Establishment of ES cell-derived biological pacemaker using ion channel and its application to bradycardia arrhythmias

2010 Fiscal Year Final Research Report

• Project/Area Number: 20590866
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Circulatory organs internal medicine
• Research Institution: Tottori University
• Principal Investigator: HISATOME Ichiro Tottori University, 大学院・医学系研究科, 教授 (60211504)
• Co-Investigator(Kenkyū-buntansha): SHIRAYOSHI Yasuaki 鳥取大学, 大学院・医学系研究科, 准教授 (90249946) ; YAMAMOTO Yasutaka 鳥取大学, 大学院・医学系研究科, 助教 (20362882) ; MIAKE Junichiro 鳥取大学, 医学部附属病院, 助教 (40372677)
• Project Period (FY): 2008 – 2010
• Keywords: ペースメーカ / ES細胞 / イオンチャネル

Research Abstract

We are attempting to establish the cardiac pacemaker cells derived from embryonic stem (ES) cells. To visualize cardiac pacemaker cells in differentiating embryoid bodies (EBs), EGFP gene was knocked in at HCN4 locus, which encodes ion channels responsible for pacemaker activity. In the present study, we conducted to establish EGFP-knock in ES cell line at HCN4 locus. PCR screening revealed two positive clones as possible knock in ES cell line. Southern blot analysis showed that one of them, H7 clone, is correctly targeted at HCN4 locus. After induction to cardiac differentiation through the formation of EBs in H7 clone, the expression of GFP was specifically localized at their contracting region in the outgrowth of EBs. Analysis of cell sorting revealed that 0.1～0.3% of differentiated H7 cells in EBs were GFP positive and that 70～90% of the sorted GFP positive cells were morphologically homogenous and beat spontaneously. Patch clamp analysis showed that spontaneously beating cells revealed the comparable electrophysiological properties with cardiac pacemaker cells, such as automaticity and If current. These results assured that H7 cell line was suitable to investigate and purify cardiac pacemaker cells.

Research Products

• [Journal Article] Roles of hyperpolarization-activated current If in sinoatrial node pacemaking : insights from bifurcation analysis of mathematical models Am J Physiol (2010)
  • Author(s): Kurata Y, Matsuda H, Hisatome I, Shibamoto T
  • Journal Title: Heart Circ Physiol 298 Pages: H1748-H1760
• [Journal Article] Identification, isolation and characterization of HCN4-positive pacemaking cells derived from murine embryonic stem cells during cardiac differentiation (2010)
  • Author(s): Morikawa K, Bahrudin U, Miake J, Igawa O, Kurata Y, Nakayama Y, Shirayoshi Y, Hisatome I
  • Journal Title: Pacing Clin Electrophysiol 33 Pages: 290-303
• [Journal Article] Different distribution of Cav3.2 and Cav3.1 transcripts encoding T-type Ca^<2+> channels in the embryonic heart of mice (2010)
  • Author(s): Mizuta E, Shirai M, Arakawa K, Hidaka K, Miake J, Ninomiya H, Kato M, Shigemasa C, Shirayoshi Y, Hisatome I, Morisaki T
  • Journal Title: Biomed Res 31 Pages: 301-305
• [Journal Article] Transcriptional activation of SAP97 by HSF-1 stabilizes Kv1.5 in HL-1 cells (2010)
  • Author(s): YK Ting, K Morikawa, Y Kurata, PL Li, U Bahrudin, E Mizuta, M Kato, J Miake, Y Yamamoto, A Yoshida, M Murata, T Inoue, A Nakai, G Shiota, K Higaki, E Nanba, H Ninomiya, Y Shirayoshi, I Hisatome
  • Journal Title: British J Pharmacol (in press)
• [Journal Article] Reciprocal Control of hERG Stability by Hsp70 and Hsc70 with Implication for Restoration of LQT2 Mutant Stability (2010)
  • Author(s): Peili Li, Haruaki Ninomiya, Yasutaka Kurata, Masaru Kato, Junichiro Miake, Yasutaka Yamamoto, Osamu Igawa, Akira Nakai, Katsumi Higaki, Futoshi Toyoda, Jie Wu, Minoru Horie, Hiroshi Matsuura, Akio Yoshida, Yasuaki Shirayoshi, Masayasu Hiraoka, Ichiro Hisatome
  • Journal Title: Circ Res (in press)
• [Journal Article] Fenofibrate enhances neovascularization in a murine ischemic hindlimb model. (2009)
  • Author(s): Katayama A, Yamamoto Y, Tanaka K, Matsubara K, Sugitani M, Fujihara S, Harada S, Kaetsu Y, Yoshida A, Hisatome I.
  • Journal Title: J Cardiovasc Pharmacol. 54(5) Pages: 399-404
• [Journal Article] Synergistic effects of autologous cell and hepatocyte growth factor gene therapy for neovascularization in a murine model of hindlimb ischemia. (2009)
  • Author(s): Yamamoto Y, Matsuura T, Narazaki G, Sugitani M, Tanaka K, Maeda A, Shiota G, Sato K, Yoshida A, Hisatome I.
  • Journal Title: Am J Physiol Heart Circ Physiol. 297(4) Pages: H1329-H1336
• [Journal Article] Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathway. (2009)
  • Author(s): Kawazoe S, Ikeda N, Miki K, Shibuya M, Morikawa K, Nakano S, Oshimura M, Hisatome I, Shirayoshi Y.
  • Journal Title: Dev Growth Differ. 51(2) Pages: 81-93
• [Journal Article] Stabilizing effects of eicosapentaenoic acid on Kv1.5 channel protein expressed in mammalian cells. (2009)
  • Author(s): Koshida S, Kurata Y, Notsu T, Hirota Y, Kuang TY, Li P, Bahrudin U, Harada S, Miake J, Yamamoto Y, Hoshikawa Y, Igawa O, Higaki K, Soma M, Yoshida A, Ninomiya H, Shiota G, Shirayoshi Y, Hisatome I.
  • Journal Title: Eur J Pharmacol. 604(1-3) Pages: 93-102
• [Journal Article] Pendrin is a novel autoantigen recognized by patients with autoimmune thyroid diseases. (2009)
  • Author(s): Yoshida A, Hisatome I, Taniguchi S, Shirayoshi Y, Yamamoto Y, Miake J, Ohkura T, Akama T, Igawa O, Shigemasa C, Kamijo K, Ikuyama S, Caturegli P, Suzuki K.
  • Journal Title: J Clin Endocrinol Metab. 94(2) Pages: 442-448
• [Journal Article] Long-term reliability of AAI mode pacing in patients with sinus node dysfunction and low Wenckebach block rate. (2008)
  • Author(s): Adachi M, Igawa O, Yano A, Miake J, Inoue Y, Ogura K, Kato M, Iitsuka K, Hisatome I.
  • Journal Title: Europace. 10(2) Pages: 134-137
• [Journal Article] Changes of HCN gene expression and I(f) currents in Nkx2.5-positive cardiomyocytes derived from murine embryonic stem cells during differentiation. (2008)
  • Author(s): Yano S, Miake J, Mizuta E, Manabe K, Bahrudin U, Morikawa K, Arakawa K, Sasaki N, Igawa O, Shigemasa C, Yamamoto Y, Morisaki T, Hidaka K, Kurata Y, Yoshida A, Shiota G, Higaki K, Ninomiya H, Lee JK, Shirayoshi Y, Hisatome I.
  • Journal Title: Biomed Res. 29(4) Pages: 195-203
• [Journal Article] Functional stabilization of Kv1.5 protein by Hsp70 in mammalian cell lines. (2008)
  • Author(s): Hirota Y, Kurata Y, Kato M, Notsu T, Koshida S, Inoue T, Kawata Y, Miake J, Bahrudin U, Li P, Hoshikawa Y, Yamamoto Y, Igawa O, Shirayoshi Y, Nakai A, Ninomiya H, Higaki K, Hiraoka M, Hisatome I.
  • Journal Title: Biochem Biophys Res Commun. 372(3) Pages: 469-474
• [Journal Article] Regional difference in dynamical property of sinoatrial node pacemaking : role of Na+ channel current Biophys J. (2008)
  • Author(s): Kurata Y, Matsuda H, Hisatome I, Shibamoto T.
  • Journal Title: 95(2) Pages: 951-977
• [Remarks] ホームページ等
• [Remarks] 新聞報道やテレビでの報道
• [Patent(Industrial Property Rights)] 新規ペースメーカ (2010)
  • Inventor(s): 久留一郎、白吉安昭、三明淳一朗
  • Industrial Property Rights Holder: 大学法人鳥取大学
  • Industrial Property Number: 国際特許出願,PCT/JP2010/066952
  • Filing Date: 2010-09-29
• [Patent(Industrial Property Rights)] 新規ペースメーカ (2009)
  • Inventor(s): 久留一郎、白吉安昭、三明淳一朗
  • Industrial Property Rights Holder: 大学法人鳥取大学
  • Industrial Property Number: 国内特許出願,特願2009-226760
  • Filing Date: 2009-09-30