HCN and Cav3 ion Channels Confer Automaticity of Embryonic Stem Cell-derived Cardiomyocytes

¹ Laboratory of Stem Cell Differentiation, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Japan., First Department of Surgery, University of Toyama Graduated School of Medicine, Toyama, Japan
² Department of Physiology, Faculty of Medicine, Jichi Medical University, Tochigi, Japan
³ Laboratory of Stem Cell Differentiation, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Japan.
⁴ Department of Physiology, Graduate School of Medicine, Kyoto University, Japan
⁵ First Department of Surgery, University of Toyama Graduated School of Medicine, Toyama, Japan
⁶ Laboratory of Stem Cell Differentiation, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Japan., PRESTO, Japan Science and Technology Agency, Japan

^* To whom correspondence should be addressed. E-mail: juny{at}frontier.kyoto-u.ac.jp.

	Abstract

Regeneration of cardiac pacemakers is an important target of cardiac regeneration. Previously, we developed a novel embryonic stem (ES) cell differentiation system which could trace cardiovascular differentiation process at the cellular level. In the present study, we examined expressions and functions of ion channels in ES cell-derived cardiomyocytes during their differentiation and identified 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, HCN1, 4 and Cav3.1, 3.2 were significantly decreased in purified cardiomyocytes at Flk-d23.5 compared to at Flk-d9.5, whereas expression of an atrial and ventricular ion channel, Kir2.1, 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 with those of the native mouse sino-atrial node except relatively deep maximal diastolic potential and faster maximal upstroke velocity. Though 60 % of myocytes at Flk-d23.5 revealed almost the same properties with those at Flk-d9.5, 40 % 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 cardiomyoctyes. Controlled regulation of these ion channels should be required to generate complete biological pacemakers.

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