The Effect And Underlying Mechanism Of Targeted Ablation Of Cardiac Sympathetic Neurons On Cardiac Remodeling In A Canine Model Of Myocardial Infarction

BackgroundSudden cardiac death(SCD)is still one of the major causes of human life and health,and most patients suffering SCD die directly from malignant ventricular arrhythmia after myocardial infarction(MI).In recent years,a large number of studies have shown that the increase of cardiac sympathetic nerves tone is the main cause of malignant arrhythmia and SCD in MI patients.Moreover,the inhibition of sympathetic nerve function can significantly reduce the occurrence of ventricular arrhythmias.In addition,autonomic nervous system dysfunction is the main characteristic of patients with heart failure,which is manifested as increased sympathetic tone,decreased vagal activity and excessive activation of neuroendocrine system.Sympathetic hyperactivation plays a vital role in adverse cardiac remodeling and HF progression.Cardiac sympathetic resection and blockade of stellate ganglion have been proved to be effective methods to prevent ventricular arrhythmias and cardiac dysfunction in HF patients.Beta blockers are the most widely used drugs for inhibiting sympathetic activity,also the main drugs to suppress the post-mi ventricular arrhythmias and improve the prognosis of patients with HF.However,in addition to the long-term use,some patients have contraindications or are difficult to tolerate.In addition,some new autonomic neuromodulatory therapies have emerged in recent years,such as vagus nerve stimulation,spinal nerve stimulation,aortic sinus baroreceptor stimulation.Although basic studies have shown that they are effective in preventing ischemia-induced ventricular arrhythmias,the results of clinical trials results are not satisfactory.The CTB-SAP used in this study is a drug which could ablate cardiac sympathetic neurons.Saporin(SAP)is a ribosomal inactivating protein that bindsto and inactivates ribosomes,disabling the cell’s protein synthetic machinery and causing the cell to die over a period of hours to days through apoptotic mechanisms.SAP can be linked to retrogradely transported molecules like cholera toxin B subunit(CTB)that binds to specific membrane components that are differentially expressed on nerve cells.Specifically,SAP conjugated to CTB(CTB-SAP),which binds to GM1 gangliosides located in the plasma membranes of sympathetic preganglionic neurons(SPNs),could be used to selectively ablate SPNs,and thereby reduce the cell number of post-ganglionic neurons of stellate ganglion by injecting CTB-SAP in the stellate ganglion.Therefore,the purpose of this study was to investigate the effects of targeted ablation of cardiac sympathetic neurons on post-infarcted ventricular electrical remodeling,and to determine its underlying mechanism,in order to provide a new treatment strategy for post-infarcted ventricular arrhythmias and adverse cardiac remodeling and HF progression.Part I:Targeted ablation of cardiac sympathetic neurons improves ventricular electrical remodeling in a canine model of chronic myocardial infarctionObjective:.The purpose of this study was to evaluate the cardiac electrophysiologic effects of targeted ablation of cardiac sympathetic neurons(TACSN)and to determine its underlying mechanism in a canine model of chronic MI.Methods:Thirty-eight anesthetized dogs were randomly assigned into the sham-operated(sham),MI and MI-TACSN groups,respectively.The MI model was prepared by intravascular injection of gelatin sponge particles to left anterior descending(LAD)coronary artery.After 1 week of the first surgery,TACSN was induced by injecting cholera toxin B subunit-saporin compound in the left stellate ganglion(LSG).Five weeks after surgery,heart rate variability(HRV)was determined by analyzed standard diviation of normal-to-normal intervals(SDNN)and spectral power,including LF(nu),HF(nu)and LF/HF,on a 5-minute ECG recording.Neural electrical signals of LSG were recorded using a Powerlab data acquisition system,and Labchart 8 software was applied to analyze the LSG neural activity,characterized by amplitude and frequency.LSG function was evaluated by the maximal changes of systolic blood pressure in response to high frequency(20Hz,0.1ms pulse duration)stimulation to the LSG.Programmed stimulation was delivered to measure the effective refractory period(ERP)of infarct border zone,remote area,left ventricular base(LVB),right ventricular apex(RVA),right ventricular base(RVB)and median area between RVA and RVB(RVM).A custom-made Ag-AgCl catheter was applied at these six zones to record the epicardial monophasic action potential(MAP)to determine APD90.A dynamic pacing method was performed to obtain the MAP and APD altemans cycle length,and Origin 9 software was used to construct APD restitution curves by plotting APD90 versus diastolic interval(DI).Burst stimulation was supplied to infarct border zone to measure ventricular fibrillation threshold(VFT).Cardiac sympathetic innervation was determined with immunofluorescence staining of growth associated protein-43(GAP43)and tyrosine hydroxylase(TH).Enzyme-linked immunosorbent assay kits were used to measure serum NE,BNP)and NGF levels.The neuronal number of LSG was determined by using Cresyl violet staining.Results:Compared with sham group,SDNN and HF(nu)were decreased,whereas LF(nu)and LF/HF were significantly increased in the MI group.however,TACSN significantly resulted in a significant reduction in LF(nu)and LF/HF and increase in SDNN and HF(nu)in comparison with MI group.Compared with sham group,LSG neural activity was significantly heightened in the MI group,characterized by increased frequency and amplitude of LSG discharge,and maximal systolic BP changes were also increased during stimulation to LSG at 20-70 volts in MI group.Compared with MI group,TACSN significantly induced a significant decrease in LSG neural activity and maximal systolic BP changes at the same stimulation voltage.Moreover,compared with MI group,TACSN significantly prolonged corrected QTc interval,decreased Tpeak-Tend interval,prolonged ventricular ERP and APD90 at all six zones,decreased ERP dispersion and APD90 dispersion,decreased the max slope(Smax)of APD restitution curves at infarct border zone,decreased the sptial Smax dispersion,suppressed the APD alternans,increased ventricular fibrillation threshold.In addition,compared with MI group,TACSN significantly reduced serum NE,NGF and BNP levels,reduced the counts of LSG neurons,and decreased the densities of GAP43 and TH-positive nerve fibers in the infarcted border zone.Conclusion:TACSN attenuates sympathetic remodeling and improves ventricular electrical remodeling in the chronic phase of MI.These data suggest that TACSN may be a novel approach to treating ventricular arrhythmias.Part II:Targeted ablation of cardiac sympathetic neurons attenuates adverse ventricular structure remodeling in a canine model of chronic myocardial infarctionObjective:The aim of this study was to investigate whether TACSN could suppress myocardial infarction MI-induced adverse cardiac structure remodeling and heart failure progression,and determine its underlying mechanism.Methods:38 dogs were randomly assigned into the sham-operated(n=8),MI(n=15)and MI-TACSN(n=15)groups,respectively.The MI model was prepared by intravascular injection of gelatin sponge particles to left anterior descending coronary artery.TACSN was induced by injecting cholera toxin B subunit-saporin compound in the stellate ganglia after 1 week of MI.5 weeks after MI induction,Transthoracic echocardiography was performed to determine LVEDD,LVESD,LVESV,LVESV,LVEF,LVFS.Left ventricular hemodynamic parameters,including LVEDP,LVESP and max dp/dt,were measured by inserting an angiographic catheter into left ventricle.Heart sections of infarct border zone were stained with hematoxylin-eosin(HE)and picrosirius red(PSR)to determine the cross-sectional areas of myocytes and the extent of myocardial collagen deposition.Sympathetic innervation of infarcted border zone was assessed by immunofluorescence staining of tyrosine hydroxylase(TH)and growth associated protein-43(GAP43).The neuronal number of LSG was determined by using Cresyl violet staining.Quantitative real-time PCR(qT-PCR)analyses were applied to determine the mRNA expression of myocardial fibrosis markers,including collagen Ⅰ,collagen Ⅲ,transform growth factor-β(TGF-β)and connective tissue growth factor(CTGF).Western blotting analyses were applied to determine the protein expression levels of collagen Ⅰ,collagen Ⅲ,TGF-β,CTGF and β1-adrenergic receptor(β1AR).Enzyme-linked immunosorbent assay kits were used to measure serum and tissue NE,NGF,ANP,BNP,Ang Ⅱ,hs-CRP and ALD levels.Results:Ccompared with MI group,LVEF,LVFS,LVESP and maximal dp/dt of left ventricle were significantly increased,while LVESD,LVESV and LVEDP were significantly decreaded in the MI-TACSN group.Moreover,compared with MI group,TACSN dramatically decreased the cardiomyocyte section area and collagen deposition volume,reduce the mRNA and protein expression levels of Collagen I,Collagen Ⅲ,TGF-β and CTGF at infarct border zone.All these findings indicated that TACSN could improve cardiac post-infarcted structure remodeling.Furthermore,TACSN alleviated the growth associated protein-43 and tyrosine hydroxylase positive nerve densities,restored the protein expression of β1-adrenergic receptor of left ventricular myocardium at infarct border zone,and reduced the number of neurons of LSG.In addition,compared with sham group,MI group exhibited a significantly increase in serum and cardiac tissue NE,NGF,Ang II,ALD,ANP,BNP,hs-CRP levels,however,these biomarkers levels were dramatically decreased to nearly normal levels in the MI-TACSN group,which indicated that TACSN might significantly attenuated sympathetic tone and renin angiotensin aldosterone system(RAAS)activity,and improve cardiac function.Conclusion:These findings indicate that TACNS may have a beneficial effect on adverse post-infarction remodeling and heart failure progression,which may be at least in part due to the attenuation of both sympathetic remodeling and cardiac neuroendocrine system.