Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker.
Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker.
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DOI：
10.1371/journal.pone.0138193
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Ito H
中科院分区：
文献类型：
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作者：
Saito Y;Nakamura K;Yoshida M;Sugiyama H;Ohe T;Kurokawa J;Furukawa T;Takano M;Nagase S;Morita H;Kusano KF;Ito H
生物起搏器的建立有望解决机械起搏器的持续问题，包括电池寿命和电磁干扰问题。超极化激活的环核苷酸门控（HCN）通道有趣电流（If）的增强和内向整流钾（Kir）通道内向整流钾电流（IK 1）的衰减是生物起搏器产生的关键。因此，我们产生了HCN 4过表达的小鼠胚胎干细胞（mESC）和诱导心肌细胞，原来显示穷人的IK 1电流，我们调查是否HCN 4过表达的mESC衍生的心肌细胞（mESC-CM）功能作为一个生物起搏器在体外。将兔Hcn 4基因转染mESCs，筛选稳定的克隆。mESC-CM通过胚状体产生，并在无血清/葡萄糖和补充乳酸盐的条件下纯化。大约90%的纯化细胞肌钙蛋白I免疫染色阳性。在mESC-CM中，编码Kir2.1的Kcnj 2基因的表达水平低于成年小鼠心室中的表达水平，Kir2.1是产生负责稳定静息膜电位的IK 1电流所必需的。HCN 4过表达mESC-CM表达的HCN 4基因水平比非过表达mESC-CM高约3倍。还证实了编码T型钙通道并在窦房结中产生舒张期去极化的Cacna 1h基因的表达。此外，在细胞中表达冲动传导所需的基因，包括编码形成间隙连接的连接蛋白的连接蛋白40、连接蛋白43和连接蛋白45基因，以及编码钠通道的Scn 5a基因。HCN 4过表达的mESC-CM显示出比非过表达的mESC-CM显著更大的I f电流和更快的自发搏动。HCN 4过表达mESC-CM的搏动率对伊伐布雷定（一种IF抑制剂）和异丙肾上腺素（一种β-肾上腺素能受体激动剂）有反应。人诱导多能干细胞衍生的心肌细胞（hiPSC-CM）与由mESC-CM组成的聚集体的共培养导致细胞的同步收缩。与HCN 4过表达mESC-CM的聚集体共培养的hiPSC-CM的搏动率显著高于未处理的hiPSC-CM和与非过表达mESC-CM的聚集体共培养的hiPSC-CM的搏动率。我们产生了HCN 4过表达mESC-CM，其表达冲动传导所需的基因，显示出快速的自发搏动，响应于IF抑制剂和β-肾上腺素能受体激动剂，并且在与其他可兴奋细胞的体外共培养系统中具有起搏能力。结果表明，这些细胞可以应用于生物起搏器。
Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (I f) flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (I K1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor I K1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro. The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of I K1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger I f currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an I f inhibitor, and to isoproterenol, a beta-adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of non-treated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs. We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an I f inhibitor and beta-adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.