title: Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker.
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.
  
  
  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 non-treated 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.
authors.affiliation: Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Saito
authors.affiliation: Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Nakamura
authors.affiliation: Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Yoshida
authors.affiliation: Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sugiyama
authors.affiliation: Sakakibara Heart Institute of Okayama, Okayama, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Ohe
authors.affiliation: Department of Bio-informational Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Kurokawa
authors.affiliation: Department of Bio-informational Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Furukawa
authors.affiliation: Department of Physiology, Kurume University School of Medicine, Kurume, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Takano
authors.affiliation: Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagase
authors.affiliation: Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan; Department of Cardiovascular Therapeutics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Morita
authors.affiliation: Department of Cardiovascular Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan.
authors.forename: Kengo F
authors.initials: KF
authors.lastname: Kusano
authors.affiliation: Department of Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ito
mesh_terms: D000818:Animals; D001683:Biological Clocks; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000378:metabolism*; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000378:metabolism*; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011817:Rabbits
chemical_list: C578706:Hcn4 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels