Elucidation of the underlining molecular mechanisms of mitochondrial DNA and hear failure

阐明线粒体 DNA 和听力衰竭的基本分子机制

• 批准号: 21790733
• 负责人: IDE Tomomi
• 金额: $ 2.75万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2010
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21790733/
• 关键词: ミトコンドリア; リモデリング; NFAT; 心不全; 酸化ストレス; ミトコンドリアDNA

In the present study, we aimed to the three points ;1) To determine whether mitochondrial DNA copy number is an direct factor for the regulation of remodeling after myocardial infarction.2) To clarify the underling mechanisms how the copy number of mitochondrial DNA regulate intercellular remodeling signal induced by Angiotensin II or endotherine-1.3) To establish a method to increase mitochondrial DNA.First of all, it was verified whether the cardiac remodeling is ameliorated by using Twinkle overexpression to determine the role of mitochondria DNA. Overexpression of Twinkle, a mitochondrial DNA helicase, suppressed cardiac remodeling after myocardial infarction as well as Tfam transgenic mice. As a result, the survival rate of mice after infarction was dramatically improved just as observed in Tfam mice. However, the transciption and replication of mitochondria DNA is decreased in Tfam transgenic mice whereas increased in Twinkle transgenic mice. From those data, we speculated the im … More portance and the impact of mitochondria DNA copy number as a protector against cardial remodeling. In order to establish a method to increase mitochondria DNA by exogenously administered Tfam, we prepared recombinant human TFAM protein by GST fusion gene purification protocol. Recombinant TFAM was recruited into mitochondria of cardiac myocytes, and there were no morphological changes in mitochondria observed by electron microscopy.A treatment with TFAM dose-dependently increased the mtDNA copy number maximum about 2-folds, and inhibited mitochondrial reactive oxygen species generation.Next, we examined Tfam and increased mitochondria DNA on the intracellular signaling for pathological cardiac hypertrophy and remodeling.Next we investigated the effects of TFAM on the nuclear factor of activated T cell (NFAT) signaling, which is a major transcriptional factor regulating pathological hypertrophy and remodeling. TFAM inhibited NFAT nuclear translocation induced by angiotensin II (AngII) and endothelin 1 (ET-1) significantly. TFAM also suppressed AngII and ET-1-induced NFAT transcriptional activity and NFAT-dependent gene expression. Finally TFAM inhibited subsequent morphological hypertrophy of cardiac myocytes induced by AngII and ET-1. In addition, intravenously administered recombinant TFAM was recruited into the myocardium in mice, and increased the myocardial mtDNA copy number about 1.8-folds. Conclusion : Recombinant TFAM increases the mtDNA copy number, and attenuates AngII and ET-1-induced hypertrophy of cardiac myocytes via inhibiting NFAT signaling. Recombinant TFAM might be useful as a novel therapeutic strategy for cardiac hypertrophy and failure. Less

在本研究中，我们主要针对以下三点：1)确定线粒体DNA拷贝数是否是心肌梗死后心肌重构的直接调节因素；2)阐明线粒体DNA拷贝数调控血管紧张素Ⅱ或血管内毒素诱导的细胞间重构信号的机制。3)建立一种增加线粒体DNA含量的方法。首先，通过Twinkle过表达来确定线粒体DNA的作用，以验证是否改善了心肌重构。线粒体DNA解旋酶Twinkle的过表达抑制了心肌梗死后的心脏重构以及Tfam转基因小鼠。结果，与Tfam小鼠观察到的一样，脑梗塞后小鼠的存活率显著提高。然而，Tfam转基因小鼠线粒体DNA的转录和复制减少，而Twinkle转基因小鼠线粒体DNA转录和复制增加。根据这些数据，我们推测IM…更重要的是线粒体DNA拷贝数作为心脏重塑的保护者的影响。为了建立外源性TFAM增加线粒体DNA的方法，我们采用GST融合基因纯化技术制备了重组人TFAM蛋白。重组TFAM被募集到心肌细胞线粒体中，电子显微镜下未见线粒体形态变化。TFAM剂量依赖性地使线粒体DNA拷贝数增加约2倍，并抑制线粒体活性氧的产生。其次，我们检测了TfAM和增加线粒体DNA对病理性心肌肥大和重构的细胞内信号转导的影响。其次，我们研究了TFAM对调节病理性肥大和重构的主要转录因子激活T细胞核因子(NFAT)信号转导的影响。TFAM显著抑制血管紧张素II(AngII)和内皮素-1(ET-1)诱导的NFAT核移位。TFAM还抑制AngiI和ET-1诱导的NFAT转录活性和NFAT依赖基因的表达。最终，TFAM抑制血管紧张素Ⅱ和ET-1诱导的心肌细胞形态肥大。此外，静脉注射重组TFAM后，小鼠心肌线粒体DNA拷贝数增加约1.8倍。结论：重组TFAM通过抑制NFAT信号转导，增加线粒体DNA拷贝数，减轻Ang II和ET-1诱导的心肌细胞肥大。重组TFAM有望成为治疗心肌肥厚和衰竭的新策略。较少

项目成果

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• 发表时间: 2010
• 期刊: Nucleic Acids Res 38
• 作者: Yikallio E.Ylikallio E;Page JL;Xu X;Lampinen M;Bepler G;Ide T;Tyynismaa H;Weiss RS;Suomalainen A
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