Overexpression of connexin 45 in rat mesenchymal stem cells improves the function as cardiac biological pacemakers.
Overexpression of connexin 45 in rat mesenchymal stem cells improves the function as cardiac biological pacemakers.
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发表时间：
2010-06
影响因子：
6.1
通讯作者：
Min Tong;Xiang-jun Yang;Bao-yu Geng;Lian-hua Han;Yafeng Zhou;Xin Zhao;Hong-Xia Li
中科院分区：
文献类型：
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作者：
Min Tong;Xiang-jun Yang;Bao-yu Geng;Lian-hua Han;Yafeng Zhou;Xin Zhao;Hong-Xia Li
背景：通过增强向内去极化电流来制造生物起搏器已经进行了广泛的研究。然而，研究主要集中在诱导自发活动，并没有充分解决如何改善起搏器功能。在这项研究中，我们试图通过改变大鼠间充质干细胞（MSCs）中的连接蛋白表达，使其表型与天然窦结起搏器细胞相似，从而改善起搏器功能。方法：通过慢病毒载体转染心脏起搏器基因-超极化激活的环核苷酸门控通道4 （HCN4）对MSCs进行转导，以产生生物起搏器。用电压箝位法记录HCN4（+）间充质干细胞的搞笑电流（I(f)）。通过质粒pDsRED2-N1-Gja7-RFP转染MSCs，实现了连接蛋白45（基因Gja7）的过表达。使用抗连接蛋白43和抗连接蛋白45抗体进行双重免疫标记来识别间隙连接通道。在与新生大鼠心室肌细胞共培养的MSCs中，测定了转基因MSCs对心肌细胞兴奋性的影响。用电流钳法记录了新生大鼠心室肌细胞的自发动作电位。结果在HCN4（+）间充质干细胞中检测到高水平的时间和电压依赖性向内超极化电流，该电流对4 mmol/L Cs（+）敏感，证实HCN4在间充质干细胞中作为I(f)通道。在CX45(+) MSCs中同时检测到连接蛋白43和连接蛋白45。与未转染的对照MSCs共培养的肌细胞的搏动频率为每分钟（82 +/- 8）次（n = 5），而与HCN4(+) MSCs共培养的肌细胞的搏动频率为每分钟（129 +/- 11）次（n = 5）。与HCN4和连接蛋白45共转染的MSCs共培养的肌细胞搏动频率最高，为（147 +/- 9）次/分钟（n = 5）。结论在体外表达HCN4的MSCs中，过表达connexin45并形成异质connexin45 / connexin43缝隙连接通道可改善其心脏生物起搏器功能。
BACKGROUND Extensive research toward creating a biological pacemaker by enhancement of inward depolarizing current has been performed. However, studies have mainly focused on inducing spontaneous activity and have not adequately addressed ways to improve pacemaker function. In this study we attempted to improve pacemaker function by altering connexin expression in rat mesenchymal stem cells (MSCs) to a phenotype similar to native sinus node pacemaker cells. METHODS To generate a biological pacemaker, MSCs were transduced with a cardiac pacemaker gene-hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), via transfection with a lentiviral vector. Funny current (I(f)) in HCN4(+) MSCs was recorded by voltage-clamp. Overexpression of connexin 45 (gene Gja7) in MSCs was achieved by transfection with the plasmid pDsRED2-N1-Gja7-RFP. Double-immunolabelling with anti-connexin 43 and anti-connexin 45 antibodies were used to identify the gap junction channels. The effects of the genetically modified MSCs on cardiomyocyte excitability were determined in MSCs cocultured with neonatal rat ventricular myocytes. Spontaneous action potentials of neonatal rat ventricular myocytes were recorded by current-clamp. RESULTS High level time- and voltage-dependent inward hyperpolarization current that was sensitive to 4 mmol/L Cs(+) was detected in HCN4(+) MSCs, confirming that HCN4 acted as I(f) channels in MSCs. Connexin 43 and connexin 45 were simultaneously detected in CX45(+) MSCs. Beating frequency was (82 +/- 8) beats per minute (n = 5) in myocytes cocultured with non-transfected control MSCs, versus (129 +/- 11) beats per minute (n = 5) in myocytes cocultured with HCN4(+) MSCs. Myocytes cocultured with MSCs cotransfected with HCN4 and connexin 45 had the highest beating frequency at (147 +/- 9) beats per minute (n = 5). CONCLUSION These findings demonstrate that overexpression of connexin 45 and subsequent formation of heteromeric connexin 45/connexin 43 gap junction channels in HCN4 expressing MSCs can improve their function as cardiac biological pacemakers in vitro.