Effect Of Selective Ubiquitin Ligase Nedd4-2 Inhibitory Peptide On HERG Potassium Channel Degradation And Its Pharmacological Action

Long QT syndromes（LQTs）are characterized by prolonged QT interval and abnormal T wave,which is prone to malignant arrhythmias such as torsade de pointe,and leading to recurrent syncope,cardiac arrest and even sudden death.Ion channel gene mutations cause inherited LQTs.Cardiac diseases such as pathological myocardial hypertrophy,heart failure associated with electrophysiological remodeling and drugs affecting channel function can lead to acquired LQTs.K⁺currents are the main current of cardiac action potential repolarization.Human ether-a-go-go-related gene（hERG）encodes the pore-forming subunit of the channel underlying I_Kr,which play a pivotal role for the shape and duration of action potential.A reduction in hERG currents due to either genetic defects,or adverse drug effects can lead to hereditary or acquired LQTs.Previous studies have found that decrease of I_Krin pathological cardiac hypertrophy and heart failure contributes to the delayed cardiac repolarization and thereby involve in the arrhythmogenesis.It is known that the ion channel function depends on two factors,one is channel dynamics including conductance and open probability,and the other is the channel density in the plasma membrane.The latter depends on a balance between forward trafficking and retrograde internalization/degradation.E3 ubiquitin ligase Nedd4-2 plays a key role in ubiquitin mediated degradation of channel proteins.Nedd4-2 specifically binds to channel proteins containing conserved leucine/proline tyrosine（L/PY）sequence to initiate membrane channel degradation.It is known that the main ion channels involved in depolarization and repolarization of action potential,such as the rapidly activated delayed rectifying potassium channel I_Kr（hERG）,the slowly activated delayed rectifying potassium channel I_Ks（KCNQ1/KCNE1）and the late sodium channel I_Na（Nav1.5）all contain PY sequences,which can be recognized by Nedd4-2.Our previous experimental results showed that the increased expression of Nedd4-2 and ubiquitin degradation of hERG protein may be important reasons for the decrease of I_Krduring pathological cardiac hypertrophy,Therefore,we designed short peptide-A based on the PY sequence of hERG channel which binding to the ubiquitin ligase Nedd4-2 and competitively inhibit the binding of Nedd4-2 to the hERG protein.It maybe lead to reducing the ubiquitin degradation of hERG protein and counteracting LQTs induced by pathological electrical remodeling.Our previous preliminary experiments have shown that short peptide-A can reduce degradation of hERG channel protein.On this basis,we designed synthesized peptide-B.In this project we intends to use flow cytometry or molecular biology technologies in heterologous expression cells or human induced pluripotent stem cell-derived cardiomyocytes to further validate inhibitory peptides influence on hERG protein degradation,and whether the inhibitory peptides have the effect on the other ion channels which contain PY sequence.we evaluate the selective effect of inhibitory peptides in further reserch,To observe inhibitory peptides pharmacological effects on hERG function down regulation which caused by different reasons.To comprehensively evaluate the development prospects of the ubiquitin ligase Nedd4-2 inhibitory peptide（simplified as inhibitory peptide below）.The results provide experimental basis for the new drug development of ubiquitin ligase Nedd4-2 inhibitory peptide.Part One The effects of two inhibitory peptides on hERG channelObjective:To observe the effect of two inhibitory peptides（A and B）on hERG channel and the selectivity.Method:1.Effect of inhibitory peptides on the expression of hERG membrane protein:the Nedd4-2 plasmid was transiently transfected into HEK 293 cells（hERG-HEK）which were stably expressing hERG.Western blot was used to detect hERG protein.The effects of different concentrations of inhibitory peptide B on hERG protein and the same concentration of two inhibitory peptides（A and B）on hERG protein were observed.2.Effect of inhibitory peptides on the expression of hERG current:Whole cell patch clamp method was used to detect the effect of two inhibitory peptides（A and B）on hERG current,and compared the effects of two inhibitory peptides（A and B）on I_Krcurrent in transfected Nedd4-2 plasmid hERG-HEK cells,and the current density（PA/PF）was analyzed by cell membrane capacitance.3.Dynamic degradation of hERG membrane protein:Construction of hERG plasmid with extracellular BBS tag（specific binding to BTX）and intracellular C-terminal YFP fluorescence,After transfection into HEK293cells,BTX-CY5 staining was performed,CY5 fluorescence reflects the membrane channel protein of hERG and YFP fluorescence represents the total protein of hERG.Brefeldin A（BFA）blocked the membrane process of hERG.Flow cytometry was used to observe the ratio of both YFP and CY5 positive cells,it could represent the expression of membrane mature protein at different time points after BFA incubation.The effect of inhibitory peptide on the dynamic degradation of hERG membrane protein was further observed.4.Effect on the expression of other ion channels:In HEK293 cells which were stably expressing KCNQ1/KCNE1 or Nav1.5,and Nedd4-2 plasmids were transiently transfected.Western blot was used to detect the expression of KCNQ1 and Nav1.5,respectively.The effects of two inhibitory peptides（A and B）were observed.5.Physiological effect of inhibitory peptides on hERG protein:In HEK293 cells which were stably expressing hERG,Western blot was used to detect hERG protein,The effects of two inhibitory peptides（A and B）were observed.Results:1.Two inhibitory peptides had membrane penetrating sequence.Confocal microscope observation showed that the inhibitory peptide B（100 n M,200n M,500 n M）with three concentration gradient could be successfully introduced into cells and the Tritc fluorescence intensity changed with the concentration gradient.There was no significant difference in fluorescence intensity between two inhibitory peptides（A and B）at the same concentration（500 n M）.2.Western blot results showed that hERG-HEK293 cells incubated with different concentrations（100 n M,200 n M,500 n M）of inhibitory peptide B could inhibit the decrease of hERG protein caused by Nedd4-2 overexpression in a concentration dependent manner.At the same concentration（500 n M）,inhibitory peptides（A and B）could significantly restore the expression of hERG protein to the control level（P<0.05）,while random scrambled peptides had no significant effect（P>0.05）.Inhibitory peptide B was slightly better than A,but there was no significant difference between them.3.Whole cell patch clamp results showed that overexpression of Nedd4-2significantly reduced hERG current,The tail current density was measured at+50 m V the con group current:70.88±2.25 p A/p F,the Nedd4-2 group:13.07±1.78p A/p F,（P<0.01）The same concentration（500 n M）of inhibitory peptides（A and B）could significantly restore and increase hERG current（A:61.41±1.49 p A/p F;B:61.56±2.96 p A/PF,P<0.01）,However,the two random scrambled peptides had no significant effect（A:14.31±1.48 p A/p F;B:15.75±2.55 p A/p F,P>0.05）.4.Effect on the dynamic degradation of hERG membrane protein.Compared with the control group which incubated with BFA alone,Nedd4-2overexpression significantly increased the degradation of mature hERG protein,especially after 12 h（P<0.05）.At 20 h,the proportion of both YFP and CY5 positive cells in Nedd4-2 group was significantly lower than that in control group（P<0.05）,The inhibitory peptide could significantly increase the proportion of both YFP and CY5 positive cells at 12h and 20h（P<0.05）.These results further suggest that the inhibitory peptide competes with Nedd4-2 for the internalization and degradation of hERG channel protein.5.Effect on the expression of other ion channels.In HEK293 cells which were stably expressing KCNQ1/KCNE1,and Nedd4-2 plasmids transient transfection could significantly reduce the expression of KCNQ1 protein,but inhibitory peptides（A and B）had no significant improvement on KCNQ1protein（P>0.05）.In HEK293 cells which were stably expressing Nav1.5,inhibitory peptides（A and B）also had no significant effect on Nav1.5 protein expression which were decreased by Nedd4-2（P>0.05）.6.Physiology effect on hERG protein.Inhibition peptides（A and B）had no significant effect on the physiological state of hERG-HEK293 cells（P>0.05）.Conclusion:Inhibitory peptides（A and B）can effectively inhibit the down regulation of hERG protein by Nedd4-2.There was no statistical difference between the inhibitory peptides（A and B）.and it had no obvious effect on the KCNQ1 and Nav1.5 channel protein which also inhibited by the ubiquitin ligase Nedd4-2.It is suggested that its effect is selective.Part Two Preventive effect of ubiquitin ligase Nedd4-2 inhibitory peptide on pathological hERG downregulationObjective:It is known that some congenital mutations of hERG gene lead to the decrease of membrane protein quantity,which can lead to hereditary LQT.The down-regulation of hERG function in pathological cardiac hypertrophy leads to acquired LQT.In this part,we observed the effect of inhibitory peptides on the decrease of membrane protein caused by hERG mutation in HEK293 cells,and observed the effect of inhibitory peptides on cell electrophysiology in hi PSC-CMs pathological cardiac hypertrophy model（acquired LQT）.Method:1.Effects of inhibitory peptides on hERG membrane proteins of hERG mutants,in HEK293 cells co transfected with wild type hERG and mutant hERG（M124T、A422T、R534C）plasmids,Western blot detected the effect of two inhibitory peptides（A and B）on the expression of mature hERG channel protein（155KD）.2.The establishment of hi PSC-CMs hypertrophy model,Immuno--fluorescence was used to observe cardiomyocyte markers（α-Actin and c Tn T）and it verify the purity of hi PSC-CMs.Endothelin-1（ET-1）10 n M administrate for 24 hours,q RT-PCR was used to detected the m RNA expression changes of cell hypertrophy markers ANP、BNP andβ-MHC.Immunofluorescence was used to observe the changes of cardiac hypertrophy marker BNP,and Leica software was used to measure the ROI value of BNP.Tritc fluorescent probe labeled with F-actin of cardiomyocytes.Image J software was used to measure cell surface area（CSA）of cardiomyocytes.3.Effect of inhibitory peptides on cell electrophysiology:Using cardioexcyte 96 real-time monitoring system,hi PSC-CMs is divided into Control group（Con）,Enthelin-1 group（ET-1）,Scrambled peptide A group（Scrambled peptide A）,Scrambled peptide B group（Scrambled peptide B）,Inhibitory peptide A group（Inhibitory peptide A）and Inhibitory peptide B group（Inhibitory peptide B）.Endothelin-1（ET-1）was given 10 n M for 24 h,and the treatment group was given inhibitory peptides（A and B）（500 n M）for preventive treatment at same time.The scrambled peptide groups were given the same concentration of scrambled peptides.We observed the changes of FPDc（reflected the repolarization time of action potential）and arrhythmias,such as early after depolarization（EAD）,rolling EAD and irregular beating phenomenons.Results:1.Inhibitory peptides（A and B）had no significant effect on mature hERG protein of HEK293 cells which co transfected with WT-hERG and three mutant hERG plasmids（M124T,A422T,R534C）（P>0.05）.2.Immunofluorescence experiments showed that the myocardial specific markersα-Actin and c Tn T could be significantly expressed in hi PSC-CMs,and the purity of cardiomyocytes was over 90%.Compared with the control group,the m RNA expressions of ANP and BNP were significantly increased after ET-1 incubation（P<0.05）,and the m RNA expression ofβ-MHC showed an increasing trend,but did not reach the statistically significant level（P>0.05）.The fluorescence quantification expression of BNP increased significantly（P<0.01）.CSA was significantly larger than that in control group（P<0.05）.3.The change rate of FPDc in hi PSC-CMs,FPDc of ET-1 group was significantly increased than control group（P<0.05）.ET-1 administration induced cardiac pathological hypertrophy in hi PSC-CMs,it significantly delayed cell repolarization and induced arrhythmias such as early after depolarization（EAD）,rolling EAD and irregular beating phenomenons.The combination of inhibitory peptides and ET-1 for 24h could reduce the prolongation of repolarization and arrhythmias.Conclusion:Inhibitory peptides had no significant effect on mature hERG protein of HEK293 cells which co transfected with WT-hERG and mutant hERG plasmids（M124T,A422T,R534C）.ET-1 administration resulted in pathological hypertrophy and prolonged FPDc which was reflected repolarization in hi PSC-CMs.Inhibitory peptides can effectively prevent FPDc prolongation and arrhythmias,it suggested that inhibitory peptides has antagonistic effect on acquired LQT.Part Three Effects of ubiquitin ligase Nedd4-2 inhibitory peptides on ECG parameters of guinea pigs in vivoObjective:To observe the effects of inhibitory peptides（A and B）on the surface ECG of guinea pigs.Methods:Adult guinea pigs were injected with inhibitory peptide via interphalangeal vein at the dose of 0.231mg/kg,once every other day for 2weeks.Lead II ECG was recorded under isoflurane anesthesia before and after inhibitory peptides administration,and the changes of ECG parameters were measured and analyzed.Results:1.Effects of inhibitory peptides on QTc.Body surface electrocardiogram QTc was not significantly changed after 2 weeks of inhibitory peptides injection（P>0.05）.2.Effects of inhibitory peptides on PR intervals.Body surface electrocardiogram PR intervals was not significantly changed after 2 weeks of inhibitory peptides injection（P>0.05）.3.Effects of inhibitory peptides on Tp-e.Body surface Tp-e was not significantly changed after 2 weeks of inhibitory peptides injection（P>0.05）.Conclusion:The inhibitory peptides（A and B）had no significant effect on the QTc,PR intervals and Tp-e in body surface ECG.These results indicate that inhibitory peptides（A and B）have physiological safety.Summary:In this project,molecular biology and flow cytometry technologies were used to observe the preventive effect of the self-designed inhibitory peptides on the decrease of hERG membrane protein in heterologous expression cells and human-induced pluripotent stem cell derived cardiomyocytes.The results showed that two inhibitory peptides could prevent the decrease of hERG membrane protein induced by Nedd4-2,and could significantly prevent the delay of repolarization and arrhythmia in human cardiomyocyte hypertrophy model（acquired LQT cell model）.However,it had no effect on the decrease of membrane proteins caused by several heritable hERG channel mutations.In addition,the inhibitory peptides had no effects on KCNQ1 and Nav1.5channels,and had no significant effects on the whole animal surface ECG.The results provide experimental basis for the new drug development of ubiquitin ligase Nedd4-2 target.