The functional development of excitation-contraction coupling during cardiomyogenesis in mouse embryonic stem cells

小鼠胚胎干细胞心肌发生过程中兴奋-收缩耦合的功能发育

• 批准号: 13670037
• 负责人: KAWANO Seiko
• 金额: $ 2.11万
• 依托单位: Tokyo Medical & Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670037/
• 关键词: ES cell; cardiomyocyte; excitation-contraction coupling; IP3 receptor; Ryanodine receptor; Ca^<2+> signaling pathway; Na^+ Ca^<2+> exchanger; Na^+・K^+ pump; 胚性幹細胞; Caシグナル伝達; plasma membrane Ca pump; Na-Ca exchanger; Na-K pump; 幹細胞; カルシュウム

Mouse embryonic stem (ES) cells differentiate to all cell lineages including cardiomyocytes. In this study, we investigated the functional development of excitation-contraction coupling during cardiomyogenesis in mouse embryonic stem cells. During the differentiation to cardiomyocytes from ES cells (Cell line D3), the Ca^<2+> signaling pathway in each stage was examined using Ca^<2+> or Na^+ imaging, patch clamp techniques and RT-PCR. Our results showed that in undifferentiated ES cells Ca^<2+> was released from ER by the stimulation of G-protein coupled receptors. Using RT-PCR, mRNA for IP3 type I, II and III were detected, confirming IP3-induced Ca^<2+> release (IICR). On the other hand, function of ryanodine receptors or voltage-operated Ca^<2+> channels (VOCC) could not be detected, indicating no function of Ca^<2+>-induced Ca^<2+> release (CICR). For the Ca^<2+> entry pathway, store-operated Ca^<2+> entry (ISOC) was observed. From these results we conclude that Ca^<2+> entry through plasma membrane is mainly mediated by ISOC with little contribution of VOCCs. In this study, we also found that Na^+-Ca^<2+> exchangers and plasma membrane pumps functioned as Ca^<2+> extrusion system. The function of Na^+-K^+ pump examined using ouabain sensitivity could be detected in ES cells. During the differentiation to cardiomyocytes, IICR function was observed in all stages. In the 7-day differentiated cells, CICR could be observed. Both T-type and L-type Ca^<2+> channel were recorded by patch clamp experiments. Ouabain-sensitive Na^+-K^+ pump functions were increased with the differentiation. In whole cell patch clamp, Na^+-K^+ pump currents could be recorded in cells from at 8-day-differentiated stage but not earlier stages. Thus, we clarified the Ca^<2+> signaling pathways in undifferentiated mouse ES cells for the first time in this study. However, the further studies are needed to explore the mechanisms how these signaling pathways differentiate or develop.

小鼠胚胎干（ES）细胞分化为包括心肌细胞在内的所有细胞谱系。在这项研究中，我们研究了小鼠胚胎干细胞心肌形成过程中兴奋-收缩偶联的功能发育。在ES细胞（细胞系D3）向心肌细胞分化的过程中，利用Ca^<2+>或Na^+成像、膜片钳技术和RT-PCR检测了各个阶段的Ca^<2+>信号通路。我们的结果表明，在未分化的ES细胞中，通过G蛋白偶联受体的刺激，Ca^<2+>从ER中释放出来。使用RT-PCR，检测到IP 3 I、II和III型的mRNA，证实了IP 3诱导的Ca^2+释放（IICR）。另一方面，未检测到兰尼碱受体或电压操纵性Ca^<2+>通道（VOCC）的功能，表明Ca^<2+>诱导的Ca^<2 +>释放（CICR）没有功能。对于Ca^2+进入途径，观察到钙池操纵的Ca^2+进入（ISOC）。从这些结果我们可以得出结论，Ca^2+的跨膜进入主要是由ISOC介导的，VOCCs的贡献很小。本研究还发现Na^+-Ca^2+交换器和质膜泵具有Ca^2+挤出系统的功能。用哇巴因敏感性检测ES细胞的Na^+-K^+泵功能。在向心肌细胞分化的过程中，在所有阶段都观察到IICR功能。在分化7天的细胞中，可观察到CICR。采用膜片钳技术记录T型和L型Ca^<2+>通道。哇巴因敏感的Na^+-K^+泵功能随着分化而增强。在全细胞膜片钳技术中，Na^+-K^+泵电流可在分化8天的细胞中记录到，但在分化更早的细胞中不能记录到。因此，我们在本研究中首次阐明了未分化小鼠ES细胞中的Ca^<2+>信号通路。然而，这些信号通路的分化和发展机制还需要进一步的研究。

项目成果

Seiko Kawano et al.: "Ca^<2+> Oscillations regulated by Na^+-Ca^<2+> Exchanger and Plasma Membrane Ca^<2+> Pump Induce Fluctuations of Membrane Currents and Potentials in Human Mesenchymal Stem Cells"Cell Calcium. (in press). (2003)

Seiko Kawano 等人：“Ca^<2> 由 Na^-Ca^<2> 交换器和质膜 Ca^<2> 泵调节的振荡诱导人间充质干细胞中膜电流和电位的波动”细胞钙。

Tagami M., Ichinose S., Yamagata K., Fujino H., Shoji S., Hiraoka M., Kawano S.: "Genetic and ultrastructural demonstration of strong reversibility in human mesenchymal stem cell"Cell and Tissue Research. (in press). (2003)

Tagami M.、Ichinose S.、Yamagata K.、Fujino H.、Shoji S.、Hiraoka M.、Kawano S.：“人类间充质干细胞强可逆性的遗传和超微结构证明”细胞和组织研究。

Seiko Kawano et al.: "Characterization of Ca2+ signaling pathways in human mesenchymal stem cells"Cell Calcium. 32. 165-174 (2002)

Seiko Kawano 等人：“人间充质干细胞中 Ca2 信号通路的表征”细胞钙。

Seiko Kawano et al.: "Ca^<2+> Oscillations regulated by Na^+ -Ca^<2+> Exchanger and Plasma Membrane Ca^<2+> Pump Induce Fluctuations of Membrane Currents and Potentials in Human Mesenchymal Stem Cells"Cell Calcium. (in press). (2003)

Seiko Kawano 等人：“Ca^<2> 由 Na^-Ca^<2> 交换器和质膜 Ca^<2> 泵调节的振荡诱导人间充质干细胞中膜电流和电位的波动”细胞钙。

Hirayama Y., Kuruma A., Hiraoka M., Kawano S: "Ca^<2+>-activated Cl^-current is enhanced by acidosis and contributes to the shortening of action potential duration in rabbit ventricular myocytes"Japanese Journal of Physiology. 52. 293-300 (2002)

Hirayyama Y.、Kuruma A.、Hiraoka M.、Kawano S：“Ca^2-激活的Cl^-电流因酸中毒而增强，并有助于缩短兔心室肌细胞的动作电位持续时间”《日本生理学杂志》。