膜片钳原位检测HCN4基因修饰的MSCs移植至大鼠心脏后的细胞电生理变化
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摘要
背景与目的电子心脏起搏器是目前临床上有效治疗缓慢性心律失常的常规方法，但存在费用昂贵、电池寿命有限、起搏器缺乏对神经激素的自动反应性等不足，且可能出现出血、感染等并发症。近年来，随着对起搏细胞离子通道基因的了解进一步深入，利用生物学及其相关技术，对受损的心脏起搏点进行修复或替代，重建心脏“生物起搏点”，使心脏的起搏和传导功能得以恢复，已成为当前心脏电生理学界研究的热点。
起搏电流(current funny，If)是窦房结细胞舒张期自动除极化的主要决定因素，并在心率控制及神经递质对心率的调节中起重要作用；超极化激活的环化核苷酸门控(hyperpolarization-activated cyclic nucleotide-gated, HCN)通道基因家族编码的HCN通道则是If形成的分子基础。目前已有利用HCN基因修饰的间充质干细胞(Mesenchymalstem cells, MSCs)移植进入房室传导阻滞的动物模型中成功创建生物起搏点的报道，结果令人鼓舞，但有一些关键的问题仍亟待解决。首先，在体研究发现生物起搏的频率低下(约40~50bpm，也有低至30bpm的情况)，显著低于与体外心肌细胞共培养时的起搏频率，是否起搏电流的特性发生了改变？具体原因尚不清楚；其次，MSCs具有多向分化的潜能，其移植到宿主心脏后，仅作为一个载体工具，还是分化为具备功能的心肌样细胞发挥起搏作用？以上问题对于移植细胞能否在体内稳定、持久的发挥起搏功能至关重要。
然而，由于缺乏对移植细胞直接进行原位功能检测的手段，目前多数研究主要是通过免疫组织化学检测移植细胞的表型变化，以及通过宏观的心电图、三维电解剖标测系统(CARTO)等无创检查对移植细胞的起搏功能进行评估。但移植细胞表型的改变并不等同于具备了心肌细胞样的功能；无创检查的空间分辨力有限，不能对“体内干细胞和心肌细胞如何交互作用影响心脏功能”进行精确描述。因此，目前的研究尚不能真正阐明移植细胞在宿主心脏中发挥起搏功能及起搏功能发生改变的机制。
众所周知，起搏电流(funny current, If)是起搏细胞发挥功能的基础，而膜片钳技术是检测细胞膜离子通道电流的最根本、有效的方法。利用膜片钳技术针对移植细胞进行原位的电生理学研究，将有助于阐明起搏细胞的电生理学特性及其与宿主心肌细胞交互作用的机制。对移植细胞进行原位膜片钳检测需要在组织切片上进行，但目前尚未发现利用成年动物心肌组织切片膜片钳技术原位检测移植干细胞电生理特性及其与宿主心肌细胞交互作用功能的报道。本实验拟利用组织片膜片钳技术，对同种异体移植到宿主心脏中的mHCN4基因修饰的大鼠MSCs(HCN4-MSCs)进行原位电生理学研究。
方法
1.取健康SD大鼠骨髓，经密度梯度离心和贴壁培养获得MSCs，在体外扩增、纯化。
2.构建mHCN4的表达载体pLenti6.3-mHCN4-IRES2-EGFP(LV-HCN4-EGFP)及空载体pLenti6.3-IRES2-EGFP(LV-EGFP)，分别转染第三代的MSCs，5~7天后利用免疫组化方法及膜片钳方法检测起搏离子通道蛋白及电流的表达。
3.将MSCs同种移植到SD大鼠心脏中创建细胞移植模型，在此基础上建立适合于膜片钳检测的心肌组织切片方法。
4.将SD大鼠分为两组(每组各10只)：1)实验组移植转染mHCN4的MSCs(HCN4-MSCs)；2)体内对照组移植仅转染EGFP的MSCs。各组大鼠在细胞移植4周后处死，取心脏进行切片，对移植细胞进行原位的膜片钳检测。同时，另设两个组作为体外对照：1)HCN4-MSCs体外培养1周组，2)HCN4-MSCs体外培养4周组。在本研究中，上述4组分别被定义如下：实验组，体内对照组，体外对照组(1)及体外对照组(2)。利用膜片钳技术对各组细胞的起搏电流(If)进行检测，并对各组细胞记录到的If电流特性进行比较和分析。并检测实验组及体内对照组移植细胞的动作电位、钠电流及钙电流。
5.在全细胞膜片钳封接的基础上，结合荧光黄染料转运的实验方法，检测移植细胞与宿主心肌细胞之间的缝隙连接通讯功能。
6.膜片钳检测结束后，将心肌组织用4%多聚甲醛常规固定、石蜡包埋、切片，利用免疫荧光方法检测移植细胞HCN4及EGFP蛋白的表达；利用免疫组化的方法检测缝隙连接蛋白43(connexin43, CX43)的表达及分布，以及移植区域IgG的表达及CD3~+T细胞的浸润。
结果
1.本实验分离纯化的MSCs高表达CD29和CD44抗原(高达99%以上)，而CD34和CD45抗原表达与同型对照没有显著差异；并且在特定的诱导条件下，MSCs可以分化为心肌样细胞、成骨细胞及脂肪细胞，证实其多向分化潜能。以上特性符合目前公认的MSCs标准。
2.在MOI=10的条件下，慢病毒对MSCs的转染效率可以达到67.0±6.6%(n=5)，MSCs形态及活性未受明显影响。转染“pLenti6.3-mHCN4-IRES2-EGFP”的MSCs同时表达EGFP及mHCN4蛋白，并记录到可被4mM CsCl可逆性阻断的超极化激活的内向电流，证实为起搏电流If；而转染“pLenti6.3-IRES2-EGFP”的MSCs仅表达EGFP，未记录到起搏电流If。
3.通过改良的心肌组织切片方法，获得了活性良好、背景荧光低的心肌组织切片。在切片中，心肌细胞结构完整、横纹清晰可见；移植的MSCs呈簇状分布在心肌细胞的缝隙之间，细胞呈类圆形、短梭形，形态完整、表面光滑，与心肌细胞紧密连接。在荧光条件下，心肌组织自发荧光背景较低，可以观察到EGFP标记的MSCs。通过全细胞膜片钳方式，分别记录到了组织片中心肌细胞及移植的MSCs细胞的膜电流，证实细胞活性良好，完全能够满足针对移植MSCs进行原位膜片钳检测的需要。
4.实验组移植的HCN4-MSCs在宿主心脏中能存活4周以上。实验组与体外对照组(1)及体外对照组(2)的HCN4-MSCs表达超极化激活的起搏电流(If)。体内对照组的MSCs不表达If。
(1)实验组与体外对照组(1)及体外对照组(2)相比，三组间If电流幅值无显著差异[分别为-1007.4±132.3pA (n=14)、-1012.5±187.3pA (n=16)及-1025.5±200.9pA (n=15)，P>0.05]。三组间细胞膜电容比较有显著性差异(P值均<0.05)，其中实验组(6.8±1.2pF， n=14)<体外对照组(1)(25.0±5.6pF,n=16)<体外对照组(2)(32.4±4.8pF，n=15)。三组间电流密度分别为-151.7±26.1pA/pF (n=14)，-41.6±7.7pA/pF (n=16)及-31.7±4.0pA/pF (n=15)，差异亦达显著性(P值均<0.05)。
(2)实验组If电流的激活阈电位为-46.7±5.9mV(n=20)，明显低于体外对照组(1)(-40.6±4.4mV，n=20)及体外对照组(2)(-41.3±5.2mV，n=22)，P值均<0.05；而两个体外对照组之间无显著差异(P=0.181)。
(3)与体外对照组(1)及体外对照组（2）If电流的半激活电压[分别为-94.8±7.8mV, n=16；-96.4±6.6mV, n=15]相比，实验组If电流的半激活电压(-107.8±14.6mV,n=14)分别负移了大约13mV及11mV，差异达到显著性(P值均<0.05)。而两个体外对照组之间无显著差异(P=0.553)。
(4)实验组的If电流激活曲线的斜率为-13.7±1.7(n=14)，与体外对照组(1)(-11.5±1.8,n=16)及体外对照组(2)(-11.2±2.2,n=15)相比，均达到显著差异(P值均<0.05)，而两个体外对照组之间无显著差异(P=0.717)。
(5)在指令电压为-140mV时，实验组If电流的激活时间常数为509.8±66.6ms(n=6)，与体外对照组(1)(405.6±64.5ms， n=8)及体外对照组(2)(427.5±62.2ms，n=7)相比，有更缓慢激活的趋势，但三组之间的差异未达显著性(P=0.051)。
(6)三组If电流的翻转电位分别为实验组-32.3±3.3(n=8)，体外对照组(1)-29.5±3.9(n=11)，体外对照组(2)-30.9±3.0(n=10)，三者之间无统计学差异(P>0.05)。
(7)3μM异丙肾上腺素能够使实验组If电流激活曲线的半激活电压正移约9.7mV(从-106.5±9.5mV到-96.8±8.7mV, n=4; P<0.05)，但对激活曲线的斜率影响不明显(从-12.8±2.1到-12.9±1.8，n=4; P>0.05)。
5.在实验组、体内对照组及两个体外对照组中，均未能在MSCs上记录到去极化激活的内向INa或ICa-L电流，亦未能记录到动作电位。在膜片钳全细胞记录模式的基础上进行的荧光染料转运实验中，未发现荧光素黄染料从被检测的HCN4-MSCs(n=6)扩散到周围的心肌细胞中。
6.免疫组化检测结果显示，只有极少数的移植HCN4-MSCs表达CX43，且CX43以点状的模式随意分布于细胞的接触界面上，与心肌细胞的CX43分布(局限在闰盘)显著不同。移植区域未见IgG的表达及CD3+T细胞的浸润。
结论
1.本实验建立了一种能够满足膜片钳检测需要的心肌组织切片方法，并在此基础上成功对移植到宿主心肌组织中的起搏细胞进行了原位的电生理学研究。
2. mHCN4基因修饰的大鼠MSCs(HCN4-MSCs)同种移植到宿主心脏中能存活4周以上，并持续表达超极化激活的起搏电流(If)。体内对照组的MSCs(MSCs-EGFP)不表达If。
3.移植的HCN4-MSCs表达的If电流幅值与体外对照组相似，但膜电容及电流密度与体外对照组细胞有显著差异。与体外对照组的细胞相比，移植的HCN4-MSCs的If电流激活阈电位及半激活电压显著负移(差异达显著性)，激活曲线斜率增大(差异达显著性)，激活时间常数延长(但差异未达显著性)，翻转电位无显著差异。
4.移植后4周，HCN4-MSCs及MSCs-EGFP均不表达去极化激活的内向钠电流及钙电流，未记录到动作电位。MSCs与宿主心肌细胞形成缝隙连接耦合的比率低下。
5.移植后4周，在MSCs的移植区域，未观察到体液免疫排斥及细胞免疫排斥现象。
Background and Aims
The present therapy for bradyarrhythmias consists of the use of electronic pacemakersto sustain the heart rate. However, such devices are not optimal because of the high cost,limited battery life, the lack of a biological response to enable adaptation toneurotransmitter changes following changes in physiological conditions, and undesirablecomplications, such as hemorrhage and bacterial infection. With improved understanding ofthe genetic determinants of ion channel function in pacemaker cells, the attention of anincreasing number of researchers is turning to the field of biological pacing, which couldreplace the traditional electronic pacemaker in the treatment of bradyarrhythmias.
The funny current (If) of sino-atrial node cells, which plays a key role in the process ofpacemaker generation and in the modulation of autonomic transmitters for heart rate, isgenerated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Thus far,proof-of-concept data have been generated in recent experiments, in which transplantedmesenchymal stem cells (MSCs) carrying HCN genes generated pacemaker activity inmodels of complete heart block. However, There are some key issues still to be unclear. Onthe one hand, it was shown in the above studies that the beating rate ranged from about40to50bpm, which was significantly lower than that of cardiomyocytes cocultured withMSCs transfected with HCN in vitro. The specific mechanism underlying this phenomenonremains fragmented. On the other hand, MSCs have the potential for multi-directionaldifferentiation, which raised concerns about whether MSCs used only as a gene deliverysystem could acquire the functional attributes of excitable cells via the hostmicroenvironment-mediated induction of differentiation. The above issues is crucial for thetransplanted cells to play a stable and long-lasting pacing function in vivo.
Nevertheless, due to the lack of direct experimental access to the implanted cells in situ,current knowledge of the changes in electrophysiological characteristics and the mechanisms underlying the pacemaker activity of engrafted HCN gene-transfected MSCs invivo remains unclear. In previous studies, the functional examination of engrafted MSCsrelied chiefly on immunohistochemical endpoints or data from electrocardiograms (ECG)and optical action potentials(APs) mapping. But the expression of a cardiac myocyte-likephenotype is not equivalent to obtaining cardiac myocyte-like function, and the noninvasiveexamination is subject to their restricted spatial resolution and being difficult to confirm thegeneration of APs.
Because ion channels form the basis for engrafted cells to generate pacemaker functionin vivo and because the patch-clamp technique is the most fundamental and effectivemethod for detecting ion channel currents, we performed in situ patch-clamp recording ofengrafted MSCs using vital ventricular slices and investigated the related ionic currents oftheir pacing action, as well as their gap junctional communication with host cardiomyocytes,to provide more insight into electrophysiological properties of engrafted cells and itsinteraction with endogenous myocytes. To the best of our knowledge, this is the first reportdescribing in situ patch-clamp recording on engrafted cells to examine their ioncharacteristics.
Method
1. Bone marrow specimens were extracted. MSCs were isolated by gradientcentrifugation and its character of adherence to culture plates, and further amplified andpurified in vitro.
2. The lentiviral vector pLenti6.3-IRES2-EGFP (which is designated as LV-EGFP) andpLenti6.3-mHCN4-IRES2-EGFP(which is designated as LV-HCN4-EGFP) wereconstructed and produced. Passage-3rMSCs were transfected with LV-HCN4-EGFP orLV–EGFP at multiplicities of infection (MOIs) of10. After being transfected for5~7days,rMSCs were tested for the expression of EGFP and mHCN4protein by usingImmunohistochemical method and patch-clamp technique.
3. MSCs were delivered into host heart to build a cell transplantation modle, and aneffective method was established to prepare viable ventricular slices to meet therequirements for patch-clamp detection.
4. SD rats were divided into the following two groups: i) experimental group, were transplanted with mHCN4-EGFP-transfected rMSCs, and ii) in vivo control group, weretransplanted with EGFP-transfected rMSCs. The rats were sacrificed at4weeks after cell implantation and the engrafted MSCs were investigated using in situpatch-clamp recording. In addition, as in vitro controls, the recording was also performed inthe following two groups: i) freshly mHCN4-transfected rMSCs before prolonged culture,and ii) after parallel culture for4weeks in vitro. Throughout the report, these four groupsare designated as follows: experimental group, in vivo control group, in vitro(i) and invitro(ii), respectively. The pacemaker current (If) of the cells in each group were detectedusing patch-clamp technique, and the current characteristics of each group were furthercompared and analyzed. In addition, action potential and other current associated with thepacing function, such as sodium current and L-type calcium current, also were detected.
5. Gap junctional intercellular communication between engrafted rMSCs and residentcardiomyocytes was determined by the use of Lucifer yellow dye on the basis of the wholecell patch clamp sealing.
6. Myocardial tissue adjacent to the sections detected using the patch-clamp techniquewas fixed in4%paraformaldehyde, cryopreserved overnight, and embedded in paraffin.Serial sections (5-μm-thick) were cut from the tissue. The immunofluorescence method wasused for detection of HCN4and EGFP protein expression in the transplanted cells, while theimmunohistochemical method was used for the detection of expression and distribution ofconnexin43(CX43), as well as the presence of IgG antibody and CD3+T cells in thetransplantation area.
Results
1. In this experiment, purified MSCs express the surface molecules CD29and CD44inthe absence of CD34, CD45, which was assessed by fluorescence-activated cell sorteranalysis, and have the capacity for differentiation to osteoblasts, adipocytes, and myocardialcells in vitro. The above characteristics are in line with the currently accepted standard ofMSCs.
2. At a MOI of10, the transfection efficiencies of rMSCs were67.0±6.6%(n=5),while MSCs morphology and activity was unaffected. EGFP-transfected rMSCsdemonstrated EGFP but not HCN4protein expression, whereas mHCN4-EGFP-transfected rMSCs expressed both EGFP and HCN4protein. The results of patch clamp recording showthat Ifwas elicited from mHCN4-EGFP-transfected rMSCs, but not from EGFP-transfectedrMSCs.
3. Based on the improved method of preparation of ventricular slices, we obtainedviable ventricular slices with clean surfaces, low fluorescence background and maintainedthe structure integrity of cells. Clusters of transplanted rMSCs distributed within theinterstitial compartment of cardiomyocytes remained round-shaped, and a small number ofthe cells exhibited a short spindle, arranged in parallel with the myocardial cells.Futhermore, membrane currents of myocardial cells and MSCs were successfully recordedby using patch-clamp recording of ventricular slices, which suggested that the viableventricular slices were fully able to meet the requirements for patch-clamp detection.
4. Patch-clamp experiments results suggested that the allograftedmHCN4-EGFP-transfected rMSCs of experimental group survived in the host heart for over4weeks, that they expressed Ifwith a similar amplitude but with a more negative activationcompared with parallel mHCN4-transfected rMSCs cultured in vitro(i) and in vitro(ii),while the EGFP-transfected rMSCs of in vivo control group did not express Ifcurrents.
(1) There were trends toward larger current densities (at-140mV) inmHCN4-EGFP-transfected rMSCs in situ (-151.7±26.1pA/pF, n=14) compared with invitro(i) cells (-41.6±7.7pA/pF,n=16) and in vitro(ii) cells (-31.7±4.0pA/pF, n=15) andtoward smaller membrane capacities in situ (6.8±1.2pF, n=14) compared with in vitro(i)(25.0±5.6pF,n=16) and in vitro(ii)(32.4±4.8pF, n=15), and for any two of the three sets ofdata, the differences were significant (P<0.05for each). However, there were no significantdifferences in current amplitudes (at-140mV) among the three groups (in situ,-1007.4±132.3pA, n=14; in vitro(i),-1012.5±187.3pA, n=16; in vitro(ii),-1025.5±200.9pA, n=15; P=0.957).
(2) All of the mHCN4-EGFP-transfected rMSCs studied in vitro exhibited thresholdvalues between-40and-50mV [in vitro(i), mean value-40.6±4.4mV (n=20), vs. invitro(ii), mean value-41.3±5.2mV (n=22); P=0.181]. In comparison, in20engraftedmHCN4-EGFP-transfected rMSCs studied in situ, the threshold of Ifactivation varied from-40to-90mV (mean value-46.7±5.9mV), and both of the differences reached statisticalsignificance (P<0.05for each) when compared with in vitro(i) and in vitro(ii), respectively.
(3) The half-maximal activation voltage for Ifcurrent in situ (-107.8±14.6mV,n=14)was more negative, respectively, compared with that of in vitro(i)(-94.8±7.8mV, n=16;P<0.05) and that of in vitro(ii)(-96.4±6.6mV, n=15; P<0.05). However, the differencebetween the groups of in vitro(i) and in vitro(ii) did not reach significance (P=0.553).
(4) Similarly, the slope values of the voltage dependences in situ (-13.7±1.7mV,n=14) was different from that in vitro(i)(-11.5±1.8mV, n=16; P<0.05) and that invitro(ii)(-11.2±2.2mV, n=15; P<0.05), but there was also no significant difference betweenthe two groups in vitro(P=0.717).
(5) At-140mV, the time constant for Ifactivation was509.8±66.6ms (n=6) for insitu cells compared with405.6±64.5(n=8) for in vitro(i) cells and427.5±62.2ms (n=7) forin vitro(ii) cells, but the difference among the three groups did not reach statisticalsignificance(P=0.051).
(6) The reversal potentials were-32.3±3.3mV (n=8) in situ and-29.5±3.9mV (n=11)in vitro(i) and-30.9±3.0mV (n=10) in vitro(ii), respectively. There was no significantdifference among the three groups (P=0.288).
(7) In the presence of3μM isoproterenol, the half-maximum activation voltage inengrafted MSCs was shifted approximately9.7mV (from-106.5±9.5mV to-96.8±8.7mV,n=4; P<0.05) in the positive direction, whereas its slope was not significantly modified(from-12.8±2.1to-12.9±1.8mV).
5. Neither sodium currents nor calcium currents were elicited inmHCN4-EGFP-transfected rMSCs in situ (n=10),in vitro(i)(n=12) and in vitro(ii)(n=15).Similarly, an action potential was not evoked in all groups of cells in the current-clampconfiguration. The results were similar in EGFP-transfected rMSCs in situ (n=10). Inaddition, in the fluorescent dye transfer experiments conducted on the basis of the wholecell patch clamp recording, dye did not transfer from those investigated rMSCs (n=6) to theadjacent myocardial cells or other transplanted cells.
6. Immunohistochemical results show that only a few rMSCs in vivo expressedconnexin43in a immature manner, which was randomly distributed at the contact interfacesbetween engrafted MSCs and host myocytes in a punctate pattern and without properalignment, significantly different from that of myocardial cells (confined to the intercalateddisc). In addition, there was no binding of rat IgG to the surface of rMSCs and no significant infiltration by CD3+T lymphocytes in the area of cell transplantation.
Conclusions
1. we established a improved method of preparation of ventricular slices, andsuccessfully performed in situ patch-clamp recording of engrafted MSCs using vitalventricular slices and investigated the related ionic currents of their pacing action, as well astheir gap junctional communication with host cardiomyocytes.
2. Patch-clamp experiments results suggested that the allografted mHCN4-transfectedrMSCs of experimental group survived and expressed Ifcurrents in the host heart for over4weeks, while the EGFP-transfected rMSCs of in vivo control group did not express Ifcurrents.
3. The allografted mHCN4-transfected rMSCs of experimental group expressed Ifwitha similar amplitude but with a more negative activation compared with parallelmHCN4-transfected rMSCs cultured in vitro(i) and in vitro(ii), characterized by a significantnegative shift of activation threshold potential and half-maximal activation voltage, andaccompanied by a seemingly slower time course with time constants. However, there wereno significant differences of reversal potential among the three groups.
4. At4weeks after cell implantation, in all of the four groups of cells, neither sodiumnor calcium currents were subsequently elicited, and APs were not evoked in any group ofcells. In addition, the transplanted MSCs exhibited a low incidence of gap junctionalcoupling with host cardiomyocytes.
5. At4weeks after cell implantation, humoral and cellular immune phenomena werenot observed in the area of MSCs transplantation.
起搏电流(current funny，If)是窦房结细胞舒张期自动除极化的主要决定因素，并在心率控制及神经递质对心率的调节中起重要作用；超极化激活的环化核苷酸门控(hyperpolarization-activated cyclic nucleotide-gated, HCN)通道基因家族编码的HCN通道则是If形成的分子基础。目前已有利用HCN基因修饰的间充质干细胞(Mesenchymalstem cells, MSCs)移植进入房室传导阻滞的动物模型中成功创建生物起搏点的报道，结果令人鼓舞，但有一些关键的问题仍亟待解决。首先，在体研究发现生物起搏的频率低下(约40~50bpm，也有低至30bpm的情况)，显著低于与体外心肌细胞共培养时的起搏频率，是否起搏电流的特性发生了改变？具体原因尚不清楚；其次，MSCs具有多向分化的潜能，其移植到宿主心脏后，仅作为一个载体工具，还是分化为具备功能的心肌样细胞发挥起搏作用？以上问题对于移植细胞能否在体内稳定、持久的发挥起搏功能至关重要。
然而，由于缺乏对移植细胞直接进行原位功能检测的手段，目前多数研究主要是通过免疫组织化学检测移植细胞的表型变化，以及通过宏观的心电图、三维电解剖标测系统(CARTO)等无创检查对移植细胞的起搏功能进行评估。但移植细胞表型的改变并不等同于具备了心肌细胞样的功能；无创检查的空间分辨力有限，不能对“体内干细胞和心肌细胞如何交互作用影响心脏功能”进行精确描述。因此，目前的研究尚不能真正阐明移植细胞在宿主心脏中发挥起搏功能及起搏功能发生改变的机制。
众所周知，起搏电流(funny current, If)是起搏细胞发挥功能的基础，而膜片钳技术是检测细胞膜离子通道电流的最根本、有效的方法。利用膜片钳技术针对移植细胞进行原位的电生理学研究，将有助于阐明起搏细胞的电生理学特性及其与宿主心肌细胞交互作用的机制。对移植细胞进行原位膜片钳检测需要在组织切片上进行，但目前尚未发现利用成年动物心肌组织切片膜片钳技术原位检测移植干细胞电生理特性及其与宿主心肌细胞交互作用功能的报道。本实验拟利用组织片膜片钳技术，对同种异体移植到宿主心脏中的mHCN4基因修饰的大鼠MSCs(HCN4-MSCs)进行原位电生理学研究。
方法
1.取健康SD大鼠骨髓，经密度梯度离心和贴壁培养获得MSCs，在体外扩增、纯化。
2.构建mHCN4的表达载体pLenti6.3-mHCN4-IRES2-EGFP(LV-HCN4-EGFP)及空载体pLenti6.3-IRES2-EGFP(LV-EGFP)，分别转染第三代的MSCs，5~7天后利用免疫组化方法及膜片钳方法检测起搏离子通道蛋白及电流的表达。
3.将MSCs同种移植到SD大鼠心脏中创建细胞移植模型，在此基础上建立适合于膜片钳检测的心肌组织切片方法。
4.将SD大鼠分为两组(每组各10只)：1)实验组移植转染mHCN4的MSCs(HCN4-MSCs)；2)体内对照组移植仅转染EGFP的MSCs。各组大鼠在细胞移植4周后处死，取心脏进行切片，对移植细胞进行原位的膜片钳检测。同时，另设两个组作为体外对照：1)HCN4-MSCs体外培养1周组，2)HCN4-MSCs体外培养4周组。在本研究中，上述4组分别被定义如下：实验组，体内对照组，体外对照组(1)及体外对照组(2)。利用膜片钳技术对各组细胞的起搏电流(If)进行检测，并对各组细胞记录到的If电流特性进行比较和分析。并检测实验组及体内对照组移植细胞的动作电位、钠电流及钙电流。
5.在全细胞膜片钳封接的基础上，结合荧光黄染料转运的实验方法，检测移植细胞与宿主心肌细胞之间的缝隙连接通讯功能。
6.膜片钳检测结束后，将心肌组织用4%多聚甲醛常规固定、石蜡包埋、切片，利用免疫荧光方法检测移植细胞HCN4及EGFP蛋白的表达；利用免疫组化的方法检测缝隙连接蛋白43(connexin43, CX43)的表达及分布，以及移植区域IgG的表达及CD3~+T细胞的浸润。
结果
1.本实验分离纯化的MSCs高表达CD29和CD44抗原(高达99%以上)，而CD34和CD45抗原表达与同型对照没有显著差异；并且在特定的诱导条件下，MSCs可以分化为心肌样细胞、成骨细胞及脂肪细胞，证实其多向分化潜能。以上特性符合目前公认的MSCs标准。
2.在MOI=10的条件下，慢病毒对MSCs的转染效率可以达到67.0±6.6%(n=5)，MSCs形态及活性未受明显影响。转染“pLenti6.3-mHCN4-IRES2-EGFP”的MSCs同时表达EGFP及mHCN4蛋白，并记录到可被4mM CsCl可逆性阻断的超极化激活的内向电流，证实为起搏电流If；而转染“pLenti6.3-IRES2-EGFP”的MSCs仅表达EGFP，未记录到起搏电流If。
3.通过改良的心肌组织切片方法，获得了活性良好、背景荧光低的心肌组织切片。在切片中，心肌细胞结构完整、横纹清晰可见；移植的MSCs呈簇状分布在心肌细胞的缝隙之间，细胞呈类圆形、短梭形，形态完整、表面光滑，与心肌细胞紧密连接。在荧光条件下，心肌组织自发荧光背景较低，可以观察到EGFP标记的MSCs。通过全细胞膜片钳方式，分别记录到了组织片中心肌细胞及移植的MSCs细胞的膜电流，证实细胞活性良好，完全能够满足针对移植MSCs进行原位膜片钳检测的需要。
4.实验组移植的HCN4-MSCs在宿主心脏中能存活4周以上。实验组与体外对照组(1)及体外对照组(2)的HCN4-MSCs表达超极化激活的起搏电流(If)。体内对照组的MSCs不表达If。
(1)实验组与体外对照组(1)及体外对照组(2)相比，三组间If电流幅值无显著差异[分别为-1007.4±132.3pA (n=14)、-1012.5±187.3pA (n=16)及-1025.5±200.9pA (n=15)，P>0.05]。三组间细胞膜电容比较有显著性差异(P值均<0.05)，其中实验组(6.8±1.2pF， n=14)<体外对照组(1)(25.0±5.6pF,n=16)<体外对照组(2)(32.4±4.8pF，n=15)。三组间电流密度分别为-151.7±26.1pA/pF (n=14)，-41.6±7.7pA/pF (n=16)及-31.7±4.0pA/pF (n=15)，差异亦达显著性(P值均<0.05)。
(2)实验组If电流的激活阈电位为-46.7±5.9mV(n=20)，明显低于体外对照组(1)(-40.6±4.4mV，n=20)及体外对照组(2)(-41.3±5.2mV，n=22)，P值均<0.05；而两个体外对照组之间无显著差异(P=0.181)。
(3)与体外对照组(1)及体外对照组（2）If电流的半激活电压[分别为-94.8±7.8mV, n=16；-96.4±6.6mV, n=15]相比，实验组If电流的半激活电压(-107.8±14.6mV,n=14)分别负移了大约13mV及11mV，差异达到显著性(P值均<0.05)。而两个体外对照组之间无显著差异(P=0.553)。
(4)实验组的If电流激活曲线的斜率为-13.7±1.7(n=14)，与体外对照组(1)(-11.5±1.8,n=16)及体外对照组(2)(-11.2±2.2,n=15)相比，均达到显著差异(P值均<0.05)，而两个体外对照组之间无显著差异(P=0.717)。
(5)在指令电压为-140mV时，实验组If电流的激活时间常数为509.8±66.6ms(n=6)，与体外对照组(1)(405.6±64.5ms， n=8)及体外对照组(2)(427.5±62.2ms，n=7)相比，有更缓慢激活的趋势，但三组之间的差异未达显著性(P=0.051)。
(6)三组If电流的翻转电位分别为实验组-32.3±3.3(n=8)，体外对照组(1)-29.5±3.9(n=11)，体外对照组(2)-30.9±3.0(n=10)，三者之间无统计学差异(P>0.05)。
(7)3μM异丙肾上腺素能够使实验组If电流激活曲线的半激活电压正移约9.7mV(从-106.5±9.5mV到-96.8±8.7mV, n=4; P<0.05)，但对激活曲线的斜率影响不明显(从-12.8±2.1到-12.9±1.8，n=4; P>0.05)。
5.在实验组、体内对照组及两个体外对照组中，均未能在MSCs上记录到去极化激活的内向INa或ICa-L电流，亦未能记录到动作电位。在膜片钳全细胞记录模式的基础上进行的荧光染料转运实验中，未发现荧光素黄染料从被检测的HCN4-MSCs(n=6)扩散到周围的心肌细胞中。
6.免疫组化检测结果显示，只有极少数的移植HCN4-MSCs表达CX43，且CX43以点状的模式随意分布于细胞的接触界面上，与心肌细胞的CX43分布(局限在闰盘)显著不同。移植区域未见IgG的表达及CD3+T细胞的浸润。
结论
1.本实验建立了一种能够满足膜片钳检测需要的心肌组织切片方法，并在此基础上成功对移植到宿主心肌组织中的起搏细胞进行了原位的电生理学研究。
2. mHCN4基因修饰的大鼠MSCs(HCN4-MSCs)同种移植到宿主心脏中能存活4周以上，并持续表达超极化激活的起搏电流(If)。体内对照组的MSCs(MSCs-EGFP)不表达If。
3.移植的HCN4-MSCs表达的If电流幅值与体外对照组相似，但膜电容及电流密度与体外对照组细胞有显著差异。与体外对照组的细胞相比，移植的HCN4-MSCs的If电流激活阈电位及半激活电压显著负移(差异达显著性)，激活曲线斜率增大(差异达显著性)，激活时间常数延长(但差异未达显著性)，翻转电位无显著差异。
4.移植后4周，HCN4-MSCs及MSCs-EGFP均不表达去极化激活的内向钠电流及钙电流，未记录到动作电位。MSCs与宿主心肌细胞形成缝隙连接耦合的比率低下。
5.移植后4周，在MSCs的移植区域，未观察到体液免疫排斥及细胞免疫排斥现象。
Background and Aims
The present therapy for bradyarrhythmias consists of the use of electronic pacemakersto sustain the heart rate. However, such devices are not optimal because of the high cost,limited battery life, the lack of a biological response to enable adaptation toneurotransmitter changes following changes in physiological conditions, and undesirablecomplications, such as hemorrhage and bacterial infection. With improved understanding ofthe genetic determinants of ion channel function in pacemaker cells, the attention of anincreasing number of researchers is turning to the field of biological pacing, which couldreplace the traditional electronic pacemaker in the treatment of bradyarrhythmias.
The funny current (If) of sino-atrial node cells, which plays a key role in the process ofpacemaker generation and in the modulation of autonomic transmitters for heart rate, isgenerated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Thus far,proof-of-concept data have been generated in recent experiments, in which transplantedmesenchymal stem cells (MSCs) carrying HCN genes generated pacemaker activity inmodels of complete heart block. However, There are some key issues still to be unclear. Onthe one hand, it was shown in the above studies that the beating rate ranged from about40to50bpm, which was significantly lower than that of cardiomyocytes cocultured withMSCs transfected with HCN in vitro. The specific mechanism underlying this phenomenonremains fragmented. On the other hand, MSCs have the potential for multi-directionaldifferentiation, which raised concerns about whether MSCs used only as a gene deliverysystem could acquire the functional attributes of excitable cells via the hostmicroenvironment-mediated induction of differentiation. The above issues is crucial for thetransplanted cells to play a stable and long-lasting pacing function in vivo.
Nevertheless, due to the lack of direct experimental access to the implanted cells in situ,current knowledge of the changes in electrophysiological characteristics and the mechanisms underlying the pacemaker activity of engrafted HCN gene-transfected MSCs invivo remains unclear. In previous studies, the functional examination of engrafted MSCsrelied chiefly on immunohistochemical endpoints or data from electrocardiograms (ECG)and optical action potentials(APs) mapping. But the expression of a cardiac myocyte-likephenotype is not equivalent to obtaining cardiac myocyte-like function, and the noninvasiveexamination is subject to their restricted spatial resolution and being difficult to confirm thegeneration of APs.
Because ion channels form the basis for engrafted cells to generate pacemaker functionin vivo and because the patch-clamp technique is the most fundamental and effectivemethod for detecting ion channel currents, we performed in situ patch-clamp recording ofengrafted MSCs using vital ventricular slices and investigated the related ionic currents oftheir pacing action, as well as their gap junctional communication with host cardiomyocytes,to provide more insight into electrophysiological properties of engrafted cells and itsinteraction with endogenous myocytes. To the best of our knowledge, this is the first reportdescribing in situ patch-clamp recording on engrafted cells to examine their ioncharacteristics.
Method
1. Bone marrow specimens were extracted. MSCs were isolated by gradientcentrifugation and its character of adherence to culture plates, and further amplified andpurified in vitro.
2. The lentiviral vector pLenti6.3-IRES2-EGFP (which is designated as LV-EGFP) andpLenti6.3-mHCN4-IRES2-EGFP(which is designated as LV-HCN4-EGFP) wereconstructed and produced. Passage-3rMSCs were transfected with LV-HCN4-EGFP orLV–EGFP at multiplicities of infection (MOIs) of10. After being transfected for5~7days,rMSCs were tested for the expression of EGFP and mHCN4protein by usingImmunohistochemical method and patch-clamp technique.
3. MSCs were delivered into host heart to build a cell transplantation modle, and aneffective method was established to prepare viable ventricular slices to meet therequirements for patch-clamp detection.
4. SD rats were divided into the following two groups: i) experimental group, were transplanted with mHCN4-EGFP-transfected rMSCs, and ii) in vivo control group, weretransplanted with EGFP-transfected rMSCs. The rats were sacrificed at4weeks after cell implantation and the engrafted MSCs were investigated using in situpatch-clamp recording. In addition, as in vitro controls, the recording was also performed inthe following two groups: i) freshly mHCN4-transfected rMSCs before prolonged culture,and ii) after parallel culture for4weeks in vitro. Throughout the report, these four groupsare designated as follows: experimental group, in vivo control group, in vitro(i) and invitro(ii), respectively. The pacemaker current (If) of the cells in each group were detectedusing patch-clamp technique, and the current characteristics of each group were furthercompared and analyzed. In addition, action potential and other current associated with thepacing function, such as sodium current and L-type calcium current, also were detected.
5. Gap junctional intercellular communication between engrafted rMSCs and residentcardiomyocytes was determined by the use of Lucifer yellow dye on the basis of the wholecell patch clamp sealing.
6. Myocardial tissue adjacent to the sections detected using the patch-clamp techniquewas fixed in4%paraformaldehyde, cryopreserved overnight, and embedded in paraffin.Serial sections (5-μm-thick) were cut from the tissue. The immunofluorescence method wasused for detection of HCN4and EGFP protein expression in the transplanted cells, while theimmunohistochemical method was used for the detection of expression and distribution ofconnexin43(CX43), as well as the presence of IgG antibody and CD3+T cells in thetransplantation area.
Results
1. In this experiment, purified MSCs express the surface molecules CD29and CD44inthe absence of CD34, CD45, which was assessed by fluorescence-activated cell sorteranalysis, and have the capacity for differentiation to osteoblasts, adipocytes, and myocardialcells in vitro. The above characteristics are in line with the currently accepted standard ofMSCs.
2. At a MOI of10, the transfection efficiencies of rMSCs were67.0±6.6%(n=5),while MSCs morphology and activity was unaffected. EGFP-transfected rMSCsdemonstrated EGFP but not HCN4protein expression, whereas mHCN4-EGFP-transfected rMSCs expressed both EGFP and HCN4protein. The results of patch clamp recording showthat Ifwas elicited from mHCN4-EGFP-transfected rMSCs, but not from EGFP-transfectedrMSCs.
3. Based on the improved method of preparation of ventricular slices, we obtainedviable ventricular slices with clean surfaces, low fluorescence background and maintainedthe structure integrity of cells. Clusters of transplanted rMSCs distributed within theinterstitial compartment of cardiomyocytes remained round-shaped, and a small number ofthe cells exhibited a short spindle, arranged in parallel with the myocardial cells.Futhermore, membrane currents of myocardial cells and MSCs were successfully recordedby using patch-clamp recording of ventricular slices, which suggested that the viableventricular slices were fully able to meet the requirements for patch-clamp detection.
4. Patch-clamp experiments results suggested that the allograftedmHCN4-EGFP-transfected rMSCs of experimental group survived in the host heart for over4weeks, that they expressed Ifwith a similar amplitude but with a more negative activationcompared with parallel mHCN4-transfected rMSCs cultured in vitro(i) and in vitro(ii),while the EGFP-transfected rMSCs of in vivo control group did not express Ifcurrents.
(1) There were trends toward larger current densities (at-140mV) inmHCN4-EGFP-transfected rMSCs in situ (-151.7±26.1pA/pF, n=14) compared with invitro(i) cells (-41.6±7.7pA/pF,n=16) and in vitro(ii) cells (-31.7±4.0pA/pF, n=15) andtoward smaller membrane capacities in situ (6.8±1.2pF, n=14) compared with in vitro(i)(25.0±5.6pF,n=16) and in vitro(ii)(32.4±4.8pF, n=15), and for any two of the three sets ofdata, the differences were significant (P<0.05for each). However, there were no significantdifferences in current amplitudes (at-140mV) among the three groups (in situ,-1007.4±132.3pA, n=14; in vitro(i),-1012.5±187.3pA, n=16; in vitro(ii),-1025.5±200.9pA, n=15; P=0.957).
(2) All of the mHCN4-EGFP-transfected rMSCs studied in vitro exhibited thresholdvalues between-40and-50mV [in vitro(i), mean value-40.6±4.4mV (n=20), vs. invitro(ii), mean value-41.3±5.2mV (n=22); P=0.181]. In comparison, in20engraftedmHCN4-EGFP-transfected rMSCs studied in situ, the threshold of Ifactivation varied from-40to-90mV (mean value-46.7±5.9mV), and both of the differences reached statisticalsignificance (P<0.05for each) when compared with in vitro(i) and in vitro(ii), respectively.
(3) The half-maximal activation voltage for Ifcurrent in situ (-107.8±14.6mV,n=14)was more negative, respectively, compared with that of in vitro(i)(-94.8±7.8mV, n=16;P<0.05) and that of in vitro(ii)(-96.4±6.6mV, n=15; P<0.05). However, the differencebetween the groups of in vitro(i) and in vitro(ii) did not reach significance (P=0.553).
(4) Similarly, the slope values of the voltage dependences in situ (-13.7±1.7mV,n=14) was different from that in vitro(i)(-11.5±1.8mV, n=16; P<0.05) and that invitro(ii)(-11.2±2.2mV, n=15; P<0.05), but there was also no significant difference betweenthe two groups in vitro(P=0.717).
(5) At-140mV, the time constant for Ifactivation was509.8±66.6ms (n=6) for insitu cells compared with405.6±64.5(n=8) for in vitro(i) cells and427.5±62.2ms (n=7) forin vitro(ii) cells, but the difference among the three groups did not reach statisticalsignificance(P=0.051).
(6) The reversal potentials were-32.3±3.3mV (n=8) in situ and-29.5±3.9mV (n=11)in vitro(i) and-30.9±3.0mV (n=10) in vitro(ii), respectively. There was no significantdifference among the three groups (P=0.288).
(7) In the presence of3μM isoproterenol, the half-maximum activation voltage inengrafted MSCs was shifted approximately9.7mV (from-106.5±9.5mV to-96.8±8.7mV,n=4; P<0.05) in the positive direction, whereas its slope was not significantly modified(from-12.8±2.1to-12.9±1.8mV).
5. Neither sodium currents nor calcium currents were elicited inmHCN4-EGFP-transfected rMSCs in situ (n=10),in vitro(i)(n=12) and in vitro(ii)(n=15).Similarly, an action potential was not evoked in all groups of cells in the current-clampconfiguration. The results were similar in EGFP-transfected rMSCs in situ (n=10). Inaddition, in the fluorescent dye transfer experiments conducted on the basis of the wholecell patch clamp recording, dye did not transfer from those investigated rMSCs (n=6) to theadjacent myocardial cells or other transplanted cells.
6. Immunohistochemical results show that only a few rMSCs in vivo expressedconnexin43in a immature manner, which was randomly distributed at the contact interfacesbetween engrafted MSCs and host myocytes in a punctate pattern and without properalignment, significantly different from that of myocardial cells (confined to the intercalateddisc). In addition, there was no binding of rat IgG to the surface of rMSCs and no significant infiltration by CD3+T lymphocytes in the area of cell transplantation.
Conclusions
1. we established a improved method of preparation of ventricular slices, andsuccessfully performed in situ patch-clamp recording of engrafted MSCs using vitalventricular slices and investigated the related ionic currents of their pacing action, as well astheir gap junctional communication with host cardiomyocytes.
2. Patch-clamp experiments results suggested that the allografted mHCN4-transfectedrMSCs of experimental group survived and expressed Ifcurrents in the host heart for over4weeks, while the EGFP-transfected rMSCs of in vivo control group did not express Ifcurrents.
3. The allografted mHCN4-transfected rMSCs of experimental group expressed Ifwitha similar amplitude but with a more negative activation compared with parallelmHCN4-transfected rMSCs cultured in vitro(i) and in vitro(ii), characterized by a significantnegative shift of activation threshold potential and half-maximal activation voltage, andaccompanied by a seemingly slower time course with time constants. However, there wereno significant differences of reversal potential among the three groups.
4. At4weeks after cell implantation, in all of the four groups of cells, neither sodiumnor calcium currents were subsequently elicited, and APs were not evoked in any group ofcells. In addition, the transplanted MSCs exhibited a low incidence of gap junctionalcoupling with host cardiomyocytes.
5. At4weeks after cell implantation, humoral and cellular immune phenomena werenot observed in the area of MSCs transplantation.
引文
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