The development of a diagnostic method for detecting heart defects based on spontaneous sarcomeric oscillations (SPOC) profiles from human iPS cell-derived cardiomyocytes

• Project/Area Number: 24650213
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Neurophysiology and muscle physiology
• Research Institution: Waseda University
• Principal Investigator: OHKI TAKASHI 早稲田大学, 理工学術院, 講師 (80443480)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2013: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2012: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 心筋 / 筋収縮 / SPOC / アデノウイルス / 拡大型心筋症 / 心筋分化 / ヒトiPS細胞 / アクチン / 拡張型心筋症 / iPS細胞（人工多能性幹細胞）

Outline of Final Research Achievements

SPOC (spontaneous sarcomeric oscillations) is a characteristic state of the contractile system of striated muscle that exists between the states of relaxation and contraction. We analyzed the SPOC properties in human iPSC-derived cardiomyocytes expressing E101K mutant actin causing hypertrophic cardiomyopathy．Cardiomyocytes expressing E101K mutant showed that the sarcomere length (SL) amplitude decreases by ~37 % compared to cells expressing the wild-type actin. To investigate the molecular mechanism underlying the SL amplitude decrease, E101K mutant actin was expressed by adenovirus vector in the stable C2C12 cell line expressing the adenovirus E1 gene. Actin-activated ATPase measurements using the expressed actins showed that the E101K mutation increased the Kactin for actin 3-fold, whereas the Vmax values were similar. These results suggest that reduction of the actomyosin interaction by E101K actin mutant leads to decreased SL amplitude in the SPOC state.

Research Products

• [Journal Article] Cell-sized spherical confinement induces the spontaneous formation of contractile actomyosin rings in vitro (2015)
  • Author(s): Miyazaki, M., Chiba, M., Eguchi, H., Ohki, T., and Ishiwata, S.
  • Journal Title: Nat. Cell Biol. Volume: 17 Issue: 4 Pages: 480-489
  • DOI: 10.1038/ncb3142
  • Peer Reviewed
• [Journal Article] Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin-AcGFP in Z-discs (2014)
  • Author(s): Shintani, S., Oyama, K., Kobirumaki, F., Shimozawa, F., Ohki, T., Ishiwata, S., and Fukuda, N.
  • Journal Title: J. Gen. Physiol. Volume: 143 Issue: 4 Pages: 513-524
  • DOI: 10.1085/jgp.201311118
  • Peer Reviewed
• [Journal Article] Improvement of the yields of recombinant actin and myosin V-HMM in the insect cell/baculovirus system by the addition of nutrients to the high-density cell culture (2012)
  • Author(s): Ohki, T., Ishiwata, S., et al.
  • Journal Title: J. Muscle Res. Cell Motil Volume: 33 Issue: 5 Pages: 351-358
  • DOI: 10.1007/s10974-012-9323-8
  • Peer Reviewed
• [Presentation] 細胞サイズ液滴内でのアクトミオシンリングの自発形成と収縮 (2014)
  • Author(s): 宮崎牧人、千葉雅隆、大木高志、石渡信一
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 札幌コンベンションセンター
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] アクチニンで架橋された二次元アクチンネットワークのミオシン依存的な形態変化 (2014)
  • Author(s): 江口宙輝、宮崎牧人、大木高志、石渡信一
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 札幌コンベンションセンター
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] ヒト遺伝性難聴(DFNA20/26)γアクチン変異体とミオシンとの相互作用 (2013)
  • Author(s): 大木 高志、石渡 信一
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 国立京都国際会館
• [Presentation] Expression of recombinant beta-actin in the high-density insect cell culture (2012)
  • Author(s): Takashi Ohki, Shin'ichi Ishiwata
  • Organizer: 日本生物物理学会第50回年会
  • Place of Presentation: 名古屋大学 東山キャンパス