LQT-related Calmodulin Mutant E141G Attenuated The CDI Of Ca_V1.2 Channel And The Repair Role Of Novel Peptides

Objective:Long QT syndrome?LQTS?is a hereditary or acquired disease.It is clinically manifested as cardiac dysfunction,prolonged action potential duration,prolonged QT interval,occasional recurrent syncope and epilepsy,arrhythmia,and even sudden death.However,the heart structure not change abnormally.Clinical research on a large number of patients with LQTS have shown that genetic mutations of calcium channels,potassium channels,sodium channels,and calmodulin?CaM?can cause LQTS.In particular,LQTS caused by CaM mutation is a new discovery in recent years.CaM is encoded by three different genes?CALM1,CALM2,CALM3?,and mutations in any of these genes can cause LQTS.CaM is an important calciumion(Ca²⁺)sensor.When CaM combined with Ca²⁺,it forms a Ca²⁺/CaM complex to play physiological functions role such as signal transduction in tissue cells,and it have important regulatory effects on many cell functions,enzyme systems,and myocardial ion channels?Ca_V1.2 calcium channels,Na_V1.5 sodium channels,K_V1.5 potassium channels?,and ryanodine receptor 2?RyR2?and other activities.CaM is also involved in the growth and proliferation of tumor cells and inducing interventions on receptors for multiple drugs.The ability of CaM mutant of binding to Ca²⁺is weakened or lost,which affects the regulation of calcium ion channels.Therefore,these mutants are often used to study the pathogenesis of LQTS and the mechanism of Ca²⁺-related regulation of calcium channels.L-type calcium channels?LTCC?are mainly expressed in cardiomyocytes.It controls the response of cell Ca²⁺to changes in membrane potential and regulates the occurrence of cardiac action potential,the secretion of hormones,the secretion and release of neurotransmitters,excited contraction coupling and other processes.LTCC is composed of four subunits??₁,?,?/?and??.The?₁ subunit is the main functional unit and the main pathway for Ca²⁺to enter the cardiac cells.The?₁ subunit is divided into Ca_V1.1,Ca_V1.2,Ca_V1.3,Ca_V1.4 calcium channels according to the amino acid sequence.The combination of Ca²⁺/CaM and myocardial Ca_V1.2 channels play an important role in various physiological and pathological states of the body.The C-terminus and N-terminus of the Ca_V1.2 channel are CaM binding sites,and the interaction with CaM participates in the regulation of Ca²⁺-dependent facilitation?CDF?and Ca²⁺-dependent inactivation?CDI?process.Studies have shown that both the CDF and CDI processes require the participation of Ca²⁺/CaM.The interaction of Ca²⁺/CaM complexes with Ca_V1.2 channels controls the opening and closing of calcium channels and regulates many physiological processes by controlling the entry of Ca²⁺.CaM mutations can cause a variety of diseases,such as LQTS,catecholaminergic polymorphic ventricular tachycardia?CPVT?,arrhythmias,myocardial hypertrophy,primary ventricular fibrillation,and sudden death.It is known that the CaM mutant associated with LQTS can inhibit the binding of CaM and Ca²⁺,weaken the CDI effect of Ca_V1.2 channel,and eventually lead to cardiac dysfunction,prolonged depolarization of action potential,prolonged QT interval of electrocardiogram,and clinical manifestations of LQTS.According to reports,LQTS-related CaM mutations include E141G,F142L,D130V,D130G,D96V,etc.Among them,E141G is a key mutation site,because CaM-E141G is the first mutant currently to be found that not only abnormally regulated adult Na_V1.5 sodium channels but also affect Ca_V1.2 calcium channel,while other mutation sites generally only affect the regulation of Ca_V1.2 calcium channels and infant Na_V1.5 sodium channels,and will not affect the role of RyR2.In previous studies,the focus has been on the changes in the binding affinity of CaM mutants to Ca²⁺,but the changes in the interactions with Ca_V1.2 channels are currently unknown.With the increasing development of biotechnology and peptide synthesis technology,more and more peptide drugs have been applied to clinical research.It has the characteristics of small adverse reactions,high safety,and easy synthesis.It is mainly used to treat cancer,tumors,metabolism dysfunction,cardiovascular disease,etc.Therefore,we have also tried to repair LQTS with peptides.In this study,CaM-E141G was selected as the research object,and GST pull-down technology,electrophysiological patch-clamp technology,langendorff heart perfusion technology and whole animal model experiment technology were used to explore the changes in the binding of CaM mutant to Ca_V1.2 calcium channels and the mechanism of abnormal regulation of the channel.Two new peptides were constructed to repair the prolongation of the QT interval caused by CaM mutant,and provide a certain theoretical basis for further exploration of the relationship between CaM and cardiovascular diseases.Methods:We used site-directed mutation technology to mutate wild-type CaM to CaM-E141G,and applied GST pull-down experiments to explore the interaction between CaM-E141G and Ca_V1.2 calcium channels?CT1;PreIQ-IQ;PreIQ;IQ;NT?.And we compare it with CaM-WT to observe the changes in the binding of the mutant to the Ca_V1.2 calcium channel and the degree of influence of the mutant on different motifs on the channel.Next,we use electrophysiological patch-clamp?inside-out mode?technology to study the electrophysiological effects of CaM-E141G on Ca_V1.2 calcium channels,and to analyze the changes in channel activity and channel opening and closing time under different Ca²⁺concentrations.According to the related amino acid sequences of binding and functional regulation of CaM/Ca²⁺and Ca_V1.2 calcium channels,we design and compound two novel peptides using solid-phase synthesis,and then investigate the changes of ECG in animal be resulted from E141G mutation and the possibility of peptide therapy using langendorff heart perfusion technique.We detect relevant ECG parameters,mRNA expression of related proteins.Finally,the whole animal model was studied,and the LQTS animal model is established by using peptides to explore the CaM-E141G mutation abnormal ECG and the possible repairing role of peptides.Results:1?The combined effect of CaM-E141G and CaV1.2 calcium channels.The results of GST pull-down showed that the binding of CaM-E141G to Ca_v1.2 calcium channel is protein concentration-and Ca²⁺concentration-depended.There is significantly weakened binding of CaM-E141G to CT1?PreIQ-IQ?PreIQ?IQ and NT,the maximum binding amount?Bmax?was reduced by 17.71-59.26%at a high Ca²⁺concentrations which compared with CaM-WT.The binding affinity of CaM-E141G to NT,PreIQ and IQ on Ca_V1.2 calcium channel are decreased,and the binding characteristics are changed.2?The electrophysiological effects of CaM-E141G to Ca_v1.2 calcium channel.The results of patch clamp experiments showed that Ca²⁺/CaM-E141G inhibited the Ca_V1.2 calcium channel activities by 57.77%,and the process of CDI was inhibited at500 nM concentration of Ca²⁺;the opening and closing times of the channel have also changed:CaM-E141G does not change the fast opening time of Ca_V1.2 calcium channel,but the fast closing time is decreased from 1.35±0.09 ms to 1.03±0.09 ms?P<0.05,n=5?;the slow opening time is extended from 4.60±0.72 ms to 7.69±0.45 ms?P<0.01,n=5?,and the slow closing time does not change,but the number of closed channels are decreased significantly.These results indicate that when the Ca²⁺concentration is 500nM,CaM-E141G prolongs the opening time of the Ca_V1.2 channel in the inside-out mode,that is,the Ca²⁺-dependent inactivation decreased.3?Effect of CaM-E141G-derived peptide CM4 on QT interval of electrocardiogram which from the isolated heart of guinea pig and the repair of peptide CM4-R.The electrocardiogram monitoring results of Langendorff cardiac perfusion showed that the QT interval of the electrocardiogram was significantly longer?P<0.01?with the occurred of ventricular Arrhythmia?55.56%?in the CM4 group?n=9?;and there was no significantly different in QT interval of the electrocardiogram between the CM4-R group?n=6?and the CM4+CM4-R group?n=6?compared with the control group?KH solution,n=4?.The results indicate that the peptide CM4-R has a certain repair effect on the QT interval extension caused by CM4.4?Effect of peptide CM4 on the expression of CaM,CaMKII and Ca_V1.2 mRNA in myocardium of different groups and the repairing effect of CM4-R in the Langendorff heart perfusion experiment.We used qRT-PCR to detect the expression of CaM and CaMKII mRNA in different groups of myocardial tissues.The results showed that the expression of CaM in the CM4group?n=9?was significantly lower than that in the control group?0.9%NaCl,n=4??P<0.01?,CM4+CM4-R group?P<0.05,n=6?and CM4-R group?P<0.05,n=6?;the expression of CaMKII in the CM4 group was significantly lower than the control group.There were no significantly different of the expressions of CaM and CaMKII in the CM4+CM4-R group and the CM4-R group compared with the control group.The results indicate that CM4 reduces the expression of CaM and CaMKII,and CM4-R has a certain repair effect on this abnormality at the mRNA level.We used qRT-PCR to detect the expression of Ca_v1.2 mRNA in different groups of myocardial tissues.The results showed that there was no statistical difference in the expression of Ca_V1.2 in the CM4 group,CM4+CM4-R group,and CM4-R group compared with the control group.The result shows that CM4 and CM4-R do not affect the mRNA expression of Ca_V1.2.5?Effect of CaM-E141G-derived peptide CM4 on QT interval of electrocardiogram in mice and the repair of peptide CM4-R.The results of the in vivo electrocardiogram test of the mice showed that the QT interval of the electrocardiogram of the mice in the CM4 group?n=6?was significantly longer?P<0.01?and there was no significant difference in QT interval of the electrocardiogram between the CM4+CM4-R group?n=8?and the CM4-R group?n=7?compared with the control group?0.9%NaCl,n=14?.The result indicates that the peptide CM4-R has no effect on the QT interval of the electrocardiogram and can repair the QT interval prolongation caused by CM4.6?Effect of peptide CM4 on the expression of CaM and Ca_V1.2 protein in animal experiments and repair of peptide CM4-R.Western blot was used to detect the expression of CaM protein in myocardial tissue in different groups,the result showed that compared with the control groups?0.9%NaCl,n=14?,the expression of CaM in the CM4 group?n=6?was significantly reduced?P<0.05?,and there was no significant difference in the expression of CaM between the CM4-R group?n=7?and the CM4-R+CM4group?n=8?.The result shows that CM4 can down-regulate the expression of CaM protein,and CM4-R has a certain repaired effect on this change.We also used western blot to detect the expression of Ca_V1.2 protein in myocardial tissue in different groups.The results showed that compared with the control group?0.9%NaCl,n=14?,there were no statistical difference in the CM4 group?n=6?,CM4-R group?n=7?and the CM4-R+CM4 group?n=8?of the expression of protein Ca_V1.2?₁ subunit,suggesting the expression of Ca_V1.2 protein is not affected by CM4 and CM4-R.Conclusion:1.Mutations in the Ca²⁺binding domain of CaM will reduce the bingding density between CaM and Ca_V1.2 calcium channels.In particular,the binding characteristics of CaM-E141G to PreIQ,IQ,NT protein fragments of Ca_V1.2 calcium channel have changed significantly.2.As the concentration of Ca²⁺is 500 nM,CaM-E141G can change the opening and closing time of Ca_V1.2 calcium channel,which increases its activity and decreases the process of Ca²⁺-dependent inactivation?CDI?.3.CaM-E141G-derived peptide CM4 prolongs the QT interval of the electrocardiogram in isolated heart of guinea pig,and down-regulates the expression of CaM and CaMKII at mRNA level in myocardial tissue.CM4-R has a repairing effect on this change.4.CaM-E141G-derived peptide CM4 prolongs the QT interval of the electrocardiogram in mice,and down-regulates the expression of CaM at the protein level in myocardial tissue.CM4-R can repair this change.