Effect Of Puerarin On Action Potential Of Human Hypertrophic Cardiomyocytes And Its Mechanism

A variety of cardiovascular diseases can lead to myocardial hypertrophy.Hypertrophic cardiomyocytes with the decompensated stage,besides the decline of systolic and diastolic function,may also have electrophysiological disorders,which can induce arrhythmia.It is particularly important to develop drugs that can not only treat myocardial hypertrophy but also arrhythmia.Puerarin has a variety of pharmacological activities.Previous studies have confirmed that puerarin can inhibit myocardial hypertrophy.However,the anti-arrhythmic effect of puerarin,especially on human hypertrophic cardiomyocytes,has not been reported.Objective:To explore the effect of Puerarin on electrophysiological activity of human hypertrophic cardiomyocytes.Methods:Epithelioid cells were extracted from urine of healthy people.Sendai virus carrying Oct3/4,Sox2,Klf4 and c-Myc genes was transfected into epithelioid cells.Human Induced pluripotent stem cells(hIPSCs)were obtained by reprogramming and their pluripotency was detected.Induced pluripotent stem cells(IPSCs)were cultured by monolayers,and the human induced pluripotent stem cells were differentiated into cardiomyocytes by sequential regulation method,namely adding WNT signaling pathway activators and inhibitors at different stages of cell growth.Then,the local ET-1 concentration in patients with myocardial hypertrophy was simulated to significantly increase,and the Hypertrophic cardiomyocyte model was established by adding ET-1 into the culture medium to induce the hypertrophy of myocardial cells.The safety of Puerarin on myocardial cells was detected by real-time unlabeled cell analyzer.Finally,whole-cell patch clamp technique was used to determine the effect of Puerarin on electrophysiological activity of hypertrophic cardiomyocytes.Results:Epithelioid cells were successfully extracted from human urine.Immunofluorescence staining with alkaline phosphatase and pluripotent markers was performed on hIPSCs obtained.Teratoma formation test was also carried out to confirm the pluripotency of hIPSCs.Chromosome karyotype analysis of the obtained hIPSCs showed that their karyotypes were normal.HIPSCs could differentiate into cardiomyocytes and express troponin(TNNT2)and a-Actinin as well as myocardial specific genes NKX2-5,MYL2,MYH6 and MYH7.Human cardiomyocytes were induced by ET-1 and hypertrophy was observed,BNP and a-actinin were significantly increased,and MYH7/MYH6 ratio was increased.The results of real-time unlabeled cell analyzer indicated that puerarin with series of concentration had little effect on cell index,beating rate and amplitude of cardiomyocytes.In hypertrophic cardiomyocyte group and puerarin post-treatment group,hiPSC-CMs APD90 was slightly prolonged,APD50 and APD30 were significantly prolonged,and the whole duration of action potential of cardiomyocyte was prolonged.There was no significant difference in depolarization amplitude(APA)of action potential at phase 0,but the maximum depolarization velocity(Vmax)decreased significantly at phase 0.Compared with the control group,the peak current density of sodium channel decreased significantly in hypertrophic cardiomyocyte group.Puerarin group and puerarin post-treatment group could not improve the current of sodium channel in hypertrophic cardiomyocytes.However,puerarin pretreatment can improve the sodium current in hypertrophic cardiomyocytes.There was no significant difference in activation curve,inactivation curve V50,Slope and recovery curve Tau between each group.Conclusion:The obtained hIPSCs can be directionally differentiated into cardiomyocytes,of which electrophysiology is highly similar to that of adult ventricular myocytes.Puerarin can improve the maximum depolarization rate of action potential at phase 0 of hypertrophic cardiomyocytes,which probably result from increasing the peak density of INa.