Hyperpolarization-activated cyclic nucleotide-gated channels and T-Type calcium channels confer automaticity of embryonic stem cell-derived cardiomyocytes
Hyperpolarization-activated cyclic nucleotide-gated channels and T-Type calcium channels confer automaticity of embryonic stem cell-derived cardiomyocytes
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DOI：
10.1634/stemcells.2006-0388
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发表时间：
2007-11-01
期刊：
影响因子：
5.2
通讯作者：
Yamashita, Jun K.
中科院分区：
文献类型：
--
作者：
Yanagi, Kentoku;Takano, Makoto;Yamashita, Jun K.
心脏起搏器的再生是心脏再生的重要靶点。以前，我们开发了一种新的胚胎干（ES）细胞分化系统，可以在细胞水平上跟踪心血管分化过程。在本研究中，我们研究了ES细胞来源的心肌细胞在分化过程中离子通道的表达和功能，并确定了赋予其自律性的离子通道。ES细胞来源的Flk 1（+）中胚层细胞在OP 9基质细胞上产生自发搏动的心肌细胞。在Flk 1（+）细胞培养物（Flk-d9.5）的第9.5天观察到的自发搏动集落在Flk-d23.5时显著减少。与Flk-d9.5相比，在Flk-d23.5纯化的心肌细胞中，起搏细胞中超极化激活的环核苷酸门控（HCN）1和-4以及电压门控钙通道（Cav）3.1和-3.2的离子通道表达显著降低，而心房和心室离子通道内向整流钾通道（Kir）2.1的表达没有变化。阻断HCNs和Cav离子通道可显著抑制心肌细胞集落的搏动速率。电生理研究表明，在Flk-d9.5自发搏动的心肌细胞表现出几乎相似的功能，除了相对深的最大舒张电位和更快的最大上行速度的本地小鼠窦房结。虽然在Flk-d23.5时约60%的肌细胞显示出与Flk-d9.5时几乎相同的性质，但约40%的肌细胞显示出HCN损失和Cav 3电流降低，并停止自发搏动，Kir 2. 1没有显著增加。因此，HCN和Cav 3离子通道应负责维持ES细胞衍生的心肌细胞的自律性。这些离子通道的受控调节应该是产生完整的生物起搏器所必需的。
Regeneration of cardiac pacemakers is an important target of cardiac regeneration. Previously, we developed a novel embryonic stem (ES) cell differentiation system that could trace cardiovascular differentiation processes at the cellular level. In the present study, we examine expressions and functions of ion channels in ES cell-derived cardiomyocytes during their differentiation and identify ion channels that confer their automaticity. ES cell-derived Flk1(+) mesoderm cells give rise to spontaneously beating cardiomyocytes on OP9 stroma cells. Spontaneously beating colonies observed at day 9.5 of Flk1(+) cell culture (Flk-d9.5) were significantly decreased at Flk-d23.5. Expressions of ion channels in pacemaker cells hyperpolarization-activated cyclic nucleotidegated (HCN)l and -4 and voltage-gated calcium channel (Cav)3.1 and -3.2 were significantly decreased in purified cardiomyocytes at Flk-d23.5 compared with at Flk-d9.5, whereas expression of an atrial and ventricular ion channel, inward rectifier potassium channel (Kir)2.1, did not change. Blockade of HCNs and Cav ion channels significantly inhibited beating rates of cardiomyocyte colonies. Electrophysiological studies demonstrated that spontaneously beating cardiomyocytes at Flk-d9.5 showed almost similar features to those of the native mouse sinoatrial node except for relatively deep maximal diastolic potential and faster maximal upstroke velocity. Although similar to 60% of myocytes at Flk-d23.5 revealed almost the same properties as those at Flk-d9.5, similar to 40% of myocytes showed loss of HCN and decreased Cav3 currents and ceased spontaneous beating, with no remarkable increase of Kir2.1. Thus, HCN and Cav3 ion channels should be responsible for the maintenance of automaticity in ES cell-derived cardiomyocytes. Controlled regulation of these ion channels should be required to generate complete biological pacemakers.