Study On Cardiac Toxicity Evaluation Of Cardiomyocytes Differentiated By Human Embryonic Stem Cells

Background and objectiveDrug-induced cardiotoxicity isa major concern of pharmaceutical industry and regulatory agency.International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use(ICH), FDA and CFDA haspromulgated a number of rules and guidelines to improve the evaluation study for drug-induced cardiotoxicity. However, current experimental models still lack of validity and rationality, so we need to establish accurate and reliable methods to evaluate drug-induced cardiotoxicity, and it has important significancein the early stages of research and development of drugs for pharmaceutical industry and regulatory agency.Studies related to human embryonic stem cell-derived cardiomyocytes(h ESC-CM) in recent years suggest h ESC-CM could be considered as a potential evaluation model for evaluation of drug-induced cardiotoxicity.In the present experiments, we selected several positive drugs with different toxic mechanisms and investigated their effects on h ESC-CM in cell function and electrophysiological features. At the same time, we used functional evaluationmethod to assesse the cardiotoxicity of compound AMethods and Results1. Evaluation for function of h ESC-CMFour drugs with different cardiotoxicity were selected as references. They were: ①doxorubicin(anthracycline anticancer drug), imatinib(the tyrosine kinase inhibitor), isoproterenol(β receptor agonist) andverapamil(calcium channel blocker). These drugs were incubated with h ESC-CMrespectively. The real-time cell analysis system and high content cell imaging technology were used to monitor the changes of h ESC-CM’s beat rate, amplitude, beat rate regularity index and mitochondrial membrane potential after 1 h, 4 h, 24 h, 48 h, 72 h, and 96 h treatment.Further more,the cardiac toxicity of compound A was evaluatedby this method.Resultsindicated that there were obvious decrease in cell’s beat rate, amplitude and beat rate regularity, mitochondrial membrane potential in all concentrations doxorubicin treated h ESC-CM. Doxorubicin showed a strong inhibition on h ESC-CM function. When h ESC-CM incubated with imatinib(20 μmol·L-1), h ESC-CM’s amplitude, beat rate and beat rate regularity index, mitochondrial membrane potential were significant inhibited.too.These suggested that at high concentration imatinib had inhibitory effects on h ESC-CM function. However, effects of isoproterenol were different. It decreased cells’ amplitude but increased beat rate. With the prolongation of drug action, the effect of isoproterenol reduced. When treated with verapamil, cells’ amplitude and beat rate decreased rapidly.The indexes was decreased at the early stage, but recovered from the48 h of incubation. Cell’s beat rate regularity, mitochondrial membrane potential didn’t change in isoproterenol and verapamil treated h ESC-CM groups. When treated with the testing compound A, cell’s amplitude, beat rate, beat rate regularity of h ESC-CMs were decreased and showed a dose-time dependence. There was no change with mitochondrial membrane potential.2.Evaluation for electrophysiology of h ESC-CMThis part also selected four cardiotoxicity drugs of different mechanisms: DOX, IMA, ISO and VER. We used standard whole-cell patch-clamp technique to detect the changes of cardiomyocytes’ action potential, h ERG potassium current, Nav1.5 current, as well as L-Ca2+channel current after drugs incubated with h ESC-CM. Establish new methods to assess the electrophysiology function toxicity of drugs on these basics.The results showed: ①h ESC-CMs’ action potential duration,action potential amplitude and resting potentialwere shortening after DOX treatment. There was a dose-response relation.However, max dv/dt, Nav1.5 current, L-Ca2+ current and h ERG potassium currenthad no significant changes.②IMA significantly decresed the Nav1.5 currentsof h ESC-CM, IC50 was 2.00 ± 0.49 μmol·L-1.L-Ca2+current was also inhibited, IC50 was 23.4 ± 9.8 μmol·L-1. However, IMA had no significant effect on the h ERG potassium current, RP and Max dv/dt. APD50, APD90, APA were shortened with increasing concentrations of IMA. Our resultssuggested that IMA could shorten h ESC-CMs’ action potential, which might be related to its Nav1.5 and L-Ca2+current inhibition.③ISO had small effect on Nav1.5 current of h ESC-CMs, IC50 was more than 100 μmol·L-1. However it could activate L-Ca2+current. On the other hand, h ERG potassium current was inhibited.ISO had no effect on the action potential.④VER hada minor effect on the Nav1.5 current, IC50 was 147.3±5.4 μmol·L-1.L-Ca2+ current was strongly inhibited, IC50 of which was 7.1 ± 1.5 μmol·L-1.For h ERG potassium current, VERshowed a significantly effect with it and IC50 is 3.5 ± 1.0 μmol·L-1. APD50, APD90, APA, RP and Max dv/dt were significantly decreased as increasing of its concentration, which made the action potential duration decrease. These results demonstratedthat VER was a multi-ion channel blockers and has significant effectson electrophysiology function of h ESC-CM. ConclusionsOur study had established the new methods to evaluate drug-induced cardiomyocyte toxicity including effects on electrophysiological function by usinghuman embryonic stem cells-derived cardiomyocytes. h ESC-CM combined with the Real-time cell analysis and high content cellular imaging technology can evaluate new compounds’ toxicity.h ESC-CMis an ideal model to evaluate drug-induced cardiotoxicity.