慢病毒转染HCN4基因构建生物起搏细胞的体外研究
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摘要
背景:植入电子起搏器已成为治疗症状性缓慢性心律失常的首选方法,并取得很大的成功。然而,有限的电池寿命、缺乏对神经激素自动反应性、儿童期植入起搏器后电极不能随身体的发育而相应变长等缺点使得人们迫切需要一种更理想的治疗方法。国内外的学者研究认为利用基因治疗和细胞治疗构建生物起搏在不久的将来可能会成为电子起搏器最为理想的替代方法。然而目前有关生物起搏的体内实验都没有超过2周,这是否与目前研究者所选用的基因以及基因载体有关?慢病毒表达系统因其基因组可以整合于宿主基因组中,长时间、稳定表达外源基因并具有较低的免疫原性,受到广泛关注。所以本实验我们使用5-aza(5-氮胞苷)诱导骨髓干细胞向心肌样细胞转化,同时将HCN4(hyperpolarization-activated cyclic nucleotide-gated,超极化激活环核苷酸门控)起搏基因使用慢病毒转染进心肌样骨髓基质干细胞,观察在体外能否构建生物起搏细胞,并能长期表达,从而为进一步在体研究生物起搏打下基础。
第一部分猪骨髓间充质干细胞(MSCs)的分离、培养及5-aza诱导其转化为心肌样细胞的研究
第一节猪骨髓间充质干细胞(MSCs)的分离、培养
目的:建立猪骨髓间充质干细胞体外培养方法,为5-aza诱导转化为心肌样细胞提供材料。
方法:猪的骨髓MSCs分离纯化后,流式细胞仪检测细胞周期。
结果:体外培养的原代MSCs 10～14d达到融合,细胞周期显示73%的细胞处于G_0/G_1期。
结论:根据黏附特性分离的猪骨髓MSCs在体外条件下可生长扩增,可用于5-aza诱导转化为心肌样细胞。
第二节5-aza体外诱导MSCs分化为心肌样细胞的研究
目的:建立猪骨髓间充质干细胞体外转化为心肌样细胞的方法,为构建生物起搏细胞提供新材料。
方法:猪的骨髓MSCs分离纯化后,用5-aza诱导后,用抗结蛋白(desmin)、肌球蛋白重链(MHC)、心肌特异性肌钙蛋白I(cTnI)、连接蛋白-43(Cx-43)进行免疫细胞化学染色鉴定。
结果:体外培养的第二代MSCs经5-aza诱导后,部分MSCs呈梭形,阳性表达desmin、MHC、cTnI和Cx-43。
结论:根据黏附特性分离的猪骨髓MSCs在体外条件下可分化为心肌样细胞,可用于构建生物起搏细胞。
第二部分穿梭质粒pCDH1-GFP-HCN4的构建及HCN4重组慢病毒的包装
第一节穿梭质粒pCDH1-GFP-HCN4的构建
目的:将全长约3.6kb的hHCN4目的基因片段从Dr Stieber教授惠赠的pCDNA3-hHCN4质粒卸载下来,最终装载到带有GFP绿色荧光蛋白标记的去内毒素穿梭质粒pCDH1-MCS1-EF1-copGFP上。
方法:用Eco RI和XbaⅠ从pCDNA3-hHCN4质粒切下hHCN4目的片段连接到Puc19载体中,再用EcoRI和HindⅢ从质粒Puc19切下目的片段连接到pcDNA3.1(A)载体中,再用XbaⅠ单酶从质粒pcDNA3.1(A)双切到pCDH1-MCS1-EF1-copGFP中。用XbaⅠ或者Eco RI鉴定,将阳性克隆质粒送上海生工测序,并将构建的目的基因质粒转染293细胞和猪骨髓间充质干细胞。
结果:XbaⅠ及Eco RI鉴定表明,PCR的产物和克隆扩增的产物的电泳结果该基因的大小约为3.6kb;阳性克隆质粒测序结果与GENEBANK目的基因HCN4序列完全相同;构建的目的基因质粒转染293细胞和原代的猪骨髓间充质干细胞后,均发现有强烈的绿色荧光的出现。
结论:成功将全长hHCN4目的基因片段从pCDNA3-hHCN4质粒卸载下来,装载到去内毒素穿梭质粒pCDH1-MCS1-EF1-copGFP上,可用于下一步慢病毒的包装。
第二节HCN4重组慢病毒的包装
目的:使用慢病毒包装系统(pPACKH1-Lentivector Packaging Kit)建立稳定的HCN4重组慢病毒。
方法:实验过程参照SBI的Lentivector Expression System的操作手册进行。将构建好的慢病毒包装质粒混合物混合后感染293T细胞及心肌样骨髓基质干细胞。
结果:构建的慢病毒包装质粒混合物转染293细胞和心肌样猪骨髓间充质干细胞后,均发现有强烈的绿色荧光的出现,细胞感染效率可以达到90%以上。
结论:成功建立稳定的hHCN4重组慢病毒,该病毒载体有很高的转染效率。第三部分慢病毒转染基质干细胞构建起搏样细胞的体外研究
目的:使用hHCN4重组慢病毒载体转染心肌样骨髓基质干细胞获得稳定过表达HCN4基因的起搏样干细胞。
方法:慢病毒转染心肌样骨髓基质干细胞实验过程参照SBI的LentivectorExpression System的操作手册进行;稳定转染细胞继续培养两周后行Western blot鉴定蛋白的表达并行real-time RT-PCR实时荧光定量检测HCN4目的基因的表达情况;全细胞膜片钳记录转染细胞HCN4通道电流的表达;转染细胞体外长期培养观察细胞的转染状态。
结果:病毒感染48小时后,目的细胞出现大量的荧光;Western blot检测结果见图,可见一约170kDa条带,与克隆hHCN4通道蛋白分子量相符;RT-PCR显示慢病毒转染的细胞hHCN4-RNA是原代细胞的123倍;膜片钳记录在转染细胞中可记录到明显的电压与时间依赖性的超极化内向电流,即If电流;将转染的心肌样骨髓基质干细胞培养至2个月时,显微镜下发现细胞呈群体性搏动,20℃室温下的搏动频率为15±1次/分。该病毒载体有很高的转染效率,在体外已获得至少8个月的阳性表达,8个月后因为细胞污染未进一步观察。
结论:hHCN4重组慢病毒转染心肌样骨髓基质干细胞获得了稳定过表达HCN4基因的起搏样干细胞。这种基因修饰后的心肌样干细胞有可能成为一种有效的生物起搏种子细胞,并期待在缓慢性心律失常的治疗技术上实现新突破。
第四部分稳定转染hHCN4起搏通道亚型的电生理特点
目的:全细胞膜片钳技术记录稳定转染pCDH1-GFP-HCN4的心肌样骨髓基质干细胞,了解hHCN4起搏通道亚型的电生理特点。
方法:全细胞膜片钳技术记录稳定转染pCDH1-GFP-HCN4的心肌样骨髓基质干细胞,观察hHCN4通道电流、通道电流激活曲线及电压依赖性激活时间常数动力学曲线;改变细胞外液K~+、Na~+浓度,观察K~+、Na~+对I_(hHCN4)电流的影响;以河豚毒素(TTX,I_(Na)阻断剂)、四乙胺(TEA,I_K阻断剂)、环磷酸腺苷(cAMP)、异丙基肾上腺素(ISO)、乙酰胆碱(Ach)、铯(Cs~+)、ZD7288(If电流特异性阻断剂)和胺碘酮分别灌流,观察它们对I_(hHCN4)电流的影响。
结果:
1.大多数稳定转染的心肌样骨髓干细胞可记录到约-80mV开始激活的一系列超极化内向离子流(I _(hHCN4_电流),该电流激活缓慢,呈电压、时间依赖性。电流的激活电位(V_(th))、半最大激活电位(V_(1/2))分别为-83±8mV、-108±2mV,斜率为-11.2±0.6 mV(n=10)。钳制电压-140mV时,激活时间常数为0.66±0.1s;钳制电压-110mV时,激活时间常数为3.1±0.7s。I_(hHCN4)的反转电位21.2±2.3mV(n=10),P_(Na)/P_K为0.23。
2.钳制电位-140mV,外液K~+浓度30 mmol/L,Na~+浓度由110 mmol/L降为50mmol/L,I_(hHCN4)电流密度分别为-300±25pA/pF~(-1)、-87±10 pA/pF~(-1)(n=8,p＜0.001),减少73%;外液Na~+浓度110 mmol/L,K~+浓度由30 mmol/L降为5 mmol/L,I_(hHCN4)电流密度分别为-302±30pA/pF~(-1)、-244±19 pA/pF~(-1)(n=8,p＜0.05),减少20%。
3.TTX和TEA灌流10min,对I_(hHCN4)没有影响。
4.1mmol/L cAMP灌流10min,I_(hHCN4)无明显变化,电极内液中加入终浓度为1mmol/L的cAMP,I_(hHCN4)激活加快,激活时间加快,V_(1/2)由-109±2mV变为-92±2mV,向正向移动17mV(n=10,p＜0.05),激活阈值及最大激活电流没有明显改变
5.1μmol/L ISO灌流10min,I_(hHCN4)略加快。1μmol/L Ach灌流10min,I_(hHCN4)无明显改变。
6.低浓度Cs~+可阻滞I_(hHCN4),冲洗后抑制作用完全恢复,半效抑制浓度(IC50)为128.8±28.1μmol/L;较低浓度ZD7288可阻滞I_(hHCN4),冲洗后抑制作用不能恢复,IC50为23.8±5.7μmol/L。
7.胺碘酮可阻滞I_(hHCN4),冲洗后抑制作用无明显恢复,IC50为1.34±0.33μmol/L;胺碘酮使各钳制电压下I_(hHCN4)激活缓慢。
结论:重组HCN4慢病毒转染心肌样干细胞,其表达的I_(hHCN4)电流具有天然If电流特点,表现为电压与时间依赖性的超极化内向电流、cAMP直接激活、Na~+/K~+离子混合通透,Cs~+、ZD7288可以阻断I_(hHCN4);胺碘酮可以抑制I_(hHCN4),这些不仅为起搏通道HCN4基因治疗缓慢性心律失常提供理论基础,同时也为胺碘酮治疗由于起搏电流异常增加所致的快速型心律失常提供实验依据。
Background:The implantation of electronic devices has become the preferred treatment for symptomatic bradyarrhythmias with excellent success and minimal morbidity. The shortcomings of electronic pacemakers include limited battery life, need for lead implantation into heart, which in most cases can no longer be removed, and lack of response to autonomic and physiologic demands on the heart. Furthermore, cardiac pacing in the pediatric age still needs improvements, as the devices cannot follow the somatic growth of the little patients and have to be changed over the years. Nonetheless, the ideal therapy for these disorders may be the development of a biological solution allowing reconstitution of the physiological electrical activity of the cardiac conduction system with the same plasticity and adaptability to the human body and to the physiology of the cardiovascular system. At present, molecular approaches to the development of a biological pacemaker are a conceptually attractive alternate treatment modality for bradyarrhythmias. In this field, both gene and stem cell therapies represent new and promising strategies for the development of a biological pacemaker. This paper will focus on hyperpolarization-activated cyclic nucleotide-gated channel HCN4 gene lentiviral transfection to create biological-pacemaker cells in vitro.
PartⅠ
Culture of Mesenchymal Stem Cells from Porcine Bone Marrow and Transformation to Myogenic Cell Induced by 5-aza
Section A
Culture of Mesenchymal Stem Cells from Swine Bone Marrow
Objective: By establishing a method of the culture of swine mesenchymal stem cells (MSCs) from bone marrow, a new cell source for the cardiomyocytes induced by 5-aza.
Methods: MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with flowcytometer.
Results: The adherent, fibroblast-like cells were confluent in single layer after plating for 10～12 days. The cell cycle analysis showed that 73% of MSCs was in G_0/G_1 phase.
Conclusion: Porcine MSCs can be isolated from postnatal bone marrow through their adherent ability. It is suggesting that MSCs may be a new cell source for the cardiomyocytes induced by 5-aza.
Section B
Mesenchymal Stem Cells Transformed to Myogenic Cell Induced by 5-azacytidine inVitro
Objective: By establishing a method for the culture of porcine mesenchymal stem cells(MSCs) from bone marrow and the transformation to myogenic cells in vitro, a new cell source to create biological-pacemaker cells in vitro.
Methods: MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. After being co-cultured with 5-azacytidine (5-aza) for 24h, the cultured cells were evaluated by immunohistochemical stains.
Results: After being co-cultured with 5-aza, some of MSCs became spindle-like and were found to be stained positively with desmin, MHC, cTnI, and Cx-43.
Conclusion: Porcine MSCs can be isolated from postnatal bone marrow through their adherentability. Myogenic cells can be generated from MSCs in vitro. It is suggesting that MSCs may be a new cell source to create biological-pacemaker cells .
PartⅡ
Construct the Shuttle Plasmid pCDHl-GFP-HCN4 and Pack theRecombined Lentivirus
Section A
Construct the Shuttle Plasmid pCDH1-GFP-HCN4
Objective: HCN4 gene fragment to be unloaded from plasmid pCDNA3-hHCN4 generously provided by Dr Stieber, and to be cloned into the shuttle plasmid pCDHl-GFP-HCN4. HCN4 gene fragment to be unloaded from plasmid pCDNA3-hHCN4 generously provided by Dr Stieber, and to be cloned into the shuttle plasmid pCDH1-GFP-HCN4.
Methods: HCN4 gene fragment was unloaded from plasmid pCDNA3-hHCN4 and cloned into plasmid Puc19 by Enzymes Eco RI and XbaⅠ; then the target gene was cutted and cloned into plasmid pcDNA3.1 (A) by Enzymes Eco RI and HindⅢ. In the end, HCN4 gene fragment was cutted into plasmid pCDH1-MCS1 -EF1-copGFP by Enzymes XbaⅠ. Double Enzymes XbaⅠand EcoR I verified approach and analysising the sequence were used to confirm the positive plasmid. The positive plasmid was then transfected into 293 cells and the MCSc.
Results: HCN4 gene fragment was cloned into the plasmid pCDH1-MCS1 -EF1-copGFP confirmed by Enzymes XbaⅠand EcoRI and the sequence in the positive plasmid was confirmed by comparison with the published gene bank; GFP was observed in the transfected 293 cells and the MSCs under the fluorescent microscope.
Conclusion: HCN4 gene fragment was successfully unloaded from plasmid pCDNA3-hHCN4 cloned into the shuttle plasmid pCDHl-GFP-HCN4 which would be constructed lentivirus in the next step.
Section B
Pack the Recombined Lentivirus
Objective: Using the pPACKH1-Lentivector Packaging Kit to construct HCN4 recombined lentivirus.
Methods: HCN4 recombined lentivirus was constructed according to operating manual of Lentivector Expression System. pPACKHl- Lentivector Packaging Kit with three plasmids was used to transfect MSCs and 293 T cells. After 48 hours culturing, GFP (green) was detected by the fluorescence microscope.
Results: The recombined lentivirus was used to transfect MSCs and 293 T cells and GFP was observed in the transfected 293 cells and the MSCs under the fluorescent microscope. The efficiency of infection was above 90 percents.
Conclusion:The recombined lentivirus was successfully constructed and had high efficiency of infection.
PartⅢ
Using an HIV-Based Lentiviral Vector to Transfect Mesenchymal Stem Cells to Create Biological-Pacemaker Cells in Vitro
Objective: Using HCN4 recombined lentivirus to transfect Mesenchymal stem cells to make them Stably overexpressing HCN4 to create biological-pacemaker cells in vitro.
Methods: MSCs were transfected by HCN4 recombined lentivirus according to operating manual of Lentivector Expression System. After two weeks' culturing, GFP (green) was detected by the fluorescence microscope. Protein was extracted for Western blot analysis and real time RT-PCR was carried out using superscript one step RT-PCR with Promega Taq kits and Sybergreen. GAPDH was used as a housekeeping gene. Whole-cell patch clamp to study membrane currents in control MSCs and those transfected with hHCN4 and the state of transfected MSCs long-term cultured were observed.
Results: After 48 hours culturing, GFP (green) was detected in transfected MSCs by the fluorescence microscope. Protein expression by hHCN4 was confirmed by comparison with the published one according Western blot analysis. The expression intensity of HCN4 in MSCs remained strong 2 weeks later with detected by real time RT-PCR. Whole-cell patch clamp recorded an inward current in response to hyperpolarization which was time-dependent and voltage-dependent. Spontaneous beating in cell group was found in the microscope and the beating rate was 15±lbpm after the transfected MSCs were cultered for 2 months.
Conclusion: Biological-pacemaker cells were successfully created by HCN4 recombined lentivirus to transfect Mesenchymal stem cells, which were stably overexpressed HCN4 in vitro.
PartⅣ
Electrophysiological characterization of the expressed hHCN4 channel inthe MSCs transfected with lentiviris
Objective: Whole-cell patch clamp to study membrane currents in the MSCs transfected with hHCN4 and the electrophysiology feature of hHCN4 channel.
Methods: Whole-cell patch clamp was studied the electrophysiology feature of membrane currents in the MSCs transfected with hHCN, such as activation curves of hHCN4 current, curve of voltage dependence of activation kinetics and effect of extracellular K~+、Na~+、TTX、TEA、cAMP、ISO、Ach、Cs~+、ZD7288 and amiodarone on the HCN4 currents.
Results:
1.In whole-cell voltage-clamp mode, hyperpolarizing voltage steps negative to -80 mV induced inward currents in hHCN4 expressing cells. It began to slowly active in voltage and time dependent manner without deactivation.The membrane potential of threshold voltage (V_(th)) and half-maximal activation (V_(1/2)) obtained by fits to Boltzmann equations was-83±8mV and-108±2mV (n=10) for the hHCN4 current. The hHCN4 activation time constants ranged from 0.66±0.1s (n = 10) at -140 mV to 3.1±0.7s (n = 10) at -110 mV. The reversal potential was -21.2±2.3mV(n=10). The relative permeability ratio for Na~+ versus K~+ (P_(Na)/P_K), as determined by the Goldmann-Hodgkin-Katz equation, was 0.23.
2.At -140mV, I_(hHCN4) current density decrease from -300±25pA/pF~(-1) to -87±10 pA/pF~(-1) with reducing the extracellular Na~+ concentration from 110 mmol/Lto 50 mmol/L when the extracellular K~+ concentration was 30 mmol/L and decrease from -302±30pA/pF~(-1) to -244±19 pA/pF~(-1) with reducing the extracellular K~+ concentration from 30 mmol/Lto 5 mmol/L when the extracellular Na~+ concentration was 110 mmol/L.
3. Neither current was sensitive to TTX nor TEA, two classic potassium channel blockers.
4. In whole-cell mode measurements, I_(hHCN4) did not change in the presence of 1 mM cAMP in the bath solution for 10 minutes. But 1 mmol/L cAMP increased the hHCN4 current by shifting the activation curves 17 mV in the positive direction when the cell exposed to 1 mM cAMP intracellular solution. The V_(1/2) values for the hHCN4 current were -109±2mV in the absence and -92±2mV in the presence of 1 mM cAMP in the intracellular solution for 10 minutes (n=10, p<0.05).
5.Application of 1μmol/L ISO to the extracellular solution resulted in lightly accelerated I_(hHCN4)·And I_(hHCN4) did not change in the presence of 1 umol/L Ach in the bath solution for 10 minutes.
6. The I_(hHCN4) was blocked by low concentrations of extracellular Cs~+. The IC50 was 128.8±28.1μmol/L and the block was released by (extracellular) washout. The I_(hHCN4) was blocked by low concentrations of extracellular ZD7288 but the block could not be released by (extracellular) washout.
7. Amiodarone decreased the peak current of I_(hHCN4) and could block the current. The IC50 was 1.34±0.33μmol/L and the block could not be released by (extracellular) washout. The hHCN4 activation time constants prolonged at every clamp voltage.
Conclusion:The current of expressed hHCN4 channel in the MSCs transfected with lentivirus has the electrophysiological characterization of native If, that is, activation upon membrane hyperpolarization, slow activation in voltage and time dependent manner without deactivation, combine-conduction of Na~+ and K~+, enhancement by cAMP and blockage by Cs~+、ZD7288 and amiodarone. This experment provides theory ground for biological-pacemaker study in treatment of arrhythmia.
第一部分猪骨髓间充质干细胞(MSCs)的分离、培养及5-aza诱导其转化为心肌样细胞的研究
第一节猪骨髓间充质干细胞(MSCs)的分离、培养
目的:建立猪骨髓间充质干细胞体外培养方法,为5-aza诱导转化为心肌样细胞提供材料。
方法:猪的骨髓MSCs分离纯化后,流式细胞仪检测细胞周期。
结果:体外培养的原代MSCs 10～14d达到融合,细胞周期显示73%的细胞处于G_0/G_1期。
结论:根据黏附特性分离的猪骨髓MSCs在体外条件下可生长扩增,可用于5-aza诱导转化为心肌样细胞。
第二节5-aza体外诱导MSCs分化为心肌样细胞的研究
目的:建立猪骨髓间充质干细胞体外转化为心肌样细胞的方法,为构建生物起搏细胞提供新材料。
方法:猪的骨髓MSCs分离纯化后,用5-aza诱导后,用抗结蛋白(desmin)、肌球蛋白重链(MHC)、心肌特异性肌钙蛋白I(cTnI)、连接蛋白-43(Cx-43)进行免疫细胞化学染色鉴定。
结果:体外培养的第二代MSCs经5-aza诱导后,部分MSCs呈梭形,阳性表达desmin、MHC、cTnI和Cx-43。
结论:根据黏附特性分离的猪骨髓MSCs在体外条件下可分化为心肌样细胞,可用于构建生物起搏细胞。
第二部分穿梭质粒pCDH1-GFP-HCN4的构建及HCN4重组慢病毒的包装
第一节穿梭质粒pCDH1-GFP-HCN4的构建
目的:将全长约3.6kb的hHCN4目的基因片段从Dr Stieber教授惠赠的pCDNA3-hHCN4质粒卸载下来,最终装载到带有GFP绿色荧光蛋白标记的去内毒素穿梭质粒pCDH1-MCS1-EF1-copGFP上。
方法:用Eco RI和XbaⅠ从pCDNA3-hHCN4质粒切下hHCN4目的片段连接到Puc19载体中,再用EcoRI和HindⅢ从质粒Puc19切下目的片段连接到pcDNA3.1(A)载体中,再用XbaⅠ单酶从质粒pcDNA3.1(A)双切到pCDH1-MCS1-EF1-copGFP中。用XbaⅠ或者Eco RI鉴定,将阳性克隆质粒送上海生工测序,并将构建的目的基因质粒转染293细胞和猪骨髓间充质干细胞。
结果:XbaⅠ及Eco RI鉴定表明,PCR的产物和克隆扩增的产物的电泳结果该基因的大小约为3.6kb;阳性克隆质粒测序结果与GENEBANK目的基因HCN4序列完全相同;构建的目的基因质粒转染293细胞和原代的猪骨髓间充质干细胞后,均发现有强烈的绿色荧光的出现。
结论:成功将全长hHCN4目的基因片段从pCDNA3-hHCN4质粒卸载下来,装载到去内毒素穿梭质粒pCDH1-MCS1-EF1-copGFP上,可用于下一步慢病毒的包装。
第二节HCN4重组慢病毒的包装
目的:使用慢病毒包装系统(pPACKH1-Lentivector Packaging Kit)建立稳定的HCN4重组慢病毒。
方法:实验过程参照SBI的Lentivector Expression System的操作手册进行。将构建好的慢病毒包装质粒混合物混合后感染293T细胞及心肌样骨髓基质干细胞。
结果:构建的慢病毒包装质粒混合物转染293细胞和心肌样猪骨髓间充质干细胞后,均发现有强烈的绿色荧光的出现,细胞感染效率可以达到90%以上。
结论:成功建立稳定的hHCN4重组慢病毒,该病毒载体有很高的转染效率。第三部分慢病毒转染基质干细胞构建起搏样细胞的体外研究
目的:使用hHCN4重组慢病毒载体转染心肌样骨髓基质干细胞获得稳定过表达HCN4基因的起搏样干细胞。
方法:慢病毒转染心肌样骨髓基质干细胞实验过程参照SBI的LentivectorExpression System的操作手册进行;稳定转染细胞继续培养两周后行Western blot鉴定蛋白的表达并行real-time RT-PCR实时荧光定量检测HCN4目的基因的表达情况;全细胞膜片钳记录转染细胞HCN4通道电流的表达;转染细胞体外长期培养观察细胞的转染状态。
结果:病毒感染48小时后,目的细胞出现大量的荧光;Western blot检测结果见图,可见一约170kDa条带,与克隆hHCN4通道蛋白分子量相符;RT-PCR显示慢病毒转染的细胞hHCN4-RNA是原代细胞的123倍;膜片钳记录在转染细胞中可记录到明显的电压与时间依赖性的超极化内向电流,即If电流;将转染的心肌样骨髓基质干细胞培养至2个月时,显微镜下发现细胞呈群体性搏动,20℃室温下的搏动频率为15±1次/分。该病毒载体有很高的转染效率,在体外已获得至少8个月的阳性表达,8个月后因为细胞污染未进一步观察。
结论:hHCN4重组慢病毒转染心肌样骨髓基质干细胞获得了稳定过表达HCN4基因的起搏样干细胞。这种基因修饰后的心肌样干细胞有可能成为一种有效的生物起搏种子细胞,并期待在缓慢性心律失常的治疗技术上实现新突破。
第四部分稳定转染hHCN4起搏通道亚型的电生理特点
目的:全细胞膜片钳技术记录稳定转染pCDH1-GFP-HCN4的心肌样骨髓基质干细胞,了解hHCN4起搏通道亚型的电生理特点。
方法:全细胞膜片钳技术记录稳定转染pCDH1-GFP-HCN4的心肌样骨髓基质干细胞,观察hHCN4通道电流、通道电流激活曲线及电压依赖性激活时间常数动力学曲线;改变细胞外液K~+、Na~+浓度,观察K~+、Na~+对I_(hHCN4)电流的影响;以河豚毒素(TTX,I_(Na)阻断剂)、四乙胺(TEA,I_K阻断剂)、环磷酸腺苷(cAMP)、异丙基肾上腺素(ISO)、乙酰胆碱(Ach)、铯(Cs~+)、ZD7288(If电流特异性阻断剂)和胺碘酮分别灌流,观察它们对I_(hHCN4)电流的影响。
结果:
1.大多数稳定转染的心肌样骨髓干细胞可记录到约-80mV开始激活的一系列超极化内向离子流(I _(hHCN4_电流),该电流激活缓慢,呈电压、时间依赖性。电流的激活电位(V_(th))、半最大激活电位(V_(1/2))分别为-83±8mV、-108±2mV,斜率为-11.2±0.6 mV(n=10)。钳制电压-140mV时,激活时间常数为0.66±0.1s;钳制电压-110mV时,激活时间常数为3.1±0.7s。I_(hHCN4)的反转电位21.2±2.3mV(n=10),P_(Na)/P_K为0.23。
2.钳制电位-140mV,外液K~+浓度30 mmol/L,Na~+浓度由110 mmol/L降为50mmol/L,I_(hHCN4)电流密度分别为-300±25pA/pF~(-1)、-87±10 pA/pF~(-1)(n=8,p＜0.001),减少73%;外液Na~+浓度110 mmol/L,K~+浓度由30 mmol/L降为5 mmol/L,I_(hHCN4)电流密度分别为-302±30pA/pF~(-1)、-244±19 pA/pF~(-1)(n=8,p＜0.05),减少20%。
3.TTX和TEA灌流10min,对I_(hHCN4)没有影响。
4.1mmol/L cAMP灌流10min,I_(hHCN4)无明显变化,电极内液中加入终浓度为1mmol/L的cAMP,I_(hHCN4)激活加快,激活时间加快,V_(1/2)由-109±2mV变为-92±2mV,向正向移动17mV(n=10,p＜0.05),激活阈值及最大激活电流没有明显改变
5.1μmol/L ISO灌流10min,I_(hHCN4)略加快。1μmol/L Ach灌流10min,I_(hHCN4)无明显改变。
6.低浓度Cs~+可阻滞I_(hHCN4),冲洗后抑制作用完全恢复,半效抑制浓度(IC50)为128.8±28.1μmol/L;较低浓度ZD7288可阻滞I_(hHCN4),冲洗后抑制作用不能恢复,IC50为23.8±5.7μmol/L。
7.胺碘酮可阻滞I_(hHCN4),冲洗后抑制作用无明显恢复,IC50为1.34±0.33μmol/L;胺碘酮使各钳制电压下I_(hHCN4)激活缓慢。
结论:重组HCN4慢病毒转染心肌样干细胞,其表达的I_(hHCN4)电流具有天然If电流特点,表现为电压与时间依赖性的超极化内向电流、cAMP直接激活、Na~+/K~+离子混合通透,Cs~+、ZD7288可以阻断I_(hHCN4);胺碘酮可以抑制I_(hHCN4),这些不仅为起搏通道HCN4基因治疗缓慢性心律失常提供理论基础,同时也为胺碘酮治疗由于起搏电流异常增加所致的快速型心律失常提供实验依据。
Background:The implantation of electronic devices has become the preferred treatment for symptomatic bradyarrhythmias with excellent success and minimal morbidity. The shortcomings of electronic pacemakers include limited battery life, need for lead implantation into heart, which in most cases can no longer be removed, and lack of response to autonomic and physiologic demands on the heart. Furthermore, cardiac pacing in the pediatric age still needs improvements, as the devices cannot follow the somatic growth of the little patients and have to be changed over the years. Nonetheless, the ideal therapy for these disorders may be the development of a biological solution allowing reconstitution of the physiological electrical activity of the cardiac conduction system with the same plasticity and adaptability to the human body and to the physiology of the cardiovascular system. At present, molecular approaches to the development of a biological pacemaker are a conceptually attractive alternate treatment modality for bradyarrhythmias. In this field, both gene and stem cell therapies represent new and promising strategies for the development of a biological pacemaker. This paper will focus on hyperpolarization-activated cyclic nucleotide-gated channel HCN4 gene lentiviral transfection to create biological-pacemaker cells in vitro.
PartⅠ
Culture of Mesenchymal Stem Cells from Porcine Bone Marrow and Transformation to Myogenic Cell Induced by 5-aza
Section A
Culture of Mesenchymal Stem Cells from Swine Bone Marrow
Objective: By establishing a method of the culture of swine mesenchymal stem cells (MSCs) from bone marrow, a new cell source for the cardiomyocytes induced by 5-aza.
Methods: MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with flowcytometer.
Results: The adherent, fibroblast-like cells were confluent in single layer after plating for 10～12 days. The cell cycle analysis showed that 73% of MSCs was in G_0/G_1 phase.
Conclusion: Porcine MSCs can be isolated from postnatal bone marrow through their adherent ability. It is suggesting that MSCs may be a new cell source for the cardiomyocytes induced by 5-aza.
Section B
Mesenchymal Stem Cells Transformed to Myogenic Cell Induced by 5-azacytidine inVitro
Objective: By establishing a method for the culture of porcine mesenchymal stem cells(MSCs) from bone marrow and the transformation to myogenic cells in vitro, a new cell source to create biological-pacemaker cells in vitro.
Methods: MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. After being co-cultured with 5-azacytidine (5-aza) for 24h, the cultured cells were evaluated by immunohistochemical stains.
Results: After being co-cultured with 5-aza, some of MSCs became spindle-like and were found to be stained positively with desmin, MHC, cTnI, and Cx-43.
Conclusion: Porcine MSCs can be isolated from postnatal bone marrow through their adherentability. Myogenic cells can be generated from MSCs in vitro. It is suggesting that MSCs may be a new cell source to create biological-pacemaker cells .
PartⅡ
Construct the Shuttle Plasmid pCDHl-GFP-HCN4 and Pack theRecombined Lentivirus
Section A
Construct the Shuttle Plasmid pCDH1-GFP-HCN4
Objective: HCN4 gene fragment to be unloaded from plasmid pCDNA3-hHCN4 generously provided by Dr Stieber, and to be cloned into the shuttle plasmid pCDHl-GFP-HCN4. HCN4 gene fragment to be unloaded from plasmid pCDNA3-hHCN4 generously provided by Dr Stieber, and to be cloned into the shuttle plasmid pCDH1-GFP-HCN4.
Methods: HCN4 gene fragment was unloaded from plasmid pCDNA3-hHCN4 and cloned into plasmid Puc19 by Enzymes Eco RI and XbaⅠ; then the target gene was cutted and cloned into plasmid pcDNA3.1 (A) by Enzymes Eco RI and HindⅢ. In the end, HCN4 gene fragment was cutted into plasmid pCDH1-MCS1 -EF1-copGFP by Enzymes XbaⅠ. Double Enzymes XbaⅠand EcoR I verified approach and analysising the sequence were used to confirm the positive plasmid. The positive plasmid was then transfected into 293 cells and the MCSc.
Results: HCN4 gene fragment was cloned into the plasmid pCDH1-MCS1 -EF1-copGFP confirmed by Enzymes XbaⅠand EcoRI and the sequence in the positive plasmid was confirmed by comparison with the published gene bank; GFP was observed in the transfected 293 cells and the MSCs under the fluorescent microscope.
Conclusion: HCN4 gene fragment was successfully unloaded from plasmid pCDNA3-hHCN4 cloned into the shuttle plasmid pCDHl-GFP-HCN4 which would be constructed lentivirus in the next step.
Section B
Pack the Recombined Lentivirus
Objective: Using the pPACKH1-Lentivector Packaging Kit to construct HCN4 recombined lentivirus.
Methods: HCN4 recombined lentivirus was constructed according to operating manual of Lentivector Expression System. pPACKHl- Lentivector Packaging Kit with three plasmids was used to transfect MSCs and 293 T cells. After 48 hours culturing, GFP (green) was detected by the fluorescence microscope.
Results: The recombined lentivirus was used to transfect MSCs and 293 T cells and GFP was observed in the transfected 293 cells and the MSCs under the fluorescent microscope. The efficiency of infection was above 90 percents.
Conclusion:The recombined lentivirus was successfully constructed and had high efficiency of infection.
PartⅢ
Using an HIV-Based Lentiviral Vector to Transfect Mesenchymal Stem Cells to Create Biological-Pacemaker Cells in Vitro
Objective: Using HCN4 recombined lentivirus to transfect Mesenchymal stem cells to make them Stably overexpressing HCN4 to create biological-pacemaker cells in vitro.
Methods: MSCs were transfected by HCN4 recombined lentivirus according to operating manual of Lentivector Expression System. After two weeks' culturing, GFP (green) was detected by the fluorescence microscope. Protein was extracted for Western blot analysis and real time RT-PCR was carried out using superscript one step RT-PCR with Promega Taq kits and Sybergreen. GAPDH was used as a housekeeping gene. Whole-cell patch clamp to study membrane currents in control MSCs and those transfected with hHCN4 and the state of transfected MSCs long-term cultured were observed.
Results: After 48 hours culturing, GFP (green) was detected in transfected MSCs by the fluorescence microscope. Protein expression by hHCN4 was confirmed by comparison with the published one according Western blot analysis. The expression intensity of HCN4 in MSCs remained strong 2 weeks later with detected by real time RT-PCR. Whole-cell patch clamp recorded an inward current in response to hyperpolarization which was time-dependent and voltage-dependent. Spontaneous beating in cell group was found in the microscope and the beating rate was 15±lbpm after the transfected MSCs were cultered for 2 months.
Conclusion: Biological-pacemaker cells were successfully created by HCN4 recombined lentivirus to transfect Mesenchymal stem cells, which were stably overexpressed HCN4 in vitro.
PartⅣ
Electrophysiological characterization of the expressed hHCN4 channel inthe MSCs transfected with lentiviris
Objective: Whole-cell patch clamp to study membrane currents in the MSCs transfected with hHCN4 and the electrophysiology feature of hHCN4 channel.
Methods: Whole-cell patch clamp was studied the electrophysiology feature of membrane currents in the MSCs transfected with hHCN, such as activation curves of hHCN4 current, curve of voltage dependence of activation kinetics and effect of extracellular K~+、Na~+、TTX、TEA、cAMP、ISO、Ach、Cs~+、ZD7288 and amiodarone on the HCN4 currents.
Results:
1.In whole-cell voltage-clamp mode, hyperpolarizing voltage steps negative to -80 mV induced inward currents in hHCN4 expressing cells. It began to slowly active in voltage and time dependent manner without deactivation.The membrane potential of threshold voltage (V_(th)) and half-maximal activation (V_(1/2)) obtained by fits to Boltzmann equations was-83±8mV and-108±2mV (n=10) for the hHCN4 current. The hHCN4 activation time constants ranged from 0.66±0.1s (n = 10) at -140 mV to 3.1±0.7s (n = 10) at -110 mV. The reversal potential was -21.2±2.3mV(n=10). The relative permeability ratio for Na~+ versus K~+ (P_(Na)/P_K), as determined by the Goldmann-Hodgkin-Katz equation, was 0.23.
2.At -140mV, I_(hHCN4) current density decrease from -300±25pA/pF~(-1) to -87±10 pA/pF~(-1) with reducing the extracellular Na~+ concentration from 110 mmol/Lto 50 mmol/L when the extracellular K~+ concentration was 30 mmol/L and decrease from -302±30pA/pF~(-1) to -244±19 pA/pF~(-1) with reducing the extracellular K~+ concentration from 30 mmol/Lto 5 mmol/L when the extracellular Na~+ concentration was 110 mmol/L.
3. Neither current was sensitive to TTX nor TEA, two classic potassium channel blockers.
4. In whole-cell mode measurements, I_(hHCN4) did not change in the presence of 1 mM cAMP in the bath solution for 10 minutes. But 1 mmol/L cAMP increased the hHCN4 current by shifting the activation curves 17 mV in the positive direction when the cell exposed to 1 mM cAMP intracellular solution. The V_(1/2) values for the hHCN4 current were -109±2mV in the absence and -92±2mV in the presence of 1 mM cAMP in the intracellular solution for 10 minutes (n=10, p<0.05).
5.Application of 1μmol/L ISO to the extracellular solution resulted in lightly accelerated I_(hHCN4)·And I_(hHCN4) did not change in the presence of 1 umol/L Ach in the bath solution for 10 minutes.
6. The I_(hHCN4) was blocked by low concentrations of extracellular Cs~+. The IC50 was 128.8±28.1μmol/L and the block was released by (extracellular) washout. The I_(hHCN4) was blocked by low concentrations of extracellular ZD7288 but the block could not be released by (extracellular) washout.
7. Amiodarone decreased the peak current of I_(hHCN4) and could block the current. The IC50 was 1.34±0.33μmol/L and the block could not be released by (extracellular) washout. The hHCN4 activation time constants prolonged at every clamp voltage.
Conclusion:The current of expressed hHCN4 channel in the MSCs transfected with lentivirus has the electrophysiological characterization of native If, that is, activation upon membrane hyperpolarization, slow activation in voltage and time dependent manner without deactivation, combine-conduction of Na~+ and K~+, enhancement by cAMP and blockage by Cs~+、ZD7288 and amiodarone. This experment provides theory ground for biological-pacemaker study in treatment of arrhythmia.
引文
1.Lindolfo SM, Nance B. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. British Journal of Hematology, 2003, 123:702-711.
2.Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve, 1995,18:1412-1416.
3.Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluriporency of mesenchymal stem cells derived from adult marrow. Nature, 2002,418:41-49.
4.Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997,276:71-74.
5.Woodburry D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J neurosic Res,2000,61:364-370.
6. Pittenger MF, Mackay AM, Jaiswal SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284:143-147.
7. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol,2000,28(8):875-884.
8. Conger PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J cell physiol, 1999,181:67-74.
9. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate in fracted myocardium. Nature, 2001,410:701-705.
10. Peterson BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science, 1999,284:1168-1170.
11. Fukuda K. Reprogramming of bone marrow mesenchymal stem cells into cardiomyo- cytes. C. R. Biologies, 2002(325):1027-1038.
1.Ye NS, Zhang RL. Zhao YF. et al Effect of 5-azacytidine on the protein expression of porcine b ne marrow mesenchymal stem cells in vitro. Genomics Proteomics Bioinformatics 2006; 4(1):18-25.
2.Wakitani S, Saito T, Caplan AL Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve, 1995,18:1412-1416.
3.Liu Y, Song J, Liu W, et al. Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res,2003, 58: 460-466.
4.Panteghini M. Present issues in the determination of troponins and other markers of cardiac damage. Clin Biochem 2000; 33(3): 161-166.
5.Moscoso I, centmo A, Jopez E, et al. Diferentiation in vitro of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation. Transplant Proc 2005; 37(1):481-482.
6.Condorelli G, Borello U, De Angelis L, et al. Cardiomyocytes induce endothelial cells to transdifferentiate into cardiac muscle: Implications for myocardium regeneration. Proc Natl Acad Sci, USA. 2001, 98(19):10733-10738.
7.Wang J, Shumtim D, Galipeau J, et al .Marrow stromal cells for cellular cardiomyoplasty:feasibility and potential clinical advantages. J Thorac Cardiovasc Surg, 2000,120:999-1006.
8.RubioD, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res. 2005, 65: 3035-3039.
9.Xu M, Wan i M, Dai YS, et al. Diferentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes. Circulation, 2004, 110: 2658-2665. 14.
10.Tomita S, Li RK, Weisel RD, et al .Autologous transplantation of bone marrow cells improves damaged heart functions. Circulation, 1999,100(Suppl II):245-256.
11 .Makino S, Fukuda K, Miyoshi S, et al .Cardiomyocytes can be generated from marrow stromal cells in vitro. Journal Clinical Investigation, 1999,103:687-705.
1.El Chemaly A, Magaud C, Patri S, et al.The heart rate-lowering agent ivabradine inhibits the pacemaker current I(f) in human atrial myocytes.J Cardiovasc Electrophysiol 2007,18(11):1190-6.
2.Accili EA, Proenza C, Baruscottil M, etal. From funny current to HCN channels:20 years of excitation.News Physiol Sci 2002,17(1):32-6.
3.Herrmann S, Stieber J, Ludwig A.Pathophysiology of HCN channels.Pflugers Arch 2007,454(4):517-22.
4.DiFrancesco D, Ferroni A, Mazzanti M, et al. Properties of the hyperpolarizing- activated current (If) in cells isolated from the rabbit sino-atrial node. J Physiol (Lond) 1986,37(7):61-7.
5.Keating .MT and sanguinetti MC.Molecular and cellular mechanisms of cardiac arrhythmias.Cell 2001, 104(3):569-580.
6.Ishii TM, Takano M, Xie LH, et al. Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J Biol Chem 1999, 274(8): 12,835-39.
7.Qu J, Kryukova Y,Potapova I,et al. MiRPl modulates HCN2 channel expression and gating in cardiac myocytes.Biological Chemistry J 2004,279(40):43497-506.
8.Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc Med 2004,14:23-8.
9.Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci USA 2003,100(11):15235-40.
10.Marionneau C, Couette B, Liu J, et al. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol 2005,562(2):223-34.
11.Bucchi A, Tognati A, Milanesi R, et al. Properties of ivabradine-induced block of HCN1 and HCN4 pacemaker channels.J Physiol. 2006 , 572(Pt 2):335-46.
12.Santoro B, Liu DT, Yao H, et al.Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 1978, 93(4):717-29.
13.Whitaker GM, Angoli D, Nazzari H, et al. HCN2 and HCN4 isoforms self-assemble and co-assemble with equal preference to form functional pacemaker channels.J Biol Chem 2007,282(31):22900-9.
14.Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest 2002,111(12):1537- 45.
15.Ludwig A, Budde T, Stieber J, et al. Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2. Embo 1998,22:216-224.
1.Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 1996,272:263-267.
2.Naldini L, Blomer U, Gage F H, et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA, 1996, 92(21):11382-11388.
3.Zufferey R, Nagy D, Mandel R J, et al. Multiply attenuated lentiviral vector achieved efficient gene delivery in vivo. Nat Biotechnol, 1997, 15(9): 871-875.
4.Miyoshi H, Takshashi M, Gage F H, et al. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA, 1997,94(19):10319-10323.
5.Goldman M J, Lee P S, Yang J S, et al. Lentiviral vectors for gene therapy of cystic fibrosis. Hum Gene Thera, 1997, 8:2261-2268.
6.Parolin C, Sodroski J. A defective HIV-1 vector for gene transfer to human lymphocytes. J Mol Med, 1995, 73:279-288.
7. Lois C, Elizabeth J Hong, Shirley Pease, et al. Germline transmission and tissue-specific expression of transgenes delivered by Lentiviral vectors. Science, 2002, 295:868 -872.
8. Hofmann A, Kessler B, Ewerling S, et al. Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep, 2003,4(11): 1054-1060.
9. Ikawa M, Tanaka N, Kao WY, et al. Generation of transgenic mice using lentiviral vectors: a novel preclinical assessment of lentiviral vectors for gene therapy. Mol Ther, 2003,8 (4): 666-673.
1.Edelberg JM, Aird WC, Rosenberg RD. Enhancement of murine cardiac chronotropy by the molecular transfer of the human β2-adrenergic receptor cDNA. J Clin Invest, 1998,101: 337-343.
2.Miake J, Marba'n E, Nuss HB. Gene therapy: biological pacemaker created by gene transfer. Nature, 2002, 419: 132-133.
3.Qu J, Plotnikov AN, Danilo Jr P, et al. Expression and function of a biological pacemaker in canine heart. Circulation , 2003, 107: 1106-1109.
4.Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ,Brink PR, Cohen IS, Robinson RB, Danilo P Jr, Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptablerates. Circulation. 2004;109 (4):506-12.
5.Potapova I, Plotnikov A, Lu Z. Human mesenchymal stem cells as a gene delivery system to crate cardiac pacemakers. Circulation research. 2004;952-959.
6.Hung-Fat Tse, Tian Xue, Chu-Pak,et al. Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker HCN channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation. 2006;114:1000-1011.
7.Wang J, Salata JJ,Penna KD,et al. Stable expression and characterization of canine ether-a-go-go related gene(cerg) in HEK293 cells. Biophys J. 2001, 80: 213-214.
8.Herrmann S, Stieber J, Ludwig A.Pathophysiology of HCN channels.Pflugers Arch 2007,454(4):517-22.
9.Ya-Feng Zhou, Xiang-Jun Yang, Hong-Xia Li, et al. Mesenchymal stem cells transfected with HCN2 genes can be modified to be cardiac pacemaker cells. Med Hypotheses2007,69(5): 1093-7.
10.Ya-Feng Zhou, Xiang-Jun Yang, Hong-Xia Li. Hyperpolarization-activated cyclic nucleotide-gated channel gene: the most possible therapeutic applications in the field of cardiac biological pacemakers. Med Hypotheses 2007,69(3): 541-4.
11.Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc Med 2004,14:23-8.
1.Ishii TM, Takano M, Xie LH, et al.Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J Biol Chem 1999; 274:12,835-12,839.
2.Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc,Med, 2004,14:23-8.
3.Robinson RB, Siegelbaum SA. Hyperpolarization-activated cation currents: from molecules to physiological function. Annu Rec Physiol, 2003,65:453-480.
4.Stieber J, Herrmann S, Feil S, et al.The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embyonic heart. Proc Natl Acad Sci USA , 2003, 100: 15235-15240.
5.Pape HC. Queer current and pacemaker: the hyperpolarization -activated cation current in neurons.Annu Rec Physiol, 1996,58:299-327.
6.Keating MT and sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell ,2001;104:569-580.
7.Viscomi C, Altomare C,Bucchi A,et al. C Terminus-mediated Control of Voltage and cAMP Gating of Hyperpolarization-activated Cyclic Nucleotide-gated Channels. J Biol Chem, 2001, 276: 29930 - 29934.
8.Wang J, Chen S, Siegelbaum SA. et al. Regulation of Hyperpolarization -activated HCN Channel Gating and cAMP Modulation due to Interactions of COOH Terminus and Core Transmembrane Regions. J Gen Physiol, 2001,118:237-250.
9.Abi-Gerges N, Ji GJ, Lu ZJ, et al. Functional expression and regulation of the hyperpolarization acrivated non-selective cation currentin embryonic stem cell-derived cardiomyocytes. J Physiol, 2000,523:377-389.
10.Ludwig A, Zong X,Stieber J,et al. Two pacemaker channels from human heart with profoundly different activation kinetcs. EMBO J, 1999,18:2323-2329.
11.Macri V, Accili EA. Structural Elements of Instantaneous and Slow Gating in Hyperpolarization-activated Cyclic Nucleotide-gated Channels. J Biol Chem, 2004,279: 16832 -16846.
12.DiFrancesco. Cesium and the pacemaker current. J Cardiovasc Electrophysiol, 1995,6(12): 1152-1155.
13.BoSmith RE, Brigge I, Sturgess NC. Inhibitory actions of ZENECA ZD7288 on whole-cell hyperpolarization activated inward current (If) in guinea-pig dissociated sinoatrial node cells. Br J Pharmacol, 1993,110(1): 343-349.
14. Stieber J, Stockl G, Herrmann S,et al . Functional Expression of the Human HCN3 Channel. J Biol Chem, 2005,280: 34635 - 34643.
15.Cerbai E, Sartiani L, Depaoli P,et al. The properties of the pacemaker current I(F)in human ventricular myocytes are modulated by cardiac disease. J Mol Cell Cardiol, 2001, 33(3): 441-448.
16.Guido M, Fikret E, Ismil K,et al. Single-Channel Properties Support a Potential Contribution of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels and I_f to Cardiac Arrhythmias. Circulation, 2005,111: 399 - 404.
17.Maria FV,Nora GG,Gemma B,et al. Regional distribution of hyperpolarization-activated current (If) and hyperpolarization -activated cyclic nucleotide-gated channel mRNA expression in ventricular cells form control and hypertrophied rat hearts. J Physiol. 2003,553:395-405.
18.Cerbai E, Sartiani L, Depali P, et al. The properties of the pacemaker current If in human vertricular myocytes are modulated by cardiac disease. J Mol Cell Cardiol.2001,33:441-448.
19. Waldo A, Camm JA, deRuyter H, et al for the SWORD investigators. Effect of d-sotalol on mortality in patients with left ventricular dysfunction after recent and remote myocardial infarction. Lancet, 1996,348:7-12
20.Cairns JA, Connolly SJ, Roberts R, et al. Randomised trial of outcome after myocardial infarction in patients with frequent or repetitive ventricular premature depolarisations: CAMIAT. Lancet,1997,349:675-682.
21.Julian DG, Camm AJ,Frangin G, et al.Randomised trial of effect of amiodarone on mortality in patients with left-ventricular dysfunction after recent myocardial infarction: EMIAT. Lancet,1997,439:667-674.
22.Amiodarone Trials Meta-Analysis Investigators. Effect of prophyactic amiodarone on mortality after acute myocardial infarction and in congestive heart failure: meta-analysis of individual data from 6500 patients in randomized trials. Lancet,1997,350:1417-1424.
1.Seifert R,Scholten A,Gauss R,et al.Molecular characterization of a slowly gating human hyperpolarization- activated channel predominantly expressed in thalamus, heart, and testis[J].PNAS,1999,96:9391.
2.Keating MT and sanguinetti MC.Molecular and cellular mechanisms of cardiac arrhythmias.Cell 104:569-580,2001.
3.DiFrancesco D.Dual allosteric modulation of pacemaker(f) channels by cAMP and voltage in rabbit SA node[J].J physiol(Lond),l999,515:367.
4.Qu J, Kryukova Y,Potapova I,et al.MiRPl modulates HCN2 channel expression and gating in cardiac myocytes[J].Biological Chemistry J,2004,279(40):43497.
5.Brown HF, DiFrancesco D. Voltage clamp investigations of currents underlying pacemaker activity in rabbit sino-atrial node. J Physiol, 1980,308:331-51.
6.DiFrancesco D. A new interpretation of the pace-maker current in calf Purkinje fibres. J Physiol, 1981,314:359-76.
7.DiFrancesco D. A study of the ionic nature of the pacemaker current in calf Purkinje fibres. J Physiol, 1981,314:377-93.
8.Yanagihara K, Irisawa H. Inward current activated during hyperpolarization in the rabbit sino atrial node cell.Pflugers Arch, 1980, 385:11-19.
9.Accili EA, Proenza C, Baruscottil M, etal. From funny current to HCN channels:20 years of excitation[J].News Physiol Sci,2002,17:32.
10.DiFrancesco D, Ferroni A, Mazzanti M, et al. Properties of the hyperpolarizing-activated current (I_f) in cells isolated from the rabbit sino-atrial node[J]. J Physiol(Lond),1986,377:61.
11.DiFrancesco D. Characterization of single pacemaker channels in cardiac sino-atrial node cells. Nature, 1986, 324:470-73.
12.Andreas Ludig et al. HCN channels, From genes to function. In: Douglas p . Zipes,Jose Jalife,ed: Cardiac electrophysiology, From cell to bedside. Fourth edition. Saunders.pp. 59-65.
13. Graf EM, Heubach JF, Ravens U. The hyperpolarizationactivated current If in ventricular myocytes of non-transgenic and beta2-adrenoceptor overexpressing mice. Naunyn Schmiedebergs Arch Pharmacol, 2001, 364:131 -9.
14. Ishii TM, Takano M, Xie LH, et al.: 1999. Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J Biol Chem 274:12,835-12,839.
15. Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc,Med, 2004,14:23-8.
16.Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci USA, 2003,100:15235-40.
n.Marionneau C, Couette B, Liu J, et al. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol, 2005,562:223-34.
18. Ludwig A, Budde T, Stieber J, et al.: 1998.Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2. Embo J 22:216-224.
19. Herrmann S, Stieber J, Feil S, et al.: 1999.Pacemaker channel HCN4 is required for normal cardiac function in the mouse embryo.Naunyn-Schmiedeberg's Archive of Pharmacology 367 (Suppl 1): R91.
20. Santoro B, Liu DT, Yao H, et al.: 1978. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93:717-729.
21.Sarachek NS, Leonard JL: 1999. Familial heart block and sinus bradycardia. Classification and natural history. Am J Cardiol 29:451- 458.
22.Schulze-Bahr E, Neu A, Friederich P, et al.:2002. Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest 111:1537- 1545.
23. Edelberg JM, Aird WC, Rosenberg RD. Enhancement of murine cardiac chronotropy by the molecular transfer of the human P2-adrenergic receptor cDNA. J Clin Invest, 1998, 101: 337-343.
24. Miake J, Marba'n E, Nuss HB. Gene therapy: biological pacemaker created by gene transfer. Nature, 2002, 419: 132-133.
25.Qu J, Plotnikoy AN, Danilo Jr P, et al. Expression and function of a biological pacemaker in canine heart. Circulation , 2003, 107: 1106-1109.
26. Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P Jr, Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptablerates. Circulation. 2004;109 (4):506-12.
27. Potapova I, Plotnikov A, Lu Z. Human mesenchymal stem cells as a gene delivery system to crate cardiac pacemakers. Circulation research. 2004;952-959.
28.Hung-Fat Tse, Tian Xue, Chu-Pak,et al. Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker HCN channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation. 2006;114 :1000-1011.
2.Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve, 1995,18:1412-1416.
3.Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluriporency of mesenchymal stem cells derived from adult marrow. Nature, 2002,418:41-49.
4.Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science,1997,276:71-74.
5.Woodburry D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J neurosic Res,2000,61:364-370.
6. Pittenger MF, Mackay AM, Jaiswal SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999,284:143-147.
7. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol,2000,28(8):875-884.
8. Conger PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J cell physiol, 1999,181:67-74.
9. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate in fracted myocardium. Nature, 2001,410:701-705.
10. Peterson BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic oval cells. Science, 1999,284:1168-1170.
11. Fukuda K. Reprogramming of bone marrow mesenchymal stem cells into cardiomyo- cytes. C. R. Biologies, 2002(325):1027-1038.
1.Ye NS, Zhang RL. Zhao YF. et al Effect of 5-azacytidine on the protein expression of porcine b ne marrow mesenchymal stem cells in vitro. Genomics Proteomics Bioinformatics 2006; 4(1):18-25.
2.Wakitani S, Saito T, Caplan AL Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve, 1995,18:1412-1416.
3.Liu Y, Song J, Liu W, et al. Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res,2003, 58: 460-466.
4.Panteghini M. Present issues in the determination of troponins and other markers of cardiac damage. Clin Biochem 2000; 33(3): 161-166.
5.Moscoso I, centmo A, Jopez E, et al. Diferentiation in vitro of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation. Transplant Proc 2005; 37(1):481-482.
6.Condorelli G, Borello U, De Angelis L, et al. Cardiomyocytes induce endothelial cells to transdifferentiate into cardiac muscle: Implications for myocardium regeneration. Proc Natl Acad Sci, USA. 2001, 98(19):10733-10738.
7.Wang J, Shumtim D, Galipeau J, et al .Marrow stromal cells for cellular cardiomyoplasty:feasibility and potential clinical advantages. J Thorac Cardiovasc Surg, 2000,120:999-1006.
8.RubioD, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res. 2005, 65: 3035-3039.
9.Xu M, Wan i M, Dai YS, et al. Diferentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes. Circulation, 2004, 110: 2658-2665. 14.
10.Tomita S, Li RK, Weisel RD, et al .Autologous transplantation of bone marrow cells improves damaged heart functions. Circulation, 1999,100(Suppl II):245-256.
11 .Makino S, Fukuda K, Miyoshi S, et al .Cardiomyocytes can be generated from marrow stromal cells in vitro. Journal Clinical Investigation, 1999,103:687-705.
1.El Chemaly A, Magaud C, Patri S, et al.The heart rate-lowering agent ivabradine inhibits the pacemaker current I(f) in human atrial myocytes.J Cardiovasc Electrophysiol 2007,18(11):1190-6.
2.Accili EA, Proenza C, Baruscottil M, etal. From funny current to HCN channels:20 years of excitation.News Physiol Sci 2002,17(1):32-6.
3.Herrmann S, Stieber J, Ludwig A.Pathophysiology of HCN channels.Pflugers Arch 2007,454(4):517-22.
4.DiFrancesco D, Ferroni A, Mazzanti M, et al. Properties of the hyperpolarizing- activated current (If) in cells isolated from the rabbit sino-atrial node. J Physiol (Lond) 1986,37(7):61-7.
5.Keating .MT and sanguinetti MC.Molecular and cellular mechanisms of cardiac arrhythmias.Cell 2001, 104(3):569-580.
6.Ishii TM, Takano M, Xie LH, et al. Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J Biol Chem 1999, 274(8): 12,835-39.
7.Qu J, Kryukova Y,Potapova I,et al. MiRPl modulates HCN2 channel expression and gating in cardiac myocytes.Biological Chemistry J 2004,279(40):43497-506.
8.Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc Med 2004,14:23-8.
9.Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci USA 2003,100(11):15235-40.
10.Marionneau C, Couette B, Liu J, et al. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol 2005,562(2):223-34.
11.Bucchi A, Tognati A, Milanesi R, et al. Properties of ivabradine-induced block of HCN1 and HCN4 pacemaker channels.J Physiol. 2006 , 572(Pt 2):335-46.
12.Santoro B, Liu DT, Yao H, et al.Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 1978, 93(4):717-29.
13.Whitaker GM, Angoli D, Nazzari H, et al. HCN2 and HCN4 isoforms self-assemble and co-assemble with equal preference to form functional pacemaker channels.J Biol Chem 2007,282(31):22900-9.
14.Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest 2002,111(12):1537- 45.
15.Ludwig A, Budde T, Stieber J, et al. Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2. Embo 1998,22:216-224.
1.Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 1996,272:263-267.
2.Naldini L, Blomer U, Gage F H, et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA, 1996, 92(21):11382-11388.
3.Zufferey R, Nagy D, Mandel R J, et al. Multiply attenuated lentiviral vector achieved efficient gene delivery in vivo. Nat Biotechnol, 1997, 15(9): 871-875.
4.Miyoshi H, Takshashi M, Gage F H, et al. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci USA, 1997,94(19):10319-10323.
5.Goldman M J, Lee P S, Yang J S, et al. Lentiviral vectors for gene therapy of cystic fibrosis. Hum Gene Thera, 1997, 8:2261-2268.
6.Parolin C, Sodroski J. A defective HIV-1 vector for gene transfer to human lymphocytes. J Mol Med, 1995, 73:279-288.
7. Lois C, Elizabeth J Hong, Shirley Pease, et al. Germline transmission and tissue-specific expression of transgenes delivered by Lentiviral vectors. Science, 2002, 295:868 -872.
8. Hofmann A, Kessler B, Ewerling S, et al. Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep, 2003,4(11): 1054-1060.
9. Ikawa M, Tanaka N, Kao WY, et al. Generation of transgenic mice using lentiviral vectors: a novel preclinical assessment of lentiviral vectors for gene therapy. Mol Ther, 2003,8 (4): 666-673.
1.Edelberg JM, Aird WC, Rosenberg RD. Enhancement of murine cardiac chronotropy by the molecular transfer of the human β2-adrenergic receptor cDNA. J Clin Invest, 1998,101: 337-343.
2.Miake J, Marba'n E, Nuss HB. Gene therapy: biological pacemaker created by gene transfer. Nature, 2002, 419: 132-133.
3.Qu J, Plotnikov AN, Danilo Jr P, et al. Expression and function of a biological pacemaker in canine heart. Circulation , 2003, 107: 1106-1109.
4.Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ,Brink PR, Cohen IS, Robinson RB, Danilo P Jr, Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptablerates. Circulation. 2004;109 (4):506-12.
5.Potapova I, Plotnikov A, Lu Z. Human mesenchymal stem cells as a gene delivery system to crate cardiac pacemakers. Circulation research. 2004;952-959.
6.Hung-Fat Tse, Tian Xue, Chu-Pak,et al. Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker HCN channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation. 2006;114:1000-1011.
7.Wang J, Salata JJ,Penna KD,et al. Stable expression and characterization of canine ether-a-go-go related gene(cerg) in HEK293 cells. Biophys J. 2001, 80: 213-214.
8.Herrmann S, Stieber J, Ludwig A.Pathophysiology of HCN channels.Pflugers Arch 2007,454(4):517-22.
9.Ya-Feng Zhou, Xiang-Jun Yang, Hong-Xia Li, et al. Mesenchymal stem cells transfected with HCN2 genes can be modified to be cardiac pacemaker cells. Med Hypotheses2007,69(5): 1093-7.
10.Ya-Feng Zhou, Xiang-Jun Yang, Hong-Xia Li. Hyperpolarization-activated cyclic nucleotide-gated channel gene: the most possible therapeutic applications in the field of cardiac biological pacemakers. Med Hypotheses 2007,69(3): 541-4.
11.Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc Med 2004,14:23-8.
1.Ishii TM, Takano M, Xie LH, et al.Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J Biol Chem 1999; 274:12,835-12,839.
2.Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc,Med, 2004,14:23-8.
3.Robinson RB, Siegelbaum SA. Hyperpolarization-activated cation currents: from molecules to physiological function. Annu Rec Physiol, 2003,65:453-480.
4.Stieber J, Herrmann S, Feil S, et al.The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embyonic heart. Proc Natl Acad Sci USA , 2003, 100: 15235-15240.
5.Pape HC. Queer current and pacemaker: the hyperpolarization -activated cation current in neurons.Annu Rec Physiol, 1996,58:299-327.
6.Keating MT and sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell ,2001;104:569-580.
7.Viscomi C, Altomare C,Bucchi A,et al. C Terminus-mediated Control of Voltage and cAMP Gating of Hyperpolarization-activated Cyclic Nucleotide-gated Channels. J Biol Chem, 2001, 276: 29930 - 29934.
8.Wang J, Chen S, Siegelbaum SA. et al. Regulation of Hyperpolarization -activated HCN Channel Gating and cAMP Modulation due to Interactions of COOH Terminus and Core Transmembrane Regions. J Gen Physiol, 2001,118:237-250.
9.Abi-Gerges N, Ji GJ, Lu ZJ, et al. Functional expression and regulation of the hyperpolarization acrivated non-selective cation currentin embryonic stem cell-derived cardiomyocytes. J Physiol, 2000,523:377-389.
10.Ludwig A, Zong X,Stieber J,et al. Two pacemaker channels from human heart with profoundly different activation kinetcs. EMBO J, 1999,18:2323-2329.
11.Macri V, Accili EA. Structural Elements of Instantaneous and Slow Gating in Hyperpolarization-activated Cyclic Nucleotide-gated Channels. J Biol Chem, 2004,279: 16832 -16846.
12.DiFrancesco. Cesium and the pacemaker current. J Cardiovasc Electrophysiol, 1995,6(12): 1152-1155.
13.BoSmith RE, Brigge I, Sturgess NC. Inhibitory actions of ZENECA ZD7288 on whole-cell hyperpolarization activated inward current (If) in guinea-pig dissociated sinoatrial node cells. Br J Pharmacol, 1993,110(1): 343-349.
14. Stieber J, Stockl G, Herrmann S,et al . Functional Expression of the Human HCN3 Channel. J Biol Chem, 2005,280: 34635 - 34643.
15.Cerbai E, Sartiani L, Depaoli P,et al. The properties of the pacemaker current I(F)in human ventricular myocytes are modulated by cardiac disease. J Mol Cell Cardiol, 2001, 33(3): 441-448.
16.Guido M, Fikret E, Ismil K,et al. Single-Channel Properties Support a Potential Contribution of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels and I_f to Cardiac Arrhythmias. Circulation, 2005,111: 399 - 404.
17.Maria FV,Nora GG,Gemma B,et al. Regional distribution of hyperpolarization-activated current (If) and hyperpolarization -activated cyclic nucleotide-gated channel mRNA expression in ventricular cells form control and hypertrophied rat hearts. J Physiol. 2003,553:395-405.
18.Cerbai E, Sartiani L, Depali P, et al. The properties of the pacemaker current If in human vertricular myocytes are modulated by cardiac disease. J Mol Cell Cardiol.2001,33:441-448.
19. Waldo A, Camm JA, deRuyter H, et al for the SWORD investigators. Effect of d-sotalol on mortality in patients with left ventricular dysfunction after recent and remote myocardial infarction. Lancet, 1996,348:7-12
20.Cairns JA, Connolly SJ, Roberts R, et al. Randomised trial of outcome after myocardial infarction in patients with frequent or repetitive ventricular premature depolarisations: CAMIAT. Lancet,1997,349:675-682.
21.Julian DG, Camm AJ,Frangin G, et al.Randomised trial of effect of amiodarone on mortality in patients with left-ventricular dysfunction after recent myocardial infarction: EMIAT. Lancet,1997,439:667-674.
22.Amiodarone Trials Meta-Analysis Investigators. Effect of prophyactic amiodarone on mortality after acute myocardial infarction and in congestive heart failure: meta-analysis of individual data from 6500 patients in randomized trials. Lancet,1997,350:1417-1424.
1.Seifert R,Scholten A,Gauss R,et al.Molecular characterization of a slowly gating human hyperpolarization- activated channel predominantly expressed in thalamus, heart, and testis[J].PNAS,1999,96:9391.
2.Keating MT and sanguinetti MC.Molecular and cellular mechanisms of cardiac arrhythmias.Cell 104:569-580,2001.
3.DiFrancesco D.Dual allosteric modulation of pacemaker(f) channels by cAMP and voltage in rabbit SA node[J].J physiol(Lond),l999,515:367.
4.Qu J, Kryukova Y,Potapova I,et al.MiRPl modulates HCN2 channel expression and gating in cardiac myocytes[J].Biological Chemistry J,2004,279(40):43497.
5.Brown HF, DiFrancesco D. Voltage clamp investigations of currents underlying pacemaker activity in rabbit sino-atrial node. J Physiol, 1980,308:331-51.
6.DiFrancesco D. A new interpretation of the pace-maker current in calf Purkinje fibres. J Physiol, 1981,314:359-76.
7.DiFrancesco D. A study of the ionic nature of the pacemaker current in calf Purkinje fibres. J Physiol, 1981,314:377-93.
8.Yanagihara K, Irisawa H. Inward current activated during hyperpolarization in the rabbit sino atrial node cell.Pflugers Arch, 1980, 385:11-19.
9.Accili EA, Proenza C, Baruscottil M, etal. From funny current to HCN channels:20 years of excitation[J].News Physiol Sci,2002,17:32.
10.DiFrancesco D, Ferroni A, Mazzanti M, et al. Properties of the hyperpolarizing-activated current (I_f) in cells isolated from the rabbit sino-atrial node[J]. J Physiol(Lond),1986,377:61.
11.DiFrancesco D. Characterization of single pacemaker channels in cardiac sino-atrial node cells. Nature, 1986, 324:470-73.
12.Andreas Ludig et al. HCN channels, From genes to function. In: Douglas p . Zipes,Jose Jalife,ed: Cardiac electrophysiology, From cell to bedside. Fourth edition. Saunders.pp. 59-65.
13. Graf EM, Heubach JF, Ravens U. The hyperpolarizationactivated current If in ventricular myocytes of non-transgenic and beta2-adrenoceptor overexpressing mice. Naunyn Schmiedebergs Arch Pharmacol, 2001, 364:131 -9.
14. Ishii TM, Takano M, Xie LH, et al.: 1999. Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J Biol Chem 274:12,835-12,839.
15. Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc,Med, 2004,14:23-8.
16.Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci USA, 2003,100:15235-40.
n.Marionneau C, Couette B, Liu J, et al. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J Physiol, 2005,562:223-34.
18. Ludwig A, Budde T, Stieber J, et al.: 1998.Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2. Embo J 22:216-224.
19. Herrmann S, Stieber J, Feil S, et al.: 1999.Pacemaker channel HCN4 is required for normal cardiac function in the mouse embryo.Naunyn-Schmiedeberg's Archive of Pharmacology 367 (Suppl 1): R91.
20. Santoro B, Liu DT, Yao H, et al.: 1978. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93:717-729.
21.Sarachek NS, Leonard JL: 1999. Familial heart block and sinus bradycardia. Classification and natural history. Am J Cardiol 29:451- 458.
22.Schulze-Bahr E, Neu A, Friederich P, et al.:2002. Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest 111:1537- 1545.
23. Edelberg JM, Aird WC, Rosenberg RD. Enhancement of murine cardiac chronotropy by the molecular transfer of the human P2-adrenergic receptor cDNA. J Clin Invest, 1998, 101: 337-343.
24. Miake J, Marba'n E, Nuss HB. Gene therapy: biological pacemaker created by gene transfer. Nature, 2002, 419: 132-133.
25.Qu J, Plotnikoy AN, Danilo Jr P, et al. Expression and function of a biological pacemaker in canine heart. Circulation , 2003, 107: 1106-1109.
26. Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P Jr, Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptablerates. Circulation. 2004;109 (4):506-12.
27. Potapova I, Plotnikov A, Lu Z. Human mesenchymal stem cells as a gene delivery system to crate cardiac pacemakers. Circulation research. 2004;952-959.
28.Hung-Fat Tse, Tian Xue, Chu-Pak,et al. Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker HCN channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation. 2006;114 :1000-1011.