心筋イオン輸送体の遺伝子異常に起因する致死性不整脈の新規病態メカニズムの解明

2015 Fiscal Year Annual Research Report

• Project/Area Number: 15H04823
• 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): 吉浦 孝一郎 長崎大学, 原爆後障害医療研究所, 教授 (00304931) ; 渡邊 泰秀 浜松医科大学, 医学部, 教授 (50305380) ; 牧山 武 京都大学, 医学(系)研究科(研究院), 助教 (30528302)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: 致死性不整脈 / トランスポータ / 分子心臓学

Outline of Annual Research Achievements

致死性遺伝性不整脈の研究は、QT延長症候群を中心として、心筋イオンチャネルを標的とした候補遺伝子解析に依存してきた。しかしその他の致死性遺伝性不整脈の病態解明は、この手法で解明するのは困難であり、解析の方法論と標的分子を見直す必要がある。最近、細胞内Ca制御に係わるイオン輸送体が新規QT感受性遺伝子であることが報告された。本研究では、この知見をもとに、原因不明の致死性不整脈患者に対して網羅的遺伝子解析と疾患iPS心筋細胞の機能解析をおこない、心臓突然死の予知予防への応用を目指す。
初年度は、致死性不整脈からの蘇生例のうち、心筋イオンチャネルKCNQ1, KCNH2, SCN5Aの変異が除外された15人に全エクソン解析を行った。その結果、カルモジュリン（CALM1, CALM2）のほかにNa-Ca交換体(NCX1;　SLC8A1)、Na-HCO3-交換体(SLC4A4)、Na-Kポンプ(ATP1B1)、筋小胞体Caポンプ(ATP2A2)などの細胞膜トランスポーター・ポンプの変異を発見した。さらに致死性不整脈蘇生群のターゲットエクソン解析したところ、6例(J波症候群+QT短縮症以降群1例、J波症候群2例、進行性心臓伝導障害2例、房室ブロック1例)に5つのSLC8A1変異(L218H, I409V, R517P, A519G, R841Q)を同定した。これらの変異cDNAを作成し培養繊維芽細胞にトランスフェクションし、NCX1交換体活性を測定した。また、変異タンパクの局在を調べるために変異プラスミドをHEK293細胞にトランスフェクションし、抗NCX1抗体免疫染色を行い、共焦点顕微鏡で局在を観察した。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

1.　全エクソン解析で細胞内Ca制御に係わるイオン輸送体の変異を同定している。
2.　ターゲットエクソン解析で、致死性不整脈蘇生患者６人に、Na-Ca交換体遺伝子SLC8A1変異を5個同定している。
3. 変異プラスミドを作成し、パッチクランプでNa-Ca交換体活性の電流解析を行っている。
4. 変異タンパクの細胞内局在を共焦点顕微鏡で解析した。

Strategy for Future Research Activity

今後変異NCX1の安定細胞株を作成し、45Ca取り込み能を計測する。
NCX1以外のトランスポータ遺伝子のターゲットエクソン解析、機能解析も行う。
患者末梢血からiPS心筋細胞を作成し、拍動iPS心筋細胞を用いて活動電位、Na-Ca交換電流、Na-Kポンプ電流、Caポンプ電流をパッチクランプで計測する。

Research Products

• [Int'l Joint Research] INSERM(フランス)
  • Country Name: FRANCE
  • Counterpart Institution: INSERM
• [Journal Article] Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I. (2016)
  • Author(s): Daumy X, Amarouch MY, Lindenbaum P, Bonnaud S, Charpentier E, Bianchi B, Nafzger S, Baron E, Fouchard S, Thollet A, Kyndt F, Barc J, Le Scouarnec S, Makita N, Le Marec H, Dina C, Gourraud JB, Probst V, Abriel H, Redon R, Schott JJ.
  • Journal Title: Int J Cardiol Volume: 207 Pages: 349-358
  • DOI: 10.1016/j.ijcard.2016.01.052
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Inherited bradyarrhythmia: A diverse genetic background. (2016)
  • Author(s): Ishikawa T, Tsuji Y, Makita N.
  • Journal Title: Journal of Arrhythmia Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1016/j.joa.2015.09.009
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Genetic defects in a His-Purkinje system transcription factor, IRX3, cause lethal cardiac arrhythmias. (2016)
  • Author(s): Koizumi A, Sasano T, Kimura W, Miyamoto Y, Aiba T, Ishikawa T, Nogami A, Fukamizu S, Sakurada H, Takahashi Y, Nakamura H, Ishikura T, Koseki H, Arimura T, Kimura A, Hirao K, Isobe M, Shimizu W, Miura N, Furukawa T.
  • Journal Title: Eur Heart J Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1093/eurheartj/ehv449
  • Peer Reviewed
• [Journal Article] Prognostic Significance of Premature Ventricular Contractions without Obvious Heart Diseases Determined by Standard 12-Lead Electrocardiography Considering their Morphology. (2016)
  • Author(s): Haruta D, Akahoshi M, Hida A, Sera N, Imaizumi M, Ichimaru S, Nakashima E, Takahashi I, Ohishi W, Fukae S, Maemura K.
  • Journal Title: Ann Noninvasive Electrocardiol. Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1111/anec.12275.
  • Peer Reviewed
• [Journal Article] Cardiac sodium channel mutation associated with epinephrine-induced QT prolongation and sinus node dysfunction. (2016)
  • Author(s): Chen J, Makiyama T, Wuriyanghai Y, Ohno S, Sasaki K, Hayano M, Harita T, Nishiuchi S, Yuta Yamamoto, Ueyama T, Shimizu A, Horie M, Kimura T.
  • Journal Title: Heart Rhythm Volume: 13(1) Pages: 289-98
  • DOI: 10.1016/j.hrthm.2015.08.021.
  • Peer Reviewed
• [Journal Article] The genetics underlying acquired long QT syndrome: impact for genetic screening. (2016)
  • Author(s): Itoh H, Crotti L, Aiba T, Spazzolini C, Denjoy I, Fressart V, Hayashi K, Nakajima T, Ohno S, Makiyama T, Wu J, Hasegawa K, Mastantuono E, Dagradi F, Pedrazzini M, Yamagishi M, Berthet M, Murakami Y, Shimizu W, Guicheney P, Schwartz PJ, Horie M
  • Journal Title: Eur Heart J Volume: 37(18) Pages: 1456-64.
  • DOI: 10.1093/eurheartj/ehv695
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Asymmetry of parental origin in long QT syndrome: preferential maternal transmission of KCNQ1 variants linked to channel dysfunction (2016)
  • Author(s): Itoh H, Berthet M, Fressart V, Denjoy I, Maugenre S, Klug D, Mizusawa Y, Makiyama T, Hofman N, Stallmeyer B, Zumhagen S, Shimizu W, Wilde AA, Schulze-Bahr E, Horie M, Tezenas du Montcel S, Guicheney P.
  • Journal Title: Eur J Hum Genet. Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1038/ejhg.2015.257.
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] LMNA cardiomyopathy detected in Japanese arrhythmogenic right ventricular cardiomyopathy cohort. (2016)
  • Author(s): Kato K, Takahashi N, Fujii Y, Umehara A, Nishiuchi S, Makiyama T, Ohno S, Horie M
  • Journal Title: J Cardiol Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1016/j.jjcc.2015.10.013.
  • Peer Reviewed
• [Journal Article] Novel SCN10A variants associated with Brugada syndrome (2016)
  • Author(s): Fukuyama M, Ohno S, Makiyama T, Horie M
  • Journal Title: Europace. Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1093/cvr/cvv042
• [Journal Article] Genotype-dependent differences in age of manifestation and arrhythmia complications in short QT syndrome (2015)
  • Author(s): Harrell DT, Ashihara T, Ishikawa T, Tominaga I, Mazzanti A, Takahashi K, Oginosawa Y, Abe H, Maemura K, Sumitomo N, Uno K, Takano M, Priori SG, Makita N.
  • Journal Title: Int J Cardiol Volume: 190 Pages: 393-402
  • DOI: 10.1016/j.ijcard.2015.04.090
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Functional Characterization of Rare Variants Implicated in Susceptibility to Lone Atrial Fibrillation. (2015)
  • Author(s): Hayashi K, Konno T, Tada H, Tani S, Liu L, Fujino N, Nohara A, Hodatsu A, Tsuda T, Tanaka Y, Kawashiri MA, Ino H, Makita N, Yamagishi M
  • Journal Title: Circ Arrhythm Electrophysiol Volume: 8(5) Pages: :1095-1104,
  • DOI: 10.1161/CIRCEP.114.002519
  • Peer Reviewed
• [Journal Article] Novel mutation in the alpha-myosin heavy chain gene is associated with sick sinus syndrome. (2015)
  • Author(s): Ishikawa T, Jou CJ, Nogami A, Kowase S, Arrington CB, Barnett SM, Harrell DT, Arimura T, Tsuji Y, Kimura A, Makita N.
  • Journal Title: Circ Arrhythm Electrophysiol Volume: 8(2) Pages: 400-408
  • DOI: 10.1161/CIRCEP.114.002534
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Molecular Mechanisms Underlying Urate-Induced Enhancement of Kv1.5 Channel Expression in HL-1 Atrial Myocytes (2015)
  • Author(s): Maharani N, Ting YK, Cheng J, Hasegawa A, Kurata Y, Li P, Nakayama Y, Ninomiya H, Ikeda N, Morikawa K, Yamamoto K, Makita N, Yamashita T, Shirayoshi Y, Hisatome I.
  • Journal Title: Circ J Volume: 79(12) Pages: 2659-2668
  • DOI: 10.1253/circj.CJ-15-0416
  • Peer Reviewed
• [Journal Article] Fibrosis, Connexin-43, and Conduction Abnormalities in the Brugada Syndrome (2015)
  • Author(s): Nademanee K, Raju H, de Noronha SV, Papadakis M, Robinson L, Rothery S, Makita N, Kowase S, Boonmee N, Vitayakritsirikul V, Ratanarapee S, Sharma S, van der Wal AC, Christiansen M, Tan HL, Wilde AA, Nogami A, Sheppard MN, Veerakul G, Behr ER
  • Journal Title: J Am Coll Cardiol Volume: 66(18) Pages: 1976-1986,
  • DOI: 10.1016/j.jacc.2015.08.862
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Relationships Between Clinical Characteristics and Decreased Plakoglobin and Connexin 43 Expressions in Myocardial Biopsies From Patients With Arrhythmogenic Right Ventricular Cardiomyopathy. (2015)
  • Author(s): Yoshida T, Kawano H, Kusumoto S, Fukae S, Koga S, Ikeda S, Koide Y, Abe K, Hayashi T, Maemura K.
  • Journal Title: Int Heart J. Volume: 56 Pages: 626-31
  • DOI: 10.1536/ihj.15-144.
  • Peer Reviewed / Open Access
• [Journal Article] Effective Management of Atrioventricular Interval for Paroxysmal Atrial Fibrillation That Developed After DDDR Pacemaker Implantation in a Sick Sinus Syndrome Patient. (2015)
  • Author(s): Tasaki H, Ashizawa N, Nagao S, Fukushima K, Furukawa R, Fukae S, Maemura K.
  • Journal Title: Int Heart J Volume: 56 Pages: 558-63
  • DOI: 10.1536/ihj.15-008.
  • Peer Reviewed / Open Access
• [Presentation] Electrophysiological Features of Patient-specific Human-induced Pluripotent Stem Cell-derived Cardiomyocytes Carrying the SCN5A-D1275N Mutation (2016)
  • Author(s): 早野 護、牧山 武
  • Organizer: 日本循環器学会
  • Place of Presentation: 仙台、仙台国際会議場
  • Year and Date: 2016-03-18
  • Invited
• [Presentation] 睡眠・サーカディアンリズムと循環器疾患～体内時計の視点から～ (2015)
  • Author(s): 前村浩二
  • Organizer: 第25回日本循環薬理学会
  • Place of Presentation: 奈良市、東大寺総合文化センター
  • Year and Date: 2015-12-04
  • Invited
• [Presentation] SCN5A and ventricular arrhythmias (2015)
  • Author(s): Makita N
  • Organizer: Asian Pacific Heart Rhythm Society,
  • Place of Presentation: Melbourne, Australia.
  • Year and Date: 2015-11-22
  • Int'l Joint Research / Invited
• [Presentation] Cardiac Sodium Channel Mutation Associated With Epinephrine-Induced QT Prolongation and Sinus Node Dysfunction, (2015)
  • Author(s): 陳 嘉容、牧山 武
  • Organizer: 日本不整脈学会／日本心電学会
  • Place of Presentation: 京都、京都国際会議場
  • Year and Date: 2015-07-28
  • Invited
• [Presentation] New genes for Progressive Cardiac Conduction Disease. (2015)
  • Author(s): Makita N
  • Organizer: Heart Rhythm Society
  • Place of Presentation: Boston, USA
  • Year and Date: 2015-05-14
  • Int'l Joint Research / Invited
• [Presentation] Structural and Electrophysiological Characteristic of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes, (2015)
  • Author(s): 牧山 武
  • Organizer: 日本循環器学会
  • Place of Presentation: 大阪、大阪国際会議場
  • Year and Date: 2015-04-24
  • Invited
• [Presentation] Genetic Backgrounds of Patients with Clinically Diagnosed Idiopathic Ventricular Fibrillation (2015)
  • Author(s): 糀谷 泰彦、牧山 武
  • Organizer: 日本循環器学会
  • Place of Presentation: 大阪、大阪国際会議場
  • Year and Date: 2015-04-24
  • Invited
• [Book] 不整脈症候群－遺伝子変異から不整脈治療を捉えるー (2015)
  • Author(s): 蒔田直昌
  • Total Pages: 196
  • Publisher: 南江堂
• [Book] 不整脈診療クリニカルクエスチョン200 (2015)
  • Author(s): 蒔田直昌
  • Total Pages: 333
  • Publisher: 診断と治療社