| Background:The mortality of acute myocardial infarction has fallen due to the development of reperfusion therapy.However,the morbidity of heart failure is on the rise.The adverse structural and electrical remodeling after myocardial infarction resulted in alterations in cardiac structural,fuction and electrophysiology,and led to chamber enlargement,cardiac dysfunction and malignant ventricular arrhythmias.So,attenuating or reversing ventricular remodeling could decrease the morbidity and mortality of heart failure and malignant arrhythmia to improve the outcome of patients.Glucagon-like peptide-1 is released from enteroendocrine L-cells post meals,which stimulates insulin secretion from the pancreas on glucose-dependent manner.Despite its effect on lowering blood glucose,it gains much attention because of its effect on cardiovascular system.GLP-1 is rapidly degraded by ubiquitous proteolytic enzyme dipeptidyl peptidase-4(DPP4).Exendin-4 is one of GLP-1R agonist.GLP-1R is not only expressed in pancreatic β-cells,but also in the heart,central nervous system,kidney and lung.Previous studies mainly focused on the effect of GLP-1 and GLP-1 agonists on ischemia reperfusion injury which could decrease post-ischemic damage through activating pro-survival kinase and improve energy utilization to augment post injury recovery.Although,recently study demonstrated that exendin-4 exerted cardioprotective effects on ventricular remodeling post-MI,there was no study on electrical remodeling.eNOS/cGMP/PKG signal is the downstream of GLP-1R,which could regulate calcium cycling to exert cardioprotective effect on structural and electrical remodeling.Thus we hypothesize that GLP-1 R agonist exendin-4 may attenuate ventricular structural and electrical remodeling post-MI through eNOS/cGMP/PKG signal.Therefore,we explored the effects of exendin-4 on structural and electrical remodeling and the mechanism in myocardial infarction rats model by coronary ligation.Part Ⅰ:Effects of GLP-1R Agonist Exendin-4 on Cardiac Structural Remodeling in Myocardial Infarction-Heart Failure RatsObjective:To investigate the effects of GLP-1R agonist exendin-4 on cardiac structural remodeling in myocardial infarction-heart failure rats.Methods:80 SD rats were randomly divided into four groups.Twenty-four hours after LAD ligation surgery,the rats were randomly divided into the MI group(infarcted rats treated with vehicle only);the MI+Ex-4 group(infarcted rats injected with 10μg/kg/day of exendin-4,ip;or the MI+Ex-4+Ex9-39 group(infarcted rats injected with 10 μg/kg/day of exendin-4 and 200 μg/kg/day of exendin9-39,ip).The sham group was subjected to the same surgery protocol without LAD ligation and received vehicle alone.24 hours after LAD ligation surgery,(1)using Elisa methods to detect the plasma level of GLP-1,glucose and insulin and western blot and RT-PCR methods to detect the expression of GLP-1R in the border zone of left ventricular.(2)Echocardiographic analysis was performed to assess cardiac function and left ventricular dimension.(3)Haemodynamic measurements to measure cardiac systolic and diastolic funtions and recorde systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean blood pressure(MBP).(4)Using heart weight/body weight(HW/BW)ratio,heart weight/tibia length(HW/TL)ratio and lung weight/tibia length(LW/TL)ratio to evaluate cardiac hypertrophy and heart failure.(5)Using HE staining and Masson staining to assess infarct size,cardiomyocyte hypertrophy and interstitial fibrosis.Results:(1)Compared with sham group rats,the plasma GLP-1 level and left ventricular GLP-1R expression were decreased in MI group rats.Exendin-4 treatment halted the changes,and exendin9-39 partly abolish the effects of exendin-4(all P<0.05).There were no significant difference in plasma glucose and insulin among groups.(2)Compared with sham group rats,the values of LVEF,LVFS were decreased and LVDs and LVDd were increased in MI group rats(P<0.05).After exendin-4 treatment,the above mentioned values were close to the values in sham group rats(P<0.05).What is more,exendin9-39 partly abolished the cardioprotective effects of exendin-4(P<0.05).(3)Compared with sham group,the absolute value of dp/dtmax and dp/dtmin were decreased in MI group(P<0.05),exendin-4 treatment attenuated the changes(P<0.05).However,exendin9-39 partly abolished the protection of exendin-4(P<0.05).There were no significant differences in SBP,DBP and MBP among groups.(4)Compared with sham group rats,the HW/BW ratio,HW/TL ratio,LW/TL ratio and plasma ANP level were increased in MI group rats(P<0.05).Exendin-4 treatment decreased the above mentioned ratio(P<0.05).However,exendin9-39 partly attenuated the effects of exendin-4(P<0.05).(5)Compared with sham group rats,the cross-sectional area and interstitial fibrosis were elevated in MI group rats(P<0.05).Exendin-4 treatment decreased the values of infarct size,cross-sectional area and interstitial fibrosis(P<0.05).However,exendin9-39 partly abolished the cardioprotection of exendin-4(P<0.05).Part II:Effects of GLP-1R Agonist Exendin-4 on Cardiac Electrical Remodeling in Myocardial Infarction-Heart Failure RatsObjective:To investigate the effects of GLP-1R agonist exendin-4 on cardiac structural remodeling in myocardial infarction-heart failure rats.Methods:The groups and treatment were same with part I.28 days after surgery,(I)Telemetry probes were implanted and 24 hours ambulatory electrocardiogram was recorded to analyze the incidence of arrhythmias and ECG parameters,such as RR interval,QRS interval,QT interval and QTc;(2)In Langendorff perfused hearts,using microelectrode array to record conduction velocity(CV),total activation time(TAT)and dispersion of total activation time(dispersion of TAT)in border zone of left ventricular;(3)In Langendorff perfused hearts,with electrophysiological detection to record action potential(AP);(4)Using S1 S1 pacing to induce action potential duration(APD)alternans;(5)Using Burst pacing to inducing ventricular arrhythmias(VAs)to calculate the ratio of VAs.Results:(1)Compared with sham group,MI rats displayed longer QRS interval,QT interval and QTc(P<0.05).After exendin-4 treatment,the above mentioned parameters were shorter(P<0.05),co-administration with exendin9-3 9 partly diminished the protective effect of exendin-4.There were no significant differences of RR interval among groups;(2)There were no ventricular fibrillation and sustained ventricular tachycardia in all group.Compared with sham group,MI rats had higher incidence of ventricular tachycardia and premature ventricular contraction(P<0.05).With exendin-4 treatment,the above mentioned ratios were decreased(P<0.05).However,co-administration with exendin9-39 partly abolished the effect of exendin-4(P<0.05);(3)Compared with sham group,the CV in border area of MI rats was decreased,TAT was prolonged,dispersion of TAT was increased(P<0.05).Exendin-4 treatment ameliorated the above mentioned changes(P<0.05).However,co-administration exendin9-39 partly diminished the effect of exendin-4(P<0.05);(4)Compared with sham group,the APD90 was prolonged in MI group(P<0.05).Exendin-4 attenuated the change(P<0.05),co-administration with exendin9-39 partly abolished the protective effects of exendin-4(P<0.05);(5)Compared with sham group,threshold of APD alternans was decreased in MI group(P<0.05).With exendin-4 treatment,the threshold was increased(P<0.05).However,co-administration with exendin9-39 partly diminished the effect of exendin-4;(6)Compared with sham group,the incidence of VAs and sustained VAs were elevated(P<0.05).With exendin-4 treatment,the above mentioned ratio was decreased(P<0.05).However,co-administration of exendin9-39 partly diminished the effect of exendin-4(P<0.05).Conclusion:exendin-4 treatment attenuates ventricular electrical remodeling post-MI,decreased the incidence of VAs,which may due to the effect of exendin-4 on shortening APD,elevating threshold of APD alternans,accelearating C V and making propagation homogeneity.Part Ⅲ:Mechanisms of GLP-1R Agonist Exendin-4 on Cardiac Structural and Electrical Remodeling in Myocardial Infarction-Heart Failure RatsObjective:To investigate the mechanisms of GLP-1R agonist exendin-4 on cardiac structural and electrical remodeling in myocardial infarction-heart failure rats.Methods:The groups and treatment were same with part Ⅰ.28 days after surgery,(1)Epifluorescence microscope(Lei ca Microsystems,Germany)was used to detect the Ca2+ transient amplitude(AF/FO),Ca2+ content(△F/F0,Caffeine),fractional release in isolated ventricular myocytes with Fluo-4 AM staining;(2)Epifluorescence microscope(Leica Microsystems,Germany)was used to detect calcium sparks in isolated ventricular myocytes with Fluo-4 AM staining;(3)L-type Ca2+ current I-V curve and activation curve and current density were recorded by whole-cell patch-clamp technique;(4)Calcium handling proteins SERCA2a,PLB,phosphorylated PLB,phosphorylated RyR and Cav1.2 were detected by western blot methods;(5)The expression of eNOS,cGMP,PKG and CaMKⅡ were detected by western blot or Elisa methods.Results:(1)Compared with sham group,the amplitude of calcium transients,SR calcium content were decreased and fractional release were elevated in MI ventricular myocytes(P<0.05).However,with exendin-4 treatment,the above mentioned parameters were improved(P<0.05).Co-administration with exendin9-39 partly abolished the effect of exendin-4(P<0.05);(2)Compared with sham group,the amplitude,frequency of calcium sparks and calcium leak were increased in MI ventricular myocytes(P<0.05).With exendin-4 treatment,the above mentioned parameters were decreased(P<0.05).Co-administration with exendin9-39 partly diminished the effect of exendin-4(P<0.05);(3)Compared with sham group,the I-V curve was shitted up,the peak ICa,L was decreased,V0.5 was increased(P<0.05).With exendin-4 treatment,the peak ICa,L was inreased,V0.5 was decreased(P<0.05).Co-administration with exendin9-39 partly diminished the effect of exendin-4(P<0.05);(4)Compared with sham group,the expression of SERCA2a,p-PLB,Cav1.2 were decreased,the expression of PLB、p-RyR were increased in MI group(P<0.05).With exendin-4 treatment,the expression of SERCA2a,p-PLB,Cav1.2 were increased,the expression of PLB、p-RyR were decreased(P<0.05).Co-administration with exendin9-39 partly abolished the effect of exendin-4(P<0.05);(5)Compared with sham group,the expression of eNOS,p-eNOS,cGMP and PKG were decreased,the expression of p-CaMK II was increased in MI group(P<0.05).With exendin-4 treatment,the expression of eNOS,p-eNOS,cGMP and PKG were increased,the expression of p-CaMK II was decreased(P<0.05).Co-administration with exendin9-39 partly diminished the effect of exendin-4(P<0.05).Conclusion:Exendin-4 exerts its protective effects on cardiac structural and electrical remodeling through modulating calcium handling via activation eNOS/cGMP/PKG signaling pathway and inactivation CaMKII signaling pathway. |
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