兔左心室动脉灌流楔形标本在药物潜在致心律失常风险评价中的应用
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摘要
自上世纪50年代发现抗心律失常药奎尼丁能引起心脏复极化延迟,引发严重室性心律失常即尖端扭转性室性心动过速(torsade de pointes, TdP)以来,临床越来越多的发现药物引发TdP造成心源性晕厥、甚至猝死的报道。业已发现,能够引发TdP的药物涉及各个系统治疗药物,除延迟复极的抗心律失常药外,常见药物包括抗组胺药、抗精神失常药、抗菌药、抗肿瘤药及心血管系统药物等。这些药物尽管结构各异,但共同的作用是抑制心肌细胞复极化中起关键作用的一种延迟整流钾通道(该通道的孔区由human ether-a-go-go related gene编码,简称HERG通道),延长心脏复极过程,心电图上表现QT间期的延长。QT间期延长致复极离散度增大,大大增加心脏发生室性心动过速的危险。特非那定、阿司咪唑、西沙必利、米贝拉地尔及格帕沙星等药因引发TdP被相继撤出市场。基于药物此种潜在致命性威胁的认识,人用药品注册技术要求国际协调会(ICH)于2002年11月专门发布了“人用药品延迟心室复极化(QT间期延长)潜在致心律失常作用的非临床安全评价”指导原则的讨论稿,经广泛征求意见后于2005年5月形成了最后稿,作为国际新药安全药理研究的补充指导原则(S7B)于2005年12月正式启用。
S7B指导原则要求对QT间期延长药物的非临床安全性评价包括以下4个不同功能水平的研究:1)采用离体动物或人体心脏细胞、培养心脏细胞系或克隆人离子通道的异种表达体系评价对HERG离子流的影响。2)在离体心脏标本进行动作电位参数测定,或在麻醉动物中进行能体现动作电位周期的特异性电生理参数检测。3)测定清醒或麻醉动物的ECG参数。4)在离体心脏标本或动物进行致心律失常作用测定。其中前三项包括HERG电流、动作电位复极参数以及整体动物心电图QT间期的评价均为通过延迟复极指标或参数间接判断药物引发心律失常的潜在危险。然而,由于药物延迟复极程度与致心律失常危险性高低之间的关系尚未确立,近几年,建立离体或整体敏感心律失常动物模型成为研究热点。因药物诱发TdP常常是在病理情况下,特别是心肌复极储备降低的心脏(如遗传性LQTs、心肌肥厚或心衰等),常规正常动物心脏模型难以观察到QT延长药物的致心律失常作用,有研究显示甲氧胺敏化兔模型以及慢性房室阻断犬模型可提高评价的敏感性,此外,已报道的心律失常动物模型有离体动脉灌流左室楔形标本及Langendorff灌流兔心。在上述报道的四种模型中,动脉灌流左心室楔形标本具有较高的敏感性和特异性,而被越来越多地采用,该模型还对于理解心电图复极波的细胞基础及心律失常发生机制具有重要帮助。
目前,我国对药物致心律失常危险性的认识处于起步阶段,尚未建立完善的的技术评价体系,尤其是缺乏敏感心律失常模型。
本实验拟建立冠状动脉灌流兔左心室楔形组织模型,即制作兔左心室游离壁楔形标本,经冠状动脉左回旋支灌流台式液,采用浮置玻璃微电极同步记录心内膜、心外膜下心肌细胞动作电位及跨室壁心电图,并利用此方法评价阿霉素的急性心脏毒性。该心律失常模型的建立将作为“QT延迟药物潜在致心律失常危险性”评价技术体系的一部分,用于新药,特别是创新药物非临床安全性评价。可在新药研发过程中及早发现新药潜在的致命性不良反应,从而做到合理评估药物的研发前景,对降低创新药物研发风险及预测药物严重不良反应、保障人民生命安全均具有十分重要的意义。
一兔左心室动脉灌流楔形模型的建立
目的:建立稳定、敏感的兔左心室动脉灌流楔形组织模型
方法: 3~4个月龄新西兰雌性白兔,体重2.0～2. 5kg,耳缘静脉注射肝素(200 IU/kg)抗凝、戊巴比妥钠( 30～35 mg/kg )麻醉后,迅速开胸取出心脏。先以低温停跳液(KCl为24mmol/L、不含Ca~(2+)的台氏液)从主动脉逆行灌流,将自制灌流针经主动脉左窦开口处插入冠状动脉左侧回旋支,制成以回旋支为中心轴,长约10～20 mm、宽约5～10 mm、厚约3～3.5 mm的心室肌楔型组织块。将组织块固定于恒温浴槽中,持续灌流充以纯氧的正常台氏液,温度35.7±0.1°C,灌流速度为3～4ml/min。台式液组成(mmol/L):NaCl 140、KCl 5.4、MgCl_2 1.0、CaCl_2 2.0、HEPS 10、葡萄糖10(pH7.35～7.45)。
微电极拉制器制备记录玻璃微电极,使其内充3 mol/L KCl时的电极电阻为10～20MΩ。玻璃微电极与悬浮铂金丝电极相连,经微电极放大器、多道生理信号采集系统进行信号转化、记录。以1~2mm的间隔从外膜向内膜侧心肌的动作电位(APD)进行记录,观察跨壁各层的动作电位时程特点,以确定内、外膜下心肌记录部位。
将两根玻璃微电极扎入同一平面内、外膜心肌细胞,同时在相同平面距离心室组织块内、外膜侧距离分别约为1 . 0～1. 5 cm处放置电极记录楔状组织块跨壁心电图(ECG)。获得稳定的内、外膜侧心肌细胞APD、跨壁ECG同步记录。为验证系统敏感性,分别记录延长复极但不引发TdP的胺碘酮(100μmol/L,)、延长复极且引发TdP的莫西沙星(100μmol/L)给药前及给药后30min 1Hz频率刺激下APD、ECG。计算以下参数:APD_(90)(动作电位复极90%需要时间)、跨壁复极离散度(TDR ) =内膜下细胞APD_(90)—外膜下细胞APD_(90)、QT间期(为QRS复合波的起点到T波降支终点之间的时程)、Tp-e间期(为T波的峰值与T波终点之间的时程,一般认为该时程间接反映跨壁TDR)。同时观察药物是否引发早后除极(EAD)。
结果:(1)在动脉灌流组织块标本上,从心外膜侧到内膜侧心肌细胞APD呈逐渐延长趋势,除在两例标本的中间层细胞上偶记录到平台期明显较内、外膜层长的APD外,未发现明显的M细胞层。故心内、外膜细胞APD同步记录时选择内、外各约1/3侧肌层细胞。(2)取得心内、外膜细胞APD、跨壁ECG同步记录后,分别在0min、30min、60min、90min、120min给予1Hz刺激, APD_(90)、QT间期在2h内无明显改变,表明获得了稳定的组织块灌流标本。(3)标本稳定1~1.5小时后,灌流液中分别加入胺碘酮(100μmol/L)、莫西沙星(100μmol/L)。结果显示胺碘酮延长外膜层心肌APD_(90)(由药前的277±12延长至365±16,p<0.05),同时亦延长内膜层心肌APD_(90)(由药前的329±22延长至406±19,p<0.05),但由于外膜的延长幅度大于内膜,TDR明显减小(由对照的52±13减小到40±11,p<0.05),QT间期明显延长(由药前的395±14延长至460±17, p<0.05),Tp-e间期明显减小(由对照的67±12减小到53±14,p<0.05)。所有记录标本上均未发现EAD。莫西沙星可显著延长外膜层心肌APD_(90)(由药前的287±17延长至335±21,p<0.05),同时亦延长内膜层心肌APD_(90)(由药前的335±13延长至529±18,p<0.05),其中内膜延长幅度远大于外膜的延长幅度,TDR明显增大(由对照的48±10增大到195±33 ,p<0.05),QT间期显著延长(由药前的365±20延长至547±23,p<0.05),Tp-e间期显著增大(由对照的62±14增加到221±23,p<0.05),在六例标本中有四例出现EAD。
结论:本实验建立了稳定的兔左心室动脉灌流楔形组织模型,此模型可同步记录心内、外膜下心肌细胞动作电位及跨壁组织块心电图。利用延长复极但不引发TdP的胺碘酮和延长复极且引发TdP的莫西沙星验证了系统各电生理指标的敏感性。该模型可用于QT延迟药物潜在致心律失常危险性的评价。
二、利用兔左心室动脉灌流楔形模型评价阿霉素的急性心脏毒性
目的:评价阿霉素急性应用的潜在致心律失常危险性
方法:参照第一部分实验方法制备稳定的兔左心室动脉灌流楔形组织标本,分别在1Hz、0.5Hz刺激频率下,记录阿霉素(100μmol/L)给药前及给药后20min、40min、60min APD及ECG。计算以下参数:APD_(90)(动作电位复极90%需要时间)、跨壁复极离散度(TDR ) =内膜下细胞APD_(90)—外膜下细胞APD_(90)、QT间期(为QRS复合波的起点到T波降支终点之间的时程)、Tp-e间期(为T波的峰值与T波终点之间的时程,一般认为该时程间接反映跨壁TDR),同时观察药物是否引发早后除极(EAD)。
结果:取得心内、外膜细胞APD、跨壁ECG同步记录后,灌流液中加入阿霉素,在1Hz和0.5Hz频率刺激下,灌流前和灌流20min、40min、60min后相比较,左心室壁内、外膜动作电位时程(APD)、TDR、QT间期、Tp-e间期均无显著性差异(P > 0. 05)。结果显示,在1Hz刺激频率下,阿霉素在一小时内对外膜层心肌APD_(90)无明显作用(给药前后变化分别为362±12、366±25、365±23、361±13,P > 0. 05),对心内膜亦无明显作用(给药前后变化分别为402±22、405±27、405±17、398±21,P > 0. 05),TDR无明显变化(给药前后分别为42±9、40±12、41±11、38±16,P > 0. 05),QT间期无明显变化(给药前后分别为419±25、416±22、420±12、418±13,P > 0. 05),Tp-e间期无明显变化(给药前后分别为61±13、60±10、63±15、60±11,P > 0. 05)。在0.5Hz刺激频率下,阿霉素在一小时内对外膜层心肌APD_(90)亦无明显作用(给药前后变化分别为360±16、364±13、365±11、361±20,P > 0. 05),对心内膜亦无明显作用(给药前后变化分别为401±11、405±17、407±12、397±24,P > 0. 05),TDR无明显变化(给药前后分别为41±15、40±10、41±11、37±9,P > 0. 05),QT间期无明显变化(给药前后分别为421±13、424±20、426±14、420±21,P > 0. 05),Tp-e间期无明显变化(给药前后分别为62±10、61±13、63±16、60±12,P > 0. 05)。
结论:阿霉素急性应用,在短时间内对心肌无明显致心律失常作用
There have been more and more reports about drug-induced torsade de pointes (TdP) which can lead to cardiogenic syncope or even sudden death ever since antiarrhythmic drug quinidine-induced TdP caused by repolarization delay was found in 1950s. Besides antiarrhythmic drug that can cause repolarization delay, it has been found that antihistaminics, psychotroptic drug, antiseptic, antineoplastic and cardiovascular drugs contribute to TdP as well. Although the chemical structures of these drugs are different, they can all block the delayed rectifier potassium current, Ikr (encoded by human ether-a-go-go related gene, HERG), which plays an important role in cardiac repolarization, resulting in the delayed repolarization characterized by lengthening of the QT interval on the surface ECG. QT prolongation can cause increase of transmural dispersion of repolarization (TDR), greatly increase the risk of ventricular tachycardia. Many drugs like Terfenadine, Astemizole, Cisapride, Mibefradil and Grepafloxacin have been withdrawn from the market in recent years. Based on the understanding of this drug-induced deadly threat, the International Committee for Harmonisation (ICH) issued the discussion paper of guideline:“Safety pharmacology studies for assessing the potential for delayed ventriculat repolarization: QT interval prolongation by human pharmaceuticals”in November 2002,and formed the final project after soliciting the suggestions widely in May 2005, and was officially put into use as the supplementary guideline of international drug safety pharmacology research(S7B) in December 2005.
The S7B guideline requires that the non-clinical safety assessment for drug-induced QT interval prolongation should include following 4 different functional levels: 1) Ionic currents measured in isolated animal or human cardiac myocytes, cultured cardiac cell lines, or heterologous expression systems for cloned human ion channels. 2) Action potential parameters in isolated cardiac preparations or specific electrophysilology parameters indicative of action potential duration in anesthetized animals. 3) ECG parameters in conscious or anesthetized animals. 4) Proarrhythmic effects measured in isolated cardiac preparations or animals. The first three containing the evaluation of HERG current, action potential repolarization parameters and QT interval on ECG in conscious or anesthetized animals are all judge the potential risk of drugs-induced arrhythmia indirectly through the repolarization delayed index or parameters. However, because of the uncertain relationship between the extent of drugs-induced repolarization delay and the level of drugs-induced arrhythmia risk, the development of proarrhythmic models in vitro or in vivo has become a focus recently. Because drugs-induced TdP often emerge in pathological conditions, especially in the heart with low repolarization reserves (such as congenital LQTs, cardiac hypertrophy or heart failure , etc), it is hart to find arrhythmias caused by drugs with the function of prolong QT interval in normal. Studies show that the methoxamine-sensitized rabbit model, and the chronic AV block dog model can improve the sensibility of assessment, in addition, there are also the isolated arterially perfused left ventricular wedge preparation and the Langendorff-perfused rabbit heart. In the above of four, the isolated arterially perfused left ventricular wedge preparation is being widely used for its highly sensitivity and specificity, and it can help us understand more about the cytology base of ECG repolarization waves and the mechanism of arrhythmia.
Up to now, safety evaluation and technical standards have not been set up on drug-induced arrhythmias in China, especially lack of proarrhythmic model for preclinical drug safety evaluation.
The aim of this study is to build the proarrhythmic model, in which rabbit rabbit left ventricular wedge preparations were perfused with Tyrode solution continuously via left circumflex, and the action potentials of endocardium, epicardium or transmural electrocardiogram were recorded simultaneously. Acute potential proarrhythmic risk of adriamycin was assessed in this method . As a part of experimental system for cardiac-safty assessment on proarrhythmic risk of QT prolonged drugs, the proarrhythmic model can be used in the non-clinical safety assessment for new drug, especially for innovative drugs. This study will be of importance for forecasting the adverse effect of drugs and reasonably evaluating the foreground of new pharmaceutical compounds and thus reducing drug development risk and protecting people's life safety in the field of drug research and development,.
Part.1 The building of the arterially perfused rabbit left ventricular wedge preparation
Objective: To build the steady and effective arterially perfused ventricular wedge preparation
Methods:
Female New Zealand White rabbits weighing 2.0–2.5kg,at around 3-4 months of age, were anticoagulated with heparin(200 IU/kg), anesthetized by intravenous administration of pentobarbital sodium (30–35 mg/kg). The chest was opened via a left thoracotomy, and the heart was excised and placed in a cardioplegic solution consisting of cold (4°C) cardioplegic solution. The left circum?ex branch of the coronary artery was then cannulated using a self made cannula and perfused with the cardioplegic solution. Once adequate perfusion was established, the cannula was tied with silk sutures., and make a 10 ~ 20 mm long, 5 ~ 10 mm wide, 3 ~ 3.5 mm thick ventricle muscle wedge tissue. The preparation was then placed in a small tissue bath containing normal Tyrode's solution and arterially perfused with Tyrode's solution. Perfusion speed is 3～4ml/min .The temperature of the bath and the arterial perfusate was maintained at 35.7±0.1°C.The constituents of Tyrode's solution were as follows(mmol/L): NaCl 140、KCl 5.4、MgCl2 1.0、CaCl2 2.0、HEPS 10、glucose 10(pH7.35～7.45).
Glass microelectrode (DC resistance, 10 to 20 M?) was pulled and filled with 3.0mol/L KCl and then connected to ?oating platinum silk electrodes, recorded via high-input impedance amplifier and multi-channel physiological signal acquisition system. Using floating microelectrodes record the action potential continuously with 1~2mm intervals of ventricular wall from outside to inside at the stimulatory frequency of 1Hz, explored the character of transmembrane APD and confirm the recording sites on subendocardial and subepicardial. The electrodes were placed in the Tyrode’s solution bathing the preparation, 1.0 to 1.5cm from the epicardial and endocardial surfaces of the preparation. Impalements were obtained from the endocardial and the epicardial surface of the preparation at the positions approximating the transmural axis of the ECG recording. Get the steady recording. To verify its sensibility, the action potential duration (APD),transmural dispersion of repolarization (TDR) and QT intervals were compared before and 30min after perfusion with amiodarone(100μmol/L),whitch prolong repolarization but hardly cause TdP, and moxifloxacin(100μmol/L),whitch prolong repolarization and cause TdP. APD was measured at 90% repolarization (APD_(90)). TDR was defined as the difference between the longest and the shortest repolarization times (activation time plus APD_(90)) of transmembrane action potentials recorded across the wall. The QT interval was defined as the time between QRS onset and the point at which the final downslope of the T wave crossed the baseline , Tp-e was the interval from the peak to the end of the T wave, which may correspond to the transmural dispersion of repolarization. And observe if EAD can be induced.
Results: (1)On the arterially perfused rabbit left ventricular wedge preparation, the APD increase from from the epicardial to endocardial, except longer APD was recorded in the middle of left ventricular wall in two cases, the M cell was not clearly found out. Thus the 1/3 of inside and outside are the recording sites. (2)There was no change on APD_(90)、QT interval at 0min、30min、60min、90min、120min,indicating the model was steady. (3)After 1~1.5 hours, perfuse amiodarone(100μmol/L) and moxifloxacin(100μmol/L). Amiodarone produced a prolongation in both epicardial APD_(90) (from 277±12 to 365±16,p<0.05) and endocardial APD_(90)(from 329±22 to 406±19, p<0.05) as well as the QT interval(from 395±14 to 460±17, p<0.05) on transmural ECG at the stimulatory frequency of 1Hz , Moreover ,the prolongation of APD in the endocardial region was less prominent than that in the epicardial region ,accordingly, TDR(from 52±13 to 40±11,p<0.05) and Tp-e(from 67±12 to 53±14,p<0.05)were obviously decreased. There were no EAD observed in all cases. Moxifloxacin produced a prolongation in both epicardial APD_(90) (form 287±17 to 335±21 , p<0.05) and endocardial APD_(90)(from 335±13 to 529±18,p<0.05) as well as the QT interval(from 365±20 to 547±23, p<0.05)on transmural ECG at the stimulatory frequency of 1Hz ,unlike amiodarone, the prolongation of APD in the endocardial region was more prominent than that in the epicardial region ,accordingly, TDR(from 48±10 to 195±33,p<0.05) and Tp-e(from 62±14 to 221±23,p<0.05)were obviously increased. There was EAD observed in four out of six cases.
Conclusion: The arterially perfused rabbit left ventricular wedge proarrhythmic model has been builded, in which the action potentials of endocardium, epicardium or transmural electrocardiogram were recorded simultaneously. Amiodarone (prolong repolarization but hardly cause TdP), and moxifloxacin (prolong repolarization and cause TdP) were tested used to prove that the electrophysiologic parameters were effective and the model were effective in evaluating drug induced potential arrhythmia.
Part 2 The application of the arterially perfused rabbit left ventricular wedge preparation in estimating acute heart toxicity of adriamycin
Objective: To estimate acute heart toxicity of adriamycin
Methods: the methods were the same as part 1.The action potential of endocardium, epicardium and transmural electrocardiogram were recorded simultaneously using floating microelectrodes at the stimulatory frequencies of 1Hz and 0.5Hz. The action potential duration (APD),transmural dispersion of repolarization (TDR) and QT intervals were compared before and 20、40、60min after perfusion with adriamycin(100μmol/L). APD was measured at 90% repolarization (APD_(90)). TDR was defined as the difference between the longest and the shortest repolarization times (activation time plus APD_(90)) of transmembrane action potentials recorded across the wall. The QT interval was defined as the time between QRS onset and the point at which the final downslope of the T wave crossed the baseline, Tp-e was the interval from the peak to the end of the T wave, which may correspond to the transmural dispersion of repolarization. And observe if EAD can be induced.
Results:The action potential of endocardium, epicardium and transmural electrocardiogram were recorded simultaneously, amiodarone(100μmol/L)was perfused, at the stimulatory frequencies of 1Hz and 0.5Hz , there was no obvious change in both epicardial and endocardial APD as well as ECG before and 20、40、60min after perfusion with adriamycin. At the stimulatory frequencies of 1Hz, there was no obvious change in epicardial APD_(90)( from 362±12 to 366±25、365±23、361±13ms, P > 0.05) , endocardial APD_(90)( from 402±22 to 405±27、405±17、398±21ms, P > 0.05) and QT interval(from 419±25 to 416±22、420±12、418±13ms,P > 0. 05), TDR(from 42±9 to 40±12、41±11、38±16ms,P > 0. 05)and Tp-e(from 61±13 to 60±10、63±15、60±11ms,P > 0. 05)had no obvious change, either. At the stimulatory frequencies of 0.5 Hz, there was no obvious change in epicardial APD_(90)(from 360±16 to 364±13、365±11、361±20,P > 0. 05) , endocardial APD_(90)(from 401±11 to 405±17、407±12、397±24,P > 0. 05)and QT interva(lfrom 421±13 to 424±20、426±14、420±21,P > 0. 05), TDR(from 41±15 to 40±10、41±11、37±9,P > 0. 05)and Tp-e(from 62±10 to 61±13、63±16、60±12,P > 0. 05)had no obvious change, either.
Conclusion: The risk of adriamycin-induced arrhythmia is low in a short time.
S7B指导原则要求对QT间期延长药物的非临床安全性评价包括以下4个不同功能水平的研究:1)采用离体动物或人体心脏细胞、培养心脏细胞系或克隆人离子通道的异种表达体系评价对HERG离子流的影响。2)在离体心脏标本进行动作电位参数测定,或在麻醉动物中进行能体现动作电位周期的特异性电生理参数检测。3)测定清醒或麻醉动物的ECG参数。4)在离体心脏标本或动物进行致心律失常作用测定。其中前三项包括HERG电流、动作电位复极参数以及整体动物心电图QT间期的评价均为通过延迟复极指标或参数间接判断药物引发心律失常的潜在危险。然而,由于药物延迟复极程度与致心律失常危险性高低之间的关系尚未确立,近几年,建立离体或整体敏感心律失常动物模型成为研究热点。因药物诱发TdP常常是在病理情况下,特别是心肌复极储备降低的心脏(如遗传性LQTs、心肌肥厚或心衰等),常规正常动物心脏模型难以观察到QT延长药物的致心律失常作用,有研究显示甲氧胺敏化兔模型以及慢性房室阻断犬模型可提高评价的敏感性,此外,已报道的心律失常动物模型有离体动脉灌流左室楔形标本及Langendorff灌流兔心。在上述报道的四种模型中,动脉灌流左心室楔形标本具有较高的敏感性和特异性,而被越来越多地采用,该模型还对于理解心电图复极波的细胞基础及心律失常发生机制具有重要帮助。
目前,我国对药物致心律失常危险性的认识处于起步阶段,尚未建立完善的的技术评价体系,尤其是缺乏敏感心律失常模型。
本实验拟建立冠状动脉灌流兔左心室楔形组织模型,即制作兔左心室游离壁楔形标本,经冠状动脉左回旋支灌流台式液,采用浮置玻璃微电极同步记录心内膜、心外膜下心肌细胞动作电位及跨室壁心电图,并利用此方法评价阿霉素的急性心脏毒性。该心律失常模型的建立将作为“QT延迟药物潜在致心律失常危险性”评价技术体系的一部分,用于新药,特别是创新药物非临床安全性评价。可在新药研发过程中及早发现新药潜在的致命性不良反应,从而做到合理评估药物的研发前景,对降低创新药物研发风险及预测药物严重不良反应、保障人民生命安全均具有十分重要的意义。
一兔左心室动脉灌流楔形模型的建立
目的:建立稳定、敏感的兔左心室动脉灌流楔形组织模型
方法: 3~4个月龄新西兰雌性白兔,体重2.0～2. 5kg,耳缘静脉注射肝素(200 IU/kg)抗凝、戊巴比妥钠( 30～35 mg/kg )麻醉后,迅速开胸取出心脏。先以低温停跳液(KCl为24mmol/L、不含Ca~(2+)的台氏液)从主动脉逆行灌流,将自制灌流针经主动脉左窦开口处插入冠状动脉左侧回旋支,制成以回旋支为中心轴,长约10～20 mm、宽约5～10 mm、厚约3～3.5 mm的心室肌楔型组织块。将组织块固定于恒温浴槽中,持续灌流充以纯氧的正常台氏液,温度35.7±0.1°C,灌流速度为3～4ml/min。台式液组成(mmol/L):NaCl 140、KCl 5.4、MgCl_2 1.0、CaCl_2 2.0、HEPS 10、葡萄糖10(pH7.35～7.45)。
微电极拉制器制备记录玻璃微电极,使其内充3 mol/L KCl时的电极电阻为10～20MΩ。玻璃微电极与悬浮铂金丝电极相连,经微电极放大器、多道生理信号采集系统进行信号转化、记录。以1~2mm的间隔从外膜向内膜侧心肌的动作电位(APD)进行记录,观察跨壁各层的动作电位时程特点,以确定内、外膜下心肌记录部位。
将两根玻璃微电极扎入同一平面内、外膜心肌细胞,同时在相同平面距离心室组织块内、外膜侧距离分别约为1 . 0～1. 5 cm处放置电极记录楔状组织块跨壁心电图(ECG)。获得稳定的内、外膜侧心肌细胞APD、跨壁ECG同步记录。为验证系统敏感性,分别记录延长复极但不引发TdP的胺碘酮(100μmol/L,)、延长复极且引发TdP的莫西沙星(100μmol/L)给药前及给药后30min 1Hz频率刺激下APD、ECG。计算以下参数:APD_(90)(动作电位复极90%需要时间)、跨壁复极离散度(TDR ) =内膜下细胞APD_(90)—外膜下细胞APD_(90)、QT间期(为QRS复合波的起点到T波降支终点之间的时程)、Tp-e间期(为T波的峰值与T波终点之间的时程,一般认为该时程间接反映跨壁TDR)。同时观察药物是否引发早后除极(EAD)。
结果:(1)在动脉灌流组织块标本上,从心外膜侧到内膜侧心肌细胞APD呈逐渐延长趋势,除在两例标本的中间层细胞上偶记录到平台期明显较内、外膜层长的APD外,未发现明显的M细胞层。故心内、外膜细胞APD同步记录时选择内、外各约1/3侧肌层细胞。(2)取得心内、外膜细胞APD、跨壁ECG同步记录后,分别在0min、30min、60min、90min、120min给予1Hz刺激, APD_(90)、QT间期在2h内无明显改变,表明获得了稳定的组织块灌流标本。(3)标本稳定1~1.5小时后,灌流液中分别加入胺碘酮(100μmol/L)、莫西沙星(100μmol/L)。结果显示胺碘酮延长外膜层心肌APD_(90)(由药前的277±12延长至365±16,p<0.05),同时亦延长内膜层心肌APD_(90)(由药前的329±22延长至406±19,p<0.05),但由于外膜的延长幅度大于内膜,TDR明显减小(由对照的52±13减小到40±11,p<0.05),QT间期明显延长(由药前的395±14延长至460±17, p<0.05),Tp-e间期明显减小(由对照的67±12减小到53±14,p<0.05)。所有记录标本上均未发现EAD。莫西沙星可显著延长外膜层心肌APD_(90)(由药前的287±17延长至335±21,p<0.05),同时亦延长内膜层心肌APD_(90)(由药前的335±13延长至529±18,p<0.05),其中内膜延长幅度远大于外膜的延长幅度,TDR明显增大(由对照的48±10增大到195±33 ,p<0.05),QT间期显著延长(由药前的365±20延长至547±23,p<0.05),Tp-e间期显著增大(由对照的62±14增加到221±23,p<0.05),在六例标本中有四例出现EAD。
结论:本实验建立了稳定的兔左心室动脉灌流楔形组织模型,此模型可同步记录心内、外膜下心肌细胞动作电位及跨壁组织块心电图。利用延长复极但不引发TdP的胺碘酮和延长复极且引发TdP的莫西沙星验证了系统各电生理指标的敏感性。该模型可用于QT延迟药物潜在致心律失常危险性的评价。
二、利用兔左心室动脉灌流楔形模型评价阿霉素的急性心脏毒性
目的:评价阿霉素急性应用的潜在致心律失常危险性
方法:参照第一部分实验方法制备稳定的兔左心室动脉灌流楔形组织标本,分别在1Hz、0.5Hz刺激频率下,记录阿霉素(100μmol/L)给药前及给药后20min、40min、60min APD及ECG。计算以下参数:APD_(90)(动作电位复极90%需要时间)、跨壁复极离散度(TDR ) =内膜下细胞APD_(90)—外膜下细胞APD_(90)、QT间期(为QRS复合波的起点到T波降支终点之间的时程)、Tp-e间期(为T波的峰值与T波终点之间的时程,一般认为该时程间接反映跨壁TDR),同时观察药物是否引发早后除极(EAD)。
结果:取得心内、外膜细胞APD、跨壁ECG同步记录后,灌流液中加入阿霉素,在1Hz和0.5Hz频率刺激下,灌流前和灌流20min、40min、60min后相比较,左心室壁内、外膜动作电位时程(APD)、TDR、QT间期、Tp-e间期均无显著性差异(P > 0. 05)。结果显示,在1Hz刺激频率下,阿霉素在一小时内对外膜层心肌APD_(90)无明显作用(给药前后变化分别为362±12、366±25、365±23、361±13,P > 0. 05),对心内膜亦无明显作用(给药前后变化分别为402±22、405±27、405±17、398±21,P > 0. 05),TDR无明显变化(给药前后分别为42±9、40±12、41±11、38±16,P > 0. 05),QT间期无明显变化(给药前后分别为419±25、416±22、420±12、418±13,P > 0. 05),Tp-e间期无明显变化(给药前后分别为61±13、60±10、63±15、60±11,P > 0. 05)。在0.5Hz刺激频率下,阿霉素在一小时内对外膜层心肌APD_(90)亦无明显作用(给药前后变化分别为360±16、364±13、365±11、361±20,P > 0. 05),对心内膜亦无明显作用(给药前后变化分别为401±11、405±17、407±12、397±24,P > 0. 05),TDR无明显变化(给药前后分别为41±15、40±10、41±11、37±9,P > 0. 05),QT间期无明显变化(给药前后分别为421±13、424±20、426±14、420±21,P > 0. 05),Tp-e间期无明显变化(给药前后分别为62±10、61±13、63±16、60±12,P > 0. 05)。
结论:阿霉素急性应用,在短时间内对心肌无明显致心律失常作用
There have been more and more reports about drug-induced torsade de pointes (TdP) which can lead to cardiogenic syncope or even sudden death ever since antiarrhythmic drug quinidine-induced TdP caused by repolarization delay was found in 1950s. Besides antiarrhythmic drug that can cause repolarization delay, it has been found that antihistaminics, psychotroptic drug, antiseptic, antineoplastic and cardiovascular drugs contribute to TdP as well. Although the chemical structures of these drugs are different, they can all block the delayed rectifier potassium current, Ikr (encoded by human ether-a-go-go related gene, HERG), which plays an important role in cardiac repolarization, resulting in the delayed repolarization characterized by lengthening of the QT interval on the surface ECG. QT prolongation can cause increase of transmural dispersion of repolarization (TDR), greatly increase the risk of ventricular tachycardia. Many drugs like Terfenadine, Astemizole, Cisapride, Mibefradil and Grepafloxacin have been withdrawn from the market in recent years. Based on the understanding of this drug-induced deadly threat, the International Committee for Harmonisation (ICH) issued the discussion paper of guideline:“Safety pharmacology studies for assessing the potential for delayed ventriculat repolarization: QT interval prolongation by human pharmaceuticals”in November 2002,and formed the final project after soliciting the suggestions widely in May 2005, and was officially put into use as the supplementary guideline of international drug safety pharmacology research(S7B) in December 2005.
The S7B guideline requires that the non-clinical safety assessment for drug-induced QT interval prolongation should include following 4 different functional levels: 1) Ionic currents measured in isolated animal or human cardiac myocytes, cultured cardiac cell lines, or heterologous expression systems for cloned human ion channels. 2) Action potential parameters in isolated cardiac preparations or specific electrophysilology parameters indicative of action potential duration in anesthetized animals. 3) ECG parameters in conscious or anesthetized animals. 4) Proarrhythmic effects measured in isolated cardiac preparations or animals. The first three containing the evaluation of HERG current, action potential repolarization parameters and QT interval on ECG in conscious or anesthetized animals are all judge the potential risk of drugs-induced arrhythmia indirectly through the repolarization delayed index or parameters. However, because of the uncertain relationship between the extent of drugs-induced repolarization delay and the level of drugs-induced arrhythmia risk, the development of proarrhythmic models in vitro or in vivo has become a focus recently. Because drugs-induced TdP often emerge in pathological conditions, especially in the heart with low repolarization reserves (such as congenital LQTs, cardiac hypertrophy or heart failure , etc), it is hart to find arrhythmias caused by drugs with the function of prolong QT interval in normal. Studies show that the methoxamine-sensitized rabbit model, and the chronic AV block dog model can improve the sensibility of assessment, in addition, there are also the isolated arterially perfused left ventricular wedge preparation and the Langendorff-perfused rabbit heart. In the above of four, the isolated arterially perfused left ventricular wedge preparation is being widely used for its highly sensitivity and specificity, and it can help us understand more about the cytology base of ECG repolarization waves and the mechanism of arrhythmia.
Up to now, safety evaluation and technical standards have not been set up on drug-induced arrhythmias in China, especially lack of proarrhythmic model for preclinical drug safety evaluation.
The aim of this study is to build the proarrhythmic model, in which rabbit rabbit left ventricular wedge preparations were perfused with Tyrode solution continuously via left circumflex, and the action potentials of endocardium, epicardium or transmural electrocardiogram were recorded simultaneously. Acute potential proarrhythmic risk of adriamycin was assessed in this method . As a part of experimental system for cardiac-safty assessment on proarrhythmic risk of QT prolonged drugs, the proarrhythmic model can be used in the non-clinical safety assessment for new drug, especially for innovative drugs. This study will be of importance for forecasting the adverse effect of drugs and reasonably evaluating the foreground of new pharmaceutical compounds and thus reducing drug development risk and protecting people's life safety in the field of drug research and development,.
Part.1 The building of the arterially perfused rabbit left ventricular wedge preparation
Objective: To build the steady and effective arterially perfused ventricular wedge preparation
Methods:
Female New Zealand White rabbits weighing 2.0–2.5kg,at around 3-4 months of age, were anticoagulated with heparin(200 IU/kg), anesthetized by intravenous administration of pentobarbital sodium (30–35 mg/kg). The chest was opened via a left thoracotomy, and the heart was excised and placed in a cardioplegic solution consisting of cold (4°C) cardioplegic solution. The left circum?ex branch of the coronary artery was then cannulated using a self made cannula and perfused with the cardioplegic solution. Once adequate perfusion was established, the cannula was tied with silk sutures., and make a 10 ~ 20 mm long, 5 ~ 10 mm wide, 3 ~ 3.5 mm thick ventricle muscle wedge tissue. The preparation was then placed in a small tissue bath containing normal Tyrode's solution and arterially perfused with Tyrode's solution. Perfusion speed is 3～4ml/min .The temperature of the bath and the arterial perfusate was maintained at 35.7±0.1°C.The constituents of Tyrode's solution were as follows(mmol/L): NaCl 140、KCl 5.4、MgCl2 1.0、CaCl2 2.0、HEPS 10、glucose 10(pH7.35～7.45).
Glass microelectrode (DC resistance, 10 to 20 M?) was pulled and filled with 3.0mol/L KCl and then connected to ?oating platinum silk electrodes, recorded via high-input impedance amplifier and multi-channel physiological signal acquisition system. Using floating microelectrodes record the action potential continuously with 1~2mm intervals of ventricular wall from outside to inside at the stimulatory frequency of 1Hz, explored the character of transmembrane APD and confirm the recording sites on subendocardial and subepicardial. The electrodes were placed in the Tyrode’s solution bathing the preparation, 1.0 to 1.5cm from the epicardial and endocardial surfaces of the preparation. Impalements were obtained from the endocardial and the epicardial surface of the preparation at the positions approximating the transmural axis of the ECG recording. Get the steady recording. To verify its sensibility, the action potential duration (APD),transmural dispersion of repolarization (TDR) and QT intervals were compared before and 30min after perfusion with amiodarone(100μmol/L),whitch prolong repolarization but hardly cause TdP, and moxifloxacin(100μmol/L),whitch prolong repolarization and cause TdP. APD was measured at 90% repolarization (APD_(90)). TDR was defined as the difference between the longest and the shortest repolarization times (activation time plus APD_(90)) of transmembrane action potentials recorded across the wall. The QT interval was defined as the time between QRS onset and the point at which the final downslope of the T wave crossed the baseline , Tp-e was the interval from the peak to the end of the T wave, which may correspond to the transmural dispersion of repolarization. And observe if EAD can be induced.
Results: (1)On the arterially perfused rabbit left ventricular wedge preparation, the APD increase from from the epicardial to endocardial, except longer APD was recorded in the middle of left ventricular wall in two cases, the M cell was not clearly found out. Thus the 1/3 of inside and outside are the recording sites. (2)There was no change on APD_(90)、QT interval at 0min、30min、60min、90min、120min,indicating the model was steady. (3)After 1~1.5 hours, perfuse amiodarone(100μmol/L) and moxifloxacin(100μmol/L). Amiodarone produced a prolongation in both epicardial APD_(90) (from 277±12 to 365±16,p<0.05) and endocardial APD_(90)(from 329±22 to 406±19, p<0.05) as well as the QT interval(from 395±14 to 460±17, p<0.05) on transmural ECG at the stimulatory frequency of 1Hz , Moreover ,the prolongation of APD in the endocardial region was less prominent than that in the epicardial region ,accordingly, TDR(from 52±13 to 40±11,p<0.05) and Tp-e(from 67±12 to 53±14,p<0.05)were obviously decreased. There were no EAD observed in all cases. Moxifloxacin produced a prolongation in both epicardial APD_(90) (form 287±17 to 335±21 , p<0.05) and endocardial APD_(90)(from 335±13 to 529±18,p<0.05) as well as the QT interval(from 365±20 to 547±23, p<0.05)on transmural ECG at the stimulatory frequency of 1Hz ,unlike amiodarone, the prolongation of APD in the endocardial region was more prominent than that in the epicardial region ,accordingly, TDR(from 48±10 to 195±33,p<0.05) and Tp-e(from 62±14 to 221±23,p<0.05)were obviously increased. There was EAD observed in four out of six cases.
Conclusion: The arterially perfused rabbit left ventricular wedge proarrhythmic model has been builded, in which the action potentials of endocardium, epicardium or transmural electrocardiogram were recorded simultaneously. Amiodarone (prolong repolarization but hardly cause TdP), and moxifloxacin (prolong repolarization and cause TdP) were tested used to prove that the electrophysiologic parameters were effective and the model were effective in evaluating drug induced potential arrhythmia.
Part 2 The application of the arterially perfused rabbit left ventricular wedge preparation in estimating acute heart toxicity of adriamycin
Objective: To estimate acute heart toxicity of adriamycin
Methods: the methods were the same as part 1.The action potential of endocardium, epicardium and transmural electrocardiogram were recorded simultaneously using floating microelectrodes at the stimulatory frequencies of 1Hz and 0.5Hz. The action potential duration (APD),transmural dispersion of repolarization (TDR) and QT intervals were compared before and 20、40、60min after perfusion with adriamycin(100μmol/L). APD was measured at 90% repolarization (APD_(90)). TDR was defined as the difference between the longest and the shortest repolarization times (activation time plus APD_(90)) of transmembrane action potentials recorded across the wall. The QT interval was defined as the time between QRS onset and the point at which the final downslope of the T wave crossed the baseline, Tp-e was the interval from the peak to the end of the T wave, which may correspond to the transmural dispersion of repolarization. And observe if EAD can be induced.
Results:The action potential of endocardium, epicardium and transmural electrocardiogram were recorded simultaneously, amiodarone(100μmol/L)was perfused, at the stimulatory frequencies of 1Hz and 0.5Hz , there was no obvious change in both epicardial and endocardial APD as well as ECG before and 20、40、60min after perfusion with adriamycin. At the stimulatory frequencies of 1Hz, there was no obvious change in epicardial APD_(90)( from 362±12 to 366±25、365±23、361±13ms, P > 0.05) , endocardial APD_(90)( from 402±22 to 405±27、405±17、398±21ms, P > 0.05) and QT interval(from 419±25 to 416±22、420±12、418±13ms,P > 0. 05), TDR(from 42±9 to 40±12、41±11、38±16ms,P > 0. 05)and Tp-e(from 61±13 to 60±10、63±15、60±11ms,P > 0. 05)had no obvious change, either. At the stimulatory frequencies of 0.5 Hz, there was no obvious change in epicardial APD_(90)(from 360±16 to 364±13、365±11、361±20,P > 0. 05) , endocardial APD_(90)(from 401±11 to 405±17、407±12、397±24,P > 0. 05)and QT interva(lfrom 421±13 to 424±20、426±14、420±21,P > 0. 05), TDR(from 41±15 to 40±10、41±11、37±9,P > 0. 05)and Tp-e(from 62±10 to 61±13、63±16、60±12,P > 0. 05)had no obvious change, either.
Conclusion: The risk of adriamycin-induced arrhythmia is low in a short time.
引文
1 Belardinelli L, Antzelevitch C, VosMA. Assessing predictors of drug-induced torsade de pointes. Trends Pharmacol Sci, 2003; 24: 619-625
2 Antzelevitch C.Arrhythmogenic mechanisms of QT prolongling drugs:Is QT prolongation really the problem Electrocardiol,2004;37(Suppl):S15-S24
3 Clancyce, Kassrs.Inherited and acquired vulnerability to ventricular arrhythmias : cardiac Na+ and K+ channels.Physiol Rev, 2005, 85 (1) : 33-47
4 Sicouri S , Antzelevitch C. A subpopulation of cells with unique electrophysiological properties in the deep subepicardiun of the canine ventricle: TheM cell. Circ Res, 1991; 68: 1729 - 1741
5 Mcintosh M A, Cobbe SM, S Mith GL, et al. Heterogeneous changes in action potential and intracellular Ca2+ in left ventricularmyocyte sub-types from rabbits with heart failure.Cardiovasc Res, 2000, 45 (2) : 397-409
6 Anyukhovsky E P, Sosunov E A, Feinmark S J, et al Effects of quinidine on repolarization in canine epicardium, midmyocardium, and endocardium: II In vivo study. Circulation, 1997, 96 (11) : 4019-4026
7 Antzelevitch C, Sun ZQ,Zhang ZQ,et al. Cellular and ionic mechanism underlying erythromycin induced long QT intervals and torsade de pointes . J Am Coll Cardiol, 1995; 28 (7) : 1836
8 Liu T, Brown BS, Wu Y, et al . Blinded validation of the isolated arterially perfused rabbit ventricular wedge in p reclinical assessment of drug-induced proarrhythmias. Heart Rhythm, 2006;3:948-956
9 Chen X, Cordes JS, Bradley JA, et al . Use of arterially perfused rabbit ventricular wedge in predicting arrhythmogenic potentials of drugs. Pharmacol Toxicol Methods, 2006; 54: 261-272
10 Xu X, Rials SJ, Wu Y, et al . Left ventricular hypertrophy decreasesslowly, but not rapidly activating delayed rectifier K+currents of epicardial and endocardialmyocytes in rabbits [ J ]. Cir culation , 2001;103: 1585 - 1590
1 Rohrdanz E, Obertrifter B, Ohler S,et al.Influence of Adriamycin and paraquat on antioxidant enzyme expression in primary rat hepatocytes[J]. Arch Toxicol,2000,74(4 5):231
2 Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L.Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. PharmacolRev 2004;56:185–229.
3 Singal PK, Deally CM, Weinberg LE. Subcellular effects of adriamycin in the heart: a concise review. J Mol Cell Cardiol 1987;19:817–28
4 Mukherjee S, Banerjee SK, Maulik M.Protection against acuteadriamycin inducedcardiotoxicity by garlic: role of endogenous antioxidants and inhibition of TNF alpha expression[J]. BMPharmacol,2003,3(1):16
5 Yeh ETH, Tong AT, Lenihan DJ, Yusuf SW, Swafford J, Champion C et al.. Cardiovascular complications of cancer therapy: diagnosis, pathogenesis, and management. Circulation 2004 109: 3122–3131.
6 Nousiainen T, Vanninen E, Rantala A, Jantunen E, Hartikainen J. QT dispersion and late potentials during doxorubicin therapy for non-Hodgkin’s lymphoma. J Intern Med 1999;245: 359–364.
7 Lacasse Y, Bolduc P (1992). Sudden death in leukemic patient treated with doxorubicin. Can J Cardiol 8: 53–56.
8 Wang GX, Wang YX, Zhou XB, Korth M (2001). Effects of doxorubicinol on excitation-contraction coupling in guinea pig ventricular myocytes. Eur J Pharmacol 423: 99–107.
9 Ducroq J, Moha ou Maati H, Guilbot S, Dilly S, Laemmel E, Pons-Himbert C, Faivre JF, Bois P, Stücker O, Le Grand M. Dexrazoxane protects the heart from acutedoxorubicin-induced QT prolongation:a key role for IKs. Br J Pharmacol,2010, 159:93–101.
10 Milberg P, Fleischer D, Stypmann J, Osada N, M?nning G, Engelen MA et al. (2007). Reduced repolarization reserve due to anthracycline therapy facilitates torsade de pointes induced by IKr blockers. Basic Res Cardiol 102:42–51.
11 Wang YX, Korth M (1995). Effects of doxorubicin on excitation-contraction coupling in guinea pig ventricular myocardium. Circ Res 76: 645–653.
1 Sicouri S, Antzelevitch C. A subpopulation of cells with unique electrophysiological properties in the deep subep icardiun of the canine ventricle: The M cell[ J ]. Circ Res, 1991; 68: 1729 - 1741
2 Liu D W,AntzelevitchC .Characteristics of the delayed rectifier current(IKr and IKs) in canine ventricular epicardial,midmyocardial,and endocardial myocytes .A weaker IKs contributes to the longer action potential of the M cell[J]. Circ Re s,1995,76(3):351-365
3 Cheng J, Kamiya K, Liu W, etal . Heterogeneous distribution of the two components of delayed rectifier K+ current:a potential mechanism of the proarrhythmic effects of methanesulfonanilide classⅢagents[J]. Cardiovasc Res, 1999, 43(1): l35-l47
4 Roden D M. Taking the“idio”out of“idiosyncratic”: predicting torsades de pointes[J].Pacing Clin Electrophysiol,199 8,21(5):1029-1034
5 Attila S. Farkas,Stanley Nattel.Minimizing Repolarization-Related Proarrhythmic Risk in Drug Development and Clinical Practice.Drugs 2010; 70 (5): 573-603
6 Guth BD, Germeyer S, Kolb W, et al . Developing a strategy for the nonclinical assessment of proarrhythmic risk of phar maceuticals due to prolonged ventricular repolarization[J].J Pharmacol Toxicol Methods, 2004, 49 (3) : 159-169
7 Josh IA, DM, Vohra Y . Preclinical strategies to assess QT liability and torsadogenic potential of new drugs : the role of experimental models [J]. J Electrocardiol, 2004, 37 ( Supp l) : 714
8 Gintant G A, Lmber IS J T, Mcdermott J S, et al.The canine Purkinje fiber : an in vitro model system for acquired long QT syndrome and drug-induced arrhythmogenesis [J]. J Cardiovasc Pharmacol, 2001, 37 (5) : 607-618
9 Milberg P, Rensch N, Wasmer K, et al. Transmural dispersion of repolarization as a key factor of arrhythmogenicity in a novel intact heartmodel of LQT3 [ J ]. Cardiovasc Res, 2005, 65(2):397-404
10 Yan GX,Antzelevitch C. Cellular basis for the electrocardi ographic J wave[ J ]. Circulation, 1996; 93: 372 - 379
11 Yang X B, Yang L. The study of electrophysiological mechanisms of ventricular arrhythmia in LQT2 models of the Long QT syndrome [ J ]. Chin J Cardiac Pacing Electrophysiol (中国心脏起搏与电生理杂志), 2004, 18 (4) : 282-284
12 Liu J Q, Yang Y Z, Kenneth R L. Optical mapping of the transmural dispersion of repolarization and early after depolarization in LQT2 and LQT3 models of long QT syndrome [ J ]. Chin JCardiac Arrhythmias (中华心律失常学杂志), 2004, 8 ( 4 ) :236
13 De Clerck F, Van deWater A, D Aubioul J, et al . In vivo measurement of QT prolongation, dispersion and arrhythmogenesis: application to the preclinical cardiovascular safety pharmacology of a new chemical entity [J].Fundam Clin Pharmacol,2002, 16 (2) : 125-140
14 Testa IL, Calderone V, Salvador IA, et al . QT prolongation in anaesthetized guineapigs:an experimental approach for preliminary screening of torsadogenicity of drugs and drug candidates [ J ]. J App l Toxicol, 2004, 24 (3) : 217-222
15 Noda Y, Hashimoto K . Development of a halothane-adrenaline arrhythmia model using in vivo Guinea pigs [ J ]. J PharmacolSci, 2004, 95 (2): 234-239
16 Hauser D S, Stade M, Schmidt A, et al . Cardiovascular parameters in anaesthetized guinea pigs:a safety pharmacology screening model[J].J Pharmacol Toxicol Methods, 2005, 52(1) : 106-114
17 Vaynblat M, Pagala M K, Dav Isw J, et al . Telemetrically monitored arrhythmogenic effects of doxorubicin in a dog model of heart failure[J].Pathophysiology,2003,9(4):241-248
18 Chugh S S, Johnson S B, Packer D L, et al . Altered response to ibutilide in a heart failure model [ J ]. Cardiovasc Res,2001, 49 (1) : 94-102
19 Carlsson L, Abrahamsson C, Andersson B, et al. Proarrhythmic effects of the class agent almokalant:importance of infusion rate, QT dispersion,andearly afterdepolarisations[J].Cardiovasc Res, 1993, 27: 2186-2193
20 He J Q, Ma Y, Lee Y, et al . Human embryonic stem cells develop into multiple types of cardiac myocytes:action potential characterization[J]. Circ Res, 2003, 93 (1) : 32-39
21 Nuyensd, Stengl M, Dugarmaa S, et al . Abrup t rate accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome[J].NatMed, 2001, 7 ( 9 ):1021-1027
22 Demolombe S, Lande G, Charpentier F, et al . Transgenic mice overexpressing human Kv LQT1 dominant-negative isoform. Part I : Phenotypic characterisation [J]. Cardiovasc Res,2001, 50 (2) : 314-327
23 Anne Royer S D, Aziza EL Harch I A, Khar Le Quanga, et al . Expression of human HERG K+ channels in the mouse heart exerts anti-arrhythmic activity [ J ]. Cardiovasc Res, 2005,65: 128-137
24 Gussak I, Chaitman B R, Kopecky SL, et al . Rapid ventricular repolarization in rodents:electrocardiographic manifestations, molecularmechanisms, and clinical insights[J]. J Electrocardiol, 2000, 33 (2) : 159-170
25 Ten Tusscher K H, Noble D, Noble P J, et al . A model for human ventricular tissue [ J ]. Am J Physiol Heart Circ Physiol, 2004, 286 (4) : H1573-1589
26 Liu T, Brown BS, Wu Y, et al . Blinded validation of the is olated arterially perfused rabbit ventricular wedge in p reclinical assessment of drug - induced p r oarrhythmias[ J ]. Heart Rhythm, 2006; 3: 948 -956
27 Belardinelli L, Antzelevitch C, VosMA. Assessing predictors of drug-induced torsade de pointes[ J ] . Trends Pharmacol Sci, 2003; 24: 619-625
28 Antzelevitch C. Arrhythmogenic mechanisms of QT prolongling drugs :Is QT prolongation really the problem [ J ] Electrocardiol, 2004; 37( Supp l . ) : S15 - S24
29 Mci Ntosh M A, Cobbe SM, Smith GL, et al . Heterogeneous changes in action potential and intracellular Ca2+in left ventricularmyocyte sub-typesfrom rabbits with heart failure [ J ].Cardiovasc Res, 2000, 45 (2) : 397-409
30 Yan G X,Antzelevitch C.Cellularbasisforthenormal Twaveand theelectrocar diographicm anifestation soft helong- QT syndrome Circulation.1998, 98 : 1928 - 1936
31 Yan GX,Shimizu W,Antzelevitch C.The characteristics and distribution of M cells in arterially-perfused canine left ventricular wedge preparations.Circulation,1998,98:1921-1927
32 Yan GX, Martin J Electrocardiographic T Wave: A Symbol of Transmural Dispersion of Repolarization in the Ventricles . J Cardiovasc Electrophysiol, 2003, 14 (6) : 639 - 640
33 Hevia JC, Antzelevitch C, Bárzaga FT, et al . Tpeak-Tend and Tpeak-Tend dispersion as risk factors for ventricular tachycardia ventricular fibrillati on inpatients with the Brugada Syndrome . J Am Coll Cardi ol, 2006, 47 (9) : 1828 - 1834
34 Fish JM, Di Diego JM, Nesterenko VV, et al .Epicardial activation of left ventricular wall prolongs QT interval and transmural dispersion of repolarization implications for biventricular pacing . Circulation, 2004,109 (17) : 2136 - 2142
35 Gup ta P, Patel C, Patel H,et al.Tp-e ratio as an index of arrhythmogenesis . J Electr ocardiol, 2008, 41 (6) : 567 - 574
2 Antzelevitch C.Arrhythmogenic mechanisms of QT prolongling drugs:Is QT prolongation really the problem Electrocardiol,2004;37(Suppl):S15-S24
3 Clancyce, Kassrs.Inherited and acquired vulnerability to ventricular arrhythmias : cardiac Na+ and K+ channels.Physiol Rev, 2005, 85 (1) : 33-47
4 Sicouri S , Antzelevitch C. A subpopulation of cells with unique electrophysiological properties in the deep subepicardiun of the canine ventricle: TheM cell. Circ Res, 1991; 68: 1729 - 1741
5 Mcintosh M A, Cobbe SM, S Mith GL, et al. Heterogeneous changes in action potential and intracellular Ca2+ in left ventricularmyocyte sub-types from rabbits with heart failure.Cardiovasc Res, 2000, 45 (2) : 397-409
6 Anyukhovsky E P, Sosunov E A, Feinmark S J, et al Effects of quinidine on repolarization in canine epicardium, midmyocardium, and endocardium: II In vivo study. Circulation, 1997, 96 (11) : 4019-4026
7 Antzelevitch C, Sun ZQ,Zhang ZQ,et al. Cellular and ionic mechanism underlying erythromycin induced long QT intervals and torsade de pointes . J Am Coll Cardiol, 1995; 28 (7) : 1836
8 Liu T, Brown BS, Wu Y, et al . Blinded validation of the isolated arterially perfused rabbit ventricular wedge in p reclinical assessment of drug-induced proarrhythmias. Heart Rhythm, 2006;3:948-956
9 Chen X, Cordes JS, Bradley JA, et al . Use of arterially perfused rabbit ventricular wedge in predicting arrhythmogenic potentials of drugs. Pharmacol Toxicol Methods, 2006; 54: 261-272
10 Xu X, Rials SJ, Wu Y, et al . Left ventricular hypertrophy decreasesslowly, but not rapidly activating delayed rectifier K+currents of epicardial and endocardialmyocytes in rabbits [ J ]. Cir culation , 2001;103: 1585 - 1590
1 Rohrdanz E, Obertrifter B, Ohler S,et al.Influence of Adriamycin and paraquat on antioxidant enzyme expression in primary rat hepatocytes[J]. Arch Toxicol,2000,74(4 5):231
2 Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L.Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. PharmacolRev 2004;56:185–229.
3 Singal PK, Deally CM, Weinberg LE. Subcellular effects of adriamycin in the heart: a concise review. J Mol Cell Cardiol 1987;19:817–28
4 Mukherjee S, Banerjee SK, Maulik M.Protection against acuteadriamycin inducedcardiotoxicity by garlic: role of endogenous antioxidants and inhibition of TNF alpha expression[J]. BMPharmacol,2003,3(1):16
5 Yeh ETH, Tong AT, Lenihan DJ, Yusuf SW, Swafford J, Champion C et al.. Cardiovascular complications of cancer therapy: diagnosis, pathogenesis, and management. Circulation 2004 109: 3122–3131.
6 Nousiainen T, Vanninen E, Rantala A, Jantunen E, Hartikainen J. QT dispersion and late potentials during doxorubicin therapy for non-Hodgkin’s lymphoma. J Intern Med 1999;245: 359–364.
7 Lacasse Y, Bolduc P (1992). Sudden death in leukemic patient treated with doxorubicin. Can J Cardiol 8: 53–56.
8 Wang GX, Wang YX, Zhou XB, Korth M (2001). Effects of doxorubicinol on excitation-contraction coupling in guinea pig ventricular myocytes. Eur J Pharmacol 423: 99–107.
9 Ducroq J, Moha ou Maati H, Guilbot S, Dilly S, Laemmel E, Pons-Himbert C, Faivre JF, Bois P, Stücker O, Le Grand M. Dexrazoxane protects the heart from acutedoxorubicin-induced QT prolongation:a key role for IKs. Br J Pharmacol,2010, 159:93–101.
10 Milberg P, Fleischer D, Stypmann J, Osada N, M?nning G, Engelen MA et al. (2007). Reduced repolarization reserve due to anthracycline therapy facilitates torsade de pointes induced by IKr blockers. Basic Res Cardiol 102:42–51.
11 Wang YX, Korth M (1995). Effects of doxorubicin on excitation-contraction coupling in guinea pig ventricular myocardium. Circ Res 76: 645–653.
1 Sicouri S, Antzelevitch C. A subpopulation of cells with unique electrophysiological properties in the deep subep icardiun of the canine ventricle: The M cell[ J ]. Circ Res, 1991; 68: 1729 - 1741
2 Liu D W,AntzelevitchC .Characteristics of the delayed rectifier current(IKr and IKs) in canine ventricular epicardial,midmyocardial,and endocardial myocytes .A weaker IKs contributes to the longer action potential of the M cell[J]. Circ Re s,1995,76(3):351-365
3 Cheng J, Kamiya K, Liu W, etal . Heterogeneous distribution of the two components of delayed rectifier K+ current:a potential mechanism of the proarrhythmic effects of methanesulfonanilide classⅢagents[J]. Cardiovasc Res, 1999, 43(1): l35-l47
4 Roden D M. Taking the“idio”out of“idiosyncratic”: predicting torsades de pointes[J].Pacing Clin Electrophysiol,199 8,21(5):1029-1034
5 Attila S. Farkas,Stanley Nattel.Minimizing Repolarization-Related Proarrhythmic Risk in Drug Development and Clinical Practice.Drugs 2010; 70 (5): 573-603
6 Guth BD, Germeyer S, Kolb W, et al . Developing a strategy for the nonclinical assessment of proarrhythmic risk of phar maceuticals due to prolonged ventricular repolarization[J].J Pharmacol Toxicol Methods, 2004, 49 (3) : 159-169
7 Josh IA, DM, Vohra Y . Preclinical strategies to assess QT liability and torsadogenic potential of new drugs : the role of experimental models [J]. J Electrocardiol, 2004, 37 ( Supp l) : 714
8 Gintant G A, Lmber IS J T, Mcdermott J S, et al.The canine Purkinje fiber : an in vitro model system for acquired long QT syndrome and drug-induced arrhythmogenesis [J]. J Cardiovasc Pharmacol, 2001, 37 (5) : 607-618
9 Milberg P, Rensch N, Wasmer K, et al. Transmural dispersion of repolarization as a key factor of arrhythmogenicity in a novel intact heartmodel of LQT3 [ J ]. Cardiovasc Res, 2005, 65(2):397-404
10 Yan GX,Antzelevitch C. Cellular basis for the electrocardi ographic J wave[ J ]. Circulation, 1996; 93: 372 - 379
11 Yang X B, Yang L. The study of electrophysiological mechanisms of ventricular arrhythmia in LQT2 models of the Long QT syndrome [ J ]. Chin J Cardiac Pacing Electrophysiol (中国心脏起搏与电生理杂志), 2004, 18 (4) : 282-284
12 Liu J Q, Yang Y Z, Kenneth R L. Optical mapping of the transmural dispersion of repolarization and early after depolarization in LQT2 and LQT3 models of long QT syndrome [ J ]. Chin JCardiac Arrhythmias (中华心律失常学杂志), 2004, 8 ( 4 ) :236
13 De Clerck F, Van deWater A, D Aubioul J, et al . In vivo measurement of QT prolongation, dispersion and arrhythmogenesis: application to the preclinical cardiovascular safety pharmacology of a new chemical entity [J].Fundam Clin Pharmacol,2002, 16 (2) : 125-140
14 Testa IL, Calderone V, Salvador IA, et al . QT prolongation in anaesthetized guineapigs:an experimental approach for preliminary screening of torsadogenicity of drugs and drug candidates [ J ]. J App l Toxicol, 2004, 24 (3) : 217-222
15 Noda Y, Hashimoto K . Development of a halothane-adrenaline arrhythmia model using in vivo Guinea pigs [ J ]. J PharmacolSci, 2004, 95 (2): 234-239
16 Hauser D S, Stade M, Schmidt A, et al . Cardiovascular parameters in anaesthetized guinea pigs:a safety pharmacology screening model[J].J Pharmacol Toxicol Methods, 2005, 52(1) : 106-114
17 Vaynblat M, Pagala M K, Dav Isw J, et al . Telemetrically monitored arrhythmogenic effects of doxorubicin in a dog model of heart failure[J].Pathophysiology,2003,9(4):241-248
18 Chugh S S, Johnson S B, Packer D L, et al . Altered response to ibutilide in a heart failure model [ J ]. Cardiovasc Res,2001, 49 (1) : 94-102
19 Carlsson L, Abrahamsson C, Andersson B, et al. Proarrhythmic effects of the class agent almokalant:importance of infusion rate, QT dispersion,andearly afterdepolarisations[J].Cardiovasc Res, 1993, 27: 2186-2193
20 He J Q, Ma Y, Lee Y, et al . Human embryonic stem cells develop into multiple types of cardiac myocytes:action potential characterization[J]. Circ Res, 2003, 93 (1) : 32-39
21 Nuyensd, Stengl M, Dugarmaa S, et al . Abrup t rate accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome[J].NatMed, 2001, 7 ( 9 ):1021-1027
22 Demolombe S, Lande G, Charpentier F, et al . Transgenic mice overexpressing human Kv LQT1 dominant-negative isoform. Part I : Phenotypic characterisation [J]. Cardiovasc Res,2001, 50 (2) : 314-327
23 Anne Royer S D, Aziza EL Harch I A, Khar Le Quanga, et al . Expression of human HERG K+ channels in the mouse heart exerts anti-arrhythmic activity [ J ]. Cardiovasc Res, 2005,65: 128-137
24 Gussak I, Chaitman B R, Kopecky SL, et al . Rapid ventricular repolarization in rodents:electrocardiographic manifestations, molecularmechanisms, and clinical insights[J]. J Electrocardiol, 2000, 33 (2) : 159-170
25 Ten Tusscher K H, Noble D, Noble P J, et al . A model for human ventricular tissue [ J ]. Am J Physiol Heart Circ Physiol, 2004, 286 (4) : H1573-1589
26 Liu T, Brown BS, Wu Y, et al . Blinded validation of the is olated arterially perfused rabbit ventricular wedge in p reclinical assessment of drug - induced p r oarrhythmias[ J ]. Heart Rhythm, 2006; 3: 948 -956
27 Belardinelli L, Antzelevitch C, VosMA. Assessing predictors of drug-induced torsade de pointes[ J ] . Trends Pharmacol Sci, 2003; 24: 619-625
28 Antzelevitch C. Arrhythmogenic mechanisms of QT prolongling drugs :Is QT prolongation really the problem [ J ] Electrocardiol, 2004; 37( Supp l . ) : S15 - S24
29 Mci Ntosh M A, Cobbe SM, Smith GL, et al . Heterogeneous changes in action potential and intracellular Ca2+in left ventricularmyocyte sub-typesfrom rabbits with heart failure [ J ].Cardiovasc Res, 2000, 45 (2) : 397-409
30 Yan G X,Antzelevitch C.Cellularbasisforthenormal Twaveand theelectrocar diographicm anifestation soft helong- QT syndrome Circulation.1998, 98 : 1928 - 1936
31 Yan GX,Shimizu W,Antzelevitch C.The characteristics and distribution of M cells in arterially-perfused canine left ventricular wedge preparations.Circulation,1998,98:1921-1927
32 Yan GX, Martin J Electrocardiographic T Wave: A Symbol of Transmural Dispersion of Repolarization in the Ventricles . J Cardiovasc Electrophysiol, 2003, 14 (6) : 639 - 640
33 Hevia JC, Antzelevitch C, Bárzaga FT, et al . Tpeak-Tend and Tpeak-Tend dispersion as risk factors for ventricular tachycardia ventricular fibrillati on inpatients with the Brugada Syndrome . J Am Coll Cardi ol, 2006, 47 (9) : 1828 - 1834
34 Fish JM, Di Diego JM, Nesterenko VV, et al .Epicardial activation of left ventricular wall prolongs QT interval and transmural dispersion of repolarization implications for biventricular pacing . Circulation, 2004,109 (17) : 2136 - 2142
35 Gup ta P, Patel C, Patel H,et al.Tp-e ratio as an index of arrhythmogenesis . J Electr ocardiol, 2008, 41 (6) : 567 - 574