YTAAP-13101; No. of pages: 15; 4C: Toxicology and Applied Pharmacology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap

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Gui-bo Sun a,b,1, Hong Sun a,b,1, Xiang-bao Meng a,b, Jin Hu c, Qiang Zhang c, Bo Liu c, Min Wang a,b, Hui-bo Xu c,⁎, Xiao-bo Sun a,b,⁎⁎

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

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Keywords: Aconitine Apoptosis Arrhythmia Heart Calcium overload Cardiotoxicity

Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing 100193, PR China b Peking Union Medical College, Beijing 100193, PR China c Academy of Chinese Medical Sciences of Jilin Province, Changchun, Jilin 130021, PR China

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

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 of aconitine. However, no reports are available on the role of Ca2+ in aconitine poisoning. In this study, we explored the 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 conscious freely moving rats. To investigate effects of aconitine on myocardial injury, we performed cytotoxicity assay in neonatal rat ventricular myocytes (NRVMs), as well as measured lactate dehydrogenase level in the culture medium of NRVMs and activities of serum cardiac enzymes in rats. The results showed that aconitine resulted in myocardial injury and reduced NRVMs viability dose-dependently. To confirm the pro-apoptotic effects, we performed flow cytometric detection, cardiac histology, transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The results showed that aconitine stimulated apoptosis time-dependently. The expression analysis of Ca2+ handling proteins demonstrated that aconitine promoted Ca2+ overload through the expression regulation of Ca2+ handling proteins. The expression analysis of apoptosis-related proteins revealed that pro-apoptotic protein expression was upregulated, and antiapoptotic protein BCL-2 expression was downregulated. Furthermore, increased phosphorylation of MAPK family members, especially the P-P38/P38 ratio was found in cardiac tissues. Hence, our results suggest that aconitine significantly aggravates Ca2+ overload and causes arrhythmia and finally promotes apoptotic development via phosphorylation of P38 mitogen-activated protein kinase. © 2014 Published by Elsevier Inc.

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cardiotoxic effects of aconitum plants (Fu et al., 2007; Wada et al., 2005). Previous studies have mainly focused on the cardiotoxic effects of aconitine on voltage-dependent Na+ channels (Kunze et al., 1985; Wang and Wang, 2003; Wright, 2002). However, little information is available on the role of Ca2 + in aconitine poisoning. Therefore, the present study was designed to give a clearer understanding of the importance of defective Ca2+ signaling in aconitine poisoning in vitro and in vivo. It is well known that Ca2+ overload has vital roles in the pathogenesis of heart dysfunctions, especially arrhythmia and apoptosis (Lai et al., 2011; Petersen et al., 2005; Rabkin and Kong, 2003; Soni et al., 2011). Many researchers have reported that Ca2+ plays an important role in the pathogenesis of arrhythmia and pathological cellular Ca2 + overload can lead to an arrhythmogenic state. In other words, arrhythmia is an important event that occurs during aconitine poisoning. In addition, Ca2 + overload can be involved in cardiac apoptosis and appears to be a principal mediator of apoptosis. However, the potential role of Ca2 + overload in aconitine-induced cardiotoxicity

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Introduction

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

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⁎ Correspondence to: H.-B. Xu, Academy of Chinese Medical Sciences of Jilin Province, No. 1745, Gongnongda Road, Changchun, Jilin, 130021, PR China. Fax: +86 431 86058637. ⁎⁎ Correspondence to: X.-B. Sun, Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences & Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing, 100193, PR China. Fax: +86 10 57833013. E-mail addresses: [email protected] (H. Xu), [email protected] (X. Sun). 1 Gui-bo Sun and Hong Sun contributed equally to this work.

http://dx.doi.org/10.1016/j.taap.2014.05.005 0041-008X/© 2014 Published by Elsevier Inc.

Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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Aconitine (content ≥ 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (China). The molecular weight of aconitine is 645.74. Collagenase Type II and Fura-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 dehydrogenase) were obtained from Biosino Bio-Technology and Science Incorporation (Hong Kong, China). Annexin V/propidiumiodide (PI) apoptosis detection kit was obtained from Life Technologies Corporation (Carlsbad, CA, USA). The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay kit was purchased from Roche Diagnostics (Mannheim, Germany). Primary antibodies against RyR, SERCA, NCX, BCL-2, BAX, P53, caspase-9, caspase-3, ERK, P-ERK, P38 and P-P38 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-rabbit-conjugated horseradish peroxidase antibody was purchased from Zhongshan Goldbridge Biotechnology (Beijing, China). The Bradford protein assay kit was purchased from Pierce Corporation (Rockford, USA) and super-enhanced chemiluminescence detection reagents were purchased from Applygen Technologies (Beijing, China). All of the chemical reagents were obtained from Sigma Chemical Co., Ltd (St. Louis, MO, USA).

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Animals and treatments

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Eighty adult male Wistar rats (Vital River Laboratories, Beijing, China) weighing 220 to 240 g were used and the procedures were approved by the local animal committee. These rats were kept at standard room temperature (22 ± 2 °C) and relative humidity (60% ± 10%) with a 12 h light/dark cycle. All of the rats were allowed free access to food and water ad libitum during the acclimatization and experimental period. The experiments were performed in accordance with the guidelines of the Experimental Laboratory Animal Committee of Chinese Academy of Medical Sciences and Peking Union Medical College and the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were randomly divided into the following two groups: group A as control and group B as aconitine model. Forty male Wistar rats were designated for each group. Ten rats in each group were implanted with telemetry transmitters for ECG study. After 1-week of acclimatization, the aconitine model was induced with the protocol described below. Group B orally received 0.146 mg/mL aconitine, diluted in 0.05 N hydrochloric acid, once a day by gavage at 10 mL/kg for 10 consecutive days. Group A was given vehicle instead of aconitine once a day by gavage at 10 mL/kg for 10 consecutive days. On day 3 or day 6 post-aconitine administration, 10 rats in each group were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and their hearts were removed rapidly. The left ventricle was excised for hematoxylin-eosin (HE) staining, transmission electron microscopy (TEM) and TUNEL examination. Myocardial homogenates were then prepared for Western blot analysis. On day 10 after the last administration of aconitine, blood was collected from the left ventricle and centrifuged at 800 ×g for 10 min to obtain serum, which was kept at −80 °C until analysis. Subsequently, the hearts were removed rapidly. The left ventricle was excised for HE, TEM and TUNEL examination. The myocardial homogenates were prepared for Western blot analysis.

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Measurement of beating rhythm, sarcomere shortening, and Ca2+ transients in ARVMs

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Materials

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Individual adult rat ventricular myocytes (ARVMs) were isolated from 13-week-old Wistar rats as described previously (Westfall and Borton, 2003; Westfall et al., 1997). After the rats were anesthetized with ketamine/xylazine (0.1 mL/100 g, i.p.), their hearts were removed and perfused through the aorta cannula with a series of different perfusion solutions at the rate of 6 mL/min. First, the hearts were perfused 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's solution containing with the aforementioned components except CaCl2 for 5 min, followed by a 20 min perfusion with Ca2+-free Tyrode's solution containing collagenase Type II (210.00 units/mg). After perfusion, the left ventricles were removed, minced, and filtered through a nylon mesh (300 mm). The filtered myocytes were then washed with Ca2 +-containing Tyrode's solution to restore the extracellular Ca2 + concentration to 1.2 mM. ARVMs were assayed by trypan blue exclusion assay for viability. Viability was over 80%. Only rod-shaped ARVMs with clear edges were used in this study.

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Materials and methods

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Beating rhythm, sarcomere shortening and Ca2+ transients of intact ARVMs at room temperature were assessed simultaneously upon field stimulation (0.5 Hz with 2-ms-duration, 16 V) using a video-based sarcomere contractility and calcium recording module in a SoftEdge MyoCam system (IonOptix Corporation, Milton, MA, USA). Isolated ARVMs were loaded with fura-2/AM (2 μM) for 15 min and washed twice with Ca2 +-containing Tyrode's solution after restoration of the extracellular Ca2 +. ARVMs were then placed in a Warner chamber mounted 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+ channel blocker. Beating rhythm, sarcomere shortening and Ca2 + transients were 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 baseline stabilization. Data were recorded and analyzed with IonWizard software (version 6.2.0.59).

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Electrocardiography

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Electrocardiography (ECG) recordings were taken before aconitine or vehicle treatment and 24 h after each aconitine or vehicle treatment in conscious freely moving rats. Ten rats in each group were implanted with telemetry transmitters (HD-S21) of Data Sciences International (St. Paul, MN, USA) for ECG collection. After the rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), HD-S21 transmitters were placed in the abdominal cavity of the rats and fixed to the inner peritoneal wall using silk sutures. All skin incisions were closed using wound clips under sterile conditions. After 2-week surgical recovery, ECG was measured for ten days. When we switched the mode to ON, the transmitters began to sense and transmit data. ECG data were acquired using Dataquest A.R.T. 4.31 software (Data Sciences International). The raw ECG data were then analyzed by DSI's Ponemah Physiology Platform software. This technique facilitated the collection of ECG recordings in the most reliable and efficient method without anesthetizing rats.

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Cell culture and aconitine treatment

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Isolation of adult rat ventricular myocytes and treatments

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remains largely unknown. In our research, we investigated the alterations of Ca2+ level in cardiomyocytes induced by aconitine treatment and explored whether Ca2 + overload could cause arrhythmia and trigger apoptosis.

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Primary cultures of NRVMs were prepared as described previously 190 (Gray et al., 1998). NRVMs were isolated from the hearts of 1-day-old 191

Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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Cell viability analysis

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay

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Measurement of LDH level in the culture medium of NRVMs

DNA fragmentation was detected in cardiomyocytes using the TUNEL staining kit according to the manufacturer's instructions. Frozen tissue sections were fixed with fixation solution (4% paraformaldehyde in PBS, pH 7.4, freshly prepared) for 20 min at 20 °C and washed for 30 min with PBS. They were incubated with blocking solution for 10 min at 20 °C and rinsed with PBS. The tissue sections were then incubated in permeabilization solution for 2 min on ice and rinsed twice with PBS. Then, 50 μL TUNEL reaction mixture was added to the samples which were incubated for 60 min at 37 °C in the dark and rinsed thrice with PBS. The samples were observed under a fluorescence microscope (Leica DM4000, Germany).

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

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Western blot analysis

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

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Serum analysis

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

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Flow cytometric detection in ARVMs

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The percentage of early apoptosis and necrosis was measured in isolated ARVMs using Annexin V-FITC/PI apoptosis kit for flow cytometry according to the manufacturer's brochures (Life Technologies Corporation). 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, we paced our ARVMs upon field stimulation (0.5 Hz with 2-ms-duration, 30 V) using C-Pace EP, a ARVMs stimulator designed by IonOptix Corporation (Milton, MA, USA), to keep the normal functional characteristics of ARVMs. After treatment, ARVMs were harvested and washed twice with cold PBS, and then incubated with 5 μL FITC-Annexin V and 1 μL PI working solution (100 μg/mL) for 15 min in the dark at room temperature. Cellular fluorescence was measured by flow cytometry analysis with a flow cytometer (FACS Calibur™, BD Biosciences, CA, USA).

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Histological analysis

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

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Transmission electron microscopy examination

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Preparations for TEM were made in the following manner (Dabour et 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 more than 4 h and then washed thrice in phosphate buffer. They were postfixed with 1% osmic acid for 1 h and washed thrice in phosphate buffer. Subsequently, samples were dehydrated with a graded series of ethanol from 50% to 100%, followed by infiltration of a 1:1 mixture

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Statistics

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

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Results

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

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of acetone and embedding resin for 1 h, 1:3 mixture of acetone and embedding resin for 3 h and 100% embedding resin overnight. Samples were then placed in the embedding medium 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 observed using the TEM Model Hitachi H-7000.

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Wistar rats by trypsin and collagenase digestion, purified by differential preplating, and maintained in DMEM with 10% (v/v) fetal bovine serum in a humidified incubator of 95% air and 5% CO2 at 37 °C. Two sets of experiments were performed: (1) control NRVMs; (2) NRVMs treated with indicated concentrations (0.01, 0.04, 0.16, and 0.64 μM) of aconitine for 4 h.

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The beating rhythm, sarcomere shortening, and Ca2+ transient in ARVMs 286 treated with aconitine 287 We treated freshly isolated ARVMs with aconitine (1 μM) to examine the time-course effects of aconitine on beating rhythm, and sarcomeric contractile function. Our results showed that aconitine did not affect the normal rhythm of ARVMs in the initial period of aconitine treatment, but aconitine induced continuous increases in amplitude of sarcomere shortening and peak shortening (both P b 0.01) without affecting resting sarcomere length in 4 min after 1 μM aconitine was added (Figs. 2A–C). Aconitine also induced sarcomeric diastolic dysfunction in 7 min in a time-dependent manner (Figs. 1A and C). Compared with those at 0 min, amplitude of sarcomere shortening and peak shortening significantly increased, but the resting sarcomere length significantly decreased at 7 min after aconitine perfusion was performed, suggesting the occurrence of diastolic dysfunction (P b 0.01, for all three parameters) (Figs. 2A–C). The time taken for 90% relaxation significantly decreased at 7 min compared with that at 0 min, indicating

Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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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 Ca 2 + transient. The red line indicates the addition of 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). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

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faster relaxation of ARVMs (P b 0.01) (Fig. 2H). Most importantly, the rhythm of ARVMs became faster and arrhythmic. The beating rate was 15 beats/10 s at 7 min, which was two times higher than those at 0 min (Fig. 2I). These changes suggested that the normal rhythm and diastolic function of ARVMs were disrupted by aconitine in 7 min in a time-dependent manner. Cytoplasmic Ca2+ transient was determined in fura-2 loaded ARVMs to evaluate the time-course effects of aconitine on intracellular Ca2+ homeostasis. Our results showed that aconitine significantly increased resting Ca2 + ratio, amplitude of Ca2 + ratio and amplitude/resting calcium in 4 min after 1 μM aconitine was added (P b 0.01, for all three parameters) (Figs. 2D–F). Aconitine also induced sustained increases in cytoplasmic Ca2 + ratio in 7 min in a time-dependent manner (Figs. 1B and D). Compared with those at 0 min, the resting Ca2 + ratio, amplitude of Ca2 + ratio, and amplitude/resting calcium increased significantly at 7 min after aconitine perfusion was performed (P b 0.01, for all three parameters) (Figs. 2D–F). These changes

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suggested that aconitine leads to Ca2+ overload in ARVMs in 7 min in 320 a time-dependent manner. 321 The beating rhythm, sarcomere shortening, and Ca2+ transient in ARVMs 322 treated with verapamil 323 We treated freshly isolated ARVMs with L-type Ca2+ channel blocker verapamil (10 μM) to examine the time-course effects of verapamil on beating rhythm, and sarcomeric contractile function. Our results showed that verapamil did not affect the normal beating rhythm of ARVMs in the whole period of verapamil treatment (Fig. 4 I), but verapamil induced continuous decreases in amplitude of sarcomere shortening and peak shortening (both P b 0.01) in 7 min after verapamil was added (Figs. 3A and C; Figs. 4B and C). The time taken for 90% relaxation significantly increased in 7 min compared with that at 0 min, indicating slower relaxation of ARVMs (P b 0.01) (Fig. 4H). These changes suggested that the normal beating rhythm of ARVMs was not disrupted and the sarcomeric

Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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Fig. 2. Properties of sarcomere shortening and Ca2+ transient in left ventricular myocytes 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 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.

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contractile function decreased continually by verapamil in 7 min in a time-dependent manner. Cytoplasmic Ca2+ transient was determined in fura-2 loaded ARVMs to 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, for all three parameters) in 4 min after verapamil was added (Figs. 4D–F). Verapamil also induced sustained decreases in cytoplasmic Ca2+ ratio in 7 min in a time-dependent manner (Figs. 3B and D). Compared with those at 0 min, the resting Ca2 + ratio, amplitude of Ca2 + ratio, and amplitude/resting calcium decreased significantly at 7 min after verapamil 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 by verapamil in a time-dependent manner.

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Fig. 3. Sarcomere shortening and Ca2+ transient were recorded simultaneously from the left ventricular myocytes after verapamil perfusion using SoftEdge MyoCam system. (A) Time course of 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) 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). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

The beating rhythm, sarcomere shortening, and Ca2+ transient in ARVMs 350 treated with verapamil + aconitine 351 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 of aconitine on sarcomere shortening, coadministration of verapamil and aconitine led to the similar continuous increases in amplitude of sarcomere shortening and peak shortening (both P b 0.01) without affecting resting sarcomere length in 7 min (Figs. 6A–C). The amplitude of sarcomere shortening and peak shortening in the verapamil + aconitine group increased to a smaller extent than that in the aconitine group at different time points. Most importantly, in the verapamil + aconitine group the duration of normal beating rhythm of ARVMs was maintained

Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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longer and the disrupted contractile function appeared later than those in the aconitine group (Figs. 5A and C). The beating rhythm of ARVMs in the verapamil + aconitine group became arrhythmic and the contractile function was disrupted until 12 min after coadministration of verapamil and aconitine (Figs. 5A and C; Fig. 6 I). Cytoplasmic Ca2+ transient was determined in fura-2 loaded ARVMs to examine the time-course effects of verapamil + aconitine on intracellular Ca2 + homeostasis. Compared with the time-course effects of single administration of aconitine on intracellular Ca2 + homeostasis, coadministration of verapamil and aconitine led to the similar but smaller 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 amplitude/resting calcium in the verapamil + aconitine group increased to a smaller extent than those in the aconitine group at different time points. Most importantly, in the verapamil + aconitine group the duration of normal calcium transient was maintained longer and the calcium overload appeared later than in the aconitine group (Figs. 5B and D). The rhythm of calcium transient in the verapamil + aconitine group became faster and the calcium overload appeared until 12 min (Fig. 5D; Fig. 6D).

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Fig. 4. Properties of sarcomere shortening and Ca2+ transient in left ventricular myocytes after verapamil (10 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 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.

Electrocardiography

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In our study, ventricular tachycardia and ventricular premature beats were confirmed in conscious freely moving rats by ECG after aconitine treatment. Data analysis showed that ST segment height was 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 aconitine treatment for 7 days, the significance of the ECG parameters declined because of the extreme abnormality in ECG (Fig. 7A). No abnormality in the ECG of the control group was observed throughout the study.

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The harmful effects of aconitine-induced cytotoxicity in NRVMs were detected by MTT assay. Aconitine exhibited strong dosedependent cytotoxic effects (Fig. 8A). The survival rate decreased to 94 ± 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.

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Fig. 5. Sarcomere shortening and Ca2+ transient were recorded simultaneously from the left ventricular myocytes after verapamil + aconitine perfusion using SoftEdge MyoCam 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 addition of 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). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

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To assay the effects of aconitine on cardiac injury, we measured the level of cardiac enzyme LDH which was a generally accepted indicator of cell injury in the supernate of cultured NRVMs. Aconitine increased LDH levels 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, and 0.64 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 inconsistent with its harmful effect on cell viability, which was assessed by MTT assay.

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

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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 treatment compared with the control group. CK content approximately increased by 2-fold after aconitine treatment. AST serum concentration of the model group increased by approximately 1.4-fold compared with the control group.

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In normal ARVMs, phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane. However, in apoptotic ARVMs, PS is located on the outer leaflet of the plasma membrane. Annexin V, which has a high affinity for PS, labeled with a fluorophore can identify apoptotic ARVMs by binding to PS. In the normal control group, the percentage of apoptotic ARVMs was 5.2%; however, the percentage of apoptotic ARVMs increased up to 75.9% in the aconitine model group (Fig. 9). SB203580 significantly inhibited the percentage of apoptotic ARVMs compared with the

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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) 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 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.

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

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An overall view of apoptosis in the heart tissues at the light microscopy level is shown in Fig. 10. In the control group, no obvious cellular degeneration and apoptosis were found, whereas severe myocardial damage and apoptosis were observed in the aconitine model group. Time-course studies of pathological damage suggested that apoptotic levels were not significantly changed before aconitine treatment, but markedly increased during aconitine treatment. These apoptotic changes were characterized by the increase of apoptotic cardiomyocytes, intense infiltration with neutrophil granulocytes, and morphological abnormalities of cardiomyocytes and myocardial tissues including darker (hyperchromatic) and more crowded nuclei, condensation of cytoplasm in apoptotic cardiomyocytes, disorganization of myofibrillar arrays.

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

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myocytes compared with the control group. TEM images showed anomaly of the nucleus and increase of heterochromatin, mitochondrial disorganization of cristae, irregularity and disappearance of myofilaments and disruption of sarcomeres (Figs. 11C and D).

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TUNEL staining assay

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

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

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

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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 as mean ± S.D. (n = 10). *P b 0.05 compared with the control group; **P b 0.01 compared 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, and 0.64 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 kit according to the manufacturer's instructions. (C) Concentrations of CK, LDH, and AST in cardiac tissues. 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.

Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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P b 0.05, for caspase-9 and caspase-3 on day 6; P b 0.01, for caspase-9 and caspase-3 on day 10) (Figs. 13B and C). The ratio of BCL-2/Bax was also significantly decreased compared with the control group (P b 0.01) (Fig. 13B). The protein expression of important members (ERK, P-ERK, P38 and P-P38) in the MAPK family of cardiac tissues showed that aconitine increased the expression and phosphorylation of ERK and P38 compared

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time. SERCA protein level was significantly inhibited on days 6 and 10 post-aconitine treatment (both P b 0.05) (Fig. 13A). The expression results of essential apoptosis-related proteins showed that aconitine treatment (1.46 mg/kg, 10 mL/kg, once a day for 10 days) upregulated the expression of P53, BAX, caspase-9, and caspase-3, as well as downregulated the expression of BCL-2 on days 6 and 10 post-aconitine treatment (P b 0.01, for P53 on days 6 and 10;

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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 as mean ± S.D. (n = 6). **P b 0.01 compared with the control group. ##P b 0.01 compared with the aconitine model group.

Fig. 10. Time course of pathological changes in cardiac tissues examined by HE staining. (A) Control. (B) Day 3. (C) Day 6. (D) Day 10. Scale bar: 500 μm. Aconitine induced cardiac injury in a time-dependent manner. On day 6 and day 10 post-aconitine administration, there were obvious pathological changes in cardiac tissues.

Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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

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overload could cause arrhythmia and trigger apoptosis through p38 MAPK signaling pathway. It is well known that Ca2 + is a ubiquitous intracellular signal molecule responsible for controlling several cellular processes, such as proliferation, differentiation, development and cell death (Bers, 2008; Giorgi et al., 2012; Griffiths, 2009; Schaub et al., 2006). Many studies have demonstrated that cellular Ca2+ is implicated in the pathogenesis of heart dysfunctions, especially arrhythmia and apoptosis (Biagioli et al., 2008; Ermak and Davies, 2002; Heinzel et al.,

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

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Fig. 11. TEM images of cardiac tissues in rats. Panels A and B: control group; Panels C and D: aconitine model 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.

Fig. 12. Aconitine promoted myocardial apoptotic responses determined by TUNEL labeling. (A) Time course of apoptotic responses in cardiac tissues. The nuclei of apoptotic cells with FITC are shown in green; cell nuclei counterstained with DAPI were shown in blue (scale bar: 50 μm). (B) The TUNEL apoptotic index was determined by calculating the ratio of TUNEL-positive cells to total cells. Values for each group are presented as mean ± S.D. (n = 6). **P b 0.01 compared with the control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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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 by Western 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 are presented as mean ± S.D. (n = 6). *P b 0.05 compared with the 0 day; **P b 0.01 compared with the 0 day. 13

Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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overload upon caspase cascades. The results showed that Ca2+ overload promoted the expression of caspase-9 and caspase-3. Furthermore, we examined the expression of ERK, P-ERK, P38, and P-P38 belonging to the MAPK family which has a pivotal function in cell proliferation, differentiation, transformation, and apoptosis to discover the downstream 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 aggravated by the upregulation of expression and phosphorylation of MAPK family members, especially the P-P38/P38 ratio. The flow cytometric detection also showed the percentage of apoptotic ARVMs in the SB203580 + aconitine group was significantly lower than that in the aconitine group. SB203580 is a specific inhibitor of P38 MAPK. Our results proved that aconitine induced ARVM apoptosis through the P38 MAPK pathway. Considered together, these results demonstrated that Ca2+ overload could exert pro-apoptotic effects on cardiomyocytes via activation and phosphorylation of P38 MAPK, which stimulated P53 and caspase-3 activation in cardiomyocytes. In conclusion, aconitine can lead to the apoptosis of cardiac myocytes in adult rats. In trying to elucidate the toxic mechanism underlying aconitine treatment, we demonstrated that aconitine promoted apoptotic response of cardiac myocytes by increasing intracellular Ca2+ level, ultimately leading to Ca2+ overload. The increases in cytosolic Ca2+ caused activation of apoptotic pathways, resulting in the upregulation of a series of pro-apoptotic proteins including P53, BAX, caspase-9, caspase-3, ERK, P-ERK, P38, and P-P38, and downregulation of anti-apoptotic protein BCL-2, finally leading to apoptotic death of cardiac myocytes. Our study presented here confirmed the underlying reasons that caused Ca2+ overload and elucidated the proapoptotic mechanism underlying aconitine-induced Ca2 + overload. These experimental findings may broaden our understanding of the toxic mechanism involved in aconitine treatment. Our observations and findings regarding the arrhythmogenic role and subsequent pro-apoptotic effect of Ca2+ overload in the development of aconitine-induced toxicity provide theoretical support for aconitine detoxification in the clinical setting. Demonstration of Ca2+ overload modulating apoptosis implied that Ca2 + handling proteins had important functions in apoptosis regulation of cardiomyocytes. Therefore, it is conceivable that therapeutic inhibition of intracellular Ca2+ level in cardiomyocytes represents a potential molecular method for the treatment of aconitine toxicity. These results provide insights into the detoxification strategy for aconitine to make it safe for the treatment of cardiac diseases. Most importantly, these results warrant further studies for aconitine as a potential treatment in the clinical setting.

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2011; Rogalska et al., 2013). In this paper, we studied the corresponding changes in sarcomere shortening and cytoplasmic Ca2+ level by comparing the effects of the presence and absence of aconitine, verapamil, or verapamil + aconitine in ARVMs, respectively. These findings suggested that Ca2+ transient and contractility in excitation–contraction coupling would change correspondingly. In addition, these results provided strong evidence for the rapid cellular action of aconitine on L-type Ca2+ channels. These results also confirmed aconitine-induced Ca2 + overload could lead to the pathogenesis of arrhythmia and Ltype Ca2 + channel blocker verapamil could delay the toxic effects of aconitine. The results of ECG in conscious freely moving rats also confirmed the arrhythmogenic effects of aconitine in vivo. In previous studies, it has been confirmed that aconitine prevents open Na+ channels from inactivating and causes persistent activation of Na+ channels, thereby leading to sustained Na+ influx (Kunze et al., 1985; Wang and Wang, 2003; Wright, 2002). Our work suggested that aconitine also induced Ca2+ overload and caused cardiac arrhythmia. Therefore, based on the fact that aconitine targets sodium channels and our present results, we suggested that in ARVMs, the reason calcium overload took place in 7 min after aconitine perfusion was because sustained depolarization of sodium channels activated L-type calcium channels. This led to the rapid increases in the intracellular calcium concentration. In the heart tissues treated with multiple aconitine treatment for 10 days, the upregulation of RyR and NCX and downregulation of SERCA suggested the temporal influence of aconitine on the expression of calcium handling proteins. So we thought that the immediate calcium overload in ARVMs was caused by sustained depolarization of sodium channels and the changes in protein expression of calcium handling proteins which were a downstream consequence of immediate calcium overload may have contributed to the chronic elevation of calcium in the heart tissues. Apoptosis has an essential function in the pathogenesis of cardiovascular diseases and contributes to the development of cardiovascular disorders (Copaja et al., 2011; Feuerstein et al., 1997; Garg et al., 2005; Kitsis and Mann, 2005). Ca2+ overload can cause activation of the intrinsic apoptotic pathway and activate a series of important signal molecules (Isomura et al., 2013; Jan et al., 2013; Singh et al., 2010). In the present study, we found that aconitine promoted the apoptotic response of hearts in vitro and in vivo. We used flow cytometry to identify apoptosis in ARVMs, and performed HE staining, TEM and TUNEL assay to identify apoptosis in cardiac tissues after varied periods of aconitine treatment. The four distinct in vitro and in vivo methods confirmed myocardial injury and myocyte apoptosis in the model group as time progressed, suggesting that Ca2 + overload resulted in apoptotic responses in time-dependent manner. To the best of our knowledge, this study is the first to investigate the pro-apoptotic toxicity of aconitine using animal experiments in vitro and in vivo. We examined the expression of apoptosis related proteins including BCL-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 of the signaling events involved in aconitine-induced apoptosis. BCL-2 family proteins have been implicated as major regulators of apoptosis in many cells (Clerk et al., 2003; Hardwick et al., 2012; Laulier and Lopez, 2012). The BCL-2/BAX ratio is important in regulating cell apoptosis. The decrease in BCL-2/BAX ratio may promote the activation and development of apoptosis. Our results showed that Ca2+ overload decreased 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). Once activated, P53 increases the transcription of pro-apoptotic genes, and may activate BAX directly. In our study, Ca2 + overload-induced apoptosis was accompanied by upregulated P53 expression. In addition, both caspase-9 (an initiator caspase) and caspase-3 (a critical effector caspase) expression were tested to determine the effects of Ca2 +

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The present work was supported by the National 973 Program (No. 2009CB522805), the National Major Scientific and Technological Special 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), and the Natural Science Foundation of Jilin Province (No. 201015110).

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Please cite this article as: Sun, G., et al., Aconitine-induced Ca2 + overload causes arrhythmia and triggers apoptosis through p38 MAPK signaling pathway in rats..., Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.005

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