The Application Of Arterially Perfused Rabbit Left Ventricular Wedge Preparation In Evaluation Of Drug Induced Potential Arrhythmia

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 Many drugs like Terfenadine, Astemizole, Cisapride, Mibefradil and Grepafloxacin have been withdrawn from the market in recent years.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 preparationObjective: To build the steady and effective arterially perfused ventricular wedge preparationMethods: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 1²mm 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 (APD₉₀). TDR was defined as the difference between the longest and the shortest repolarization times (activation time plus APD₉₀) 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 APD₉₀,QT interval at 0min,30min,60min,90min,120min,indicating the model was steady. (3)After 1¹.5 hours, perfuse amiodarone(100μmol/L) and moxifloxacin(100μmol/L). Amiodarone produced a prolongation in both epicardial APD₉₀ (from 277±12 to 365±16,p<0.05) and endocardial APD₉₀(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 APD₉₀ (form 287±17 to 335±21 , p<0.05) and endocardial APD₉₀(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 adriamycinObjective: To estimate acute heart toxicity of adriamycinMethods: 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 (APD₉₀). TDR was defined as the difference between the longest and the shortest repolarization times (activation time plus APD₉₀) 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 APD₉₀( from 362±12 to 366±25,365±23,361±13ms, P > 0.05) , endocardial APD₉₀( 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 APD₉₀(from 360±16 to 364±13,365±11,361±20,P > 0. 05) , endocardial APD₉₀(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.