Hyperpolarization-activated cyclic nucleotide-gated channels and T-type calcium channels confer automaticity of embryonic stem cell-derived cardiomyocytes.
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 nucleotide-gated (HCN)1 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 approximately 60% of myocytes at Flk-d23.5 revealed almost the same properties as those at Flk-d9.5, approximately 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.
Laboratory of Stem Cell Differentiation, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Kentoku
K
Yanagi
Makoto
M
Takano
Genta
G
Narazaki
Hideki
H
Uosaki
Takuhiro
T
Hoshino
Takahiro
T
Ishii
Takurou
T
Misaki
Jun K
JK
Yamashita
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D001683:Biological Clocks / Q000502:physiology*; D020747:Calcium Channels, T-Type / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000378:metabolism* / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism / Q000502:physiology*
D020747:Calcium Channels, T-Type; D054815:Cyclic Nucleotide-Gated Cation Channels