Toxicology and Applied Pharmacology 279 (2014) 8–22

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Toxicology and Applied Pharmacology

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Aconitine-induced Ca2+ overload causes arrhythmia and triggersapoptosis through p38 MAPK signaling pathway in rats

Gui-bo Sun a,1, Hong Sun a,1, Xiang-bao Meng a, Jin Hu b, Qiang Zhang b, Bo Liu b, Min Wang a,Hui-bo Xu b,⁎, Xiao-bo Sun a,⁎⁎a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy ofMedical Sciences & Peking Union Medical College, Beijing, 100193, PR Chinab Academy of Chinese Medical Sciences of Jilin Province, Changchun, Jilin 130021, PR China

⁎ Correspondence to: H.-B. Xu, Academy of Chinese MeNo. 1745, Gongnongda Road, Changchun, Jilin, 130021, PR⁎⁎ Correspondence to: X.-B. Sun, Institute of MedicinalChinese Academy of Medical Sciences & Peking UniMalianwaNorth Road, Haidian District, Beijing, 100193, PR

E-mail addresses: xhb_6505@163.com (H. Xu), sun_xi1 Gui-bo Sun and Hong Sun contributed equally to this

http://dx.doi.org/10.1016/j.taap.2014.05.0050041-008X/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 December 2013Revised 1 May 2014Accepted 10 May 2014Available online 17 May 2014

Keywords:AconitineApoptosisArrhythmiaHeartCalcium overloadCardiotoxicity

Aconitine is a major bioactive diterpenoid alkaloid with high content derived from herbal aconitum plants.Emerging evidence indicates that voltage-dependent Na+ channels have pivotal roles in the cardiotoxicity ofaconitine. However, no reports are available on the role of Ca2+ in aconitine poisoning. In this study, we exploredthe importance of pathological Ca2+ signaling in aconitine poisoning in vitro and in vivo. We found that Ca2+

overload lead to accelerated beating rhythm in adult rat ventricular myocytes and caused arrhythmia in con-scious freely moving rats. To investigate effects of aconitine on myocardial injury, we performed cytotoxicityassay in neonatal rat ventricular myocytes (NRVMs), as well asmeasured lactate dehydrogenase level in the cul-turemedium of NRVMs and activities of serum cardiac enzymes in rats. The results showed that aconitine result-ed inmyocardial injury and reducedNRVMs viability dose-dependently. To confirm the pro-apoptotic effects, weperformed flow cytometric detection, cardiac histology, transmission electron microscopy and terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The results showed that aconitinestimulated apoptosis time-dependently. The expression analysis of Ca2+ handling proteins demonstrated thataconitine promoted Ca2+ overload through the expression regulation of Ca2+ handling proteins. The expressionanalysis of apoptosis-related proteins revealed that pro-apoptotic protein expression was upregulated, and anti-apoptotic protein BCL-2 expression was downregulated. Furthermore, increased phosphorylation of MAPK fam-ilymembers, especially the P-P38/P38 ratiowas found in cardiac tissues. Hence, our results suggest that aconitinesignificantly aggravates Ca2+ overload and causes arrhythmia and finally promotes apoptotic development viaphosphorylation of P38 mitogen-activated protein kinase.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Aconitum plants have been widely used to treat various diseases,such as shock caused by acute myocardial infarction, coronary heartdisease and angina pectoris in China for thousands of years (Liou et al.,2005; Shaheen et al., 2005; Singhuber et al., 2009). Numerous herbalmedicines containing aconitum plants as main ingredients have beenformulated. However, the high cardiotoxicity of these compoundsseverely limits their clinical use (Chan, 2012; Kong et al., 2012;Lin et al., 2004). Aconitine, a major bioactive diterpenoid alkaloid

dical Sciences of Jilin Province,China. Fax:+86 431 86058637.Plant Development (IMPLAD),on Medical College, No. 151,China. Fax: +86 10 57833013.

aobo163@163.com (X. Sun).work.

derived from aconitum plants, reportedly contributes primarily to thecardiotoxic effects of aconitum plants (Fu et al., 2007; Wada et al.,2005). Previous studies have mainly focused on the cardiotoxic effectsof aconitine on voltage-dependent Na+ channels (Kunze et al., 1985;Wang and Wang, 2003; Wright, 2002). However, little information isavailable on the role of Ca2+ in aconitine poisoning. Therefore, thepresent study was designed to give a clearer understanding of theimportance of defective Ca2+ signaling in aconitine poisoning in vitroand in vivo.

It is well known that Ca2+ overload has vital roles in the pathogen-esis of heart dysfunctions, especially arrhythmia and apoptosis(Lai et al., 2011; Petersen et al., 2005; Rabkin and Kong, 2003; Soniet al., 2011). Many researchers have reported that Ca2+ plays an impor-tant role in the pathogenesis of arrhythmia and pathological cellularCa2+ overload can lead to an arrhythmogenic state. In other words,arrhythmia is an important event that occurs during aconitine poison-ing. In addition, Ca2+ overload can be involved in cardiac apoptosisand appears to be a principal mediator of apoptosis. However, thepotential role of Ca2+ overload in aconitine-induced cardiotoxicity

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remains largely unknown. In our research, we investigated the alter-ations of Ca2+ level in cardiomyocytes induced by aconitine treatmentand explored whether Ca2+ overload could cause arrhythmia andtrigger apoptosis.

Materials and methods

Materials

Aconitine (content ≥ 98%) was purchased from the National Insti-tute for the Control of Pharmaceutical and Biological Products (China).The molecular weight of aconitine is 645.74. Collagenase Type II andFura-2/AM were purchased from Life Technologies Corporation(Carlsbad, CA, USA). The kits for determining total creatine kinase(CK), aspartate aminotransferase (AST), and LDH (lactate dehydroge-nase) were obtained from Biosino Bio-Technology and Science Incorpo-ration (Hong Kong, China). Annexin V/propidiumiodide (PI) apoptosisdetection kit was obtained from Life Technologies Corporation (Carls-bad, CA, USA). The terminal deoxynucleotidyl transferase-mediateddUTP-biotin nick end labeling (TUNEL) assay kit was purchased fromRoche Diagnostics (Mannheim, Germany). Primary antibodies againstRyR, SERCA, NCX, BCL-2, BAX, P53, caspase-9, caspase-3, ERK, P-ERK,P38 and P-P38 were obtained from Santa Cruz Biotechnology (SantaCruz, CA, USA). The anti-rabbit-conjugated horseradish peroxidaseantibody was purchased from Zhongshan Goldbridge Biotechnology(Beijing, China). The Bradford protein assay kit was purchased fromPierce Corporation (Rockford, USA) and super-enhanced chemi-luminescence detection reagents were purchased from ApplygenTechnologies (Beijing, China). All of the chemical reagents wereobtained from Sigma Chemical Co., Ltd (St. Louis, MO, USA).

Animals and treatments

Eighty adult male Wistar rats (Vital River Laboratories, Beijing,China) weighing 220 to 240 g were used and the procedures were ap-proved by the local animal committee. These rats were kept at standardroom temperature (22± 2 °C) and relative humidity (60%± 10%) witha 12 h light/dark cycle. All of the rats were allowed free access to foodand water ad libitum during the acclimatization and experimentalperiod. The experiments were performed in accordancewith the guide-lines of the Experimental Laboratory Animal Committee of ChineseAcademy of Medical Sciences and Peking Union Medical College andthe principles and guidelines of the National Institutes of Health Guidefor the Care and Use of Laboratory Animals.

Rats were randomly divided into the following two groups: groupA as control and group B as aconitine model. Forty male Wistar ratswere designated for each group. Ten rats in each group were implantedwith telemetry transmitters for ECG study.

After 1-week of acclimatization, the aconitine model was inducedwith the protocol described below. Group B orally received0.146 mg/mL aconitine, diluted in 0.05 N hydrochloric acid, once aday by gavage at 10 mL/kg for 10 consecutive days. Group A wasgiven vehicle instead of aconitine once a day by gavage at 10 mL/kgfor 10 consecutive days.

On day 3 or day 6 post-aconitine administration, 10 rats in eachgroup were anesthetized with sodium pentobarbital (50 mg/kg, i.p.),and their hearts were removed rapidly. The left ventricle was excisedfor hematoxylin-eosin (HE) staining, transmission electron microscopy(TEM) and TUNEL examination. Myocardial homogenates were thenprepared for Western blot analysis. On day 10 after the last administra-tion of aconitine, blood was collected from the left ventricle and centri-fuged at 800 ×g for 10 min to obtain serum, which was kept at−80 °Cuntil analysis. Subsequently, the hearts were removed rapidly. Theleft ventricle was excised for HE, TEM and TUNEL examination. Themyocardial homogenates were prepared for Western blot analysis.

Isolation of adult rat ventricular myocytes and treatments

Individual adult rat ventricular myocytes (ARVMs) were isolatedfrom 13-week-old Wistar rats as described previously (Westfall andBorton, 2003; Westfall et al., 1997). After the rats were anesthetizedwith ketamine/xylazine (0.1 mL/100 g, i.p.), their hearts were removedand perfused through the aorta cannula with a series of differentperfusion solutions at the rate of 6 mL/min. First, the hearts wereperfused with Ca2+-containing Tyrode's solution [NaCl, 137; KCl, 5.4;MgCl2, 1.2; HEPES, 10; glucose, 10; and CaCl2, 1.2 (in mM)] for 2 min.Subsequently, the hearts were perfused with Ca2+-free Tyrode'ssolution containing with the aforementioned components exceptCaCl2 for 5min, followed by a 20min perfusionwith Ca2+-free Tyrode'ssolution containing collagenase Type II (210.00 units/mg). After perfu-sion, the left ventricles were removed, minced, and filtered through anylon mesh (300 mm). The filtered myocytes were then washed withCa2+-containing Tyrode's solution to restore the extracellular Ca2+

concentration to 1.2mM. ARVMswere assayed by trypan blue exclusionassay for viability. Viability was over 80%. Only rod-shaped ARVMswithclear edges were used in this study.

Measurement of beating rhythm, sarcomere shortening, and Ca2+

transients in ARVMs

Beating rhythm, sarcomere shortening and Ca2+ transients of intactARVMs at room temperature were assessed simultaneously upon fieldstimulation (0.5 Hz with 2-ms-duration, 16 V) using a video-basedsarcomere contractility and calcium recording module in a SoftEdgeMyoCam system (IonOptix Corporation, Milton, MA, USA). IsolatedARVMs were loaded with fura-2/AM (2 μM) for 15 min and washedtwice with Ca2+-containing Tyrode's solution after restoration of theextracellular Ca2+. ARVMs were then placed in a Warner chambermounted on the stage of an inverted microscope (Olympus, IX-70)and superfused with Ca2+-containing Tyrode's solution with aconitine(1 μM), verapamil (10 μM) or verapamil (10 μM) + aconitine (1 μM)at the rate of 1.5 mL/min. Verapamil is a specific L-type Ca2+ channelblocker. Beating rhythm, sarcomere shortening and Ca2+ transientswere recorded in 10 min intervals after the addition of aconitine(1 μM), verapamil (10 μM) or verapamil (10 μM) + aconitine (1 μM).Before measurement, isolated ARVMs were given 10 min for baselinestabilization. Data were recorded and analyzed with IonWizardsoftware (version 6.2.0.59).

Electrocardiography

Electrocardiography (ECG) recordings were taken before aconitineor vehicle treatment and 24 h after each aconitine or vehicle treatmentin conscious freely moving rats. Ten rats in each group were implantedwith telemetry transmitters (HD-S21) of Data Sciences International(St. Paul, MN, USA) for ECG collection. After the rats were anesthetizedwith sodium pentobarbital (50 mg/kg, i.p.), HD-S21 transmitters wereplaced in the abdominal cavity of the rats and fixed to the inner perito-neal wall using silk sutures. All skin incisions were closed using woundclips under sterile conditions. After 2-week surgical recovery, ECG wasmeasured for ten days. When we switched the mode to ON, the trans-mitters began to sense and transmit data. ECG data were acquiredusing Dataquest A.R.T. 4.31 software (Data Sciences International). Theraw ECG data were then analyzed by DSI's Ponemah PhysiologyPlatform software. This technique facilitated the collection of ECGrecordings in themost reliable and efficientmethodwithout anesthetiz-ing rats.

Cell culture and aconitine treatment

Primary cultures of NRVMs were prepared as described previously(Gray et al., 1998). NRVMs were isolated from the hearts of 1-day-old

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Wistar rats by trypsin and collagenase digestion, purified by differentialpreplating, andmaintained in DMEMwith 10% (v/v) fetal bovine serumin a humidified incubator of 95% air and 5% CO2 at 37 °C. Two sets ofexperiments were performed: (1) control NRVMs; (2) NRVMs treatedwith indicated concentrations (0.01, 0.04, 0.16, and 0.64 μM) ofaconitine for 4 h.

Cell viability analysis

Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. NRVMs were plated on 96-wellplates at a density of 1 × 104 cells/well. NRVMs were treated withdifferent concentrations (0.01, 0.04, 0.16, and 0.64 μM) of aconitine for4 h. Subsequently, 20 μL MTT (5 mg/mL) was added to each well,which was subsequently incubated for 4 h. The medium was thenremoved, and the formazan crystals were dissolved with dimethylsulfoxide. Absorbance was read at 570 nm on a microplate reader(MQX 200, BioTek Instruments, Winooski, VT, USA).

Measurement of LDH level in the culture medium of NRVMs

NRVMs were cultured in six-well plates at 3 × 105 cells/well. Thesupernate was collected at different concentrations (0.01, 0.04, 0.16,and 0.64 μM) of aconitine treatments to measure the LDH level usingthe detection kit according to the manufacturer's instructions.

Serum analysis

We measured serum samples using kits (Biosino Bio-Technologyand Science Incorporation, Hong Kong, China) to determine serumcardiac enzymes (CK, AST, and LDH) through an automatic biochemicalanalyzer (Hitachi 7600, Tokyo, Japan).

Flow cytometric detection in ARVMs

The percentage of early apoptosis and necrosis was measured inisolated ARVMs using Annexin V-FITC/PI apoptosis kit for flow cytome-try according to the manufacturer's brochures (Life TechnologiesCorporation). The isolated ARVMs were treated with aconitine (1 μM)or SB203580 (1 μM) + aconitine (1 μM) for 12 h in 6-well plates.SB203580 is a specific inhibitor of P38 MAPK. During treatment, wepaced our ARVMs upon field stimulation (0.5 Hz with 2-ms-duration,30 V) using C-Pace EP, a ARVMs stimulator designed by IonOptix Corpo-ration (Milton, MA, USA), to keep the normal functional characteristicsof ARVMs. After treatment, ARVMs were harvested and washed twicewith cold PBS, and then incubated with 5 μL FITC-Annexin V and 1 μLPIworking solution (100 μg/mL) for 15min in the dark at room temper-ature. Cellular fluorescence was measured by flow cytometry analysiswith a flow cytometer (FACS Calibur™, BD Biosciences, CA, USA).

Histological analysis

Five-micrometer-thick sections of formalin-fixed and paraffinembedded cardiac tissues were processed routinely for HE stainingand examined under a light microscope (CKX41, Olympus, Tokyo,Japan) by a pathologist blinded to the groups studied.

Transmission electron microscopy examination

Preparations for TEM were made in the following manner (Dabouret al., 2005). Samples were cut into small pieces (0.8 mm3–1.0 mm3).The small samples were first fixed with 2.5% glutaraldehyde for morethan 4 h and then washed thrice in phosphate buffer. They werepostfixed with 1% osmic acid for 1 h and washed thrice in phosphatebuffer. Subsequently, samples were dehydrated with a graded seriesof ethanol from 50% to 100%, followed by infiltration of a 1:1 mixture

of acetone and embedding resin for 1 h, 1:3 mixture of acetone andembedding resin for 3 h and 100% embedding resin overnight. Sampleswere then placed in the embeddingmedium and heated at 70 °C for 9 h.Ultrathin sample sections (50 nm) were stained with uranyl acetate,followed by lead citrate for 15 min after which they were observedusing the TEM Model Hitachi H-7000.

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling assay

DNA fragmentation was detected in cardiomyocytes using theTUNEL staining kit according to the manufacturer's instructions. Frozentissue sections were fixed with fixation solution (4% paraformaldehydein PBS, pH 7.4, freshly prepared) for 20 min at 20 °C and washed for30 min with PBS. They were incubated with blocking solution for10 min at 20 °C and rinsed with PBS. The tissue sections were thenincubated in permeabilization solution for 2 min on ice and rinsedtwice with PBS. Then, 50 μL TUNEL reaction mixture was added to thesampleswhichwere incubated for 60min at 37 °C in thedark and rinsedthrice with PBS. The samples were observed under a fluorescencemicroscope (Leica DM4000, Germany).

Western blot analysis

Total tissue proteins were isolated in 10 mM Tris-HCl (pH 7.4), 150mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40,10 mM DTT, and 0.5% sodium deoxycholate and protease inhibitorCocktail Set I. Protein concentration was determined using the Bradfordprotein assay kit. Up to 50 mg of protein was loaded onto sodiumdodecyl sulfate polyacrylamide gel electrophoresis gels, transferred topolyvinylidene difluoride membrane, and blocked with 5% nonfatdry milk in 1× Tris-buffered saline (TBS), and 0.1% Tween 20 for 1 h.Incubation with the primary antibody from Santa Cruz Biotechnologywas performed overnight at 4 °C. Incubation with a 1:5000 secondaryanti-rabbit-conjugatedhorseradish peroxidase antibodywasperformedat 37 °C for 1 h. After washing in 1× TBS, and 0.1% Tween-20, proteinbands were visualized by super-enhanced chemiluminescence detec-tion reagents and exposed to Kodak film.

Statistics

Quantitative data were expressed as mean ± S.D. and compared byANOVA with post hoc comparisons or by Kruskal–Wallis test whenvariances were heterogeneous. Data were considered significant whenP b 0.05.

Results

The beating rhythm, sarcomere shortening, and Ca2+ transient in ARVMstreated with aconitine

We treated freshly isolated ARVMs with aconitine (1 μM) toexamine the time-course effects of aconitine on beating rhythm, andsarcomeric contractile function. Our results showed that aconitine didnot affect the normal rhythm of ARVMs in the initial period of aconitinetreatment, but aconitine induced continuous increases in amplitude ofsarcomere shortening and peak shortening (both P b 0.01) withoutaffecting resting sarcomere length in 4 min after 1 μM aconitine wasadded (Figs. 2A–C). Aconitine also induced sarcomeric diastolicdysfunction in 7 min in a time-dependent manner (Figs. 1A and C).Compared with those at 0 min, amplitude of sarcomere shorteningand peak shortening significantly increased, but the resting sarcomerelength significantly decreased at 7 min after aconitine perfusion wasperformed, suggesting the occurrence of diastolic dysfunction (P b 0.01,for all three parameters) (Figs. 2A–C). The time taken for 90% relaxationsignificantly decreased at 7min comparedwith that at 0min, indicating

Fig. 1. Sarcomere shortening and Ca2+ transient were recorded simultaneously from the left ventricular myocytes after aconitine perfusion using SoftEdge MyoCam system.(A) Time course of sarcomere shortening. The red line indicates the addition of aconitine (1 μM). (B) Time course of Ca2+ transient. The red line indicates the addition ofaconitine (1 μM). (C) Representative traces of sarcomere shortening are shown at different time points (0, 4, and 7 min) with the same time interval (5 s). (D) Representativetraces of Ca2+ transient are shown at different time points (0, 4, and 7 min) with the same time interval (5 s).

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faster relaxation of ARVMs (P b 0.01) (Fig. 2H). Most importantly, therhythm of ARVMs became faster and arrhythmic. The beating rate was15 beats/10 s at 7 min, which was two times higher than those at 0min (Fig. 2I). These changes suggested that the normal rhythm anddiastolic function of ARVMs were disrupted by aconitine in 7 min in atime-dependent manner.

Cytoplasmic Ca2+ transientwas determined in fura-2 loadedARVMsto evaluate the time-course effects of aconitine on intracellular Ca2+

homeostasis. Our results showed that aconitine significantly increasedresting Ca2+ ratio, amplitude of Ca2+ ratio and amplitude/restingcalcium in 4 min after 1 μM aconitine was added (P b 0.01, for allthree parameters) (Figs. 2D–F). Aconitine also induced sustainedincreases in cytoplasmic Ca2+ ratio in 7 min in a time-dependentmanner (Figs. 1B and D). Compared with those at 0 min, the restingCa2+ ratio, amplitude of Ca2+ ratio, and amplitude/resting calciumincreased significantly at 7min after aconitine perfusionwas performed(P b 0.01, for all three parameters) (Figs. 2D–F). These changes

suggested that aconitine leads to Ca2+ overload in ARVMs in 7 min ina time-dependent manner.

The beating rhythm, sarcomere shortening, and Ca2+ transient in ARVMstreated with verapamil

We treated freshly isolated ARVMswith L-type Ca2+ channel blockerverapamil (10 μM) to examine the time-course effects of verapamil onbeating rhythm, and sarcomeric contractile function. Our results showedthat verapamil did not affect the normal beating rhythm of ARVMs in thewhole period of verapamil treatment (Fig. 4 I), but verapamil inducedcontinuous decreases in amplitude of sarcomere shortening and peakshortening (both P b 0.01) in 7 min after verapamil was added (Figs. 3Aand C; Figs. 4B and C). The time taken for 90% relaxation significantly in-creased in 7 min compared with that at 0 min, indicating slower relaxa-tion of ARVMs (P b 0.01) (Fig. 4H). These changes suggested that thenormal beating rhythm of ARVMs was not disrupted and the sarcomeric

Fig. 2. Properties of sarcomere shortening andCa2+ transient in left ventricularmyocytes after aconitine (1 μM)perfusion. (A) Resting sarcomere length (μm). (B) Amplitude of sarcomere shortening (μm). (C) Peaking shortening (% of sarcomere length).(D) Resting calcium ratio (F340/F380). (E) Amplitude of calcium ratio (F340/F380). (F) Amplitude/resting calcium(%). (G) Time taken for 50% relaxation (ms). (H) Time taken for 90% relaxation (ms). (I) Beating rate (beats/10 s). Values for each group arepresented as mean ± S.D. (n = 10). *P b 0.05 compared with the control group; **P b 0.01 compared with the control group.

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Fig. 3. Sarcomere shortening andCa2+ transientwere recorded simultaneously from the left ventricularmyocytes after verapamil perfusion using SoftEdgeMyoCam system. (A) Time courseof sarcomere shortening. The red line indicates the addition of verapamil (10 μM). (B) Time course of Ca2+ transient. The red line indicates the addition of verapamil (10 μM). (C) Represen-tative traces of sarcomere shortening are shown at different time points (0, 4, and 7min)with the same time interval (5 s). (D) Representative traces of Ca2+ transient are shown at differenttime points (0, 4, and 7 min) with the same time interval (5 s).

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contractile function decreased continually by verapamil in 7 min in atime-dependent manner.

Cytoplasmic Ca2+ transientwas determined in fura-2 loadedARVMsto evaluate the time-course effects of verapamil on intracellular Ca2+

homeostasis. Verapamil significantly decreased the resting Ca2+ ratio,amplitude of Ca2+ ratio and amplitude/resting calcium (P b 0.05, forall three parameters) in 4 min after verapamil was added (Figs. 4D–F).Verapamil also induced sustained decreases in cytoplasmic Ca2+ ratioin 7 min in a time-dependent manner (Figs. 3B and D). Comparedwith those at 0 min, the resting Ca2+ ratio, amplitude of Ca2+ ratio,and amplitude/resting calciumdecreased significantly at 7min after ve-rapamil perfusion was performed (P b 0.01, for all three parameters)(Figs. 4D–F). These changes suggested that the intracellular Ca2+

homeostasis of ARVMs was disrupted and decreased continually byverapamil in a time-dependent manner.

The beating rhythm, sarcomere shortening, and Ca2+ transient in ARVMstreated with verapamil + aconitine

We treated freshly isolated ARVMs with verapamil (10 μM) +aconitine (1 μM) to examine the time-course effects of verapamil +aconitine on beating rhythm, and sarcomeric contractile function.Compared with the time-course effects of single administration ofaconitine on sarcomere shortening, coadministration of verapamil andaconitine led to the similar continuous increases in amplitude of sarco-mere shortening and peak shortening (both P b 0.01) without affectingresting sarcomere length in 7 min (Figs. 6A–C). The amplitude of sarco-mere shortening and peak shortening in the verapamil + aconitinegroup increased to a smaller extent than that in the aconitine group atdifferent time points. Most importantly, in the verapamil + aconitinegroup theduration of normal beating rhythmof ARVMswasmaintained

Fig. 4. Properties of sarcomere shortening and Ca2+ transient in left ventricularmyocytes after verapamil (10 μM) perfusion. (A) Resting sarcomere length (μm). (B) Amplitude of sarcomereshortening (μm). (C) Peaking shortening (% of sarcomere length). (D) Resting calcium ratio (F340/F380). (E) Amplitude of calcium ratio (F340/F380). (F) Amplitude/resting calcium (%).(G) Time taken for 50% relaxation (ms). (H) Time taken for 90% relaxation (ms). (I) Beating rate (beats/10 s). Values for each group are presented asmean±S.D. (n=10). *P b 0.05 comparedwith the control group; **P b 0.01 compared with the control group.

14 G. Sun et al. / Toxicology and Applied Pharmacology 279 (2014) 8–22

longer and the disrupted contractile function appeared later thanthose in the aconitine group (Figs. 5A and C). The beating rhythm ofARVMs in the verapamil + aconitine group became arrhythmic andthe contractile function was disrupted until 12 min after coadmin-istration of verapamil and aconitine (Figs. 5A and C; Fig. 6 I).

Cytoplasmic Ca2+ transientwas determined in fura-2 loadedARVMsto examine the time-course effects of verapamil + aconitine on intra-cellular Ca2+ homeostasis. Compared with the time-course effects ofsingle administration of aconitine on intracellular Ca2+ homeostasis,coadministration of verapamil and aconitine led to the similar butsmaller continuous increases in resting Ca2+ ratio, amplitude of Ca2+

ratio and amplitude/resting calcium in 7 min (P b 0.05 or P b 0.01)(Figs. 6D–F). The resting Ca2+ ratio, amplitude of Ca2+ ratio and ampli-tude/resting calcium in the verapamil + aconitine group increased to asmaller extent than those in the aconitine group at different time points.Most importantly, in the verapamil + aconitine group the duration ofnormal calcium transient was maintained longer and the calcium over-load appeared later than in the aconitine group (Figs. 5B and D). Therhythm of calcium transient in the verapamil + aconitine group becamefaster and the calcium overload appeared until 12 min (Fig. 5D; Fig. 6D).

Electrocardiography

In our study, ventricular tachycardia and ventricular prematurebeats were confirmed in conscious freely moving rats by ECG afteraconitine treatment. Data analysis showed that ST segment heightwas significantly elevated on days 5 and 7 post-aconitine treatment(P b 0.05, for day 5; P b 0.01, for day 7) (Fig. 7E). After aconitinetreatment for 7 days, the significance of the ECG parametersdeclined because of the extreme abnormality in ECG (Fig. 7A). Noabnormality in the ECG of the control group was observed throughoutthe study.

Cell viability

The harmful effects of aconitine-induced cytotoxicity in NRVMswere detected by MTT assay. Aconitine exhibited strong dose-dependent cytotoxic effects (Fig. 8A). The survival rate decreased to94 ± 3.76%, 87 ± 2.21%, 79 ± 2.43%, and 71 ± 5.64% with 0.01, 0.04,0.16, and 0.64 μM aconitine, respectively.

Fig. 5. Sarcomere shortening and Ca2+ transient were recorded simultaneously from the left ventricular myocytes after verapamil+ aconitine perfusion using SoftEdgeMyoCam system.(A) Time course of sarcomere shortening. The red line indicates the addition of verapamil (10 μM)+ aconitine (1 μM). (B) Time course of Ca2+ transient. The red line indicates the additionof verapamil (10 μM) + aconitine (1 μM). (C) Representative traces of sarcomere shortening are shown at different time points (0, 4, and 7 min) with the same time interval (5 s).(D) Representative traces of Ca2+ transient are shown at different time points (0, 4, and 7 min) with the same time interval (5 s).

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LDH level in the culture medium of NRVMs

To assay the effects of aconitine on cardiac injury, we measured thelevel of cardiac enzyme LDHwhichwas a generally accepted indicator ofcell injury in the supernate of cultured NRVMs. Aconitine increased LDHlevels in the culture medium in a concentration-dependent manner(Fig. 8B; P b 0.05 for 0.01, and 0.04 μM; P b 0.01 for 0.16, and0.64 μM). The LDH levels increased to 422.98 ± 31.76, 485.39 ±52.60, 539.06 ± 27.27, and 624.93 ± 14.31 U/L with 0.01, 0.04,0.16, and 0.64 μM aconitine, respectively. This effect was incon-sistent with its harmful effect on cell viability, which was assessedby MTT assay.

Serum cardiac enzymes

Activities of the serum cardiac enzymes (CK, LDH, and AST) signifi-cantly increased after the 10th treatment of aconitine (P b 0.05 for

AST; P b 0.01 for CK and LDH) (Fig. 8C). Of these three parameters,more than 3-fold increase in LDH was found after aconitine treatmentcompared with the control group. CK content approximately increasedby 2-fold after aconitine treatment. AST serum concentration of themodel group increased by approximately 1.4-fold compared with thecontrol group.

Flow cytometric detection

In normal ARVMs, phosphatidyl serine (PS) is located on thecytoplasmic surface of the cell membrane. However, in apoptoticARVMs, PS is located on the outer leaflet of the plasma membrane.Annexin V, which has a high affinity for PS, labeled with a fluorophorecan identify apoptotic ARVMs by binding to PS.

In the normal control group, the percentage of apoptotic ARVMswas5.2%; however, the percentage of apoptotic ARVMs increased up to75.9% in the aconitine model group (Fig. 9). SB203580 significantlyinhibited the percentage of apoptotic ARVMs compared with the

Fig. 6. Properties of sarcomere shortening and Ca2+ transient in left ventricular myocytes after verapamil (10 μM)+ aconitine (1 μM). (A) Resting sarcomere length (μm). (B) Amplitudeof sarcomere shortening (μm). (C) Peaking shortening (% of sarcomere length). (D) Resting calcium ratio (F340/F380). (E) Amplitude of calcium ratio (F340/F380). (F) Amplitude/restingcalcium (%). (G) Time taken for 50% relaxation (ms). (H) Time taken for 90% relaxation (ms). (I) Beating rate (beats/10 s). Values for each group are presented as mean ±S.D. (n = 10). *P b 0.05 compared with the control group; **P b 0.01 compared with the control group.

16 G. Sun et al. / Toxicology and Applied Pharmacology 279 (2014) 8–22

aconitine group (P b 0.01). The percentage of apoptotic ARVMsdecreased to 27.8% in the SB203580 + aconitine group.

HE staining

An overall view of apoptosis in the heart tissues at the lightmicrosco-py level is shown in Fig. 10. In the control group, no obvious cellular de-generation and apoptosis were found, whereas severe myocardialdamage and apoptosis were observed in the aconitine model group.Time-course studies of pathological damage suggested that apoptoticlevels were not significantly changed before aconitine treatment, butmarkedly increased during aconitine treatment. These apoptotic changeswere characterized by the increase of apoptotic cardiomyocytes, intenseinfiltration with neutrophil granulocytes, and morphological abnor-malities of cardiomyocytes and myocardial tissues including darker(hyperchromatic) andmore crowded nuclei, condensation of cytoplasmin apoptotic cardiomyocytes, disorganization of myofibrillar arrays.

Transmission electron microscopy

Aconitine treatment induced a significant amount of apoptosisand extensive ultrastructural changes in the morphology of cardiac

myocytes compared with the control group. TEM images showedanomaly of the nucleus and increase of heterochromatin,mitochondrialdisorganization of cristae, irregularity and disappearance of myofila-ments and disruption of sarcomeres (Figs. 11C and D).

TUNEL staining assay

Apoptosis in cardiac tissues was further examined using TUNELstaining assay which identified specifically apoptotic cells. Aconitinetreatment significantly increased the number of apoptotic cells com-pared with the control group (Fig. 12A). The percentage of TUNEL-positive staining cells increased from 4.0% ± 1.3% to 58.0% ± 2.2%(P b 0.01) (Fig. 12B).

The expression of Ca2+ handling and apoptosis-related proteins in rats

RyR and NCX protein levels increased obviously over time in theaconitine model group, reflecting the upregulation of RyR and NCXprotein expression. RyR and NCX protein levels significantly increasedon days 6 and 10 post-aconitine treatment (P b 0.05, for day 6; Pb 0.01, for day 10) (Fig. 13A). Contrary to the changes in RyR and NCXprotein levels, SERCA protein level was inhibited by aconitine over

Fig. 7. Effects of aconitine treatment on ECG and its parameters in conscious freely moving rats. (A) The arrhythmias monitored in ECG using implantable telemetry. (B) RR interval (ms).(C) QRS (ms). (D)Heart rate (beats/min). (E) ST segment height (mV). Values for each group are presented asmean± S.D. (n=10). *P b 0.05 comparedwith the control group; **P b 0.01compared with the control group.

Fig. 8. Effects of aconitine on cardiac injury in myocardial cells and heart tissues. (A) Evaluation of cytotoxicity in NRVMs was detected at different concentrations (0.01, 0.04, 0.16, and0.64 μM) by MTT assay. (B) LDH level in the supernate of cultured NRVMs. LDH level was detected at different concentrations (0.01, 0.04, 0.16, and 0.64 μM) using the detection kitaccording to the manufacturer's instructions. (C) Concentrations of CK, LDH, and AST in cardiac tissues. Values for each group are presented asmean± S.D. (n= 10). *P b 0.05 comparedwith the control group; **P b 0.01 compared with the control group.

17G. Sun et al. / Toxicology and Applied Pharmacology 279 (2014) 8–22

Fig. 9. Effects of aconitine on apoptosis in ARVMs. ARVMs stained with FITC-Annexin V-PI were measured by flow cytometry. Values of apoptotic ratios for each group are presented asmean ± S.D. (n = 6). **P b 0.01 compared with the control group. ##P b 0.01 compared with the aconitine model group.

18 G. Sun et al. / Toxicology and Applied Pharmacology 279 (2014) 8–22

time. SERCA protein level was significantly inhibited on days 6 and 10post-aconitine treatment (both P b 0.05) (Fig. 13A).

The expression results of essential apoptosis-related proteinsshowed that aconitine treatment (1.46 mg/kg, 10 mL/kg, once a dayfor 10 days) upregulated the expression of P53, BAX, caspase-9, andcaspase-3, as well as downregulated the expression of BCL-2 on days6 and 10 post-aconitine treatment (P b 0.01, for P53 on days 6 and 10;

Fig. 10. Time course of pathological changes in cardiac tissues examined byHE staining. (A) Cona time-dependent manner. On day 6 and day 10 post-aconitine administration, there were obv

P b 0.05, for caspase-9 and caspase-3 on day 6; P b 0.01, for caspase-9and caspase-3 on day 10) (Figs. 13B and C). The ratio of BCL-2/Baxwas also significantly decreased compared with the control group(P b 0.01) (Fig. 13B).

The protein expression of important members (ERK, P-ERK, P38 andP-P38) in the MAPK family of cardiac tissues showed that aconitineincreased the expression and phosphorylation of ERK and P38 compared

trol. (B) Day 3. (C) Day 6. (D) Day 10. Scale bar: 500 μm. Aconitine induced cardiac injury inious pathological changes in cardiac tissues.

Fig. 11. TEM images of cardiac tissues in rats. Panels A andB: control group; Panels C andD: aconitinemodel group; tissues in Panels A (×5000) and B (×15000) are from the control group.Tissues show regular structure, mitochondria, and myofilaments; tissues in Panels C (×5000) and D (×12000) are from the aconitine model group. Tissues show altered mitochondria,irregular myofilaments, and disrupted sarcomeres.

19G. Sun et al. / Toxicology and Applied Pharmacology 279 (2014) 8–22

with the control group. The P-P38/P-38 ratio in the aconitine group wassignificantly higher than that in the control group on days 6 and 10 post-aconitine treatment (both P b 0.01) (Fig. 13D).

Discussion

In the present study, we examined the effects of aconitine on intra-cellular Ca2+ homeostasis and determined aconitine-induced Ca2+

Fig. 12. Aconitine promoted myocardial apoptotic responses determined by TUNEL labeling. (AFITC are shown in green; cell nuclei counterstained with DAPI were shown in blue (scale bTUNEL-positive cells to total cells. Values for each group are presented as mean ± S.D. (n = 6)

overload could cause arrhythmia and trigger apoptosis through p38MAPK signaling pathway. It is well known that Ca2+ is a ubiquitousintracellular signal molecule responsible for controlling several cellularprocesses, such as proliferation, differentiation, development and celldeath (Bers, 2008; Giorgi et al., 2012; Griffiths, 2009; Schaub et al.,2006).Many studies have demonstrated that cellular Ca2+ is implicatedin the pathogenesis of heart dysfunctions, especially arrhythmia andapoptosis (Biagioli et al., 2008; Ermak and Davies, 2002; Heinzel et al.,

) Time course of apoptotic responses in cardiac tissues. The nuclei of apoptotic cells withar: 50 μm). (B) The TUNEL apoptotic index was determined by calculating the ratio of. **P b 0.01 compared with the control group.

Fig. 13. Effects of aconitine on the expression of Ca2+ handling and apoptosis-related proteins in cardiac tissues. (A) Time course of the expression of Ca2+ handling proteins detected byWestern blots. (B) Time course of the expression of BCL-2, BAX,and P53 detected by Western blots. (C) Time course of the expression of Caspase-9 and Caspase-3 detected by Western blots. (D) Time course of the expression of ERK, P-ERK, P38, and P-P38 detected by Western blots. Values for each group arepresented as mean ± S.D. (n = 6). *P b 0.05 compared with the 0 day; **P b 0.01 compared with the 0 day.

20G.Sun

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2011; Rogalska et al., 2013). In this paper, we studied the correspondingchanges in sarcomere shortening and cytoplasmic Ca2+ level by com-paring the effects of the presence and absence of aconitine, verapamil,or verapamil + aconitine in ARVMs, respectively. These findings sug-gested that Ca2+ transient and contractility in excitation–contractioncoupling would change correspondingly. In addition, these resultsprovided strong evidence for the rapid cellular action of aconitine onL-type Ca2+ channels. These results also confirmed aconitine-inducedCa2+ overload could lead to the pathogenesis of arrhythmia and L-type Ca2+ channel blocker verapamil could delay the toxic effectsof aconitine. The results of ECG in conscious freely moving rats alsoconfirmed the arrhythmogenic effects of aconitine in vivo.

In previous studies, it has been confirmed that aconitine preventsopen Na+ channels from inactivating and causes persistent activationof Na+ channels, thereby leading to sustained Na+ influx (Kunzeet al., 1985;Wang andWang, 2003;Wright, 2002). Our work suggestedthat aconitine also induced Ca2+ overload and caused cardiac arrhyth-mia. Therefore, based on the fact that aconitine targets sodium channelsand our present results, we suggested that in ARVMs, the reason calci-um overload took place in 7 min after aconitine perfusion was becausesustained depolarization of sodium channels activated L-type calciumchannels. This led to the rapid increases in the intracellular calciumconcentration. In the heart tissues treatedwithmultiple aconitine treat-ment for 10 days, the upregulation of RyR andNCX and downregulationof SERCA suggested the temporal influence of aconitine on the expres-sion of calcium handling proteins. So we thought that the immediatecalcium overload in ARVMs was caused by sustained depolarization ofsodium channels and the changes in protein expression of calciumhandling proteins which were a downstream consequence of immedi-ate calcium overload may have contributed to the chronic elevation ofcalcium in the heart tissues.

Apoptosis has an essential function in the pathogenesis of cardiovas-cular diseases and contributes to the development of cardiovasculardisorders (Copaja et al., 2011; Feuerstein et al., 1997; Garg et al., 2005;Kitsis and Mann, 2005). Ca2+ overload can cause activation of the in-trinsic apoptotic pathway and activate a series of important signalmolecules (Isomura et al., 2013; Jan et al., 2013; Singh et al., 2010). Inthe present study, we found that aconitine promoted the apoptoticresponse of hearts in vitro and in vivo. We used flow cytometry toidentify apoptosis in ARVMs, and performed HE staining, TEM andTUNEL assay to identify apoptosis in cardiac tissues after varied periodsof aconitine treatment. The four distinct in vitro and in vivo methodsconfirmed myocardial injury and myocyte apoptosis in the modelgroup as time progressed, suggesting that Ca2+ overload resultedin apoptotic responses in time-dependent manner. To the best of ourknowledge, this study is the first to investigate the pro-apoptotic toxic-ity of aconitine using animal experiments in vitro and in vivo.

We examined the expression of apoptosis related proteins includingBCL-2 family proteins (BCL-2 and BAX), P53, two essential caspases(caspase-9 and caspase-3), and MAPKs (ERK, P-ERK, P-38, and P-P38)in heart tissues by Western blot analysis to gain further evidence ofthe signaling events involved in aconitine-induced apoptosis. BCL-2family proteins have been implicated as major regulators of apoptosisin many cells (Clerk et al., 2003; Hardwick et al., 2012; Laulier andLopez, 2012). The BCL-2/BAX ratio is important in regulating cellapoptosis. The decrease in BCL-2/BAX ratio may promote the activationand development of apoptosis. Our results showed that Ca2+ overloaddecreased the BCL-2/BAX ratio compared with the control group.Nuclear transcription factor P53 is another key regulator of Ca2+

overload-mediated apoptosis (Aylon and Oren, 2007; Hickman et al.,2002; Schuler and Green, 2005; Vousden and Prives, 2009). Onceactivated, P53 increases the transcription of pro-apoptotic genes, andmay activate BAX directly. In our study, Ca2+ overload-inducedapoptosis was accompanied by upregulated P53 expression. In addition,both caspase-9 (an initiator caspase) and caspase-3 (a critical effectorcaspase) expression were tested to determine the effects of Ca2+

overload upon caspase cascades. The results showed that Ca2+ overloadpromoted the expression of caspase-9 and caspase-3. Furthermore, weexamined the expression of ERK, P-ERK, P38, and P-P38 belonging tothe MAPK family which has a pivotal function in cell proliferation,differentiation, transformation, and apoptosis to discover the down-stream signal molecules for the activation of apoptosis via Ca2+

overload (Cano and Mahadevan, 1995; Kocieniewski et al., 2012;Krifka et al., 2012). Our results suggested that apoptosis was aggravatedby the upregulation of expression and phosphorylation of MAPK familymembers, especially the P-P38/P38 ratio. The flow cytometricdetection also showed the percentage of apoptotic ARVMs in theSB203580 + aconitine group was significantly lower than that inthe aconitine group. SB203580 is a specific inhibitor of P38 MAPK.Our results proved that aconitine induced ARVM apoptosis through theP38 MAPK pathway. Considered together, these results demonstratedthat Ca2+ overload could exert pro-apoptotic effects on cardiomyocytesvia activation and phosphorylation of P38 MAPK, which stimulated P53and caspase-3 activation in cardiomyocytes.

In conclusion, aconitine can lead to the apoptosis of cardiacmyocytes in adult rats. In trying to elucidate the toxic mechanismunderlying aconitine treatment, we demonstrated that aconitinepromoted apoptotic response of cardiac myocytes by increasing intra-cellular Ca2+ level, ultimately leading to Ca2+ overload. The increasesin cytosolic Ca2+ caused activation of apoptotic pathways, resulting inthe upregulation of a series of pro-apoptotic proteins including P53,BAX, caspase-9, caspase-3, ERK, P-ERK, P38, and P-P38, and down-regulation of anti-apoptotic protein BCL-2, finally leading to apoptoticdeath of cardiac myocytes. Our study presented here confirmed theunderlying reasons that caused Ca2+ overload and elucidated the pro-apoptotic mechanism underlying aconitine-induced Ca2+ overload.These experimental findings may broaden our understanding of thetoxic mechanism involved in aconitine treatment.

Our observations and findings regarding the arrhythmogenic roleand subsequent pro-apoptotic effect of Ca2+ overload in the develop-ment of aconitine-induced toxicity provide theoretical support foraconitine detoxification in the clinical setting. Demonstration of Ca2+

overload modulating apoptosis implied that Ca2+ handling proteinshad important functions in apoptosis regulation of cardiomyocytes.Therefore, it is conceivable that therapeutic inhibition of intracellularCa2+ level in cardiomyocytes represents a potential molecular methodfor the treatment of aconitine toxicity. These results provide insightsinto the detoxification strategy for aconitine tomake it safe for the treat-ment of cardiac diseases. Most importantly, these results warrantfurther studies for aconitine as a potential treatment in the clinicalsetting.

Statement of conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The present work was supported by the National 973 Program(No. 2009CB522805), the National Major Scientific and TechnologicalSpecial Project for “Significant New Drugs Formulation” (Grant Nos.2012ZX09501001-004, 2010ZX09401-305-47, and 2009ZX09102-104),the National Natural Science Foundation of China (No. 81173589), andthe Natural Science Foundation of Jilin Province (No. 201015110).

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