Application Of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes In The Assessment Of Drug-induced Proarrhythmic Risk

Objective: Ion channels are responsible for the currents that generate the cardiac action potential.Drug interactions with cardiac ion channels delay the action potential repolarization,leading to a prolonged QT interval in the surface electrocardiogram(ECG).It is a main reason for the failure of new drug development and withdrawal from the market.Therefore,detecting the effects of new chemical entity(NCE)on ion channels of cardiomyocytes and assessing the potential risk of proarrhythmia is essential for early safety screening of new drug development.Recently,it have been reported that human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)have similar electrophysiological characteristic to that of adult cardiomyocytes,which can solve the problem of species difference between animals.hiPSC-CMs are also more sensitive to drugs than heterologously expressed cell lines.Therefore,it has been proposed a new model of "Comprehensive in Vitro Proarrhythmia Assay(CiPA)" recently by Food and Drug Administration(FDA)cooperated with Health and Environmental Science Institute(HESI)and other international organizations.It is an important part of the application of human induced pluripotent stem cell-derived cardiomyocytes in assessment of arrhythmia at the cellular level.It is expected to be a new strategy for early cardiac safety screening.At present,there is no internationally commercial hiPSC-CMs in China,and only a few companies are developing hiPSC-CMs.The evaluation system based on the electrophysiological indicators of hiPSC-CMs has not been reported.In this respect,we intend to use hiPSC-CMs to establish a new screening technology for predicting drug-induced arrhythmia,and it is expected to discover potentially adverse reaction of new drugs in the early phase.It has a great significance to evaluate the development prospect of drugs rationally,especially for the development of innovative drugs.Methods:1.Immunofluorescence staining: The cells of day 7 were subjected to immunofluorescence imaging of cTNT and ?-actin,and scanned under confocal microscopy.The hiPSC-CMs were verified by fluorescence expression.2.RT-PCR: Total RNA of hiPSC-CMs was extracted by a RNAprep pure Micro Kit.Expression of hERG,KCNQ1,KCNE1,Nav1.5 and Cav1.2 was detected by RT-PCR after reverse transcription.3.Detection of CardioExcyte 96: hiPSC-CMs were thawed according to the steps provided by the supplier,and cultured on the CardioExcyte 96 sensor plate,which coated fibronectin.The cell suspension were counted by trypan blue staining and was planted into the sensor plate at 30,000/well.The main parameters such as base imp(represent cell adhesion degree),BR(beating rate)and FPDc(max)(BR corrected field potential duration)were monitored every 3 hours,and is under continuous monitoring until the above parameters are stable.After stabilization,a total of 12 drugs(recommended by the FDA)classified into high,medium,and low-risk drugs were added.Each drug was set at three concentrations: high,medium and low.Each concentration was set to three replicate wells.Data of continuous monitoring after 24 h administration(monitoring every 5 min in the first 2 h,then monitoring every 1 h)were calculated to response the change of the field potential.Results:1.Verification of hiPSC-CMs: It was found that the cells adhered to a large form shape after day 2 and day 7 under an inverted microscope.A single cell layer with uniform distribution was observed starting to beat synchronously in the culture dish.After fluorescence staining of cells on day 7,fluorescence of myocardial specific markers(cTNT and ?-actin)were observed under confocal microscopy.RT-PCR results showed that expression of hERG,KCNQ1,KCNE1,Nav1.5,and Cav1.2.Above all,it could be determined that the cells were cardiomyocytes.2.Background data of field potential: After recovery 7 days,the parameters of field potential(base imp,BR and FPDc(max))were stable and drugs could be administered.The parameter of BR was 60-80/min and FPDc(max)was 0.1-0.4 s,which was basically consistent with the foreign reports.3.The case of drug-induced arrhythmia: There are three types of arrhythmia including the early depolarization,rolling early depolarization and irregular beating.Rolling early depolarization and irregular beating are called triggered activity(TA).Any of the above types of waveforms will be judged as arrhythmia.Middle and low concentration of high risk drugs including bepridil,dofetilide,quinidine and sotalol haved induced arrhythmia,while occurrence of arrhythmia were observed in high concentration of intermediatic and low risk drugs,including chlorpromazine,cisapride,terfenadine,ondansetron,diltiazem,ranolazine and verapamil except for mexiletine.4.The case of drug-induced arrest: A pause that occurs more than 3 hours after administration is recorded as arrest.High-risk drugs were prone to occur arrest,except for sotalol,medium-risk drugs were prone to arrest at high concentrations and low-risk drugs have hardly arrest except for mexiletine.5.The change of field potential: Statistics of BR and FPDc(max)were collected by using self-comparison method,and the values were obtained after administration within 2 h and 24 h in stable status compared with the values before administration.The rate of change of the pore parameters was finally calculated by counting the mean and standard deviation.There was no significant concentration-dependent change of BR and FPDc(max).6.Comparison with international data: Most of the data was consistent with the data from other international agencies,but there was a little difference in a small amount of data.Conclusion: Domestic commercial hiPSC-CMs are sensitive to drug reaction.In terms of occurrence of arrhythmia,it can be distinguished between high-risk drugs and low-intermediactic-risk drugs,but intermediactic risk drugs and low risk drugs cannot be distinguished.It can be distinguished into three types of risk level after the combination of arrhythmia and arrest.