Elucidation of pathophysiological mechanisms and the development of innovative therapies for lethal arrhythmias caused by calmodulin mutations

2019 Fiscal Year Annual Research Report

• Project/Area Number: 18K15847
• Research Institution: Kyoto University
• Principal Investigator: 山本 雄大 京都大学, 医学研究科, 研究員 (10802762)
• Project Period (FY): 2018-04-01 – 2020-03-31
• Keywords: カルモジュリン / 不整脈 / iPS細胞 / QT延長症候群 / アンチセンス核酸 / 遺伝子治療

Outline of Annual Research Achievements

カルモジュリン遺伝子変異によって引き起こされるQT延長症候群（LQTS）やカテコラミン誘発性多形性心室頻拍（CPVT）等の致死性不整脈疾患や神経疾患はカルモジュリノパチーと呼ばれる新たな疾患概念として注目されている。本研究では表現型の異なる3症例（CALM2-E46K変異: CPVT・神経疾患, CALM2-N98S変異: LQTS・CPVT, CALM2-D134H変異: LQTS）の患者よりiPS細胞を樹立し、解析を行っている。樹立したiPS細胞モデルを用いて前年に引き続き、病態解明として患者由来iPS細胞モデルにおける緩徐活性型遅延整流性K電流（IKs）の解析、また新規治療法の検討としてアンチセンス核酸を用いた新規治療法の検討を行った。
CALM2-N98S変異症例では複数の患者において運動時の失神やQT間隔の延長などKCNQ1遺伝子変異を原因とするLQT1に類似した表現型が認められている。N98S変異iPS細胞モデルにおいてパッチクランプ法により、Iks電流の解析を行ったところ、健常人由来のコントロールと比較してイソプロテレノール負荷時の電流密度が小さいことが認められた。また、膜電位インジケーター用いたイメージングシステムによる活動電位の解析により、イソプロテレノールによる活動電位持続時間の短縮率が小さいことを明らかにした。現在、この研究成果を論文にまとめているところである。
新規治療法の開発として前年に培養細胞においてCALM2発現抑制効果の認められたアンチセンス核酸のiPS細胞由来心筋細胞における効果を検討した。QPCRによるCALM2遺伝子発現の解析ではiPS細胞由来心筋細胞においてもアンチセンス核酸の発現抑制を確認することができた。また電気生理学的解析においても活動電位持続時間の短縮が認められ、治療法としても効果が認められた。

Research Products

• [Journal Article] Phenotype-Based High-Throughput Classification of Long QT Syndrome Subtypes Using Human Induced Pluripotent Stem Cells (2019)
  • Author(s): Yoshinaga Daisuke、Baba Shiro、Makiyama Takeru、Shibata Hirofumi、Hirata Takuya、Akagi Kentaro、Matsuda Koichi、Kohjitani Hirohiko、Wuriyanghai Yimin、Umeda Katsutsugu、Yamamoto Yuta、Conklin Bruce R.、Horie Minoru、Takita Junko、Heike Toshio
  • Journal Title: Stem Cell Reports Volume: 13 Pages: 394-404
  • DOI: 10.1016/j.stemcr.2019.06.007
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Calmodulinopathy(カルモジュリン遺伝子関連不整脈疾患) iPS細胞技術を用いた疾患モデルの作製と遺伝子治療へのアプローチ (2019)
  • Author(s): 山本 雄大,牧山 武
  • Journal Title: 心電図 Volume: 39 Pages: 273-282
• [Presentation] Modeling Overlap Syndrome Of Long QT And Brugada Syndrome Associated With CACNA1C-E1115K Which Alters Ion Selectivity With Induced Pluripotent Stem Cells (2019)
  • Author(s): Asami Kashiwa, Takeru Makiyama, Hirohiko Kohjitani, Yimin Wuriyanghai, Yuta Yamamoto, Gao Jungshan, Hai Huang, Taisuke Ishikawa, Seiko Ohno, Futoshi Toyoda, , Seiichi Sato, Minoru Horie , Naomasa Makita, Takeshi Kimura.
  • Organizer: 40th Heart Rhythm Scientific Sessions
  • Int'l Joint Research
• [Presentation] Automatic Computational Action Potential Analyzing System Enables Accurate Drug Response Interpretation and Prediction in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes (2019)
  • Author(s): Hirohiko Kohjitani, Shigeya Kouda, Yukiko Himeno, Takeru Makiyama, Asami Kashiwa, Yimin Wuriyanghai, Yuta Yamamoto, Minoru Horie, Takeshi Kimura, Akinori Noma, Akira Amano.
  • Organizer: 40th Heart Rhythm Scientific Sessions
  • Int'l Joint Research
• [Presentation] Structural Insights into Catecholaminergic Polymorphic Ventricular Tachycardia-associated RyR2 Mutant Channels using a Three-dimensional In Silico Model. (2019)
  • Author(s): J. Gao, T. Makiyama, S. Ohno, Y. Yamamoto, Y. Wuriyanghai, H. Kohjitani, A. Kashiwa, H. Huang, K. Katou, M. Horie, T. Kimura.
  • Organizer: European Society of Cardiology Congress 2019
  • Int'l Joint Research
• [Presentation] KCNQ1 Channel Dysfunction in human induced pluripotent stem cell-derived cardiomyocytes carrying a CALM2-N98S mutation associated with long QT syndrome (2019)
  • Author(s): Yuta Yamamoto, Takeru Makiyama, Yimin Wuriyanghai, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Huang Hai, Takanori Aizawa, Tomohiko Imamura, Taisuke Ishikawa, Seiko Ohno, Kazuhisa Chonabayashi, Hideki Motomura, Yoshinori Yoshida, Minoru Horie, Naomasa Makita, Takeshi Kimura
  • Organizer: American Heart Association Scientific Sessions 2019
  • Int'l Joint Research
• [Presentation] Morphological and electrophysiological characterization of homozygous lamin A/C knockout human induced-pluripotent stem cell-derived cardiomyocytes. American Heart Association Scientific Sessions 2019 (2019)
  • Author(s): Yimin Wuriyanghai, Takeru Makiyama, Yuta Yamamoto, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Hai Huang, Takanori Aizawa, Tomohiko Imamura, Seiko Ohno, Minoru Horie, Takeshi Kimura
  • Organizer: American Heart Association Scientific Sessions 2019
  • Int'l Joint Research
• [Presentation] Response of KCNQ1 to Adrenergic Stimulation was Impaired in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes Carrying a CALM2-N98S Mutation Associated with Long QT Syndrome. (2019)
  • Author(s): Yuta Yamamoto, Takeru Makiyama, Yimin Wuriyanghai, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Huang Hai, Takanori Aizawa, Tomohiko Imamura, Taisuke Ishikawa, Seiko Ohno, Kazuhisa Chonabayashi, Hideki Motomura, Yoshinori Yoshida, Minoru Horie, Naomasa Makita, Takeshi Kimura
  • Organizer: 12th Asia Pacific Heart Rhythm Society Scientific Session (APHRS2019)
  • Int'l Joint Research
• [Presentation] Algorithmic Auto-Recreation System of hiPSC-CMs Simulation and Prediction of Drug Testing. (2019)
  • Author(s): Hirohiko Kohjitani, Shigeya Kouda, Yukiko Himeno, Takeru Makiyama, Asami Kashiwa, Yimin Wuriyanghai, Yuta Yamamoto, Minoru Horie, Takeshi Kimura, Akinori Noma, Akira Amano
  • Organizer: 12th Asia Pacific Heart Rhythm Society Scientific Session (APHRS2019)
  • Int'l Joint Research
• [Presentation] Patients characteristics of idiopathic ventricular fibrillation carrying SCN5A mutations without Brugada or long QT syndrome. (2019)
  • Author(s): Takanori Aizawa, Takeru Makiyama, Hai Huang, Tomohiko Imamura, JingShan Gao, Yimin Wuriyanghai, Takeru Makiyama, Yuta Yamamoto, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Hai Huang, Wuriyanghai Yimin, Hirohiko Kohjitani, Yuta Yamamoto, Seiko Ohno, Minoru Horie
  • Organizer: 12th Asia Pacific Heart Rhythm Society Scientific Session (APHRS2019)
  • Int'l Joint Research
• [Presentation] Algorithmic Auto-Recreation System of hiPSC-CMs Simulation and Prediction of Drug Testing (2019)
  • Author(s): Hirohiko Kohjitani, Shigeya Kouda, Yukiko Himeno, Takeru Makiyama, Hai Huang, Jingshan Gao, Asami Kashiwa, Yimin Wuriyanghai, Daisuke Yoshinaga, Takeshi Kimura, Akira Amano.
  • Organizer: 第83回日本循環器学会学術集会
• [Presentation] Overlapping Sick Sinus Syndrome Associated with SCN5A Mutations in Patients with Brugada Syndrome (2019)
  • Author(s): Asami Kashiwa, Takeru Makiyama, Seiko Ohno, Hisaaki Aoki, Hirohiko Kohjitani Yimin Wuriyanghai, Yuta Yamamoto, Jingshan Gao, Huang Hai, Minoru Horie, Takeshi Kimura.
  • Organizer: 第83回日本循環器学会学術集会
• [Presentation] Three-Dimensional Structural Analysis of Mutant Ryanodine Receptor Type 2 Channels Associated with Catecholaminergic Polymorphic Ventricular Tachycardia. (2019)
  • Author(s): Jingshan Gao, Takeru Makiyama, Seiko Ohno, Yuta Yamamoto, Yimin Wuriyanghai, Hirohiko Kohjitani, Asami Kashiwa, Minoru Horie, Takeshi Kimura.
  • Organizer: 第83回日本循環器学会学術集会
• [Presentation] Modeling Overlapping Phenotypes of Long-QT and Brugada Syndrome with CACNA1C-E1115K that Alters Ion Selectivity of the Cardiac L-Type Calcium Channel Using Induced Pluripotent Stem Cells. (2019)
  • Author(s): Asami Kashiwa, Takeru Makiyama, Hirohiko Kohjitani, Yimin Wuriyanghai, Yuta Yamamoto, Jingshan Gao, Hai Huang, Taisuke Ishikawa, Seiko Ohno, Futoshi Toyoda, Seiichi Sato, Minoru Horie, Naomasa Makita, Takeshi Kimura
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Algorithmic Auto-Recreation System of hiPSC-CMs Simulation and Prediction of Drug Testing. (2019)
  • Author(s): Hirohiko Kohjitani, Shigeya Kouda, Yukiko Himeno, Takeru Makiyama, Hai Huang, Jingshan Gao, Asami Kashiwa, Yimin Wuriyanghai, Daisuke Yoshinaga, Takeshi Kimura, Akira Amano.
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Single Cell Electrophysiological Analysis of iPS Cell-derived Cardiomyocytes Generated from Long-QT Syndrome Patients Carrying a CALM2 Mutation Using a Membrane Potential Imaging System. (2019)
  • Author(s): Yuta Yamamoto, Takeru Makiyama(代発表), Yimin Wuriyanghai, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Huang Hai, Taisuke Ishikawa, Seiko Ohno, Kazuhisa Chonabayashi, Kenji Suda, Yoshinori Yoshida, Minoru Horie, Naomasa Makita, Takeshi Kimura,
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Structural Insights into Catecholaminergic Polymorphic Ventricular Tachycardia-associated RyR2 Mutant Channels using a Three-dimensional in Silico Model. (2019)
  • Author(s): Jingshan Gao, Takeru Makiyama, Seiko Ohno, Yuta Yamamoto, Yimin Wuriyanghai, Hirohiko Kohjitani, Asami Kashiwa, Hai Huang, Kouichi Katou, Minoru Horie, Takeshi Kimura.
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Phenotype-based High-throughput Classification of Long-QT Syndrome Subtypes Using Human Induced Pluripotent Stem Cells. (2019)
  • Author(s): Daisuke Yoshinaga, Shiro Baba, Takeru Makiyama, Hirofumi Shibata, Takuya Hirata, Kentaro Akagi, Koichi Matsuda, Hirohiko Kohjitani, Yimin Wuriyanghai, Katsutsugu Umeda, Yuta Yamamoto, Bruce R. Conklin, Minoru Horie, Junko Takita, Toshio Heike.
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Presentation] Complex aberrant splicing in the induce pluripotent stem cell-derived cardiomyocytes from a patient with long-QT syndrome carrying KCNQ1-A344Aspl mutation. (2019)
  • Author(s): Wuriyanghai Y, Makiyama T, Sasaki K, Kamakura T, Yamamoto Y, Hayano M, Harita T, Nishiuchi S, Chen J, Kohjitani H, Hirose S, Yokoi F, Gao J, Chonabayashi K, Watanabe K, Ohno S, Yoshida Y, Kimura T, Horie M
  • Organizer: 第66回日本不整脈心電学会学術大会
• [Patent(Industrial Property Rights)] アンチセンスオリゴヌクレオチド医薬のスクリーニング方法 (2020)
  • Inventor(s): 牧山武、山本雄大、入山友輔、近藤嘉紀、日高夕貴、石川龍太郎
  • Industrial Property Rights Holder: 牧山武、山本雄大、入山友輔、近藤嘉紀、日高夕貴、石川龍太郎
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2020-036716
• [Patent(Industrial Property Rights)] ＣＡＬＭ２のアンチセンスオリゴヌクレオチド (2020)
  • Inventor(s): 入山友輔、近藤嘉紀、日高夕貴、石川龍太郎、牧山武、山本雄大
  • Industrial Property Rights Holder: 入山友輔、近藤嘉紀、日高夕貴、石川龍太郎、牧山武、山本雄大
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2020-036715