The Molecular Mechanism Underlying The Inhibitory Effect Of Tyrosine Kinase Inhibitors On HERG Potassium Crrent

The delayed rectifier K⁺currents(I_K)in mammalian ventricular myocytes consist of rapidly and slowely activating components(I_Krr and I_Ks,respectively).The human ether-a-go-go-related gene(hERG)encodes the pore-forming subunit of the channel responsible for I_Krr which has an important influence on maintaining myocardial action potential plateau phase and phase 3repolarization.The small molecular tyrosine kinase inhibitors(TKIs)for antineoplastic therapy are hot spots in new drug research and development,but these drugs can cause clinical LQT,and even lead to sudden cardiac death.The size of the ion channel current depends on the channel dynamics and the number of functional channels on the cell membrane.Acceleration of forward transport of membrane channel protein and/or acceleration of the degradation process can reduce the number of membrane channel proteins and reduce the current.In view of post-transcriptional regulation of ion channels by tyrosine kinases in cardiomyocytes via the PI3K-SGK1 signaling pathway,we hypothesized that,in addition to directly blocking hERG potassium channel and/or regulating the channel gating,TKIs are likely to be due to inhibition of PI3K-SGK1 signaling pathway,interfering the post-transcriptional process of hERG potassium channel protein,results in a decrease in the expression of membrane hERG channel protein,which in turn inhibits hERG current.This may be an important reason of LQT caused by TKIs.Four TKIs antitumor drugs:low-risk Erlotinib(Erl),imatinib(Ima)and high-risk Nilotinib(Nil),Vandetanib(Van)which could clinically induced LQT were chosen In this study,HEK293 cells and humans heterologously expressing hERG channels Cardiomyocytes(hiPS-CMs)that induced pluripotent stem cell differentiation verified the above hypothesis.Part one The acute and chronic effects of TKIs on hERG channelObjective:T To observe and compare the acute and chronic effects of four TKIs on the potassium current of hERG in a heterologous expression system.Methods:Whole cell patch clamp technique was performed on HEK293(hERG-HEK293)cells stably transfected with hERG channels,and four TKIs(6 concentrations per drug)were extracellularly perfused for 10 min(pharmacological effect reach to the steady state)tail current drop amplitude calculation IC50 value.Then,chronically incubate the drug for 24 h and observe the chronic inhibitory effect of four TKIs on hERG current compared with the same batch of solvent control cells.Results:1.Four TKIs all showed concentration-dependent acute inhibition of hERG current.IC50 values calculated by Hill fitting were Erl:7.0?M;Ima:10.0?M;Nil:0.8?M;Van:0.8?M Obviously,Nil and Van are stronger than Erl and Ima.2.After incubating the drug with the above IC50 concentration for 24hours,the current was significantly reduced compared with the control cells.The current-voltage curve was plotted at the same drug concentration(pre-dose or control cells as 100%),the acute and chronic effects of the drug on current were compared and no significant difference was found between the two low-risk TKIs Erl and Ima,under 0 mV voltage,Erl decreased the hERG current to 51.7%±3.0%and 43.4%±2.6%,respectively;and Ima was50.0%±3.5%and 43.7%±2.6%,respectively.However,two high-risk TKIs after chronically incubated were significantly enhanced the inhibition compare to acute.Nil decreased the hERG current to 52.6%±4.2%and 31.5%±2.5%,respectively(P<0.05);Van showed 51.7%±4.1%and 31.1%±2.5%,respec-tively(P<0.05).Conclusions:Four TKIs drugs have an acute blocking effect on potassium currents in h ERG,among which Van,Nil is stronger than Erl and Ima;chronic incubation of drugs finds that Van,Nil inhibits currents significantly more than acute effects,suggesting that there are other The mechanism involved in inhibition of hERG current after chronic administration.Part twoMolecular mechanism of TKIs in down-regulating hERGchannelObjective:To investigate the effects of four TKIs on the 155-kDa protein on the membrane of hERG-HEK293 cells after chronically incubated,and to analyze the mechanism of down-regulation of 155-kDa protein by these drugs with the aid of the tool agents.Methods:On hERG-HEK293 cells,chronically incubate four TKIs and/or tool agents for 24 h to extract whole-cell protein,using hERG antibodies(can be used to detect 135-kDa immature and 155-kDa mature hERG,respectively)Channel protein)to Western-blot,155-k Da protein contents were used to reflect the effect of four TKIs on functional channel protein on the hERG cell membrane.Results:1.Four TKIs drugs showed a time-dependent decrease in the expression of mature 155-kDa proteins within 24 h of chronic incubation;at 24 h,the expression of 155-kDa protein was significantly reduced to 0.70±0.05 fold(Erl),0.65±0.04 fold(Ima),0.54±0.04 fold(Nil),and 0.50±0.05 fold(Van)of control.Two high-risk TKIs(Nil,Van)were more significantly reduced155-kDa protein expression level.Ima and Van were selected to conduct experiments to analyze the molecular mechanism of protein degradation in mature channels.2.Brefeldin A(BFA)can block the process of functional intracellular protein to membrane.After BFA(10?M)incubation,the expression of155-kDa mature protein was detected by Western blot at different times to reflect the dynamic degradation of membrane channel protein.Compared with the control cells incubated with BFA only,Ima and Van increased the degradation of mature protein in different degrees,and the effects of the 12 h latter(Ima),4 h latter(Van)were more obvious.At 24 h,the 155-kDa protein expression levels of Ima and Van were 0.78±0.05 and 0.47±0.03 fold of the control,respectively(P<0.05).3.Dynosore(Dyn)blocks the internalization process of functional protein on membrane and inhibits membrane channel degradation.Compared with the cells incubated with Dyn alone(80?M),Ima and Van significantly increased the expression of 155-kDa protein,especially with Van,which was1.70±0.08 fold higher than that of the control at 24 h(P<0.05).The results further suggest that the drug promotes the internalization and degradation of membrane channel protein.Conclusions:Chronic incubation of TKIs can reduce membrane155-kDa protein expression by accelerating membrane protein internalization and further inhibit hERG currents.Part threeThe role of CER against TKIs down-regulate the hERGpotassium channelObjective:To observe the antagonistic effect of SGK1 activator C4-ceramide(CER)on the down-regulation of 155-kDa protein expression of hERG channel and inhibition of hERG current by TKIs.Methods:On hERG-HEK293 cells,four TKIs were incubated with different doses of CER(1,3,and 6?M)for 24 h to extract whole cell protein.Western-blot was used to detect the expression of 155-kDa protein;Four TKIs co-incubate with CER for 24 h,whole cell patch clamp technique was used to record the hERG current,the antagonistic effect of CER on the inhibition of hERG potassium channel function by four TKIs was observed.Results:1.hERG-HEK293 cells incubated with CER for 24 h could significantly increase the expression of 155-k Da protein;CER(1,3,and 6?M)were incubated with four TKIs for 24 h in a concentration-dependent manner against the down-regulation of 155-kDa protein expression by TKIs.Erl,Ima,Nil,Van were respectively co-incubated with CER(6?M)for 24h,the expression levels of 155-kDa functional protein were 1.17±0.04 fold,1.18±0.05 fold,1.69±0.07 fold,1.77±0.08 fold of the control,respectively(P<0.05).2.In the presence of CER(6?M),Erl and Ima slightly increased the hERG tail current density in the range of 10 to 50 mV,but there was no significant difference;while Nil and Van increased the tail current density significantly in the range of 0 to 50 mV,there were significantly increased from 26.6±1.5 p A/p F and 26.1±2.5 pA/pF to 50.0%±1.7 pA/p F and 50.3±2.5 pA/pF in+50 mV voltage,respectively(P<0.05).Conclusions:SGK1 activator CER can inhibit the reduction of the number of hERG potassium channel and the inhibition of hERG current caused by chronic incubation of TKIs.CER has a particularly significant effect on two high-risk TKIs.Part fourThe prevention and cure effects of CER on the arrhythmiainduced by TKIs in iPS-CM cellsObjective:To verify the results of our previous experiments,and to examine whether the arrhythmogenic effects of TKIs can be sensitively detected by the human-induced pluripotent stem cell-derived cardiomyocytes(hiPS-CM)model which proposed in the"Comprehensive in vitro Proarrh-ythmia Assay(CiPA)".Methods:The Cardio-ECR was used to monitor the field potential(Field potential,FP)and cell contraction effects of the electrophysiological changes before and after administration in the same well area and at different time points.The inspection parameters of FP are field potential amplitude(FPAmp)and field potential duration(FPD),and the contraction parameters include BAmp and BR.When the percentage change absolute value of above parameters are greater than 20%,they can be determined that the drug has an effect on the cells.Results:1.The percentage change absolute values of the four parameters within24 h after Erl incubation did not reach at 20%,indicating that the safety of Erl is relatively high and it is not easy to cause arrhythmia.2.The percentage change absolute values of the electrophysiological parameters FPAmp and FPD caused by Ima at various time points were all less than 20%,indicating that the risk of Ima-induced arrhythmia was low;but the percentage change absolute values of BAmp and BR at 24 h were:35.22%±2.4%,22.2%±1.3%,which will make the cell contraction weakened,the contraction frequency is accelerated,indicating that Ima inhibit myocardial contraction.3.Nil did not change every parameter at 10 min,and induced EAD at 12h and 24 h;the absolute values of FPD and FPAmp change rate at 24 h were21.5%±1.6%and 45.3%±3.5%,respectively;the absolute value of the change rate of BAmp was 67.5%±4.3%,indicating that Nil has the potential to delay cell repolarization and arrhythmia;CER(6?M)can completely abolish Nil induced early depolarization(EAD).phenomenon.4.Van did not change the parameters in 10 min.Incubation of cells for 4h resulted in"notch"(EAD sign)and EAD.The absolute values of FPD change rate at 4 h,12 h,and 24 h were 56.4%±3.4%,57.8%±4.2%and63.6%±4.7%,which is consistent with the risk of this drug in the clinical easy to cause LQT,Td P.After CER was co-incubated with them,the absolute value of FPD change rate was significantly lower(P<0.05),and the pheno-menon of"notch"and EAD were effectively cancelled.Conclusions:Erl and Ima have lower risk of arrhythmias on hiPS-CM;Nil and Van have no acute effect on cell electrophysiological parameters,but with their duration increases(Nil 12 h,Van 4 h),both of them induced EAD,suggesting that Nil and Van have a higher risk of arrhythmia;SGK1 activator can effectively counteract the arrhythmogenic effects of Nil and Van on cell models.Summary:1.In addition to inhibiton the hERG current by blocking hERG channels and/or regulating gating,TKIs can further inhibit the expression of hERG channel protein membranes by promoting the internalization and degradation of mature proteins on membranes,further suppress hERG current.2.In the hiPS-CM cell model,Erl and Ima did not significantly alter the cells electrophysiological parameters,but Nil and Van significantly delayed cells repolarization and induced cells to produce"notch"and EAD phenomenon with the prolonged action time.The results were consistent with clinical reports of TKIs-induced arrhythmias.This shows that the hiPS-CM model can sensitively detect the arrhythmia effects of TKIs..3.SGK1 activator CER is effective against the reduction of membrane hERG channels and inhibition of hERG current caused by chronic incubation of TKIs.At the same time,in the hiPS-CM cell model,CER can effectively cancel the Nil,Van induced EAD phenomenon.The above results show that promoting the degradation of hERG membrane channel proteins is an important molecular mechanism that Nil and Van trigger LQT to generate arrhythmias;The activation of SGK1 has an antagonistic effect on arrhythmia induced by them.Our experimental results provide new ideas for potential arrhythmia risk assessment of TKIs and prevention of clinical adverse drug reactions.