Novel pathophysiology underlying cardiac conduction defect due to mutations of gap junction genes

2018 Fiscal Year Research-status Report

• Project/Area Number: 18K19550
• Research Institution: Nagasaki University
• Principal Investigator: 蒔田 直昌 長崎大学, 医歯薬学総合研究科(医学系), 教授 (00312356)
• Co-Investigator(Kenkyū-buntansha): 西井 明子 (関明子) 東京女子医科大学, 医学部, 助教 (80408608) ; 柴田 恭明 長崎大学, 医歯薬学総合研究科(医学系), 講師 (80253673) ; 本荘 晴朗 名古屋大学, 環境医学研究所, 准教授 (70262912)
• Project Period (FY): 2018-06-29 – 2021-03-31
• Keywords: ギャップ結合 / コネキシン45 / 心臓伝導障害 / 歯骨形成異常 / Crispr/Cas9ゲノム編集

Outline of Annual Research Achievements

1. コネキシン40(Cx40)変異Q58LのCrispr/cas9ヘテロノックインマウス(Cx40-Q58L-KI)は作成に成功し、生後8か月まで心電図を経過観察したが、明白な伝導障害はなかった。ヒトと違ってマウスの房室結節には別のコネキシンCx30.2が発現しているため、これがCx40の機能異常を補って、伝導障害を不顕性化している可能性が考えられた。そのためCx30,2のホモノックアウトマウス(Cx30.2-KO)をCRISPR/Cas9で作成し、Cx40-Q58L-KIと交配した(Cx40-KI/Cx30.2-KO)。2系統のマウス（Cx40-Q58L-KI、Cx40-KI/Cx30.2-KO）の遺伝型を確認ののち胚を凍結し、国立循環器病研究センター（国循）に移送した。
2. Cx45の変異R75HノックインマウスもCRISPR/Cas9法で試みたが、sgRNAやオリゴを数回変えて試したが出生しなかった。また、遺伝子編集の簡便化と迅速化を図るため、野生型マウスの自然交配により着床前胚を有する子宮～卵管に核酸とCRISPR/Cas9タンパクを注入し卵管全体に電気穿孔法を行う「iGONAD法」を試みたが、これも無効であった。

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

Cx40系統のマウスは確立したが、Cx45変異KIは出生しない。Cx45のホモKOマウスは胎生致死であることが知られているが、マウスでは胎生期の生命維持に重要な役割を担うタンパクのため、ヘテロのミスセンス変異も生まれない可能性がある。

Strategy for Future Research Activity

1. Cx40の2系統のマウス（Cx40-Q58L-KI, Cx40-KI/Cx30.2-KO)は、2019年7月の国循の新実験動物施設オープン後、凍結胚から個体復元を行い、心電図・電気生理学的解析を行う。
2. Cx45に関しては、新たな手法として、C57BL/JマウスのES細胞にCRISPR/Cas9で遺伝子編集で変異ノックインをおこない、このES細胞を受精直後の受精卵に注射して子宮に戻し、キメラ状態での個体出生を試みる。

Causes of Carryover

Cx45－KIマウスがこれまでの方法では全く出生しなかったため、その後の機能解析実験が行うことができず次年度使用額が生じた。2019年7月以降、異動先の国循の施設内機関の協力を得てもう一度マウス作成を開始する。

Research Products

• [Journal Article] Association of Genetic and Clinical Aspects of Congenital Long QT Syndrome With Life-Threatening Arrhythmias in Japanese Patients (2019)
  • Author(s): Shimizu W., Makita N. et al
  • Journal Title: JAMA Cardiol Volume: 4 Pages: 246-254.
  • DOI: 10.1001/jamacardio.2018.4925
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] HCN4 Gene Polymorphisms Are Associated With Occurrence of Tachycardia-Induced Cardiomyopathy in Patients With Atrial Fibrillation (2018)
  • Author(s): Nakano Y., Ochi H., Sairaku A., Onohara Y., Tokuyama T., Motoda C., Matsumura H., Tomomori S., Amioka M., Hironobe N., Ohkubo Y., Okamura S., Makita N., Yoshida Y., Chayama K., Kihara Y.
  • Journal Title: Circ Genom Precis Med Volume: 11 Pages: e001980.
  • DOI: 10.1161/CIRCGEN.117.001980
  • Peer Reviewed
• [Journal Article] Clinical Manifestations and Long-Term Mortality in Lamin A/C Mutation Carriers From a Japanese Multicenter Registry (2018)
  • Author(s): Nakajima K., Makita N et al.
  • Journal Title: Circ J Volume: 82 Pages: 2707-2714.
  • DOI: 10.1253/circj.CJ-18-0339
  • Peer Reviewed
• [Journal Article] HCN4 pacemaker channels attenuate the parasympathetic response and stabilize the spontaneous firing of the sinoatrial node (2018)
  • Author(s): Kozasa Y., Nakashima N., Ito M., Ishikawa T., Kimoto H., Ushijima K., Makita N., Takano M.
  • Journal Title: J Physiol Volume: 596 Pages: 809-825.
  • DOI: 10.1113/JP275303
  • Peer Reviewed
• [Journal Article] Lack of genotype-phenotype correlation in Brugada Syndrome and Sudden Arrhythmic Death Syndrome families with reported pathogenic SCN1B variants (2018)
  • Author(s): Gray B., Hasdemir C., Ingles J., Aiba T., Makita N., Probst V., Wilde A. A. M., Newbury-Ecob R., Sheppard M. N., Semsarian C., Sy R. W., Behr E. R.
  • Journal Title: Heart Rhythm Volume: 15 Pages: 1051-1057.
  • DOI: 10.1016/j.hrthm.2018.03.015
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Interaction of phase singularities on the spiral wave tail: reconsideration of capturing the excitable gap (2018)
  • Author(s): Tomii Naoki、Yamazaki Masatoshi、Arafune Tatsuhiko、Kamiya Kaichiro、Nakazawa Kazuo、Honjo Haruo、Shibata Nitaro、Sakuma Ichiro
  • Journal Title: American Journal of Physiology-Heart and Circulatory Physiology Volume: 315 Pages: H318～H326
  • DOI: 10.1152/ajpheart.00558.2017
  • Peer Reviewed
• [Presentation] 不整脈症候群で心臓が構造的に正常な患者における心筋症の原因遺伝子変異の特定 (2018)
  • Author(s): 八木原伸江, 渡部裕, 蒔田直昌, 堀江稔, 清水渉, 大野聖子, 長谷川奏恵, 相庭武司, 田中敏博, 角田達彦, 重水大智, 徹 南野
  • Organizer: 第82回日本循環器学会学術集会
• [Presentation] CaMKII as a target for novel therapy to treat lethal ventricular tachyarrhythmias. (2018)
  • Author(s): 辻幸臣, 蒔田直昌
  • Organizer: 第57回日本生体医工学学会大会
• [Presentation] CALM2関連性QT延長症候群iPSCモデルにおいて、l-cis-diltiazemは遅延したL型calciumチャネルの不活性化をさらに強める (2018)
  • Author(s): 横井文香, 牧山武, 山本雄大, 張田健志, Yimin Wuriyanghai, 早野護, 西内英, 糀谷泰彦, 廣瀬紗也子, 高景山, 大野聖子, 豊田太, 吉田善紀, 蒔田直昌, 堀江稔, 木村剛
  • Organizer: 第82回日本循環器学会学術集会
• [Presentation] Torsadogenic Action Of Late Na+ Current In Experimental Electrical Storm (2018)
  • Author(s): Yamazaki M., Tsuji Y. , Niwa R., Tomii N., Arafune T., Honjo H., Dobrev D., Nattel S., Kodama I., Sakuma I., Makita N.
  • Organizer: The 39th Heart Rhythm Society Scientific Sessions
  • Int'l Joint Research / Invited
• [Presentation] Single Cell Electrophysiological Analysis of Human iPS Cell-Derived Cardiomyocytes Generated from Long-QT Syndrome Patients Carrying a CALM2 Mutation Using a Membrane Voltage Imaging System (2018)
  • Author(s): Yamamoto Y., Makiyama T, Wuriyanghai Y., Kohjitani H., Hirose S., Gao J, Kashiwa A., Kimura T., Ishikawa T., Motomura H., Makita N, Ohno S., Chonabayashi K., Yoshida Y., Suda K., Horie M
  • Organizer: The 11th Asia Pacific Heart Rhythm Society Scientific Session
  • Int'l Joint Research / Invited
• [Presentation] The inactivation of L-type Ca2+ Channel was impaired in Human iPS Cell Model of Long-QT Syndrome with CALM2-D134H Mutation. (2018)
  • Author(s): Yamamoto Y., Makiyama T., Harita T., Hayano M., Nishiuchi S., Wuriyanghai Y., Kohjitani H., Hirose S., Yokoi F., Ishikawa T., Ohno S., Chonabayashi K., Suda K., Yoshida Y., Horie M., Makita N., Kimura T.
  • Organizer: 第82回日本循環器学会学術集会
• [Presentation] Duration Predicts Events In Caucasians With E1784K-SCN5A: The E1784K International Consortium (2018)
  • Author(s): Wijeyeratne Y. ,Makita N. et al
  • Organizer: The 39th Heart Rhythm Society Scientific Sessions
  • Int'l Joint Research / Invited
• [Presentation] Mechanisms of Electrical Storm Associated With QT Prolongation: Successful Mapping of Torsades de Pointes in Rabbits (2018)
  • Author(s): Tsuji Y., Yamazaki M., Niwa R., Tomii N., Arafune T., Honjo H., Dobrev D., Nattel S., Kodama I., Sakuma I., Makita N
  • Organizer: American Heart Association Annual Scientific Sessions 2018
  • Int'l Joint Research / Invited
• [Presentation] HCN4 Polymorphisms and Tachycardia Induced Cardiomyopathy (2018)
  • Author(s): Nakano Y., Ochi H., Onohara Y., Sairaku A., Tokuyama T., Motoda C., Matsumura H., Tomomori S., Amioka M., Hironobe N., Ohkubo Y., Okamura S., Makita N., Yoshida Y., Chayama K., Kihara Y.
  • Organizer: The 65th Annual Meeting of the Japan Heart Rhythm Society
• [Presentation] Clinical Manifestations and Long-term Mortality in Lamin A/C Mutation Carriers from the Japanese Multicenter Registry (2018)
  • Author(s): Nakajima K., Aiba T., Makiyama T., Nishiuchi S., Ohno S., Doi T., Shizuta S., Onoue K., Yagihara N., Ishikawa T., Watanabe I., Oginosawa Y., Nogami A., Aonuma K., Saitoh Y., Kimura T., Makita N., Shimizu W., Horie M., Kusano K.
  • Organizer: The 65th Annual Meeting of the Japan Heart Rhythm Society
• [Presentation] Genotype-Dependent Differences in Short QT Syndrome (2018)
  • Author(s): Makita N.
  • Organizer: The 11th Asia Pacific Heart Rhythm Society Scientific Session
  • Int'l Joint Research / Invited
• [Presentation] Atrial Conduction Defects Caused by a Connexin45 Mutation (2018)
  • Author(s): Makita N.
  • Organizer: The 11th Asia Pacific Heart Rhythm Society Scientific Session
  • Int'l Joint Research / Invited
• [Presentation] Novel Arrhythmia Syndrome Associated with Gap Junction Mutations (2018)
  • Author(s): Makita N.
  • Organizer: The 11th Asia Pacific Heart Rhythm Society Scientific Session
  • Int'l Joint Research / Invited
• [Presentation] Clinical and Genetic Basis of Calmodulinopathy (2018)
  • Author(s): Makita N.
  • Organizer: The 11th Asia Pacific Heart Rhythm Society Scientific Session
  • Int'l Joint Research / Invited
• [Presentation] Pathophysiology of Inherited Arrhythmias Associated with Ca Handling Abnormality (2018)
  • Author(s): Makita N.
  • Organizer: The 65th Annual Meeting of the Japan Heart Rhythm Society
• [Presentation] Mutation Hot-spot-based Risk Stratification In Long QT Syndrome Type 1: Data From A Nationwide Japanese LQT Registry (2018)
  • Author(s): Kashiwa A., Aiba T., Makimoto H., Yamagata K. , Keiko S. , Yagihara N., Watanabe H. , Ohno S., Hayashi K. , Sumitomo N., Yoshinaga M. , Morita H., Miyamoto Y. , Makita N. , Horie M., Yasuda S., Kusano K., Shimizu W. , The_Japanese_LQTS_registry_investigators.
  • Organizer: The 39th Heart Rhythm Society Scientific Sessions
  • Int'l Joint Research / Invited
• [Presentation] Cardiac Connexin Syndrome: A New Inherited Cardiac Conduction Disease Entity with Distinct Electrophysiological and Extracardiac Manifestations. (2018)
  • Author(s): Ishikawa T., Seki A., Hagiwara N., Makita N.
  • Organizer: 第82回日本循環器学会学術大会
• [Presentation] Rare Coding Variants in Genes Other Than SCN5A Are Minimal Genetic Burden on the Prognosis of Brugada Syndrome (2018)
  • Author(s): Ishikawa T., Mishima H., Ohno S., T. Aiba, Nakano Y., Aizawa Y., Nakajima T., Hayashi K., Murakoshi N., Yagihara N., Kimoto H., Makiyama T., Watanabe H., Morita H., Yoshiura K., Nogami A., Shimizu W., Horie M., Tanaka T., Makita N.
  • Organizer: The 65th Annual Meeting of the Japan Heart Rhythm Society
• [Presentation] Lack Of Genotype-phenotype Correlation In Brugada Syndrome And Sudden Arrhythmic Death Syndrome Families With Reported Pathogenic SCN1B Variants (2018)
  • Author(s): Gray B. , Hasdemir C. , Ingles J. , Aiba T., Makita N. , PROBST V. , Wilde A., Semsarian C. , Sy R., Behr E.
  • Organizer: The 39th Heart Rhythm Society Scientific Sessions
  • Int'l Joint Research / Invited
• [Book] 不整脈 識る・診る・治す (2018)
  • Author(s): 辻幸臣 蒔田直昌（平尾見三、小室一成編）
  • Total Pages: 376
  • Publisher: 中山書店
  • ISBN: 978-4-521-74585-5
• [Book] 循環器科の心電図 (2018)
  • Author(s): 石川泰輔 蒔田直昌（村川 裕二編）
  • Total Pages: 224
  • Publisher: 南江堂
  • ISBN: 978-4-524-23791-3