Enhancement of spontaneous activity by hcn4 overexpression in mouse embryonic stem cell-derived cardiomyocytes - a possible biological pacemaker

Yukihiro Saito, Kazufumi Nakamura, Masashi Yoshida, Hiroki Sugiyama, Tohru Ohe, Junko Kurokawa, Tetsushi Furukawa, Makoto Takano, Satoshi Nagase, Hiroshi Morita, Kengo F. Kusano, Hiroshi Itoh

Research output: Contribution to journalArticle

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Abstract

Background Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (If) flowing through hyperpolarization-Activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (IK1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro. Methods and Results The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of IK1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger If currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an If inhibitor, and to isoproterenol, a beta-Adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of nontreated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs. Conclusions We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an If inhibitor and beta-Adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.

Original languageEnglish
Article numbere0138193
JournalPLoS One
Volume10
Issue number9
DOIs
Publication statusPublished - Sep 18 2015

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Biological Clocks
biological clocks
Pacemakers
embryonic stem cells
Stem cells
Cardiac Myocytes
mice
Induced Pluripotent Stem Cells
Genes
cell aggregates
genes
Adrenergic beta-Agonists
ivabradine
Coculture Techniques
Mouse Embryonic Stem Cells
cardiomyocytes
coculture
agonists
cells
Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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Enhancement of spontaneous activity by hcn4 overexpression in mouse embryonic stem cell-derived cardiomyocytes - a possible biological pacemaker. / Saito, Yukihiro; Nakamura, Kazufumi; Yoshida, Masashi; Sugiyama, Hiroki; Ohe, Tohru; Kurokawa, Junko; Furukawa, Tetsushi; Takano, Makoto; Nagase, Satoshi; Morita, Hiroshi; Kusano, Kengo F.; Itoh, Hiroshi.

In: PLoS One, Vol. 10, No. 9, e0138193, 18.09.2015.

Research output: Contribution to journalArticle

Saito, Yukihiro ; Nakamura, Kazufumi ; Yoshida, Masashi ; Sugiyama, Hiroki ; Ohe, Tohru ; Kurokawa, Junko ; Furukawa, Tetsushi ; Takano, Makoto ; Nagase, Satoshi ; Morita, Hiroshi ; Kusano, Kengo F. ; Itoh, Hiroshi. / Enhancement of spontaneous activity by hcn4 overexpression in mouse embryonic stem cell-derived cardiomyocytes - a possible biological pacemaker. In: PLoS One. 2015 ; Vol. 10, No. 9.
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abstract = "Background Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (If) flowing through hyperpolarization-Activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (IK1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro. Methods and Results The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90{\%} of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of IK1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger If currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an If inhibitor, and to isoproterenol, a beta-Adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of nontreated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs. Conclusions We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an If inhibitor and beta-Adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.",
author = "Yukihiro Saito and Kazufumi Nakamura and Masashi Yoshida and Hiroki Sugiyama and Tohru Ohe and Junko Kurokawa and Tetsushi Furukawa and Makoto Takano and Satoshi Nagase and Hiroshi Morita and Kusano, {Kengo F.} and Hiroshi Itoh",
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TY - JOUR

T1 - Enhancement of spontaneous activity by hcn4 overexpression in mouse embryonic stem cell-derived cardiomyocytes - a possible biological pacemaker

AU - Saito, Yukihiro

AU - Nakamura, Kazufumi

AU - Yoshida, Masashi

AU - Sugiyama, Hiroki

AU - Ohe, Tohru

AU - Kurokawa, Junko

AU - Furukawa, Tetsushi

AU - Takano, Makoto

AU - Nagase, Satoshi

AU - Morita, Hiroshi

AU - Kusano, Kengo F.

AU - Itoh, Hiroshi

PY - 2015/9/18

Y1 - 2015/9/18

N2 - Background Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (If) flowing through hyperpolarization-Activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (IK1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro. Methods and Results The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of IK1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger If currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an If inhibitor, and to isoproterenol, a beta-Adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of nontreated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs. Conclusions We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an If inhibitor and beta-Adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.

AB - Background Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (If) flowing through hyperpolarization-Activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (IK1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor IK1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro. Methods and Results The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of IK1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger If currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an If inhibitor, and to isoproterenol, a beta-Adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of nontreated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs. Conclusions We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an If inhibitor and beta-Adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.

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