Adrenoceptor Responses in Human Embryonic Stem Cell-Derived Cardiomyocytes: a Special Focus on Electrophysiological Property.

Abstract

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 β-AR stimulation by isoproterenol for 24 hours in hESC-CM monolayers increased CV by approximately 50%, whereas α-AR or acute β-AR stimulation had no significant effect; chronic β-AR stimulation resulted in a significant Connexin (Cx) 43 and Nav1.5 upregulation at both protein and mRNA level. Isoproterenol-induced CV accelerating and Cx43 and Nav1.5 upregulation in hESC-CMs, which was attenuated by selective β1-adrenoceptor antagonist CGP 20712A but not selective β2-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 Nav1.5 upregulation. The AP shape in hESC-CM monolayers was less susceptible by either β-AR or α-AR stimulation. It was β1-AR not β2-AR contributing to the modification of conduction velocity among hESC-CM monolayers. Chronic β1-AR stimulation accelerates CV by upregulating Cx43 via PKA/MEK/MAPK pathway. SIGNIFICANCE STATEMENT: These 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 β1-AR not β2-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.