Novel therapy of arrhythmia targeting key domain in ryanodine receptor

2017 Fiscal Year Annual Research Report

• Project/Area Number: 15K09085
• Research Institution: Yamaguchi University
• Principal Investigator: 山本 健 山口大学, 大学院医学系研究科, 教授 (50363122)
• Co-Investigator(Kenkyū-buntansha): 小田 哲郎 山口大学, 医学部附属病院, 助教 (40569290) ; 小林 茂樹 山口大学, 大学院医学系研究科, 准教授 (90397993)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: リアノジン受容体 / 不整脈

Outline of Annual Research Achievements

CaM高親和性CaMBD：申請者らはすでにCaM BDのペプチドに点突然変異を組み込んだペプチドを28種類作成した。作成する際にはCaM BDとCaMのPDB fileからSwiss PDB viewerを用いて３次元構造を描出しながら、作成した突然変異が新たな共有結合を生み出すように設計を試みた。作成した多数のCaM BDの変異ペプチドの中からCaMへの結合が最も強いものを２種類の方法でスクリーニングした。すなわちCaM BD上にあるTypの蛍光を計測することでCaMのCaM BDへの結合をみる方法で、もう一つはCaMにクロスリンカーであるsulfo-SANPAHのsuccinimydyl基を結合させ、SRと暗所でSRと混和しUVをあててRyRにクロスリンクさせ、western blotでRyRの565kDaのRyRのバンドを抗CaM抗体で検出する。このクロスリンクをCaM BDの変異ペプチドの存在下で行いCaMのRyRへのクロスリンクを最も抑制するものがCaMに対する結合性の最も高いCaM BDの変異ペプチドであるという手法である。ともに１つの有望な変異（Pep#26）を選び出した。
CaM高親和性KI マウスの作成：選んだ変異を組み込んだCaM高親和性RyR2変異を組み込んだKIマウス作成が完了し,このマウスではRyR2とCaMの親和性が高く、リン酸化してもCaMがRyR2から外れにくいことがわかった。
CaM高親和性KI マウスの不整脈：CaM高親和性RyR2変異を組み込んだKIマウスとRyR-R2474S型CPVTマウスを掛け合わせたところ、運動負荷、および薬物負荷時の不整脈の頻度は激減した。

Research Products

• [Journal Article] Mutation-linked, excessively tight interaction between the calmodulin binding domain and the C-terminal domain of the cardiac ryanodine receptor as a novel cause of catecholaminergic polymorphic ventricular tachycardia (2018)
  • Author(s): Nishimura Shigehiko、Yamamoto Takeshi、Nakamura Yoshihide、Kohno Michiaki、Hamada Yoriomi、Sufu Yoko、Fukui Go、Nanno Takuma、Ishiguchi Hironori、Kato Takayoshi、Xu Xiaojuan、Ono Makoto、Oda Tetsuro、Okuda Shinichi、Kobayashi Shigeki、Yano Masafumi
  • Journal Title: Heart Rhythm Volume: in press Pages: in press
  • DOI: 10.1016/j.hrthm.2018.02.006
  • Peer Reviewed
• [Journal Article] CaMKII-mediated phosphorylation of RyR2 plays a crucial role in aberrant Ca 2+ release as an arrhythmogenic substrate in cardiac troponin T-related familial hypertrophic cardiomyopathy (2018)
  • Author(s): Okuda Shinichi、Sufu-Shimizu Yoko、Kato Takayoshi、Fukuda Masakazu、Nishimura Shigehiko、Oda Tetsuro、Kobayashi Shigeki、Yamamoto Takeshi、Morimoto Sachio、Yano Masafumi
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 496 Pages: 1250～1256
  • DOI: 10.1016/j.bbrc.2018.01.181
• [Journal Article] Correction of impaired calmodulin binding to RyR2 as a novel therapy for lethal arrhythmia in the pressure-overloaded heart failure (2017)
  • Author(s): Kato Takayoshi、Yamamoto Takeshi、Nakamura Yoshihide、et al
  • Journal Title: Heart Rhythm Volume: 14 Pages: 120～127
  • DOI: 10.1016/j.hrthm.2016.10.019
  • Peer Reviewed
• [Journal Article] Efficacy and Safety of an Orally Administered Selective Prostacyclin Receptor Agonist, Selexipag, in Japanese Patients With Pulmonary Arterial Hypertension (2017)
  • Author(s): Tanabe N, Ikeda S, Tahara N, Fukuda K, Hatano M, Ito H, Nakayama T, Anzai T, Hashimoto A, Inoue T, Kajinami K, Kihara Y, Kinoshita H, Kuwahara K, Murohara T, Okazaki O, Sakai S, Satoh T, Takeda Y, Takeishi Y, Taniguchi M, Watanabe H, Yamamoto T, Yamauchi-Takihara K, Yoshioka K, Sasayama S
  • Journal Title: Circ J Volume: 81 Pages: 1360-1367
  • DOI: 10.1253/circj.CJ-16-1348
  • Peer Reviewed
• [Presentation] Stabilization of CaMKII-phosphorylated RyR2Prevents the Development of Age-related Car-diomyopathy Caused by Transgenic CaMKII δ cOverexpression (2018)
  • Author(s): Yoko Sufu, Shinichi Okuda, Yoko Okamoto, Tetsuro Oda, Shigeki Kobayashi, Takeshi Yamamoto, Masafumi Yano
  • Organizer: 第82回日本循環器学会学術集会
• [Presentation] Dantrolene Prevents Right Ventricular Hypertro-phy without Reducing Pulmonary Arterial Pres-sure in Monocrotaline-Induced PAH in Mice (2018)
  • Author(s): Yoshihide Nakamura, Takeshi Yamamoto, Go Fukui, Hironori Ishiguchi, Hitoshi Uchinoumi, Tetsuro Oda, Shinichi Okuda, Shigeki Kobayashi, Masafumi Yano
  • Organizer: 第82回日本循環器学会学術集会
• [Presentation] Ryanodine Receptor Targeting Therapy InhibitsCardiomyocyte Hypertrophy and RelaxationImpairment in the Compensated Pressure-Overloaded Heart (2018)
  • Author(s): Michiaki Kono, Shigeki Kobayashi, Toshiro Kajii, Takuma Nanno, Hironori Ishiguchi, Hitoshi Uchinoumi, Tetsuro Oda, Shinichi Okuda, Takeshi Yamamoto, Masafumi Yano
  • Organizer: 第82回日本循環器学会学術集会
• [Presentation] Stabilization of Camkii-Phosphorylated Ryr2 by Dantrolene Prevents the Development of Age-Related Cardiomyopathy Caused by CamkiiδC Overexpression (2017)
  • Author(s): Yoko S Shimizu, Shinichi Okuda, Yoko Okamoto, Shigehiko Nishimura, Takayoshi Kato, Tetsuro Oda, Shigeki Kobayashi, Takeshi Yamamoto and Masafumi Yano
  • Organizer: 90th Scientific Session of American Heart Association
• [Presentation] Channel Pore Mutation of RyR2 Cause Calcium Leak Independent of FKBP12.6-RyR2 and Calmodulin-RyR2 Affinity in R4496C/+ Knock-in Mouse (2017)
  • Author(s): Hitoshi Uchinoumi, Ivanita Stefanon, Yi Yang, Mena Said, Xiaoqiong Dong, Rogerio Faustino, Jose L Puglisi, Ye Chen-Izu, Razvan L Cornea, Xander H Wehrens, Wayne S Chen, Takeshi Yamamoto, Yano Masafumi and Donald M Bers
  • Organizer: 90th Scientific Session of American Heart Association
• [Presentation] Effect of Site Dependent RyR Stabilizers; a Clue to Trace a Signal Transmission Pathway Inside the RyR Molecule (2017)
  • Author(s): Go Fukui, Takeshi Yamamoto, Michiaki Kono, Yoshihide Nakamura, Takuma Nanno, Yoriomi Hamada, Yoko Sufu, Hironori Ishiguchi, Shigehiko Nishimura, Hitoshi Uchinoumi, Tetsuro Oda, Shinichi Okuda, Shigeki Kobayashi and Masafumi Yano
  • Organizer: 90th Scientific Session of American Heart Association
• [Presentation] Dantrolene Prevents Right Ventricular Hypertrophy Without Reducing Pulmonary Arterial Pressure In Monocrotaline-induced PAH In Mice (2017)
  • Author(s): Yoshihide Nakamura, Takeshi Yamamoto, Michiaki kono, Yoriomi Hamada, Go Fukui, Yoko Sufu, Takuma Nanno, Hironori Ishiguchi, Shigehiko Nishimura, Tetsuro Oda, Shinichi Okuda, Shigeki Kobayashi, Masafumi Yano
  • Organizer: 90th Scientific Session of American Heart Association