The Effect And Mechanism Of Mesenchymal Stem Cells Overexpressing Hepatocyte Growth Factor On Ventricular Arrhythmias In A Swine Infarction Model

Backgroud:Ventricular arrhythmia(VA)is one of the clinical serious complications when suffering from myocardial infarction.Numerous studies have shown that sustained ventricular tachycardia(VT)and ventricular fibrillation(VF)resulted from ischemic heart diseases are the leading cause of sudden cardiac death(SCD).The heart undergoes a series of pathological changes,generating proarrhythmic substrates.These pathological processes include changes in anatomic,electrical and neural remodeling,which facilitate VAs formation.Past researches have come to a consensus that mesenchymal stem cells(MSCs)exhibit a potent capacity of cardiac repairmen.In addition,hepatocyte growth factor(HGF)has a potential for injury repairmen under ischemic conditions.To date,several data appear in favor of MSCs overexpressiong HGF(HGF-MSCs)for its repairment in cardiac ischemia and injured function.However,few data explain whether HGF-MSCs transplantation elevates risk of ventricular arrhythmias.Objective:To investigate the influence and potential mechanisms of HGF-MSCs transplantation on cardiac electrophysiological properties in swine with cardiac infarction.Methodology:1.Swine models establishment with cardiac infarction were based on an occulusion of distal left anterior descending artery(LAD).Balloon was fully inflated and lasted for 90 minutes after ischemia precondition for several times.Prior to model creation,two dimensional echocardiography(2D-UCG)was performed to obtain the heart baseline function.Swine models were randomly divided into three groups:HGF-MSCs group(n=9),PBS group(n=10)and MSCs group(n=9).2.Four weeks after infarction,cell delivery was conducted through thoracotomy under both respiratory and circulatory support.2D-UCG was performed again before open-chest operation.Intramyocardial injection of 2ml HGF-MSCs(5×107),MSCs(5×107)or PBS was given under direct vision.Close the thoraxes layer-by-layer.Pigs were sent to the designated farm for care.3.At additional four weeks after open-chest operation,data were collected in the following order:1)Using 2D-UCG to determine left ventricular ejection fraction(LVEF),evaluating a successful model establishment.2)Using 2-hour dynamic electrocardiography(2-h dynamic ECG)to assess heart rate variability(HRV).3)Using programmed electrical stimulation(PES)to evaluate the risk of VAs.4.Lab analysis after sacrifice:Using Masson to observe fibrosis;Using TTC staining to validate a successful model establishment;Detecting HGF expression in myocardium;Observing the expression of Cx43,tyrosine hydroxylase(TH),growth associated protein 43(GAP43)and acetylcholinesterase(AChE)and their morphological distributions;Determining apoptosis-associated protein levels(Bcl-2 and Bax);Using TUNEL to explore cardiac apoptosis.Result:1.90-minute percutaneous coronary artery occlusion resulted in a successful swine infarction model.TTC staining clearly identified the infarction and non-infarction.Masson staining showed a obvious reduction in fibrosis in the HGF-MSCs group than the MSCs group and the PBS group.In the meantime,fibrosis in HGF-MSCs group appeared less than that in MSCs group.2.2D-UCG showed a substantial decrease in LVEF before open-chest operation compared with the baseline heart function(HGF-MSCs group 50.68±3.06 vs.65.61 ±3.29;PBS group 49.92±3.09 vs.66.68±2,33;MSCs group 50.94±1.85 vs.66.23±2.90).Four weeks later after cell treatment,elevations in LVEF of HGF-MSCs group and MSCs group occurred(HGF-MSCs group 60.27±3.07 vs.50.68±3.06;P<0.05;MSCs group59.44±3.66 vs.50.94±1.85,P<0.05),but PBS group showed no significant LVEF changes(49.63±2.83 vs.49.92±3.09).3.Double immunofluorescence staining for both EdU and vWF revealed a four-week survival and vessel differentiation of HGF-MSCs in vivo,however,no cardiomyogenic differentiation was evidenced.4.HGF-MSCs group showed lower vessel density than the other two groups(GF-MSCs group vs.PBS group,25.22±2.91 vs.8.00±2.36;P<0.01;HGF-MSCs group vs MSCs group,25.22±2.91 vs.14.78±2.28,P<0.01).5.HGF-MSCs group showed reduced Bax levels and apoptosis indexes,along with increased Bcl-2 levels than the MSCs group and PBS group(P<0.05).6.Nerve immunostaining and Western Blot indicated that,TH positive density was significantly declined in the HGF-MSCs group and MSCs group respectively compared with the PBS group.However,pigs in the HGF-MSCs-treated group showed lower TH density at IBZ than those in the MSCs-treated group.GAP43 signals shared the same morphological characteristics.But AChE-positive profile didn't present any distinctions among groups.7.Cx43 remodeling,including shrinkage in size and lateralization,took palce in the PBS group.Such remodeling were both ameliorated in HGF-MSCs group and MSCs group,appearing a tendency to normal distribution pattern.Noticeably,improvement in HGF-MSCs group was more substantial than that in MSCs group.8.The HGF-MSCs group showed a substantial enhancement in rMSSD and pNN50,along with a considerabe decrease in LF and L/H ratio,compared with the MSCs group and the PBS group.However,such effects were significantly less between the MSCs group and the PBS control.9.While the arrhythmia scoring for evoked VAs in PBS-treated pigs was very high,this score was significantly reduced in the HGF-MSCs group and MSCs group(HGF-MSCs group 1.11±1.17 vs.PBS group 4.90±1.37,P<0.01;MSCs group 2.56±1.13 vs.PBS group 4.90±1.37,P<0.01)Conclusion:1.90-minute distal LAD occlusion is an effective method to establish a swine infarction model.2.Intramyocardially injected HGF-MSCs can survive for four weeks in vivo and has the potential for vessel differentiation.3.The delivered HGF-MSCs substantially promoted neovascularization at the infracted border zone(IBZ),ameliorating the local ischemia and hypoxia.4.The delivered HGF-MSCs exerted an anti-apoptotic effect and significantly suppressed the cardiac apoptosis at the IBZ,which may be one of the underlying mechanisms of proarrhythmic substrates improvement.5.The injected HGF-MSCs can significantly ameliorate sympathetic sprouting and re-innervation,decrease the sympathetic tone while elevating the para-sympathetic tone,and downregulate the susceptibility for VAs.This may be a pivotal mechanism how HGF-MSCs delivery can improve the cardiac arrhythmogenic substrates.6.HGF-MSCs engraftment can effectively ameliorate the gap junction remodeling,improve electrical coupling,thus present an anti-VAs effect.7.HGF-MSCs engraftment can reduce the induced VAs,decrease arrhythmia scoring,thus downregulate the risk of VAs.