2E). We also confirmedthat SA4503 treatment restored ATP production in the heart (P<0.05 and P<0.01 vs. TACvehicle for SA 0.3 and 1.0 mg/kg, respectively) (Fig 2F). Taken together, SA4503-induced restoration of σ1R and ATP levels positively correlates with its anti-depressive action.

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Immunohistochemical Changes in Hippocampal σ1R Expression
We next defined hippocampal regions in which σ1R is downregulated using immunohistochemistry. In CA1, σ1R was predominantly expressed in astrocytes (GFAP-positive cells) inthe molecular layer and faintly in pyramidal neurons (see DAPI-positive cells; Fig 3A). Accordingly, σ1R immunoreactivity in both astrocytes and neurons in CA1 significantly decreased by
6 weeks after TAC (Fig 3A). On the other hand, when we assessed the number of living neurosn by DAPI staining, TAC had no effect on the total number of neurons in CA1 region at 6
weeks after TAC (71.17±4.64 cells (sham) vs. 70.00±3.11 cells (TAC 6 weeks), respectively(0.35 μm×0.35 μm region)). We also confirmed that the number of GFAP-positive cells in CA1
was comparable in both TAC and sham-operated mice (16.5±1.71 cells (sham) vs. 12.25±0.85
cells (TAC 6 weeks), respectively (in a 0.35 μm×0.35 μm region)). Likewise, in the DG, σ1R was
predominantly expressed in astrocytes in both molecular layer and hilus regions of the hippocampus. σ1R levels markedly decreased by 6 weeks after TAC (Fig 3B). Finally, the total number of GFAP-positive cells in DG did not change in TAC mice (23.5±2.02 cells (sham) vs. 21.0
±2.27 cells (TAC 6 weeks), respectively (in a 0.35 μm×0.35 μm region)).
Since σ1R expression decreased primarily in hippocampal astrocytes (Fig 3A and 3B), we
confirmed the effect of SA4503 treatment on σ1R expression using immunohistochemistry.
SA4503 treatment of mice subjected to TAC rescued σ1R expression primarily in CA1 astrocytes, not in pyramidal neurons (Fig 3A). Co-administration with NE-100 did not eliminate
SA4503-mediated increases in astrocytic σ1R expression (Fig 3A). In DG, SA4503 treatment
also restored σ1R expression primarily in astrocytes (Fig 3B), and NE-100 co-administration
did not eliminate these effects (Fig 3B). Taken together, SA4503-induced restoration of hippocampal σ1R levels preferentially occurs in astrocytes.

Effect of SA4503 Treatment on Corticosterone Levels in Plasma and
Hippocampal Tissue
We next addressed potential causes of hippocampal σ1R donwnregulation following TAC.
Increases in plasma corticosterone (CORT) levels reportedly occur in depressed patients andare a factor in stress-induced depression [24–26]. Thus, we investigated CORT levels following
TAC in plasma and hippocampal tissue by ELISA. As expected, plasma CORT levels increasedin TAC mice at 4- and 6-week time points after TAC surgery (P<0.01 vs. sham for 4 and 6
weeks TAC, respectively) (Fig 4A). In hippocampal tissues, however, CORT levels were not significantly changed but tended to increase in TAC mice (1.11 ± 0.11 ng/mg tissue (sham) vs.
1.35 ± 0.16 ng/mg tissue (TAC 6 weeks) (P = 0.51) respectively.) We next investigated σ1R
expression in a CORT-induced depressive mouse model (Fig 4B–4G). CORT administration
over a 3-week period increased immobility time in forced swimming (P<0.05 vs. vehicle) (Fig
4B). CORT treatment slightly increased immobility time in tail suspension task, however, there
was no stastically significance (Fig 4C). Most importantly, CORT administration decreased
σ1R expression at 1- and 3-week time points in hippocampus, including CA1 and DG (P<0.05
vs. control for 1 week CORT in CA1 region, and P<0.01 vs. control for 3 week CORT in CA1
region and for both 1 and 3 weeks CORT in DG region) (Fig 4D and 4E).
We also asked whether hippocampal σ1Rs are downregulated after CORT administration
using immunohistochemistry. In CA1, endogenous σ1R expression markedly decreased at 1
and 3 week time points after CORT treatment in both astrocytes and neurons (Fig 4F). Likewise, in the DG, expression of endogenous σ1Rs in astrocytes also markedly decreased at 1 and3 week time points following CORT treatment (Fig 4G). These findings suggest that elevatedplasma CORT levels underlie TAC-induced σ1R downregulation, especially in hippocampal
astrocytes. Furthermore, we analyzed σ1R expression levels in heart left ventricles. CORTPLOS ONE | DOI:10.1371/journal.pone.0163992 October 14, 2016

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Fig 3. Effect of SA4503 treatment on σ1R expression in hippocampal astrocytes. A:
Immunofluorescence showing localization of σ1R (red) with the astrocyte marker GFAP (green) and DAPI
(blue) in the CA1 region 6 weeks after TAC surgery with or without drug treatment for the last 4 weeks. B:
Immunofluorescence showing localization of σ1R (red) with GFAP (green) and DAPI (blue) in DG 6 weeks
after TAC surgery with or without drug treatment for last 4 weeks. 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. Boxed regions in all panels
are magnified in adjacent right-hand panels.
doi:10.1371/journal.pone.0163992.g003

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9 / 19Transverse Aortic Constriction Leads to Depression-Like Behaviors

Fig 4. Effect of TAC surgery on plasma CORT levels. A: Measurement of plasma CORT levels in TAC
mice. F(3, 21) = 16.602, P<0.01. B: Immobility time in a forced swimming test was measured after 1 and 3
weeks of chronic CORT treatment. F(2, 24) = 4.193, P<0.05. C: Immobility time in a tail suspension test was
similarly measured. Groups consisted of 8–11 mice. F(2, 24) = 2.224, P = 0.130. D: Western blot analysis ofσ1R and β-tubulin (as a loading control) proteins in the CA1 region and dentate gyrus (DG) of vehicle and
CORT treated-mice. Immunoblotting with anti-β-tubulin antibody indicates equal protein loading. E:
Densitometric quantification of σ1R immunoreactivity (n = 6). Columns represent means ± S.E.M. F(2, 15) =8.877, P<0.01 (CA1); F(2, 15) = 10.614, P<0.01 (DG). In A, B and E, *, P<0.05 and **, P<0.01 versus shamoperated mice or vehicle-treated mice. F: Immunofluorescence staining showing localization of σ1R (red)with the astrocyte marker GFAP (green) and DAPI (blue) in CA1. G: Similar analysis in the DG. Boxedregions in F and G are magnified in adjacent right-hand panels.
doi:10.1371/journal.pone.0163992.g004

treatment for both 1 and 3 weeks failed to alter σ1R expression in heart (veh: 100 ± 6.6%,
CORT 1 week: 88.1 ± 7.1%, and CORT 3 week: 107.9 ± 9.3%) (n = 5–6). These results indicatethat increased plasma CORT in TAC mice triggers σ1R reduction in the hippocampus but notin the heart.

Effect of Mifepristone on Depression-Like Behaviors and σ1RExpression in TAC MiceNext, we asked whether pharmacological inhibition of glucocorticoid receptors would eliminate TAC-induced depressive-like behaviors and/or decrease hippocampal σ1R expression.
Treatment on TAC mice with mifepristone slightly antagonized increased immobility times
seen in forced swimming (P = 0.38) (Fig 5A), however completely abolished TAC-induced
increase in immobility time in tail suspension task (P<0.05 vs. TAC-vehicle for TAC-mifepristone) (Fig 5B). Moreover, mifepristone treatment of TAC mice significantly rescued reduced
σ1R expression seen in hippocampal CA1 and DG (P<0.01 and P<0.05 vs. TAC-vehicle for
TAC-mifepristone in CA1 and DG, respectively), but had no effect on σ1R expression in sham

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Transverse Aortic Constriction Leads to Depression-Like Behaviors

Fig 5. Effect of mifepristone on depressive-like behaviors and hippocampal σ1R expression in TAC
mice. A: Immobility time in a forced swimming test was measured 6 weeks after TAC surgery with or without
drug treatment for the last 4 weeks. F(3, 17) = 13.973, P<0.01. B: Immobility time in a tail suspension test
was similarly measured. Sham and TAC mice were treated with vehicle or mifepristone (mife) (50mg/kg) as
indicated (n = 4–7). F(3, 14) = 3.490, P<0.05. Columns represent means ± S.E.M. *, P<0.05 and **, P<0.01
versus the sham/vehicle group; #, P<0.05 versus TAC/vehicle treated group. C: Western blot analysis of σ1Rand β-tubulin (as a loading control) proteins in the CA1 region and dentate gyrus (DG) of sham and TAC
mice treated with vehicle or mifepristone. Immunoblotting with anti-β-tubulin antibody indicates equal protein
loading. D: Densitometric quantification of σ1R immunoreactivity (n = 4). Columns represent means ± S.E.M.
F(3, 16) = 9.009, P<0.01 (CA1); F(3, 20) = 4.885, P<0.05. *, P<0.05 and **, P<0.01 versus sham/vehicletreated mice. #, P<0.05 and ##, P<0.01 versus the TAC/vehicle-treated mice.
doi:10.1371/journal.pone.0163992.g005

mice (Fig 5C and 5D). These results suggest that elevated CORT levels partially function in
depression-like behaviors and decreased hippocampal σ1R expression in TAC mice.

SA4503 Treatment Ameliorates Myocardial Dysfunction in TAC Mice
We previously documented that SA4503 administration immediately after TAC surgery rescues cardiac dysfunction [8]. Here, we confirmed those findings when SA4503 as administered
starting at 2 weeks after surgery. Like our previous findings [8], TAC mice showed decreases in
LV fractional shortening (FS) and increases in left ventricular end-systolic diameter (LVESD)
and left ventricular end-diastolic diameter (LVEDD) (P<0.01 vs. sham) compared with shamoperated mice (Fig 6A–6D). SA4503 treatment dose-dependently rescued decreased FS(P<0.05 vs. TAC-vehicle for SA 0.3 and SA 1.0) (Fig 6B) and increased LVESD (P<0.05 vs.
TAC-vehicle for SA 0.1, SA 0.3 and SA 1.0) (Fig 6C). Co-administration of NE-100 abolishedSA4503-mediated amelioration of cardiac dysfunction, as indicated by decreased FS andincreased LVESD (P<0.01 vs. SA 1.0) (Fig 6B and 6C). Also, TAC mice treated with vehicle,
both SA4503 and NE100 showed significant increase in LVEDD (P<0.01 and P<0.05 vs. shamfor TAC-vehicle and SA 1.0+NE) (Fig 6D). SA4503 treatment significantly and dose-dependently rescued the HW-to-BW ratio (P<0.01 vs. TAC-vehicle for SA 1.0) (Fig 6E). Co-administration of NE-100 abolished SA4503 inhibition of TAC-induced hypertrophy, as indicated by
HW-to-BW (P<0.01 vs. TAC-vehicle for SA 1.0) (Fig 6E). We conclude that the anti-pathological hypertrophic effect of SA4503 on TAC mice is mediated by σ1R stimulation in vivo.

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Transverse Aortic Constriction Leads to Depression-Like Behaviors

Fig 6. Effect of SA4503 and NE-100 on cardiac failure and hypertrophy induced by TAC. A:
Representative M-mode echocardiograms of mice treated with or without SA4503 plus or minus NE-100. B:
Changes in the percentage of LV fractional shortening (FS%). C: Changes of left ventricular end-systolic
diameter (LVESD). D: Changes of left ventricular end-diastolic diameter (LVEDD). TAC mice were treatedwith SA4503 (0.1, 0.3 or 1.0 mg/kg) or SA4503 (1.0 mg/kg) plus NE-100 (1.0 mg/kg) as indicated. E: TACinduced cardiac hypertrophy as indicated by the heart weight/body weight (HW/BW) ratio. Groups consistedof 6–8 mice. Bars represent means ± S.E.M. F(5, 45) = 12.559, P<0.01 (FS); F(5, 45) = 10.317, P<0.01(LVESD); F(5, 45) = 3.053, P<0.05 (LVEDD);. F(5, 18) = 8.383, P<0.01 (HW/BW). *, P<0.05 and **, P<0.01
versus sham group; #, P<0.05 and ##, P<0.01 versus the TAC/vehicle-treated group; ††, P<0.01 versus the
TAC plus SA4503 (1 mg/kg)-treated group.
doi:10.1371/journal.pone.0163992.g006

SA4503 Treatment Affects Sympathetic Nervous Activity as Indicatedby Peripheral Blood Pressure
Ito et al. previously reported that suppression of sympathetic nerve activity by a different σ1Ragonist, PRE084, underlies improvement in impaired LV fractional shortening in a pressure
overload- and high-salt diet-induced HF mouse model [11]. Therefore, we asked whether TAC
surgery and SA4503 treatment altered sympathetic nervous activity. At a 6-week time period,
heart rate (HR) was comparable with or without TAC surgery and with or without chronic
SA4503 treatment over the last 4-week period (Fig 7A and 7B). Although mean blood pressure(MBP), diastolic blood pressure (DBP) and systolic blood pressure (SBP) significantly
decreased in some points among 6 weeks following TAC surgery (MBP; P<0.01 and P<0.05
vs. sham at 2- and 6-weeks after TAC, DBP; P<0.01 vs. sham at 2-weeks after TAC, SBP;
P<0.01 and P<0.05 vs. sham at 2- and 6-weeks after TAC) (Fig 7C, 7E and 7G), chronic treatment with SA4503 over the last 4 weeks did not alter MBP, DBP andSBP (Fig 7D, 7F and 7H).
These data suggested that SA4503 at doses used in this study has no effect on sympathetic
activity.

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Transverse Aortic Constriction Leads to Depression-Like Behaviors

Fig 7. Effect of SA4503 treatment on sympathetic nervous activity as indicated by peripheral bloodpressure. Measurement of peripheral blood pressure using the tail-cuff method. A, B: Heart rate (HR) was
measured 2, 4 and 6 weeks after TAC surgery (A), and at 6 weeks with or without drug treatment for the last
4 weeks (B). C, D: Mean Blood Pressure (MBP) was measured at the same time points (C) with or without
drug treatment for the last 4 weeks (D). E, F: Diastolic Blood Pressure (DBP) was similarly measured (E) withor without drug treatment for the last 4 weeks (F). G, H: Systolic Blood Pressure (SBP) was similarly
measured (G) with or without drug treatment for the last 4 weeks (H). TAC mice were treated with SA4503(0.1, 0.5 or 1.0 mg/kg) or SA4503 (1.0mg/kg) plus NE-100 (1.0 mg/kg), as indicated. Groups consisted of 6
mice. Columns represent means ± S.E.M. F(5, 28) = 0.216, P = 0.953 (HR); F(5, 28) = 1.821 (MBP); F(5, 28)= 1.144, P = 0.361 (DBP); F(5, 28) = 2.288, P = 0.730 (SBP). *, P<0.05 and **, P<0.01 versus the shamgroup.
doi:10.1371/journal.pone.0163992.g007

Discussion
Here, we showed that: 1) mice subjected to a TAC-induced hypertrophy model exhibit depression-like behaviors after a prolonged period (6 weeks); 2) these behaviors are closely associatedwith reduced σ1R expression in hippocampal astrocytes; 3) treatment of mice with the selective
σ1R agonist SA4503 ameliorates TAC-induced depression-like behaviors with concomitant
improvement of heart function; and 4) increased plasma CORT levels are among causative factors underlying σ1R downregulation and depression-like behaviors following TAC. Taken
together, we provide novel evidence for crosstalk between heart failure and depression throughthe hypothalamic pituitary-adrenal (HPA) axis (Fig 8). Our findings suggest that heart failure-

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Transverse Aortic Constriction Leads to Depression-Like Behaviors

Fig 8. Schematic representation of the hypothesis tested and results obtained. Shown is a model
depicting mechanisms underlying TAC-induced depressive-like behavior, as suggested by findingspresented here. TAC-induced cardiac σ1R downregulation induces cardiac dysfunction. At the same time,levels of peripheral pro-inflammatory cytokines and corticosterone increase, aggravating cardiac dysfunction
and downregulating brain hippocampal σ1R levels. σ1R stimulation with SA4503 blocks TAC-induced σ1R
downregulation in heart and in hippocampus.
doi:10.1371/journal.pone.0163992.g008

induced inflammation and stress trigger CORT release via this axis, perturbing σ1R expressionin brain, particularly in the hippocampus. Lower σ1R activity reduces mitochondrial ATP production, further impairing brain function and resulting in depression. Depression-like behaviors may also aggravate heart function by dysregulating sympathetic nerve activity [11].
Previous reports indicate that neuronal σ1R downregulation impairs IP3R-mediated mitochondrial Ca2+ transport and ATP production in neurons [9]. ATP serves as not only anenergy supply but also acts as an extracellular signaling molecule between neurons and glial
cells [27]. For example, in hippocampal slices, norepinephrine treatment promotes glial
cell ATP production and increases synaptic transmission efficacy [28]. SA4503 treatment
enhances IP3-mediated Ca2+ mobilization into mitochondria and mitochondrial ATP production in cardiomyocytes [8] and heart tissue in vivo (Fig 2F). Our present study reveals thatSA4503 treatment antagonizes TAC-induced reduced σ1R expression in hippocampal astrocytes and enhances ATP production. Cao et al. reported that mice lacking the IP3 receptor
type 2 (Itpr2-/-) display depression-like behaviors concomitant with reduced ATP levels in
prefrontal cortex and hippocampus [29]. They also showed that acute increases in ATP ameliorate depression-like behavior by stimulating the P2X2 receptor. These authors conclude
that reduced levels of hippocampal and cortical astrocyte-derived ATP are critical for induction of depression-like behaviors [29]. Combined with our findings, these studies indicate that
reduced ATP production followed by σ1R downregulation in hippocampal astrocytes likely
triggers TAC-induced depression-like behavior.

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Transverse Aortic Constriction Leads to Depression-Like Behaviors

We also found that TAC-dependent σ1R downregulation in astrocytes occurs primarily inhippocampal CA1 and DG regions. Ito et al. reported that treatment with a different σ1R agonist, PRE084, antagonizes reduced σ1R expression seen in brain extracts after aortic banding inmice fed a high-salt diet [11]. We have reported that several σ1R agonists such as fluvoxamine,pentazocine and SA4503 restore reduced σ1R levels following heart failure in heart left ventricles [8, 16, 30]. Co-administration of σ1R antagonist, NE100, blocked those cardioprotective
effects, whereas NE-100 administration did not block the elevated σ1R levels by SA4503 [8, 16,30]. These results are consistent with the result in hippocampus of TAC mice in this report.
Overall, improvement of depressive behabiors by SA4503 in TAC mice is mediated by σ1Rstimulation rather than restoration of σ1R levels. Thus, mechanism underlying elevation ofSA4503-induced σ1R levels remains unclear.
Unlike previous report [11], TAC with or without SA4503 treatment did not alter sympathetic nerve activity, as evaluated in our study by heart rate and blood pressure analysis. On theother hand, we found that plasma CORT levels increased at 4- and 6-week time points after
TAC with concomitant CORT increases in the brain. Furthermore, chronic treatment of TAC
mice with CORT significantly reduced hippocampal σ1R expression, especially in the CA1
region and DG (Fig 4), and mifepristone-mediated glucocorticoid receptor inhibition antagonized this effect in hippocampus (Fig 5). In support of our findings, glucocorticoid receptors
are highly expressed in rat hippocampus [31]. In studies of human cohorts exhibiting heart failure, mortality increased significantly in patients with high plasma cortisol, a known risk factor
for increased mortality by heart disease [32]. Taken together, increased plasma CORT levels
are a risk factor for both reduced hippocampal σ1R expression and TAC-induced depressionlike behaviors. However, further studies are required to define additional factors underlying
TAC-induced CORT production and CORT-induced σ1R downregulation.
Accumulating clinical evidence suggests that depression is a risk factor for cardiac disease
and that SSRI treatment reduces post-MI morbidity and mortality following heart failure [3].
Hashimoto et al. recently suggested that σ1R function links cardiovascular disease to depression[33]. Indeed, mice subjected to aortic banding and infused with the σ1R agonist PRE084 showed
suppressed sympathetic nerve activation with concomitant improvements in cardiac function[11]. However, as noted, our study suggests that SA4503 had no effect on sympathetic nerve
activity. Previously we demonstrated that SA4503 directly stimulates σ1R in cardiomyocytes,
rescuing reduced ATP levels and cardiac dysfunction [8]. Thus, although sympathetic nerve regulation by σ1R may account for some amelioration of cardiac dysfunction, our study suggeststhat glucocorticoid receptors likely have a critical function in TAC-induced depression-likebehaviors. Interestingly, depressive patients exhibit increased secretions of peripheral stress hormones, including adrenal glucocorticoids, and treatment with anti-depressants including SSRIs
normalize that secretion [1]. The plasma levels of cortisol and CORT increase in cardiovasculardisease or depressive patients [25, 34], and prescribed treatment with high-dose glucocorticoids
is associated with increased risk for cardiovascular disease [35]. A previous report also suggested
that inflammatory cytokine levels increase in cardiovascular disease or depressive patients [36,37]. Moreover, HPA axis is activated following stress with concomitant increases in inflammatory cytokine levels, and increased plasma CORT upregulate pro-inflammatory gene expression[38–41]. Dantzer et al. reported that peripheral lipopolysaccharide (LPS) administrationinduced both depression-like behavior and pro-inflammatory cytokine production in mice 24
hr later [42]. Relevant to these findings, we showed that brain CORT levels increased in parallelwith peripheral CORT levels (1.11±0.11 ng/mg tissue (sham) vs. 1.35±0.16 ng/mg tissue (TAC 6
weeks), suggestive of an inflammatory response. Overall, we conclude that peripheral inductionof pro-inflammatory cytokines by prolonged TAC activates the HPA axis, although further
studies are required to define pro-inflammatory cytokines underlying these activities.

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Transverse Aortic Constriction Leads to Depression-Like Behaviors

Depression-like behaviors induced by chronic CORT administration are often accompaniedby concomitant mitochondrial dysfunction, including reduced mitochondrial DNA content
and membrane potential [43]. In Flinders rats, a genetic model of depression, depression-likebehaviors are associated with decreased numbers of mitochondria in hippocampal neurons[44]. Likewise, overall levels of proteins, DNA, and ATP are significantly reduced in depressive
patients [45]. Importantly, σ1R, a MAM-associated protein, and ATP content markedly
decreased in hippocampus following prolonged TAC, and depression-like behaviors are closely
associated with mitochondrial dysfunction in the hippocampus. Here, we focused on σ1R as it
functions as a chaperone for the IP3 receptor, which is critical for ATP production following
mitochondrial Ca2+ transport [8, 10, 24]. Heart muscle consumes large amounts of ATP [46],and SA4503 enhances heart ATP production, thereby decreasing cardiac dysfunction. Moreover, other studies suggest that heart disease is a risk factor of neurological disease including
depression or cognitive dysfunction [3, 47].

Conclusions
Downregulation of hippocampal σ1R expression was induced by increasing plasma CORT levels following heart dysfunction. The TAC model proved useful to study mechanisms linking
cardiovascular disease and depression. We also found that SA4503, a potent and selective σ1R
agonist, ameliorates TAC-induced cardiac dysfunction and depression-like behaviors throughσ1R upregulation. SA4503 has recently been tested as a treatment for depression in stroke
patients in a phase II clinical trial (NCT00639249). Thus, SA4503 is an attractive candidate toprevent depression associated with cardiovascular disease.

Supporting Information
S1 Fig. Effect of paroxetine on depression behaviors. Results of forced swimming test (A)and tail suspension test (B) including paroxetine (Paro) treatment group were shown to compare to SA4503 effect.
(TIFF)
S2 Fig. Effects of TAC and SA4503 treatments on Bip expression. Western blot analyses of
Bip and β-tubulin (as a loading control) proteins in the CA1 region and dentate gyrus (DG) of
sham and TAC mice.
(TIFF)

Acknowledgments
This work was supported in part by grants-in-aid for Scientific Research from the Ministry of
Education, Science, Sports and Culture of Japan (KAKENHI 25293124 and 26102704 to K.F.),the Smoking Research Foundation (K.F.), and from a Research Fellowship from the Japan Society for the Promotion of Science (265570 and 244360 to Y.S. and H.T., respectively).

Author Contributions
Conceptualization: KF MSB.
Data curation: KF.
Formal analysis: MSS.
Funding acquisition: KF.

PLOS ONE | DOI:10.1371/journal.pone.0163992 October 14, 201616 / 19

Transverse Aortic Constriction Leads to Depression-Like Behaviors

Investigation: YS HT.
Methodology: HH HK CZ FH.
Project administration: KF.
Resources: CZ FH.
Software: YS.
Supervision: KF.
Validation: HT.
Visualization: YS.
Writing – original draft: YS HT.
Writing – review & editing: KF.

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PLOS ONE | DOI:10.1371/journal.pone.0163992 October 14, 201619 / 19