Adrenoceptor Responses in Human Embryonic Stem Cell-Derived Cardiomyocytes: a Special Focus on Electrophysiological Property
Adrenoceptor Responses in Human Embryonic Stem Cell-Derived Cardiomyocytes: a Special Focus on Electrophysiological Property
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人胚胎干细胞衍生的心肌细胞中的肾上腺素受体反应：特别关注电生理特性
DOI：
10.1124/jpet.120.265686
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发表时间：
2020-06-01
影响因子：
3.5
通讯作者：
Wang, Yin
中科院分区：
文献类型：
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作者：
Jiang, Weiwei;Hu, Xingjian;Wang, Yin
人胚胎干细胞来源的心肌细胞（hESC-CMs）已成为一种有前景的心血管研究细胞来源。hESC-CMs的电生理特性已被广泛研究，但对肾上腺素能受体（adrenergic receptor， AR）激活的电生理反应知之甚少。本研究旨在通过传导速度（CV）和动作电位（AP）形态表征hESC-CMs对肾上腺素能刺激的电生理反应。通过经典的分化方案获得H9 hESC-CMs，并培养成融合单层细胞。在电生理和药物刺激实验中，用光学作图法记录了各单层间的AP形状和CV。采用实时定量聚合酶链反应和Western blot检测Connexin、离子通道基因及蛋白的表达水平。慢性β - ar异丙肾上腺素刺激hESC-CM单层24小时可使CV升高约50%，而α - ar或急性（3-AR）刺激无显著影响；慢性β - ar刺激可导致连接蛋白（Cx） 43和Na-v1.5在蛋白和mRNA水平上显著上调。异丙肾上腺素诱导的CV加速和hESC-CMs中Cx43和Na-v1.5上调，选择性β(1)-肾上腺素受体拮抗剂CGP 20712A可减弱这种作用，但选择性β(2)-拮抗剂ICI 118551不能减弱这种作用。此外，蛋白激酶A （PKA）抑制剂H89、丝裂原活化蛋白激酶(MAPK)/细胞外信号调节激酶（MEK）抑制剂SB203580和MAPK抑制剂PD98059预处理可抑制异丙肾上腺素诱导的CV加速和Cx43上调，而对Na-v1.5上调无显著影响。hESC-CM单层的AP形状对β - ar或α - ar刺激的影响较小。是β 1-AR而不是β 2-AR改变了hESC-CM单层间的传导速度。慢性β 1-AR刺激通过PKA/MEK/MAPK通路上调Cx43，从而加速CV。这些数据为人类胚胎干细胞来源的心肌细胞（hESC-CMs）的电生理特性提供了新的见解，并描述了肾上腺素能受体（AR）调控动作电位形状和hESC-CM单层电传播的简明信号通路。通过蛋白激酶A/丝裂原活化蛋白激酶(MAPK)-细胞外信号调节激酶/MAPK通路上调Connexin 43，从而促进hESC-CMs的传导速度，是β 1-AR而不是β 2-AR参与了传导速度的改变。
Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have become a promising cell source for cardiovascular research. The electrophysiological characteristic of hESC-CMs has been generally studied, but little is known about electrophysiological response to adrenergic receptor (AR) activation. This study aims to characterize electrophysiological response of hESC-CMs to adrenergic stimulation in terms of the conduction velocity (CV) and action potential (AP) shape. The H9 hESC-CMs were acquired by a classic differentiation protocol and cultured to achieve confluent cell monolayers. The AP shape and CV among the monolayers were recorded using optical mapping during electrophysiological and pharmacological stimulation experiments. Quantitative real-time polymerase chain reaction and Western blot were adopted to determine the expression levels of Connexin and ion channel gene and protein. Chronic beta-AR stimulation by isoproterenol for 24 hours in hESC-CM monolayers increased CV by approximately 50%, whereas alpha-AR or acute (3-AR stimulation had no significant effect; chronic beta-AR stimulation resulted in a significant Connexin (Cx) 43 and Na-v1.5 upregulation at both protein and mRNA level. Isoproterenol-induced CV accelerating and Cx43 and Na-v1.5 upregulation in hESC-CMs, which was attenuated by selective beta(1)-adrenoceptor antagonist CGP 20712A but not selective beta(2)-antagonist ICI 118551. Moreover, pretreatment with protein kinase A (PKA) inhibitor H89, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (MEK) inhibitor SB203580, and MAPK inhibitor PD98059 suppressed the isoproterenol-induced CV accelerating and Cx43 upregulation, whereas it had no significant effect on Na-v1.5 upregulation. The AP shape in hESC-CM monolayers was less susceptible by either beta-AR or alpha-AR stimulation. It was beta 1-AR not beta 2-AR contributing to the modification of conduction velocity among hESC-CM monolayers. Chronic beta 1-AR stimulation accelerates CV by upregulating Cx43 via PKA/MEK/MAPK pathway.SIGNIFICANCE STATEMENTThese data provide new insight into the electrophysiological characteristics of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and depict a concise signaling pathway in the adrenergic receptor (AR) regulation of action potential shape and electrical propagation across hESC-CM monolayer. It is beta 1-AR not beta 2-AR contributing to the modification of conduction velocity in hESC-CMs and accelerating conduction velocity by upregulating Connexin 43 via protein kinase A/ mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase/MAPK pathway.