Novel molecular basis of cardriac conduction disturbance associated with a collagene gene mutation

2017 Fiscal Year Annual Research Report

• Project/Area Number: 15K15311
• 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): 牧山 武 京都大学, 医学研究科, 助教 (30528302)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: コラーゲン / 進行性心臓伝導障害 / ノックインマウス / CRISPR/Cas9

Outline of Annual Research Achievements

前年度までに作成したCol6A2ノックインマウスは、生後１年後まで経過を見たが、伝導異常を含めた心電図異常（心拍数、PR時間、QRS幅、QT時間）はみられなかった。そのため、別の心臓伝導障害家系の次世代シークエンスで見出したコネキシン45(Cx45)の遺伝子（GJC1）のミスセンス変異R75Hについて研究を継続した。
日本・フランス2家系の進行性心房伝導障害に同一の変異Cx45-R75Hが同定された。４人のキャリアは共通の臨床症状として、(1)房室ブロック・心房静止などの進行性心房伝導障害と (2)短顔症・屈指症・永久歯無形性などの歯牙骨格異常を示した。
この変異が心房の進行性伝導障害・心房静止と歯骨形成異常の病因であることを証明するために、変異Cx45のギャップ結合の生化学的・生理学的特性をin vitroで解析した。培養N2a細胞に強制発現させたCx45-R75Hは、ほぼ正常Cx45と同様、細胞・細胞間のギャップ結合プラークを形成した。しかし、Lucifer Yellowの隣接細胞へ移行する速度はCx45-R75Hで著しく遅延、ダブルホールセルパッチクランプ法測定した細胞間コンダクタンスも著しく低下していた。これらの結果は、Cx45-R45Hはギャップ結合に大きな構造異常をもたらすことはないが、その電気生理学的特性は著しく低下していることが判明した。
Cx45変異がもたらす心房伝導障害をin vivoで確認するために、心臓特異的コンディショナルノックアウトマウスを作成した。タモキシフェン誘導によって心臓Cx45をノックアウトすると、マウスの心電図はP波消失、PR間隔延長、心房性不整脈を示し、経食道ペーシングでも洞結節回復時間の延長が確認された。
以上の研究から、Cx45遺伝子は進行性心房伝導障害の新規の疾患遺伝子であることが証明された。

Research Products

• [Int'l Joint Research] INSERM/CNRS(フランス)
  • Country Name: FRANCE
  • Counterpart Institution: INSERM/CNRS
• [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, Hasdem C, Ingles J, Aiba T, Makita N, Probst V, Wilde A, Newbury-Ecob R, Sheppard M, Semsarian C, Sy R, Behr E
  • Journal Title: Heart Rhythm Volume: in Press Pages: in Press
  • Peer Reviewed / Int'l Joint Research
• [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 / Open Access
• [Journal Article] The Phenotypic Spectrum of a Mutation?Hotspot Responsible for the?Short QT Syndrome (2017)
  • Author(s): Hu D, Li Y, Zhang J, Pfeiffer R, Gollob MH, Healey J, Harrell DT, Makita N, Abe H, Sun Y, Guo J, Zhang L, Yan G, Mah D, Walsh EP, Leopold HB, Giustetto C, Gaita F, Zienciuk-Krajka A, Mazzanti A, Priori SG, Antzelevitch C, Barajas-Martinez H
  • Journal Title: JACC: Clinical Electrophysiology Volume: 3 Pages: 727-743.
  • DOI: 10.1016/j.jacep.2016.11.013
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Letter to the Editor (2017)
  • Author(s): Ishikawa T, Makita N, Aoki H, Saida Y
  • Journal Title: J Vet Intern Med Volume: 31 Pages: 967-968.
  • DOI: 10.1111/jvim.14781
  • Peer Reviewed / Open Access
• [Journal Article] Sick sinus syndrome with HCN4 mutations shows early onset and frequent association with atrial fibrillation and left ventricular noncompaction (2017)
  • Author(s): Ishikawa T, Ohno S, Murakami T, Yoshida K, Mishima H, Fukuoka T, Kimoto H, Sakamoto R, Ohkusa T, Aiba T, Nogami A, Sumitomo N, Shimizu W, Yoshiura KI, Horigome H, Horie M, Makita N
  • Journal Title: Heart Rhythm Volume: 14 Pages: 717-724.
  • DOI: 10.1016/j.hrthm.2017.01.020
  • Peer Reviewed
• [Journal Article] Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes (2017)
  • Author(s): Kuroda Y, Yuasa S, Watanabe Y, Ito S, Egashira T, Seki T, Hattori T, Ohno S, Kodaira M, Suzuki T, Hashimoto H, Okata S, Tanaka A, Aizawa Y, Murata M, Aiba T, Makita N, Furukawa T, Shimizu W, Kodama I, Ogawa S, Kokubun N, Horigome H, Horie M, Kamiya K, Fukuda K
  • Journal Title: Biochemistry and Biophysics Reports Volume: 9 Pages: 245-256.
  • DOI: 10.1016/j.bbrep.2017.01.002
  • Peer Reviewed
• [Journal Article] Gene-Based Risk Stratification for Cardiac Disorders in LMNA Mutation Carriers (2017)
  • Author(s): Nishiuchi S, Makiyama T, Ishikawa T, Makita N et al.
  • Journal Title: Circ Cardiovasc Genet Volume: 10 Pages: e001603.
  • DOI: 10.1161/CIRCGENETICS.116.001603
  • Peer Reviewed
• [Journal Article] Postmortem genetic analysis of sudden unexpected death in infancy: neonatal genetic screening may enable the prevention of sudden infant death (2017)
  • Author(s): Oshima Y, Yamamoto T, Ishikawa T, Mishima H, Matsusue A, Umehara T, Murase T, Abe Y, Kubo SI, Yoshiura KI, Makita N, Ikematsu K
  • Journal Title: J Hum Genet Volume: 62 Pages: 989-995.
  • DOI: 10.1038/jhg.2017.79
  • Peer Reviewed
• [Journal Article] Progressive atrial conduction system defects associated with bone malformation caused by a connexin45 mutation (2017)
  • Author(s): Seki A, Ishikawa T, Daumy X, Mishima H, Barc J, Sasaki R, Nishii K, Saito K, Urano M, Ohno S, Otsuki S, Kimoto H, Baruteau A, Thollet A, Fouchard S, Bonnaud S, Parent P, Shibata Y, Perrin JP, Le Marec H, Hagiwara N, Mercier S, Horie M, Probst V, Yoshiura KI, Redon R, Schott JJ, Makita N
  • Journal Title: J Am Coll Cardiol Volume: 70 Pages: 58-70.
  • DOI: 10.1016/j.jacc.2017.05.039
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Genotype-Phenotype Correlation of SCN5A Mutation for the Clinical and Electrocardiographic Characteristics of Probands With Brugada Syndrome: A Japanese Multicenter Registry (2017)
  • Author(s): Yamagata K, Horie M, Aiba T, Ogawa S, Aizawa Y, Ohe T, Yamagishi M, Makita N, Sakurada H, Tanaka T, Shimizu A, Hagiwara N, Kishi R, Nakano Y, Takagi M, Makiyama T, Ohno S, Fukuda K, Watanabe H, Morita H, Hayashi K, Kusano K, Kamakura S, Yasuda S, Ogawa H, Miyamoto Y, Kapplinger JD, Ackerman MJ, Shimizu W
  • Journal Title: Circulation Volume: 135 Pages: 2255-2270.
  • DOI: 10.1161/CIRCULATIONAHA.117.027983
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation (2017)
  • Author(s): Yamamoto Y, Makiyama T, Harita T, Sasaki K, Wuriyanghai Y, Hayano M, Nishiuchi S, Kohjitani H, Hirose S, Chen J, Yokoi F, Ishikawa T, Ohno S, Chonabayashi K, Motomura H, Yoshida Y, Horie M, Makita N, Kimura T
  • Journal Title: Hum Mol Genet Volume: 26 Pages: 1670-1677.
  • DOI: 10.1093/hmg/ddx073
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] A novel de novo calmodulin mutation in a 6-year-old boy who experienced an aborted cardiac arrest (2017)
  • Author(s): Takahashi K, Ishikawa T, Makita N, Takefuta K, Nabeshima T, Nakayashiro M
  • Journal Title: HeartRhythm Case Reports Volume: 3 Pages: 69-72.
  • DOI: 10.1016/j.hrcr.2016.09.004
  • Peer Reviewed / Open Access
• [Presentation] Genetic and Biophysical Basis of Calmodulinopathy, and Functional Rescue by Genome-Editing in Patient-Derived iPS Cardiomyocytes (2017)
  • Author(s): Makita N.
  • Organizer: 20th International Symposium on Calcium Binding Proteins and Calcium Function in Health and Disease
  • Int'l Joint Research / Invited
• [Presentation] Genetic Mutation of Brugada Syndrome (2017)
  • Author(s): Makita N.
  • Organizer: Heart Rhythm Society Scientific Sessions
  • Int'l Joint Research / Invited
• [Presentation] Brugada Syndrome: Basic and Clinical Updates, Advancement of Basic Research (2017)
  • Author(s): Makita N.
  • Organizer: 13th Annual Congress European Cardiac Arrhythmia Society
  • Int'l Joint Research / Invited
• [Presentation] Multinational genome-wide association study in long QT syndrome identifies a role for common genetic variation in disease susceptibility and points to a polygenic architecture in mutation-negative cases (2017)
  • Author(s): Lahrouchi N, Makita N et al.
  • Organizer: Heart Rhythm Society Scientific Sessions
  • Int'l Joint Research
• [Presentation] De novo and Familial Connexin45 Mutant R75H Causes Progressive Atrioventricular Block Associated with Craniofacial and Dentodigital Dysmorphisms (2017)
  • Author(s): Makita N, Seki A, Ishikawa T, Dawmy X, Mishima H, Barc J, Sasaki R, Nishii K, Saito K, Urano M, Ohno S, Otsuki S, Kimoto H, Baruteau A, Aurelie T, Swanny F, Stephanie B, Parent P, Shibata Y, Perrin JJ, Le Marec H, Hagiwara N, Mercier S, Horie M, Probst V, Yoshiura K, Redon R, Schott J, J.
  • Organizer: Printemps de la Cardiologie Recherche Fondamentale et Clinique
  • Int'l Joint Research / Invited
• [Presentation] Gene-Based Risk Stratification for Cardiac Disorders in LMNA Mutation Carriers (2017)
  • Author(s): Nishiuchi S, Makiyama T, Aiba T, Nakajima K, Ishikawa T, Murakoshi N, Watanabe H, Ohno S, Doi T, Shizuta S, Minamino T, Saito Y, Oginosawa Y, Nogami A, Aonuma K, Kusano K, Makita N, Shimizu W, Horie M, Kimura T.
  • Organizer: The 10th Asia Pacific Heart Rhythm Sosiety Scientific Session
  • Int'l Joint Research
• [Presentation] CALM2-D134H Mutation Associated with Long-QT Syndrome Prevents Inactivation of L-type Ca2+ Currents in Human iPS Cell Derived Cardiomyocytes. (2017)
  • Author(s): Yamamoto Y, Makiyama T, Harita T, Hayano M, Nishiuchi S, Wuriyanghai Y, Kohjitani H, Hirose S, Yokoi F, Ishikawa T, Ohno S, Chinabayashi K, Suda K, Yoshida Y, Horie M, Makita N, Kimura T.
  • Organizer: The 10th Asia Pacific Heart Rhythm Society Scientific Session
  • Int'l Joint Research
• [Presentation] Sick sinus syndrome caused by HCN4 mutations shows early onset and frequent association with atrial fibrillation and left ventricular non-compaction (2017)
  • Author(s): Ishikawa T, Ohno S, Murakami T, Yoshida K, Fukuoka T, Mishima H, Kimoto H, Aiba T, Nogami A, Sumitomo N, Shimizu W, Horigome H, Yoshiura KI, Horie M, Makita N.
  • Organizer: Heart Rhythm Society Scientific Sessions
  • Int'l Joint Research
• [Presentation] Mutation in L-type Calcium Channel Cav1.3 (CACNA1D) Underlying a Consanguineous Family Associated with Congenital AV Block and Deaf-Blindness (2017)
  • Author(s): Kimoto H, Ishikawa T, Mishima H, Hasdemir C, Yoshiura K, Makita N.
  • Organizer: The 10th Asia Pacific Heart Rhythm Society Scientific Session
  • Int'l Joint Research
• [Presentation] Gene-Based Risk Stratification for Cardiac Disorders in LMNA Mutation Carriers in Japan (2017)
  • Author(s): Nishiuchi S, Aiba T, Makiyama T, Nakajima K, Yagihara N, Ishikawa T, Onoue K, Murakoshi N, Watanabe I, Ohkubo K, Watanabe H, Ohno S, Doi T, Shizuta S, Minamino T, Saito Y, Oginosawa Y, Nogami A, Aonuma K, Kusano K, Makita N, Shimizu W, Horie M, Kimura T.
  • Organizer: American Heart Association Annual Scientific Session 2017
  • Int'l Joint Research
• [Presentation] CALM2-D134H Mutation Associated With Long-QT Syndrome Delayed Inactivation of L-type Ca2+ Currents in Human iPS Cells Derived Cardiomyocytes. (2017)
  • 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: American Heart Association Annual Scientific Session 2017
  • Int'l Joint Research
• [Presentation] lL-cis-Diltiazem Ameliorates Impaired Calcium Channel Inactivation in a Patient-Specific Stem Cell Model of Long-QT Syndrome with a Calmodulin Mutation (2017)
  • Author(s): Yokoi F, Makiyama T, Harita T, Yamamoto Y, Hayano M, Nishiuchi S, Kohjitani H, Wuriyanghai Y, Hirose S, Toyoda F, Yoshida Y, Makita N, Horie M, Kimura T.
  • Organizer: The 10th Asia Pacific Heart Rhythm Society Scientific Session
• [Presentation] Genetics of Familial Atrial Fibrillation (2017)
  • Author(s): Ishikawa T, Makita N.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] Genetic-based risk stratification for cardiac disorders in LMNA mutation carriers (2017)
  • Author(s): Nishiuchi S, Aiba T, Nakajima K, Hirose S, Kohjitani H, Yamamoto Y, Harita T, Hayano M, Sasaki K, Ishikawa T, Onoue K, Saito Y, Kusano K, Makita N, Shimizu W, Horie M, Kimura T.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] Genotype-Phenotype Correlation of SCN5A Mutations for the Clinical and Electrocardiographic Characteristics of Probands with Brugada Syndrome: A Japanese Multicenter Registry (2017)
  • Author(s): Yamagata K, Horie M, Aiba T, Ogawa S, Aizawa Y, Ohe T, Yamagishi M, Makita N, Sakurada H, Tanaka T, Shimizu A, Hagiwara N, Kishi R, Nakano Y, Takagi M, Makiyama T, Ohno S, Fukuda K, Watanabe H, Morita H, Hayashi K, Fukushima-Kusano K, Kamakura S, Yasuda S, Ogawa H, Y. M, Kapplinger J, Ackerman M, Shimizu W.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] Allele-specific ablation rescues electrophysiological abnormalities in a human iPS cell model of long-QT syndrome with a CALM2 mutation (2017)
  • Author(s): Yamamoto Y, Makiyama T, Harita T, Sasaki K, Wuriyanghai Y, Nishiuchi S, Kohjitani H, Hirose S, Ishikawa T, Motomura H, Ohno S, Yoshida Y, Horie M, Makita N, Kimura T.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] Calmodulin Mutations Responsible for Long QT Syndrome and Functional Rescue using a CRISPR/Cas9 System (2017)
  • Author(s): 蒔田直昌.
  • Organizer: 第94回日本生理学会大会
  • Invited
• [Presentation] ブルガダ症候群の予後予測を目指す変異心筋Naチャネルの機能解析 (2017)
  • Author(s): 下河舞子, 木本浩貴, 石川泰輔, 蒔田直昌.
  • Organizer: 第68回西日本生理学会
• [Presentation] Mutation Spot-sensitive Clinical Features and Risk of Life-threatening Arrhythmia in Long QT Syndrome Type 1 in Japan (2017)
  • Author(s): Aiba T, Makimoto H, 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.
  • Organizer: 第81回日本循環器学会学術集会
• [Presentation] Mutations in Desmin Gene Uncover Phenotypic Overlap between Progressive Cardiac Conduction Defect with Muscular Dystrophy and Cardiomyopathy (2017)
  • Author(s): 木本浩貴, 石川泰輔, 町田紘子, 森田宏, 住友直方, 中村一文, 伊藤浩, バーク・ジュリアン, ショット・ジョンジャック, 蒔田直昌.
  • Organizer: 第81回日本循環器学会学術集会