转染HCN4基因的犬骨髓间充质干细胞同种异体移植对传导阻滞心脏电生理变化的影响
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摘要
背景与目的:
生物起搏(Biological Pacemaker)是利用细胞技术和基因技术，将目的基因(起搏基因或可促进心脏自主节律的基因)直接导入心脏或者利用各种起搏/非起搏细胞将起搏电流传递至疾病的心脏，使其能在局部发挥其接近生理状态的起搏或提高心率的作用。本研究拟通过基因转染技术和细胞治疗技术，利用骨髓间充质干细胞(MSCs)作为传递起搏电流的细胞平台，试图在动物体内“构建”生物心脏起搏器，以探索治疗缓慢型心律失常疾病的新方法。本研究是在本课题组前期研究的基础上，结合国内外这一领域研究的最新成果，就小鼠HCN4基因(mHCN4)转染犬骨髓间充质干细胞(cMSCs)同种异体移植对完全性房室传导阻滞(CAVB)心脏电生理改变的影响进行探索。包括以下三方面内容：(1)利用携带mHCN4基因的慢病毒载体(Lentiviral-mHCN4),将mHCN4基因转染到cMSCs，体外构建可表达功能性起搏通道的起搏样干细胞(Lv-mHCN4-cMSCs)；(2)将构建好的起搏样干细胞(mHCN4-cMSCs)，同种异体移植到已植入电子心脏起搏器的CAVB模型犬心室肌内，通过监测全天心律、运动变时性研究、神经递质反应性研究、组织学和分子生物学等，评价细胞移植对传导阻滞心脏心室自律性的影响；(3)将mHCN4-cMSCs同种异体移植到CAVB模型犬心室肌内，观察其对传导阻滞心脏的致心律失常相关电生理参数的改变有无影响，以探索和评价利用mHCN4-cMSCs心室移植增强传导阻滞心脏生物起搏功能的安全性和临床应用可行性。
方法:
1.利用构建好的Lentiviral-EGFP-mHCN4慢病毒载体进行293T细胞病毒包装，将mHCN4基因转染到第2-3代cMSCs，得到Lentiviral-EGFP-mHCN4-cMSCs(HCN4组细胞，以下简称mHCN4-cMSCs)；对照组cMSCs只转染Lentiviral-EGFP，得到EGFP-cMSCs。待荧光显微镜观察到两组cMSCs稳定表达HCN4通道蛋白和绿色荧光蛋白(GFP)后，利用全细胞膜片钳技术对两组转染阳性的cMSCs进行通道电生理学研究。
2.取成年犬射频消融希氏束建立完全性房室传导阻滞(CAVB)动物模型,并在颈部皮下植入电子起搏器。1周后对模型犬进行开胸手术，将mHCN4-cMSCs (HCN4组)或EGFP-cMSCs(对照组)悬液斜行注入左心室前壁(第一对角支和第二对角支之间区域)处心外膜下,并在该处心外膜进行超速起搏，做好标记。
3.术后对各组模型犬的心室自律性和移植部位组织目的基因蛋白表达进行综合检测和分析。主要采用体表心电图QRS波形分析、24小时动态心电图心律变化记录、起搏器活动日志记录分析、其对于神经体液调节和体能运动的反应能力评估心室自律性变化；用组织病理学HE染色、免疫组织荧光染色技术和Western Blot分子生物学技术等多项技术方法对细胞移植后体内存活和目的基因蛋白表达进行检测。
4.另取健康成年犬开胸，于心外膜直视下用程序电刺激(PES)的方法测定其各局部(左室前壁拟注射细胞部位、右室前壁、左室游离壁下部)心脏电生理基础参数，包括：舒张期起搏阈值(DPT)、心室有效不应期(VERP)、心室有效不应期差值(ΔVERP)、单项动作电位时程(MAPD)、单项动作电位时程差值(ΔMAPD)、室颤阈值(VFT)。测定之后做好注射部位标记，在标记处附近注射mHCN4-cMSCs(HCN4组)、EGFP-cMSCs(cMSCs对照组)和生理盐水(盐水组)，然后关胸。
5.1周后对HCN4组和细胞对照组犬制备CAVB模型，盐水对照组不进行CAVB制作，术后每96小时检查24小时动态心电图。4周后各组再次开胸行PES测定心脏电生理参数，处死动物，取材利用组织病理学HE染色、免疫组织化学染色、western blot技术检测移植部位细胞存活和HCN4蛋白表达及移植部位边缘心室肌缝隙连接蛋白43(CX43)表达情况。
结果:
1.慢病毒载体Lentiviral-EGFP-mHCN4经包装后转染第3-4代犬骨髓间充质干细胞的转染阳性率约为65%，经免疫荧光染色技术测定其膜表面可表达HCN4蛋白，经多次传代后细胞生长状态依然良好，仍可表达HCN4蛋白。
2.利用全细胞膜片钳技术对转染阳性的细胞进行电生理研究,能够记录到类似If特性的细胞膜通道离子电流。该电流具备超极化钳制电压激活和时间依赖的特点，对细胞内cAMP反应良好，符合起搏电流If的典型特征。而EGFP对照组细胞(转染lentiviral-EGFP)检测不出类似特性的电流。
3.将mHCN4-cMSCs(HCN4组)和EGFP-cMSCs(对照组)移植到已植入电子心脏起搏器的CAVB模型犬的左心室心外膜下。在移植后第2周内，24小时动态心电图及每周体表心电图检查提示HCN4组8只犬中有6只出现起源于左室的自主心律，占总心搏数的比例高于60%；并且在第2周末时犬运动时最大心率达到最高103±12bpm(对照组为50±3bpm)，同时休息时的静息心率和平均心率也随着时间逐渐增加，到第4周时趋于稳定(基础心率55±6bpm，平均心率59±5bpm)。且明显高于对照组对应心率指标(基础心率27±2bpm，平均心率38±2bpm)。HCN4组电子起搏率从第2周开始下降，到第4周时下降至6±2%，而对照组则为57±7%。通过神经递质反应性测试证实，这一异位起搏心律对阿托品无明显反应，但对肾上腺素反应良好，同时对生理性体能运动也有良好的变时性。
4.用组织病理学、免疫荧光化学染色和分子生物学技术检测细胞移植部位的心室肌标本,证实移植的MSCs细胞能够在移植部位存活至少6周,局部未发现明显的细胞凋亡坏死和免疫排斥反应。免疫荧光组织化学研究证实异体移植的MSCs细胞能够在移植部位有效表达HCN4通道蛋白，且其表达量明显超过注射未转染基因的对照组。
5.心脏电生理参数测定结果显示犬在模型制备前各组间参数无显著差异；细胞移植+CAVB模型后4周细胞对照组QT、QTc、DPT、VERP、MAPD显著高于盐水对照组(P＜0.01)和HCN4组(P＜0.05)，ΔVERP、ΔMAPD显著高于盐水对照组(P＜0.01)，VFT低于盐水对照组(P＜0.01)和HCN4组(P＜0.05)；HCN4组ΔVERP、ΔMAPD显著高于细胞对照组(P＜0.01)，VFT高于细胞对照组(P＜0.05)。
结论:
1．利用慢病毒载体可在体外稳定转染mHCN4基因至犬骨髓间充质干细胞。转染阳性的细胞可稳定表达HCN4通道蛋白。全细胞膜片钳技术对转染阳性细胞进行膜通道电流检测，证实其在体外培养环境下可表达对cAMP反应敏感的起搏样电流IHCN4，因此我们把这种细胞称为起搏样干细胞mHCN4-cMSCs。
2．将mHCN4-cMSCs同种异体移植到完全性房室传导阻滞犬心室肌后，能在机体内存活达6周，且在体内继续表达HCN4蛋白，并可有效提高移植部位的室性自主节律，减少电子起搏器依赖，并且这种生物起搏心律具有良好的变时性和神经递质反应性。
3．健康犬在心脏传导阻滞后其心室电生理特性明显改变，各致心律失常敏感性指标均高于健康状态；在心室心外膜下移植mHCN4-cMSCs可部分减轻传导阻滞后心室电生理特性的改变，并且移植部位边缘心室肌缝隙连接蛋白的表达高于移植普通cMSCs的对照组和注射生理盐水的空白组。
4.通过转基因技术和骨髓间充质干细胞移植相结合，能够传递起搏电流至心脏，提高完全性房室传导阻滞后的心室率，且一定程度上可以减轻心动过缓心脏的电活动不稳定性。
Object
The 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 function
2.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.
Methods
1. 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.
Results
1. 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.
Conclusion
1.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.
生物起搏(Biological Pacemaker)是利用细胞技术和基因技术，将目的基因(起搏基因或可促进心脏自主节律的基因)直接导入心脏或者利用各种起搏/非起搏细胞将起搏电流传递至疾病的心脏，使其能在局部发挥其接近生理状态的起搏或提高心率的作用。本研究拟通过基因转染技术和细胞治疗技术，利用骨髓间充质干细胞(MSCs)作为传递起搏电流的细胞平台，试图在动物体内“构建”生物心脏起搏器，以探索治疗缓慢型心律失常疾病的新方法。本研究是在本课题组前期研究的基础上，结合国内外这一领域研究的最新成果，就小鼠HCN4基因(mHCN4)转染犬骨髓间充质干细胞(cMSCs)同种异体移植对完全性房室传导阻滞(CAVB)心脏电生理改变的影响进行探索。包括以下三方面内容：(1)利用携带mHCN4基因的慢病毒载体(Lentiviral-mHCN4),将mHCN4基因转染到cMSCs，体外构建可表达功能性起搏通道的起搏样干细胞(Lv-mHCN4-cMSCs)；(2)将构建好的起搏样干细胞(mHCN4-cMSCs)，同种异体移植到已植入电子心脏起搏器的CAVB模型犬心室肌内，通过监测全天心律、运动变时性研究、神经递质反应性研究、组织学和分子生物学等，评价细胞移植对传导阻滞心脏心室自律性的影响；(3)将mHCN4-cMSCs同种异体移植到CAVB模型犬心室肌内，观察其对传导阻滞心脏的致心律失常相关电生理参数的改变有无影响，以探索和评价利用mHCN4-cMSCs心室移植增强传导阻滞心脏生物起搏功能的安全性和临床应用可行性。
方法:
1.利用构建好的Lentiviral-EGFP-mHCN4慢病毒载体进行293T细胞病毒包装，将mHCN4基因转染到第2-3代cMSCs，得到Lentiviral-EGFP-mHCN4-cMSCs(HCN4组细胞，以下简称mHCN4-cMSCs)；对照组cMSCs只转染Lentiviral-EGFP，得到EGFP-cMSCs。待荧光显微镜观察到两组cMSCs稳定表达HCN4通道蛋白和绿色荧光蛋白(GFP)后，利用全细胞膜片钳技术对两组转染阳性的cMSCs进行通道电生理学研究。
2.取成年犬射频消融希氏束建立完全性房室传导阻滞(CAVB)动物模型,并在颈部皮下植入电子起搏器。1周后对模型犬进行开胸手术，将mHCN4-cMSCs (HCN4组)或EGFP-cMSCs(对照组)悬液斜行注入左心室前壁(第一对角支和第二对角支之间区域)处心外膜下,并在该处心外膜进行超速起搏，做好标记。
3.术后对各组模型犬的心室自律性和移植部位组织目的基因蛋白表达进行综合检测和分析。主要采用体表心电图QRS波形分析、24小时动态心电图心律变化记录、起搏器活动日志记录分析、其对于神经体液调节和体能运动的反应能力评估心室自律性变化；用组织病理学HE染色、免疫组织荧光染色技术和Western Blot分子生物学技术等多项技术方法对细胞移植后体内存活和目的基因蛋白表达进行检测。
4.另取健康成年犬开胸，于心外膜直视下用程序电刺激(PES)的方法测定其各局部(左室前壁拟注射细胞部位、右室前壁、左室游离壁下部)心脏电生理基础参数，包括：舒张期起搏阈值(DPT)、心室有效不应期(VERP)、心室有效不应期差值(ΔVERP)、单项动作电位时程(MAPD)、单项动作电位时程差值(ΔMAPD)、室颤阈值(VFT)。测定之后做好注射部位标记，在标记处附近注射mHCN4-cMSCs(HCN4组)、EGFP-cMSCs(cMSCs对照组)和生理盐水(盐水组)，然后关胸。
5.1周后对HCN4组和细胞对照组犬制备CAVB模型，盐水对照组不进行CAVB制作，术后每96小时检查24小时动态心电图。4周后各组再次开胸行PES测定心脏电生理参数，处死动物，取材利用组织病理学HE染色、免疫组织化学染色、western blot技术检测移植部位细胞存活和HCN4蛋白表达及移植部位边缘心室肌缝隙连接蛋白43(CX43)表达情况。
结果:
1.慢病毒载体Lentiviral-EGFP-mHCN4经包装后转染第3-4代犬骨髓间充质干细胞的转染阳性率约为65%，经免疫荧光染色技术测定其膜表面可表达HCN4蛋白，经多次传代后细胞生长状态依然良好，仍可表达HCN4蛋白。
2.利用全细胞膜片钳技术对转染阳性的细胞进行电生理研究,能够记录到类似If特性的细胞膜通道离子电流。该电流具备超极化钳制电压激活和时间依赖的特点，对细胞内cAMP反应良好，符合起搏电流If的典型特征。而EGFP对照组细胞(转染lentiviral-EGFP)检测不出类似特性的电流。
3.将mHCN4-cMSCs(HCN4组)和EGFP-cMSCs(对照组)移植到已植入电子心脏起搏器的CAVB模型犬的左心室心外膜下。在移植后第2周内，24小时动态心电图及每周体表心电图检查提示HCN4组8只犬中有6只出现起源于左室的自主心律，占总心搏数的比例高于60%；并且在第2周末时犬运动时最大心率达到最高103±12bpm(对照组为50±3bpm)，同时休息时的静息心率和平均心率也随着时间逐渐增加，到第4周时趋于稳定(基础心率55±6bpm，平均心率59±5bpm)。且明显高于对照组对应心率指标(基础心率27±2bpm，平均心率38±2bpm)。HCN4组电子起搏率从第2周开始下降，到第4周时下降至6±2%，而对照组则为57±7%。通过神经递质反应性测试证实，这一异位起搏心律对阿托品无明显反应，但对肾上腺素反应良好，同时对生理性体能运动也有良好的变时性。
4.用组织病理学、免疫荧光化学染色和分子生物学技术检测细胞移植部位的心室肌标本,证实移植的MSCs细胞能够在移植部位存活至少6周,局部未发现明显的细胞凋亡坏死和免疫排斥反应。免疫荧光组织化学研究证实异体移植的MSCs细胞能够在移植部位有效表达HCN4通道蛋白，且其表达量明显超过注射未转染基因的对照组。
5.心脏电生理参数测定结果显示犬在模型制备前各组间参数无显著差异；细胞移植+CAVB模型后4周细胞对照组QT、QTc、DPT、VERP、MAPD显著高于盐水对照组(P＜0.01)和HCN4组(P＜0.05)，ΔVERP、ΔMAPD显著高于盐水对照组(P＜0.01)，VFT低于盐水对照组(P＜0.01)和HCN4组(P＜0.05)；HCN4组ΔVERP、ΔMAPD显著高于细胞对照组(P＜0.01)，VFT高于细胞对照组(P＜0.05)。
结论:
1．利用慢病毒载体可在体外稳定转染mHCN4基因至犬骨髓间充质干细胞。转染阳性的细胞可稳定表达HCN4通道蛋白。全细胞膜片钳技术对转染阳性细胞进行膜通道电流检测，证实其在体外培养环境下可表达对cAMP反应敏感的起搏样电流IHCN4，因此我们把这种细胞称为起搏样干细胞mHCN4-cMSCs。
2．将mHCN4-cMSCs同种异体移植到完全性房室传导阻滞犬心室肌后，能在机体内存活达6周，且在体内继续表达HCN4蛋白，并可有效提高移植部位的室性自主节律，减少电子起搏器依赖，并且这种生物起搏心律具有良好的变时性和神经递质反应性。
3．健康犬在心脏传导阻滞后其心室电生理特性明显改变，各致心律失常敏感性指标均高于健康状态；在心室心外膜下移植mHCN4-cMSCs可部分减轻传导阻滞后心室电生理特性的改变，并且移植部位边缘心室肌缝隙连接蛋白的表达高于移植普通cMSCs的对照组和注射生理盐水的空白组。
4.通过转基因技术和骨髓间充质干细胞移植相结合，能够传递起搏电流至心脏，提高完全性房室传导阻滞后的心室率，且一定程度上可以减轻心动过缓心脏的电活动不稳定性。
Object
The 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 function
2.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.
Methods
1. 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.
Results
1. 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.
Conclusion
1.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.
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