Alteration Of The Ventricular Electrophysiology By Allografting Mouse HCN4Transfected Canine Mesenchymal Stem Cells Into Conduction Blocked Heart

ObjectThe field of biological pacing is entering its second decade of active investigation. Theprincple was to employ both gene-based and cell-based methodologies and taken distinctapproaches to achieving automaticity pacemaking fuction in heart with bradycardiac-arrhythmia. Here we study the ability of mesenchymalstemcells(MSCs)as deliveryplatforms which have been loaded with genes of interest to reconstruct biologicalpacemaking function in animals. This study was conducted under the long-term research ofHCN4gene and MSCs in our department and combined with the advanced achievements ofbiological pacemaker nowadays. The following list are what we had explored in this study.1.To construct pacemaker-like cells by transfection of mouse HCN4gene into caninemesenchymal stem cells(cMSCs) with pysiological pacemaking function2.To assess the capability of the mHCN4modified cMSCs to reconstruct biologicalpacemaker fuction in vivo by transplanting them in dogs with experimentally inducedcomplete heart block and electronic pacing.3.To assess the effect of mHCN4modified cMSCs transplantation on cardiacelectrophysiology in dogs with experimentally induced complete heart block and electronicpacing.Methods1. We packaged lentivirus vector (Lenti6.3-IRES2-EGFP-mHCN4or Lenti6.3-IRES2-EGFP) with293T cells to get lentivirus supernatant.Then the2-3thpassages ofcMSCs were transduced by EGFP-mHCN4or EGFP. Expression of GFP was detected byfluorescence microscope and expression of HCN4was detected by immunofluorescentstaining.Patch clamp was performed to evaluate the electrophysiological properties of thepositively transfected cells. 2. Complete atrioventricular block(CAVB) was induced in adult dogs byradiofrequency ablation of His bundle with backup electronic pacemaker implantedsubcutaneously. After1week recovery,certain amount of cMSCs transfected withmHCN4(mHCN4-cMSCs) or not(EGFP-cMSCs) were injected subepicardially into the leftventricular anterior wall of these dogs. Overdrive pacing was performed at the injectionsite,followed by marking with suture.3. The ventricular automaticity was evaluated by24-hour animal Holter,six-leadsurface ECG,electronic pacemaker log record,HRV analysis, catecholamine responsivenesstest and physical exercise responsiveness.Expression of HCN4was detected by HEstaining,immunofluorescence and Western blot.4. Cardiac electrophysiology was measured in intact canines by epicardiallyprogrammed electrical stimulation (PES) in different sites(injection sites, right ventricularanterior wall,and left ventricular free wall near apex).The following listedelectrophysiological parameters was measured through PES: diastolic pacing threshold(DPT), ventricular effective refractory period (VERP), absolute difference ofVERP(ΔVERP), monophasic action potential duration(MAPD), absolute difference ofMAPD(ΔMAPD), ventricular fibrillation threshold (VFT). Then certain amount ofmHCN4-cMSCs or normal cMSCs or saline were injected subepicardially into the leftventricular anterior wall of the animals. The injection site was marked with suture,and theanimals were divided into HCN4group, control group and saline group as the differenttreatment.5. After1-week recovery, animals of control and HCN4groups were interventionedinto complete atrioventricular block (CAVB). ECG was recorded by24-hour ECGmonitoring throughout the following4-weeks study. Then PES was performed again tomeasure the ventricular electrophysiology. Histology study and immunochemistry methodswere used to evaluate the changes in gap junction properties between canines treated withthe different substance.Results1. The transfection efficiency of passage3-4cMSCs were about65%and theexpression of HCN4was confirmed by immunofluorescent staining.And the expression ofHCN4was quite strong after several rounds of generation. 2. By using the whole cell patch clamp we recorded a time-and voltage-dependentinward current which was fully activated when hyperpolarized at-160mV and completelydeactivated at+20mV in mHCN4-cMSCs.The current was suppressed by extracellular Cs+and increased by intracellular cAMP which is similar to Ifcurrent.3. At2weeks, the maximum heart rate and the number of impulses generated from theinjection sites was much higher in dogs injected with HCN4modified MSCs dogs than incontrol dogs. Basal heart rate increased in the HCN4group and became fully stabilized byWeek4, evidenced by markedly reduced numbers of electronic pacemaker beats. At Week2,HRV during exercise was significantly higher in HCN4dogs than in controls. HRV in thecontrol group remained at a low level throughout the observation period.4. HE staining,immunofluorescence stainning and western blot proved that cMSCssurvive and express HCN4protein in situ in heart of HCN4dog.5. PES results showed that the ventricular electrophysiological parameters includingDPT,VERP,ΔVERP,MAPD and ΔMAPD were all increased at4weeks after CHBinduced,but the VFT decreased. The parameters of dogs injected with mHCN4-EGFP-cMSCs were higher comparing to the dogs injected with EGFP-cMSCs. At the sametime,ΔVERP and ΔMAPD in dogs injected with cMSCs were significantly higher than theones injected with saline.Conclusion1.Mouse HCN4can be introduced into MSCs cultured in vitro. Pacemaker current hadbeen recorded from mHCN4-cMSCs and EGFP-cMSCs by patch clamp study.2.Transplantation of mHCN4modified cMSCs provided a stable biologicalpacemaking function that allowed an appropriate chronotropic response to physical exerciseand catecholamine for up to6weeks.3.Injection of cMSCs loaded with mHCN4into myocardium can achieve biologicalpacemaking function and alleviate alteration of ventricular electrophysiological propertiesin canine with CAVB.