The Study Of Cardiomyocyte Models For Drug Induced Cardiotoxicity Screening And Mitochondrial Toxicity Prediction Based On Human Primary Cardiomyocytes

Background and objectiveCardiovascular disease is one of the most important public health problems.Cardiotoxicity is one of the most common and serious adverse drug reactions and an important risk factor for cardiovascular diseases.Improvements in the preclinical cardiac safety evaluation system are expected have a profound impact on the costs during late drug development and patients’ well-being.Mitochondrial toxicity caused by therapeutic drugs is a main cause of cardiotoxicity,which seriously threatens the patients’ lives.However,mitochondrial toxicity tests have not been incorporated into routine cardiac safety screening procedures.In addition,the optimization of evaluation models,especially the use of human derived cardiomyocyte models,will further improve the preclinical cardiac safety evaluation system.The aim of this study is to establish a human cardiomyocyte model for cardiac mitochondrial toxicity prediction,optimize the cardiac safety evaluation system,avoid drug cardiotoxicity,and reduce the burden of cardiovascular disease.Methods and resultsThis study first screened a set of clinical drugs in human primary cardiomyocytes(hPCMs)by cell viability detection and found the results was highly consistent with the clinical cardiotoxicity.Through the comparison of two human cardiomyocyte models,hPCMs and human induced pluripotent stem cell-derived cardiomyocytes(hiPSCCMs),we observed differing prediction capabilities of drug-induced cardiotoxicity.hPCMs were more sensitive to 54.5%of cardiotoxic drugs,and IC50 was up to 177.7 times lower than that of hiPSC-CMs.While hiPSC-CMs are more sensitive to 27.3%of these drugs,and IC50 is about 3 to 6 times lower than that of hPCMs.These results indicat the high sensitivity of hPCMs for cardiotoxic drugs detection.To accurately model mitochondrial toxicity induced by cardiotoxic drugs on human cardiomyocytes,we systematically evaluated the toxic responses of hPCMs induced by anti-viral agent remdesivir(RDV),through the assessment of cell activity,electrophysiology,mitochondrial content,mitochondrial membrane potential(MMP),mitochondrial permeability transition pore(mPTP),oxygen consumption rate(OCR),complex activity detection,mitochondrial reactive oxygen species(mtROS),and lactate secretion.Our results showed that RDV caused widespread mitochondrial abnormalities and prolonged action potential in hPCMs,which was consistent with the clinical response of RDV-induced QT prolongation.In contrast,hiPSC-CMs exhibited moderate mitochondrial dysfunction and no electrophysiological abnormalities.Further mechanistic studies revealed that a mitochondrial function recovery mechanism,i.e.mitophagy,in hiPSC-CMs that maintains the health of the mitochondrial network.However,this repair mechanism was absent in hPCMs,and artificially inducing mitophagy did not restore mitochondrial function.Therefore,we screened 21 potential cardioprotective compounds with different mitochondrial protective abilities by evaluating the 7 key mitochondrial parameters,including MMP,mPTP,maximal respiration,spare respiratory capacity,mitochondrial complex I activity detection,mtROS,and lactate secretion.Compounds that restored mtROS,including Edaravone(Eda),Bryostatinl(Bry),Debio025(Deb),and 7-hydroxy-3-(4’-methoxyphenyl)coumarin(C12),were identified to prevent RDV-induced AP prolongation.By contrast,neither MMP nor mPTP protection drugs had this effect,suggesting that mtROS accumulation is the molecular basis of abnormal electrical activity in hPCMs.Finally,in order to further verify the reliability of hPCMs as an effective safety screening model for cardiac mitochondrial toxicity,18 drugs with different mechanisms of mitochondrial toxicities using the same parameters.The results showed that hPCMs detected 68 mitochondrial dysfunctions which not only covered 53 mitochondrial disorders reported by other models,but also revealed 15(22%)previously unknown mitochondrial abnormalities.ConclusionsThis study firstly confirmed that hPCMs highly predicted drug induced cardiotoxicity which consistents with the clinical observation at the cellular level.Secondly,hPCMs can truly reflect the cardiac mitochondrial toxicity caused by RDV,and the lack of protection mechanism mediated by mitophagy is an important reason for extensive mitochondria damage in hPCMs.In addition,it was found for the first time that the mechanism of RDV induced cardiotoxicity was related to the excessive mtROS production,which confirmed the internal relationship between mitochondrial damage and cardiac arrhythmia.Thirdly,this study systematically elucidated the feasibility,sensitivity,and accuracy of using hPCMs as a cardiomyocyte model for pharmacological and toxicological screening in a high-throughput manner at the mitochondrial level,which provides a good example for drug cardiac safety screening.Our data are expected to facilitate the optimization of preclinical cardiac safety assessment,and ensure the economic benefits of pharmaceutical industries,patient health,as well as the prevention and control of cardiovascular diseases.