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.

肌浆网(SR)兰尼定受体(RyR2)的N端(0-600)和中央(2000-2500)两个结构域相互作用，是通道门控的调节开关。我们先前报道，K201(JTV519)通过纠正衰竭心脏中两个结构域之间的缺陷相互作用来抑制钙泄漏。在这里，我们在RyR2通道门上鉴定了这个新结构域的K201结合结构域和分子特征。石英晶体微天平分析表明，K201与重组的RyR2片段特异性结合：1741。2270在1-2750区域。通过对1741-2270片段的进一步分析，发现201与其亚片段2114-2149特异结合。以与该亚片段(DP2114-2149)匹配的多肽为载体，用甲基香豆素醋酸酯(MCA)对RyR2进行特异性标记。此外，在几个重组RyR2片段中，只有片段2234-2750被MCA特异性标记，这表明K201结合域2114-2149与结构域2234-2750结合。将DP2114-2149加入到MCA标记的SR中，干扰了结构域2114-2149和结构域2234-2750之间的相互作用，导致结构域解压缩，这从结合的MCA增加了对大尺寸荧光猝灭剂的可及性可见一斑。在衰竭心肌细胞中，自发钙放电(CASF)的频率明显高于正常心肌细胞(p&lt；0.01)，而掺入DP2114-2149可使CASF显著降低至正常水平；与K201产生的作用相同。总之，我们首次确定K201结合位点为RyR2的2114-2149结构域，阻断了2114-2149结构域与中央结构域2234-2750之间的相互作用，似乎介导了RyR2在衰竭心脏中的稳定，这可能导致一种新的抗心衰治疗策略，甚至可能是致死性心律失常。

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

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
• 通讯作者: Takahiro Tokuhisa

Scavenging free radicals by low-dose carvedilol prevents redox-dependent Ca2+ leak via stabilization of ryanodine receptor in heart failure

• DOI: 10.1016/j.jacc.2007.01.064
• 发表时间: 2007-04-24
• 期刊: JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY
• 影响因子: 24
• 作者: Mochizuki, Mamoru;Yano, Masafumi;Matsuzaki, Masunori
• 通讯作者: Matsuzaki, Masunori