| Pathological cardiac hypertrophy is a common complication of various diseases such as hypertension,myocardial ischemia,and diabetic cardiomyopathy.It is a complex pathophysiological process in which various neurohumoral factors are mediated and various cellular signaling pathways are involved.Histological hypertrophy remodeling increases compensatory cardiac output at an early stage,but continuous development can lead to decompensation of cardiac contractile function and ultimately heart failure.Accompanied by hypertrophic remodeling is myocardial electrophysiological remodeling,which greatly increases the incidence of arrhythmia,resulting in sudden cardiac death(SCD)accounting for about 50%of deaths in heart failure patients.Most of the existing antiarrhythmic drugs have potential risk of arrhythmia and are limited in use.Therefore,revealing the molecular mechanism of electrical remodeling-induced arrhythmia and finding new targets for prevention and treatment are important topics in cardiovascular research in recent years.Various experimental animal models and electrophysiological recordings of ventricular cells in patients with heart failure found that the most prominent manifestation of myocardial electrophysiological remodeling was the slow repolarization of cardiomyocytes and the prolongation of action potential duration(APD).The surface electrocardiogram showed an extension of the QT interval(belonging to the acquired LQT syndrome).The repolarization delay is prone to triggering electrical activity caused by early depolarization(EAD),and the increase of repolarization dispersion leads to excitatory reentry and ventricular tachyarrhythmia.It is known that there are various voltage-gated K+channels on the myocardial cell membrane,including transient outward K+channels(current is Ito),fast and slow delay rectifier K+channels(corresponding current components are IKr,IKs)which determine the APD and action potential morphology.Among them,Ito is the main current of the rapid repolarization phase 1 of the large animal heart including human,while in the rodent small animal is the main current component of the whole repolarization period;IKr(the channel pore subunit is encoded by the h ERG gene)and IKs(channels encoded by the subunit gene KCNQ1 and the beta subunit gene KCNE1)are the major currents of the 2-phase platform and 3-phase repolarization of large animals.In the past 10-20 years,a large number of experimental studies have found that the most likely cause of APD prolongation due to cardiac hypertrophy is the reduction of different K+current densities.Multi-cardiac hypertrophy,heart failure animals(including mice,rats,rabbits,dogs,etc.)and heart failure of human ventricular myocytes observed by different causes(including rapid ventricular pacing and high post-load),the density of Ito is reduced.In addition to Ito,IKr and IKs are also reduced in hypertrophy and heart failure.In view of the fact that K+current reduction is the main cause of cardiac hypertrophy arrhythmia,attempts have been made to increase K+current with channel openers to counteract pathological APD prolongation.Experiments have shown that selective IKs channel openers can attenuate APD in hypertrophic rabbit ventricular cells and eliminate the appearance of EAD;various IKr channel openers also show the same anti-pathological electrical remodeling effect.Our previous experiments also observed that IKr opener can effectively prevent ventricular tachycardia and ventricular fibrillation(VT/VF)caused by APD prolongation in isolated hearts.Therefore,the use of K+channel openers is expected to be a new strategy for the treatment of cardiac hypertrophy arrhythmias.However,in recent years,K+channel gain of function mutations have been found to cause short QT(SQT)syndrome with the risk of ventricular fibrillation,which seems to suggest that these K+channel openers may have a potential risk of arrhythmia in shortening the QT interval.We have recently observed that different IKr and IKs openers induce VT/VF in isolated perfused hearts.Recording the outward K+current under the action potential clamp found that these openers increased the outward repolarization current while changing the current shape.In particular,the early repolarization current increases,and the corresponding early repolarization time is shortened in the APD and ECG parameters,and the degree of shortening is highly correlated with arrhythmia.This suggests that changing the channel dynamics increases the K+repolarization current as a drug that blocks the channel to reduce K+current as a risk of arrhythmia.So,how to open up new paths to increase K+current is the key issue.It is known that the channel current depends on the channel dynamics and the number of functional channels on the cell membrane.In addition to the regulation of gene transcription and protein synthesis,recent studies have found that the transport of cell membrane channel proteins is the key factor that affects the expression of IKr and IKs channel cell membranes.The number of cell membrane channel proteins depends on two reverse transport processes:one is forward transport.The channel protein is synthesized in the endoplasmic reticulum(ER)and then transported to the Golgi apparatus and transported to the cell membrane.Second,the channel protein on the membrane is internalized into the early endosome,part of which can be recycled to the cell membrane,and the other part enters late endosomes are degraded by proteasome or lysosomal pathways.Ubiquitination is the first step in the internalization of channel membrane proteins.Experiments have shown that the E3 ubiquitin ligase member Nedd4-2 is a key molecule that initiates this reaction,and it specifically binds to the PY sequence of the target protein to make it ubiquitinated.The C-terminus of h ERG and KCNQ1 have a PY sequence,and Nedd4-2 can ubiquitinate them into early endosomes.The small G protein RAB5,which regulates the transport of vesicles,promotes the ubiquitination of Nedd4-2,while RAB11 promotes early endosomal recirculation to the cell membrane.It can be seen that the forward transport barrier or the acceleration of the degradation process can result in a decrease in the number of membrane channels.Our previous experiments showed that angiotensin II significantly down-regulated mature h ERG protein through PKC signaling pathway in heterologously expressed cells,and this effect was found to be related to the accelerated degradation of membrane mature protein.Other studies have shown that angiotensin II can significantly accelerate the internalization and degradation of BKCa(large-conductance Ca2+-activated K+channel)on the vascular membrane of arteries,which is mediated by PKC.Recently,we observed the effect of PKC subtype agonist peptide on the expression of KCNQ1/KCNE1 channel.It was found that activating traditional PKC(c PKC)had no effect on the total protein content of KCNQ1,but significantly reduced the amount of cell membrane channel protein,which was related to its increased degradation.PKC is known to be an important mediator of a variety of neurohumoral fluids causing pathological cardiac hypertrophy,and is involved in the regulation of myocardial electrophysiology.Therefore,we speculate that the increase in internalization and degradation of h ERG and KCNQ1/KCNE1 channels leads to a decrease in the number of channels,which is an important reason for the decrease of myocardial hypertrophy IKrand IKs current.Based on the above problems,we hypothesized that the increase in channel ubiquitination and the decrease in the number of membrane-forming channels are important reasons for the decrease of IKcurrent in the pathological state of cardiac hypertrophy.Inhibition of the ubiquitination process will increase the number of membrane channels and increase the IK current,effectively preventing arrhythmias caused by myocardial hypertrophy and electrical remodeling.Part one Establishment of pathological myocardial hypertrophy electrical remodeling modelObjective:To establish a model of cardiac hypertrophy and electrical remodeling in the overall level of guinea pigsMethods:Ang II was continuously administered to the guinea pig by subcutaneously placing an osmotic pump under the neck for 2 weeks.The overall function and tissue structure of the guinea pig heart were observed by small animal echocardiography and isolated tissue weighing.The myocardial cell size and degree of fibrosis were observed by HE staining and Masson staining.The m RNA content of cardiac hypertrophy-related factors was detected by RT-PCR.Body surface ECG records guinea pig in vivo ECG.Guinea pig cardiomyocytes were isolated and APD was recorded using current clamp technique.Under the condition of Langendorff perfusion,the PES electrical stimulation was induced into the heart of guinea pigs to induce arrhythmia,and the susceptibility to arrhythmia was observed.Results:Echocardiographic results showed that compared with the control group(CON),the left ventricular end-systolic anterior wall,posterior wall,and ventricular septal thickness of the model group(Ang II)were significantly increased(CON:1.67±0.024,1.63±0.025,0.93).±0.066 mm;Ang II:2.07±0.055,2.01±0.06,1.18±0.028 mm,P<0.05 vs CON);the left ventricular anterior wall,posterior wall,and ventricular septal thickness were also significantly increased(CON:1.34±0.057,1.21±0.051,0.75±0.072mm;Ang II:1.7±0.064,1.6±0.058,0.98±0.027 mm,P<0.05 vs CON)at the end of diastole.Tissue weight results showed a significant increase in heart weight and left ventricular mass in the model group compared with the control group(CON:3.35±0.021,2.01±0.038 mg/g;Ang II:4.11±0.13,2.67±0.12 mg/g,P<0.01 vs CON).The results of HE staining showed that the area of myocardial cells in the model group was significantly higher than that of the control group(CON:150.72±46.62?m2,Ang II:368.8±84.19?m2,P<0.05 vs CON);Masson staining showed that the myocardial fibrosis area of the model group was better than that of the control group.The group increased significantly(1.28±0.46%,21.78±6.4%,P<0.05 vs CON).The results of RT-PCR detection of hypertrophy related factors showed that the expression of hypertrophic related factors(ANP,?-MHC,TGF-?)in the model group was significantly higher than that of the control group(P<0.05 vs CON).The QTc interval in the model group was significantly prolonged by recording ECG in guinea pigs(10.88±0.16,12.2±0.37,P<0.05 vs CON).The guinea pig cardiomyocytes were isolated and APD was recorded.APD50 and APD90were significantly prolonged in the model group(CON:419.28±25.04,460.66±25.04ms;Ang II:731.9±49.53,821.08±42.25ms,P<0.001 vs CON).The threshold of arrhythmia in guinea pig hearts was observed by electrical stimulation under perfusion conditions.The results showed that the threshold of arrhythmia in the model group was significantly lower(172.5±11.91,110±14.14 m A,P<0.01 vs CON).Arrhythmia did not occur in the control group under the same intensity current stimulation(130 m A),but 5 of 6guinea pigs in model group occurred arrhythmia.Summary:Continuous administration of Ang II through the subcutaneous placement of the osmotic pump in the neck can cause significant hypertrophy and electrical remodeling in the left ventricle of the guinea pig,manifested as thickening of the left ventricular wall and interventricular septum,increased myocardial cell area and increased fibrosis.The QT interval of the electrocardiogram is prolonged;the APD of cardiomyocytes is prolonged,and the threshold of arrhythmia is significantly reduced.Part two Changes and causes of delayed rectifier potassium channels in cardiac hypertrophy and electrical remodelingObjective:To study the changes and intrinsic mechanisms of delayed rectifier potassium channels in cardiac hypertrophy electrical remodeling models.Methods:Guinea pig cardiomyocytes were isolated and the whole-cell patch clamp technique was used to record delayed rectifier potassium currents(IKr,IKs).The expression levels of ERG gene and KCNQ1 gene in guinea pig myocardium were detected by RT-PCR.The expression levels of ERG protein,KCNQ1/KCNE1 protein,Nedd4-2/p-Nedd4-2 and Rab11 protein in guinea pig myocardium were detected by Western Blot.The interaction between ERG,KCNQ1 and Nedd4-2 proteins and the level of ERG protein ubiquitination were analyzed by Co-IP.Results:Patch clamp results showed that there was no significant change in IKs in the model group compared with the control group,and the current density of the IKr tail was significantly reduced(CON:0.51±0.0754 p A/p F at a voltage of+60 m V,Ang II:0.28±0.0456 p A/p F,P<0.05 vs CON).RT-PCR results showed that compared with the control,there was no significant change in the expression of ERG gene in the model group,and the KCNQ1 gene was significantly increased.Western Blot results showed that there was no significant difference between KCNQ1 and KCNE1 protein expression,while ERG protein was significantly down-regulated in the model group(including mature and immature ERG proteins);Nedd4-2 and Rab11protein expression in the model group significantly upregulated,while phosphorylated Nedd4-2(p-Nedd4-2)expression was significantly reduced.Co-IP results suggested that the direct effect between ERG protein and Nedd4-2 in the myocardial tissue of the model group was enhanced compared with the control group,while the effect between KCNQ1 and Nedd4-2 was not significantly different.At the same time,the direct interaction between ERG and Ub small molecules increased,indicating that the level of ubiquitination of ERG protein was enhanced in the model group.Summary:1.In the guinea pig cardiac hypertrophy electrical remodeling model,the fast-activated delayed rectifier potassium current(rather than the slow-activated delayed rectifier potassium current)was significantly down-regulated.2.The IKr current and ERG protein were significantly decreased,while the expression of ERG gene did not change significantly,indicating that the change was caused by post-transcriptional regulation.At the same time,the expression of E3 ubiquitin ligase Nedd4-2 was significantly up-regulated in the model group,and the ubiquitination level of ERG protein was also significantly increased,indicating that the potassium current down-regulation in the model was due to the increased degradation of channel protein by ubiquitination.Part three Effect of SGK1 activation on myocardial hypertrophy and electrical remodeling in guinea pigsObjective:To observe the effect of activation of SGK1 on myocardial hypertrophy electrical remodeling in vivo.Methods:SGK1 was activated in guinea pig hearts by intraperitoneal injection of C4-CER and intravenous injection of adeno-associated virus AAV-9 expression vector containing the active SGK1(S422D)gene of the cardiac specific promoter c Tn T.And observe the effects on myocardial hypertrophy and electrical remodeling in guinea pigs.The overall function and tissue structure of the guinea pig heart were observed by small animal echocardiography and isolated tissue weighing.The methods of HE and Masson staining were used to observe the size of cardiomyocytes and degree of myocardial fibrosis.Body surface ECG records guinea pig in vivo ECG.The expression levels of ERG protein,KCNQ1 protein,Nedd4-2/p-Nedd4-2and SGK1/p-SGK1 protein in guinea pig myocardium were detected by Western Blot.Results:Echocardiography showed that the left ventricle of the model group was significantly thickened,while C4-CER did not improve the thickening.The results of tissue weighing showed that the model group had obvious heart and lung weight gain.The left ventricle and ventricular septal weight were significantly increased,while the weight of the guinea pigs in the C4-CER group was not improved,and the lung and heart weights were increased compared with the model group.The results of surface electrocardiogram showed that the model group showed obvious QT interval prolongation,while C4-CER increased the tendency of this extension.Injection of adeno-associated virus carrying active SGK1(S422D)partially reversed the hypertrophy of the left ventricular wall of the model group,but could not reverse the increase of myocardial cell area or fibrosis and the prolongation of QT interval of ECG.Westernblot results showed that both ways of activating SGK1 significantly increased the amount of phosphorylated SGK1 and ERG proteins without affecting KCNQ1 protein.Further observations showed that both C4-CER and SGK1(S422D)failed to reverse the down-regulation of p-Nedd4-2 protein in the model group,whereas the active SGK1 could reverse this over-expressed Nedd4-2 protein expression under pathological conditions of cardiac hypertrophy.While C4-CER has no such effect.Summary:Direct activation of SGK1 can inhibit NEDD4-2 and increase ERG protein expression,but it cannot effectively improve the pathological state of guinea pig cardiac hypertrophy and QT interval prolongation.Part four Effect of over-expressing mutant Nedd4-2 on myocardial hypertrophy and electrical remodeling in guinea pig myocardiumObjective:To overexpress mutant Nedd4-2 in guinea pig myocardium and observe its effect on myocardial hypertrophy and electrical remodeling.Methods:The adeno-associated virus AAV-9 expression vector containing the mutant Nedd4-2(C801S)gene and the cardiac specific promoter c Tn T was constructed.The guinea pig was injected by intravenous injection,and detection of fluorogenic activity in guinea pig tissues 4 weeks after injection,and then the model of cardiac hypertrophy was established.Cardiomyocyte suspensions and tissue sections were prepared by cryosection technique.The fluorescence intensity of cardiomyocytes and tissues was observed under fluorescence microscope.The wall thickness of guinea pigs was observed by small animal echocardiography.ECG records of body surface Guinea pigs in vivo ECG,compared QTc interval changes;Isolation of guinea pig cardiomyocytes,recording current APD and delayed rectifier potassium current using current clamp technique and whole-cell patch clamp technique;Co-IP was used.Analysis of ERG protein ubiquitination level by Co-IP.Results:Fluorescence microscopy showed that after 4 weeks of AAV-9virus injection,e GFP was mainly expressed in the heart,and no obvious fluorescence was observed in other tissues.In the heart tissue,the left ventricle expressed more than the atrium and the right ventricle.At the end of the modeling,it was found that 5 guinea pigs died in the model group,while guinea pigs overexpressing Nedd4-2(mutation)did not die.;Echocardio-graphic results showed that compared with the control group(CON),the model group(Ang II)guinea pigs showed obvious left ventricular wall and ventricular septal thickening,while the guinea pigs injected with mutant Nedd4-2 virus(Ang II+Nedd4-2(mutation))showed significant improvement in left ventricular wall and ventricular septum..The control guinea pigs injected with virus(CON+Ang II(mutation))showed no significant change compared with CON;the surface ECG results showed that the model group(Ang II)guinea pigs showed significant QTc interval prolongation compared with the control group(CON).At the same time,guinea pigs injected with mutant Nedd4-2 virus(Ang II+Nedd4-2(mutation))showed significant improvement and returned to the control level;the control guinea pigs injected with virus(CON+Ang II(mutation))showed no significant change compared with CON;Separation of cardiomyocytes and recording of APD showed that compared with the control group(CON),the model group(Ang II)guinea pigs showed obvious APD prolongation,which was characterized by prolongation of APD50 and APD90,while simultaneously injecting mutant Nedd4-2 virus(Ang II+Nedd4-2(mutation))guinea pigs showed significant improvement,APD50 and APD90 recovered to the control level;the control guinea pigs injected with virus(CON+Ang II(mutation))showed no significant change compared with CON;patch clamp results showed that the model group guinea pigs showed significant down-regulation of IKr current,while simultaneously injecting mutant Nedd4-2 virus(Ang II+Nedd4-2(mutation))guinea pigs showed significant up-regulation of IKr current and was significantly higher than the level of the CON group.Control guinea pigs injected with virus(CON+Ang II(mutation))showed no significant change compared to CON.The IKs current density did not differ significantly between the 4 groups.The results of co-immunoprecipitation showed that compared with the Ang II group,the level of ERG protein ubiquitination in guinea pig cardiomyocytes mutated by Nedd4-2 was significantly attenuated.Summary:Over-expression of Nedd4-2 mutein(C801S)in the heart can effectively reverse cardiac hypertrophy and accompanying electrical remode-ling in guinea pigs by inhibiting the ubiquitination of ERG proteins.Part five Inhibitory effect of synthetic short peptides on degradation of h ERG protein induced by Nedd4-2Objective:To observe the inhibitory effect of h ERG protein-specific PY sequence short peptide on h ERG protein degradation induced by Nedd4-2.Methods:Different amounts of Nedd4-2 plasmid were transfected into HEK293 cell line stably expressing h ERG protein.Western blot and whole cell patch clamp method were used to detect h ERG protein expression and IKrcurrent.Transfection of a certain amount of Nedd4-2 plasmid with different concentrations of PY sequence short peptides with cell-penetrating peptides(designed according to Nedd4-2 and h ERG protein binding site sequences),and laser confocal method was used to observe whether short peptides were introduced into cells.The expression of h ERG protein and IKr current were detected by Western Blot and whole cell patch clamp method,and the inhibitory effect of PY peptide on Nedd4-2 was observed.Results:Western Blot results showed that the administration of three different amounts of Nedd4-2 plasmid(500 ng,1000 ng,2000 ng)signifi-cantly reduced the expression of mature h ERG protein(155 k Da instead of135 k Da)on the membrane.Patch clamp results also showed that different amounts of Nedd4-2 plasmid significantly down-regulated IKr current(consistence:+50 m V,CON:55.83±6.56 p A/p F,500 ng:24.85±2.14 p A/p F,1000 ng:25.55±2.02 p A)/p F,2000 ng:29.55±2.22 p A/p F,P<0.001 vs CON).Among them,1000 ng Nedd4-2 and 2000 ng had no significant difference in the down-regulation of h ERG protein and IKr current,so the subsequent transfection was performed with 1000 ng of Nedd4-2 plasmid.Confocal microscopy revealed that three concentrations of PY short peptides(100 n M,200 n M,500 n M)were all introduced into the cells.Western Blot results showed that Nedd4-2 significantly reduced the amount of h ERG protein compared with the control group,and the three concentrations of PY short peptide could significantly restore the expression of h ERG protein to the control level,while the garbled peptide of the random scrambled sequence had no obvious effect.The patch clamp showed the same results.Three concentrations of PY short peptides significantly restored the IKr current to the control level,and the garbled peptide had no significant effect(at a voltage of+50 m V,CON:55.83±4.55 p A/p F,Nedd4-2:21.92±1.67 p A/p F,Scrambled peptide(500 n M):22.71±2.06 p A/p F,PY peptide(100 n M):45.31±3.63p A/p F,PY peptide(200 n M):45.75±2.55 p A/p F,PY peptide(500 n M):50.95±3.06 p A/p F).Summary:PY short peptide designed according to Nedd4-2 and h ERG protein binding site sequence can effectively inhibit the down-regulation of Nedd4-2 on h ERG protein and IKr current.Conclusion:The results of this study indicate that the increase in ubiquitination of h ERG(ERG)under pathological cardiac hypertrophy is the main mechanism leading to the decrease of IKr current.Intervention the ubiquitination-mediated channel protein degradation is expected to provide a new therapeutic target for improving pathological electrical remodeling and preventing arrhythmias. |
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