Accepted Manuscript

Accepted Manuscript Taurine–magnesium coordination compound, a potential antiarrhythmic complex, improves aconitine-induced arrhythmias through regulation of multiple ion channels Jianshi Lou, Hong Wu, Lingfang Wang, Lin Zhao, Xin Li, Yi Kang, Ke Wen, Yongqiang Yin PII: S0041-008X(18)30375-2 DOI: doi:10.1016/j.taap.2018.08.008 Reference: YTAAP 14363 To appear in: Toxicology and Applied Pharmacology Received date: 1 March 2018 Revised date: 3 August 2018 Accepted date: 14 August 2018 Please cite this article as: Jianshi Lou, Hong Wu, Lingfang Wang, Lin Zhao, Xin Li, Yi Kang, Ke Wen, Yongqiang Yin , Taurine–magnesium coordination compound, a potential anti-arrhythmic complex, improves aconitine-induced arrhythmias through regulation of multiple ion channels. Ytaap (2018), doi:10.1016/j.taap.2018.08.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Taurine-magnesium Coordination Compound, a Potential Anti-arrhythmic Complex, Improves Aconitine-induced Arrhythmias through Regulation of Multiple Ion Channels Jianshi Lou1, Hong Wu1,2, Lingfang Wang3, Lin Zhao4, Xin Li1, Yi Kang1,Ke Wen1,Yongqiang Yin1* 1 Department of Pharmacology, Tianjin Medical University, Tianjin P. R. China 2 Mudanjiang Medical University, Mudanjiang P. R. China 3 Institute of Translational Medicine, Nanchang University, Nanchang P. R. China 4 International College, Tianjin Medical University, Tianjin P. R. China *Corresponding author. Department of Pharmacology, Tianjin Medical University, Tianjin 300070, P. R. China. Fax: +862283336835 E-mail:[email protected] ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Abstract Taurine-magnesium coordination compound (TMCC) exhibits antiarrhythmic effects in cesium-chloride-and ouabain-induced arrhythmias; however, the mechanism underlying these effects on arrhythmia remains poorly understood. Here, we investigated the effects of TMCC on aconitine-induced arrhythmia in vivo and the electrophysiological effects of this compound in rat ventricular myocytes in vitro. Aconitine was used to induce arrhythmias in rats, and the dosages required to produce ventricular premature contraction (VPC), ventricular tachycardia (VT), ventricular fibrillation (VF), and cardiac arrest(CA) were recorded. Additionally, the sodium current (INa) and L-type calcium current (ICa,L) were analyzed in normal and aconitine-treated ventricular myocytes using whole-cell patch-clamp recording. In vivo, intravenous administration of TMCC produced marked antiarrhythmic effects, as indicated by the increased dose of aconitine required to induce VPC,VT,VF, and CA. Moreover, this effect was abolished by administration of sodium channel opener veratridine and calcium channel agonist Bay K8644. In vitro, TMCC inhibited aconitine-induced increases in INa and ICa,L. These results revealed that TMCC inhibited aconitine-induced arrhythmias through effects on INa and ICa,L. Key Words: taurine-magnesium coordination compound,arrhythmia,veratridine,Bay ACCEPTED MANUSCRIPT K8644, whole-cell patch-clamp technique,aconitine ACCEPTED MANUSCRIPT Introduction Life-threatening cardiac arrhythmias, such as ventricular tachycardia (VT) and ventricular fibrillation (VF), account for numerous cardiac deaths and have attracted increased attention worldwide (Wu et al., 2003). Although many recent advances have been made in the non-pharmacological management of ventricular arrhythmia (VA)(Tanawuttiwat et al., 2016), pharmacotherapy remains an important aspect of both the acute treatment and chronic prevention of VA(Schleifer et al., 2015). Amiodarone is the preferred antiarrhythmic drug for acute management of VA, with long-term use of amiodarone at the lowest effective dose preventing VA recurrence at 1 year in 78% of patients; however, this was associated with a 12% risk of clinical thyroid dysfunction and a 1% risk of hepatic or pulmonary toxicity(Lee and Tai, 1997). Therefore, development of a pharmacological antiarrhythmic treatment with fewer adverse side effects would be beneficial. Our previous research found that taurine exhibited an effective antiarrhythmic effect and enhanced the activities of other antiarrhythmic drugs. Furthermore, treatment with a combination of Mg2+ and taurine was optimal to produce an antiarrhythmic effect(Yu MK, 1994); therefore, we synthesized a taurine-magnesium coordination compound[TMCC; Mg(NH2CH2CH2SO3)2·H2O] with a molecularACCEPTED weight of 290.04 and a chemicalMANUSCRIPT structure shown in Figure 1(Yin et al., 2012). Our results showed that TMCC exhibited antiarrhythmic effects in many models, including cesium chloride-induced arrhythmias in vivo (Yin et al., 2012) and ouabain-induced arrhythmias in vivo and in vitro (Zhao et al., 2012); however, the mechanism underlying these effects on arrhythmia remains poorly understood. ACCEPTED MANUSCRIPT Aconitine is a lethal toxin produced by the aconitum plant and that displays arrhythmogenic effects, including dose-dependent induction of ventricular premature contraction (VPC), VT, Torsades de Pointes, VF, and mortality (Lu and De Clerck, 1993; Amran et al., 2004). To investigate the effects of TMCC on the heart and/or ventricular myocytes exposed to aconitine, we evaluated the antiarrhythmic effects of TMCC in vivo and its cellular mechanism of action in rat cardiomyocytes. Materials and Methods Animals SPF Wistar rats (male and female) weighing between 200 g and 250 g (certificate No. SCXK (JING) 2009-003) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).This study was performed in accordance with the Guide for the Care and Use of Experimental Animals at the Animal Center of Tianjin Medical University, and the protocol was approved by the Experimental Ethics Committee of Tianjin Medical University (Tianjin, China). Chemicals TMCC was synthesized by the Department of Chemistry at Tianjin Medical University and at a purity of 99%. The quality control procedure included determination of the magnesium contentACCEPTED by ion-emission spectroscopy MANUSCRIPT and chromatometry in order to calculate the coordination-compound percentage. Aconitine, veratridine, Bay K8644,HEPES, EGTA, Na2ATP, MgATP, type II collagenase, and CdCl2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Solutions ACCEPTED MANUSCRIPT 2+ Ca -free Tyrode’s solution contained 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose (adjusted to pH 7.4 with NaOH). Krebs solution contained 50 mM glutamic acid, 80 mMKOH,40 mMKCl,25 mM KH2PO4, 3 mM MgSO4, 20 mM taurine, 0.5 mM EGTA,10 mM HEPES, and 10 mM glucose (adjusted to pH 7.4 with KOH). The extracellular solution for sodium current (INa)determination contained 130 mM choline chloride, 5.4 mM CsCl, 0.1 mM CaCl2, 1mM MgCl2, 0.33 mM NaH2PO4, 10 mM glucose, 10 mM HEPES, 10 mM NaCl, and 0.1 mM CdCl2 (adjusted to pH 7.4 with CsOH). The pipette solution for INa determination contained 120 mM CsCl, 5 mM MgCl2, 5 mM Na2ATP, 1 mM CaCl2, 11 mM EGTA, and 10 mM HEPES (adjusted to pH 7.2 with CsOH). The extracellular solution for L-type calcium current (ICa,L) determination contained 135 mM choline chloride, 1.8 mM CaCl2, 4 mM CsCl, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose(adjusted to pH 7.4 with CsOH). The pipette solution for ICa,L determination contained 120 mM CsCl, 1 mM CaCl2, 5 mM MgCl2, 5 mM Na2ATP, 11 mM EGTA, and 10 mM HEPES (adjusted to pH 7.2 with CsOH). Establishment of the in vivo model of arrhythmia This protocol ACCEPTEDwas adapted from previous MANUSCRIPT studies(Zhao et al., 2013; Liu et al., 2014). Briefly, Wistar rats were randomly divided into different groups(10 per group) and anesthetized using 25% urethane (4 g/kg),followed by a continuous injection of TMCC (50,25, or 12.5 mg/kg), amiodarone(30 mg/kg), TMCC (50 mg/kg)+veratridine(20 µg/kg),or TMCC (50 mg/kg)+Bay K8644(15 µg/kg) at a velocity of 0.1mL/min via the ACCEPTED MANUSCRIPT caudal vein for 10min. Lead II electrocardiograms were recorded by a BL-420S Biological Data Acquisition & Analysis System (Chengdu Technology & Market Corp., Ltd., Chengdu, China). After a 5-min stabilization period, aconitine(10 μg/mL) was administered continuously via the superficial caudal vein at 0.2 mL/min, and the cumulative dose of aconitine required to induce VPC, VT, VF, and cardiac arrest(CA) was determined. Stock solutions of aconitine in dimethyl sulfoxide (DMSO) were diluted to the desired final concentrations immediately before use. DMSO at a final concentration of ≤0.1% in the bath solution had no effect on INa, ICa,L ,action potential (AP), or aconitine-induced arrhythmias (data not shown). Cell preparation Single ventricular myocytes were isolated using a previously described enzymatic dissociation method (Liu et al., 2014). Briefly, rats were anesthetized with 25% urethane (1 g/kg, intraperitoneally), and the heart was excised and rinsed in oxygenated ice-cold Tyrode’s solution. The heart was then underwent retrograde perfusion on a Langendorff apparatus with oxygenated Ca2+-free Tyrode’s solution for 5 min before the perfusate was switched to enzymatic solution (8 mg collagenase type II in 50 2+ mLCa -free Tyrode’s

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