Establishment of molecular targeting therapy for duonic heart failure bystabilizmgryanodine receptor

稳定麦草碱受体治疗双元心力衰竭分子靶向治疗的建立

• 批准号: 18390234
• 负责人: YANO Masafumi
• 金额: $ 9.96万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390234/
• 关键词: sarcoplasmic reticulum; scalcium; rvanodine receptor; heart failure; lethal arrhythmia; point mutation

Two domains within the ryanodine receptor (RyR2) of sarcoplasmic reticulum (SR) {N-terminal (0-600) and central (2000-2500) domains} was found to interact with each other as a regulatory switch for channel gating. We previously reported that K201 (JTV519) inhibits Ca^<2+> leak by correcting the defective inter-domain interaction between the two domains in failing hearts. Here, we identified the K201-binding domain and diaracterizedtherole ofthis novel domain on RyR2 channel gating.An assay using a quartz-crystal microbalance revealed that K201 specifically bound to recornbinant RyR2 fragment: 1741. 2270 in the 1-2750 region. By further analysis of the fragnent1741-2270, 201 was found to specifically bind to its sub-fragment2114-2149. Using the peptide matching this sub-fragment (DP2114-2149)as a carrier, the RyR2 was specifically labeled with methylcoumarin acetate(MCA). Moreover, of several recombinant RyR2 fragments, only fragment 2234-2750 was specifically MCA-labeled; this suggests that the K201 binding domain2114-2149 binds with domain 2234-2750. Addition of DP2114-2149 to the MCA-labeled SR interfered with the interaction between domain2114-2149 and domain2234-2750 causing domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes, the frequency of spontaneous Ca^<2+> spark (CaSF) was much higher than normal cardiomyocytes (p<0.01), whereas incorporation of DP2114-2149 markedly decreased CaSF to normal level; the same effect as that produced by K201.In conclusion, we first identified the K201-binding site as domain2114-2149 of RyR2 Interruption of the inter-domain interaction between the domain2114-2149 and central domain2234-2750 seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia.

发现肌质网（RYR2）内的两个结构域（sr）（SR）{N端（0-600）和Central（2000-2500）结构域}的两个结构域与通道门控的调节开关相互相互作用。我们先前报道说，K201（JTV519）通过纠正失败心脏中两个域之间的缺陷域间相互作用来抑制CA^<2+>泄漏。在这里，我们在RYR2通道门口上鉴定了这种新型结构域的K201结合结构域和腹泻，使用石英 - 晶体微平衡的测定表明，K201在1-2750区域特异性地与再生ryr2片段特别结合：1741。2270。通过对Fragnent1741-2270的进一步分析，发现201特别结合了其子碎片2114-2149。使用将该子碎片匹配的肽（DP2114-2149）作为载体，将RYR2特异性标记为乙酸甲酯（MCA）。此外，在几个重组RYR2片段中，只有片段2234-2750被特别标记。这表明K201结合域2114-2149与域2234-2750结合。将DP2114-2149添加到MCA标记的SR中，干扰了域2114-2149和域22234-2750之间的相互作用，从而导致结构域无拉链，这证明了绑定的MCA对大尺寸荧光淬火器的可访问性的增加。在失败的心肌细胞中，自发CA^<2+>火花（CASF）的频率高于正常的心肌细胞（P <0.01），而DP2114-2149的掺入将CASF显着降低到正常水平；与K201的产生的效果相同，我们首先将K201结合位点确定为RYR2的域2114-2149，在RYR2中断域之间的域间相互作用2114-2149和Central域22234-2750之间的域间相互作用似乎会导致Ryr2在失败中的稳定性，这可能会导致Ryr2的稳定性，这可能会导致更新的策略，也许会导致型号的策略，并且可能会导致更具效果。

项目成果

Identification of therapeutic domain within the cardiac ryanodine receptor to correct abnormal Ca2+ release in failing hearts

鉴定心脏兰尼碱受体内的治疗域以纠正衰竭心脏中异常的 Ca2 释放

• 发表时间: 2008
• 作者: Mochizuki;M;Yano;M;Oda;T;Tateishi;H;Kobayashi;S;Yamamoto;T;Ikeda;Y;Ohkusa;T;Ikemoto;N;Matsuzaki;M;Takahiro Tokuhisa;Takahiro Tokuhisa;Masafumi Yano
• 通讯作者: Masafumi Yano

ATI receptor antagonist restores cardiac ryanodine receptor function, rendering isoproterenol-induced failing heart less susceptible to Ca2+ -leak induced by oxidative stress.

ATI受体拮抗剂可恢复心脏兰尼碱受体功能，使异丙肾上腺素引起的衰竭心脏不易受到氧化应激引起的Ca2+泄漏的影响。

• 发表时间: 2006
• 期刊: Circulation Journal 70
• 作者: Mochizuki;M;Yano;M;Oda;T;Tateishi;H;Kobayashi;S;Yamamoto;T;Ikeda;Y;Ohkusa;T;Ikemoto;N;Matsuzaki;M;Takahiro Tokuhisa
• 通讯作者: Takahiro Tokuhisa

Role of ryanodine receptor in heart failure

兰尼碱受体在心力衰竭中的作用

• 发表时间: 2007
• 作者: Mochizuki;M;Yano;M;Oda;T;Tateishi;H;Kobayashi;S;Yamamoto;T;Ikeda;Y;Ohkusa;T;Ikemoto;N;Matsuzaki;M;Takahiro Tokuhisa;Takahiro Tokuhisa;Masafumi Yano;Masafumi Yano;Masafumi Yano
• 通讯作者: Masafumi Yano

Identification of target domains of the cardiac ryanodine receptor to correct channel disorder in failing hearts

• DOI: 10.1161/circulationaha.107.718957
• 发表时间: 2008-02-12
• 期刊: CIRCULATION
• 影响因子: 37.8
• 作者: Yamamoto, Takeshi;Yano, Masafumi;Matsuzaki, Masunori
• 通讯作者: Matsuzaki, Masunori

ATI receptor antagonist restores cardiac ryanodine receptor function, rendering isoproterenol-induced failing heart less susceptible to Ca2+-leak induced by oxidative stress.

ATI受体拮抗剂可恢复心脏兰尼碱受体功能，使异丙肾上腺素引起的衰竭心脏不易受到氧化应激引起的Ca2+泄漏的影响。

• 发表时间: 2006
• 期刊: Circulation Journal 70
• 作者: Mochizuki;M;Yano;M;Oda;T;Tateishi;H;Kobayashi;S;Yamamoto;T;Ikeda;Y;Ohkusa;T;Ikemoto;N;Matsuzaki;M;Takahiro Tokuhisa;Takahiro Tokuhisa
• 通讯作者: Takahiro Tokuhisa