Mechanism Underlying Proarrhythmic Effects Of QT Interval-shortening Drugs And Surrogate Markers For Prediction Of The Risk

Compound-induced QT prolongation mainly through blocking the rapid component of delayed rectifier potassium current(IKr)has been associated with the life-threatening ventricular tachyarrhythmia known as “torsades de pointes(Td P)” and thus becomes a major concern in drug development.In 2005,the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use(ICH)issued related guidelines,ICH S7 B and E14.According to the regulatory strategies,it is mandatory for new drug candidates to undergo assessment for their potential effects on cardiac electrical activity with in vitro and in vivo preclinical studies,along with clinical thorough QT(TQT)studies.Since then,different screening strategies for detecting compound-induced Td P have been evaluated.Meanwhile,extensive progress has been made in studying the congenital long QT syndromes in recent years.Therefore,drug-induced long QT and its risk for Td P are currently well known and many biomarkers have been used in an effort to establish an association for predicting production of Td P,which include IKr inhibition and QT/QTc(corrected QT interval for the effects of heart rate)prolongation,as well as an increase in transmural dispersion of repolarization and triangulation of action potential,beat-to-beat variability of repolarization and early or delayed after depolarization(EAD or DAD)of action potential.However,in contrast to drug-induced QT prolongation,a number of compounds have been found to actually shorten the action potential duration(APD)and/or QT interval in the process of drug screening.Indeed,ATP-dependent potassium channel openers,antiepileptic drugs affecting the voltage-gated sodium channels and delayed rectifier potassium current(including the rapid and slow component IKr and IKs)activators lead to QT interval shortening by accelerating cardiac repolarization.Theoretically,the shortening of APD and effective refractory period(ERP)is associated with the potential proarrhythmic risks.On the other hand,there is increasing evidence that congenital short QT syndromes(SQT)constitute a new primary electrical disease associated with a high risk of sudden cardiac death,resulting not from Td P,but rather from ventricular tachycardia(VT)/ventricular fibrillation(VF).Currently,six genetic alterations associated with SQT have been identified and they include gain-of-function mutations in three genes(KCNQ1,KCNH2 and KCNJ2)encoding cardiac potassium channels and loss-of-function mutations in genes(CACNA1C,CACNB2 and CACNA2D1)encoding the L-type calcium channel.In all these cases,mutations result in an increase in ventricular repolarizing current and thus a shortening of APD and ERP.The existence of inherited SQT suggests that drug-induced QT shortening may be proarrhythmic and some experts have expressed their concerns that drug-induced QT shortening might be equally important in new drug development.However,progress in addressing the potential proarrhythmic risk of drug-induced QT shortening has not been as rapid as drug-induced QT prolongation since little attention has been paid to drug-induced QT shortening.Nevertheless,there is evidence showing that ATP-dependent potassium channel openers,pinacidil and levcromakalim as well as IKr channel activator NS1643 induce shortening of APD and have profibrillatory effects in preclinical studies.Similarly,our previous study has reveals that IKr channel activator ICA-105574 can induce VF in isolated guinea pig heart.Obviously,the preliminary data have pointed to a relationship between drug-induced short QT and VT/VF.However,which biomarkers are predictive of drug-induced short QT remains unknown.It is known that multiple proarrhythmic parameters proposed to be closely associated with drug-induced Td P can be easily analyzed in isolated Langendorff heart and thus it is a valuable preclinical model for studying proarrhythmic liability of compounds.This study was designed to evaluate proarrhythmic risk of four QT-shortening drug/compounds including ATP-sensitive potassium channel opener pinacidil,IKr channel activators ICA-105574 and NS-1643,IKs channel activator R-L3 primarily in isolated rabbit and guinea-pig hearts.Meanwhile,proarrhythmic parameters were analyzed so as to find novel biomarkers for prediction of VT/VF induced by QT-shortening drug.Part 1 Proarrhythmic effect of QT shortening drugs in isolated guinea pig and rabbit heartsObjective: To investigate the sensitivity of guinea pig and rabbit heart to potassium channel opener.Methods: Langendorff-perfused hearts was prepared as previously described.Briefly,adult rabbits or guinea pigs were injected with heparin(1000 IU·kg-1,i.p.)and anesthetized 5–10 min later with sodium pentobarbital(30–35 mg·kg-1,i.p.).The heart was quickly removed from the animal and fixed to a Langendorff perfusion apparatus(Radnoti Inc,Monrovia,CA,USA)and retrogradely perfused at a constant flow of 6 ml·min-1 with Tyrode solution,which had the following composition(in m M): Na Cl 140;KCl 5.4;Mg Cl2 1;Ca Cl2 2;HEPES 10 and glucose 10(p H 7.4 with Na OH),and was continuously bubbled with O2 at 36±1?.The heart was allowed to beat spontaneously.Two silver electrodes were attached to the aortic root and ventricular apex to record in vitro equivalent lead II ECG waveforms.The hearts were put in the thermostatic chamber and allowed to equilibrate for a minimum of 1 h to ensure the stable ECG recordings before drug testing.If the ECG recordings displayed erratic rhythms after the equilibrium period,the heart was abandoned.The hearts were then perfused with vehicle(0.1% DMSO)for 20 min,followed by three escalating concentrations of test drugs.In this study,ECG recordings were stable during the course of an experiment(up to 6 h)when no pharmacological interventions were applied.Results: Isolated rabbit or guinea pig hearts were all commonly used preclinical model for testing proarrhythmic potential of compounds in new drug development.We first examined the proarrhythmic effect of four K+ channel openers both in guinea pig and rabbit hearts.The representative ECG recordings from two models showed that all of four different potassium channel openers induced VT/VF at higher concentrations in guinea pig hearts,whereas,at the same test concentrations they did not display profibrillatory effect in rabbit hearts except IKr channel activator,ICA-105574.The incidences of VT/VF induced by four potassium channel openers in guinea pig hearts were significantly higher than those in rabbit hearts.The results indicated that guinea pig hearts were more sensitive to potassium channel openers and thus were used in the following experiments.Conclusion: Compared with rabbit hearts,guinea pig hearts were more sensitive to potassium channel openers and thus were used in the following experiments.Part 2 Effect of QT shortening drugs on in vitro ECG parametersObjective: To evaluate the effect of potassium channel openers on ECG parameters of guinea pig.Methods: ECGs were recorded from Langendorff perfused guinea pig hearts as described in part 1.Multiple parameters were directly measured or derived from ECGs to profile the effects of compounds on cardiac electrophysiologic and proarrhythmic propensity.The mean QRS complex,QT and RR intervals were measured and averaged from 10 consecutive beats between 15–20 min or 5 min before VT/VF happening after the presence of test compound or solvent by a person who was not involved in the experiments.The JTpeak intervals from the end point of QRS complex to the peak of T wave and the Tp-e from the peak of T wave to the end of T wave,which represent the early repolarization and late repolarization respectively,were also measured.Both QT and JTpeak intervals were corrected by a parabolic equation QT(333/RR)0.601.In addition,we measured transmural dispersion of repolarization(TDR),which is reflected by alternation of JT area.JT area of each beat was defined as total planimetric area between the curve and baseline from J point to T end point as already described previously.The beat-to-beat instability(variability)of the QT interval and heart rate(HR)was defined according to the method used.The index of cardiac electrophysiological balance(i CEB)was also calculated by the ratio of QT/QRS according to a recent report.VT was defined as three or more ventricular extrasystoles in succession while VF was defined as a ventricular tachyarrhythmia without identifiable ECG.Results: 1 Both ICA-105574 and NS-1643 can enhances IKr current,with the former removing the channel inactivation and the latter attenuating channel inactivation.ICA-105574 significantly shortened QTc,JTpeak interval and i CEB(ratio of QT/QRS)in a concentration dependent manner.Meanwhile,it also concentration dependently increased transmural dispersion of repolarization(expressed as JT area).In addition,at higher concentration(10 ?M),the beat-to-beat instability(variability)of the QT interval and heart rate(HR)was increased and non-Td P like VT/ VF was detected in five out of eight hearts.Moreover,inverted T wave was noticed at higher concentration.ROC analysis showed that among the above ECG parameters,JTpeak interval,Tp-e and JT area had higher AUC values than 0.9(indicative of high predictive power for VT/VF occurrence).NS-1643 significantly decreased QTc and JTpeak interval at three test concentrations,and shortened i CEB at higher concentration(10 ?M).It also concentration-dependently increased JT area.At higher concentration(10 ?M),NS-1643 induced VT/ VF in three out of five hearts.In addition,it was NS-1643 increased PR interval at higher concentration,suggesting an inhibitory effect on atrioventricular conduction.ROC analysis showed that JTpeak interval,JT area and instability of HR had higher AUC values(indicative of high predictive power for VT/VF occurrence).2 Relative to solvent,R-L3 concentration dependently decreased QTc and JTpeak interval.Similar to NS-1643,R-L3 changed i CEB only at higher concentration(10 ?M).It concentration-dependently increased JT area(up to four-fold).At 10 ?M,R-L3 induced non-Td P like VT/ VF in two out of five hearts.Similar to NS-1643,ROC analysis showed that JTpeak interval and JT area had higher AUC values.3 After the presence of pinacidil,QTc,JTpeak interval and i CEB were all decreased in a concentration dependent manner.Meanwhile,Tp-e and JT area were significantly increased.At 20 ?M pinacidil induced non-Td P like VT/VF in four out of seven hearts.ROC analysis showed that JTpeak interval,JT area including QT had higher AUC values.Conclusion: All of the four potassium channel openers decreased QTc and JTpeak interval,along with the amplification of JT area.ROC analysis showed that early-repolarization related parameters JTpeak interval and JT area had higher predictive power for VT/VF occurrence.Part 3 Effect of QT shortening drugs on AP parameters and current during repolarizationObjective: To evaluate the effect of potassium channel openers on AP parameters of guinea pig papillary muscles.Methods: 1 The papillary muscle was paced by an electrical square pulse(0.5 Hz,1.5 times threshold intensity)generated by an electronic stimulator.After an initial stabilization period of 1 h,the transmembrane action potential(AP)from papillary muscle was recorded with a glass microelectrode filled with 3 M KCl(a tip resistance of 10–20 M?)and entered into a high-input impedance amplifier.The amplified signals were fed to and processed by a multi-channel physiological signal collecting and processing system.The APD at 30%,90% repolarization(APD30,APD90)were measured using custom-written software.Effective refractory periods(ERP)were determined by the following protocol.Briefly,the papillary muscle was stimulated alternately by eight consecutive single stimuluses and a double stimulus.The interval of the double stimulus was subsequently reduced by 5 msec until no capture was observed.This coupling interval was defined as the ERP.In addition,APD90-30 was calculated.All data above were measured by a person who was not involved in the experiments and mean data were obtained by averaging over 10 beats.2 The action potential(AP)waveform was first recorded from the ventricular cardiomyocyte under I-clamp mode applying steady-state stimulation at 1 Hz frequency.Then the AP waveform was used as the command voltage onto the cardiomyocyte to elicit the outward K+ current and inward current under V-clamp mode.Results: To explain the findings in the organ and tissue levels,we further examined the effect of potassium channel openers on outward K+ current during an action potential process by using AP clamp.The morphology of outward K current varied with the different potassium channel openers since they targeted different ion channels.The maximum increment of the outward current was located in the phase 2-repolarization except pinacidil,which increased the current in a uniformly manner in the whole repolarization process.We determined the area under the current trace(shaded)to calculate the amplification of the outward current during the early repolarization section,which was corresponding to APD30.In addition,we investigated the relationship between the amplification of the outward current and the incidence of arrhythmia,the result showed they were correlated(R=0.87).Moreover,the potassium channel openers did not effect the inward current during the repolarization duration at high concentration.Conclusion: The potassium channel openers increased the outward current especially in the early repolarization,while they did not effect the inward current.