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首页> 外文期刊>Canadian Journal of Physiology and Pharmacology >Mechanisms of TFAM-mediated cardiomyocyte protection
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Mechanisms of TFAM-mediated cardiomyocyte protection

机译:TFAM介导的心肌细胞保护机制

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摘要

Although mitochondrial transcription factor A (TFAM) is a protective component of mitochondrial DNA and a regulator of calcium and reactive oxygen species (ROS) production, the mechanism remains unclear. In heart failure, TFAM is significantly decreased and cardiomyocyte instability ensues. TFAM inhibits nuclear factor of activated T cells (NFAT), which reduces ROS production; additionally, TFAM transcriptionally activates SERCA2a to decrease free calcium. Therefore, decreasing TFAM vastly increases protease expression and hypertrophic factors, leading to cardiomyocyte functional decline. To examine this hypothesis, treatments of 1.0 mu g of a TFAM vector and 1.0 mu g of a CRISPR-Cas9 TFAM plasmid were administered to HL-1 cardiomyocytes via lipofectamine transfection. Western blotting and confocal microscopy analysis show that CRISPR-Cas9 knockdown of TFAM significantly increased proteases Calpain1, MMP9, and regulators Serca2a, and NFAT4 protein expression. CRISPR knockdown of TFAM in HL-1 cardiomyocytes upregulates degradation factors, leading to cardiomyocyte instability. Hydrogen peroxide oxidative stress decreased TFAM expression and increased Calpain1, MMP9, and NFAT4 protein expression. TFAM overexpression normalizes pathological hypertrophic factor NFAT4 in the presence of oxidative stress.
机译:尽管线粒体转录因子A(TFAM)是线粒体DNA的保护成分,也是钙和活性氧(ROS)产生的调节器,但其机制尚不清楚。心力衰竭时,TFAM显著降低,随后心肌细胞不稳定。TFAM抑制活化T细胞的核因子(NFAT),从而减少活性氧的产生;此外,TFAM转录激活SERCA2a以降低游离钙。因此,降低TFAM可显著增加蛋白酶表达和肥大因子,导致心肌细胞功能下降。为了验证这一假设,通过脂质体转染将1.0μg TFAM载体和1.0μg CRISPR-Cas9 TFAM质粒给予HL-1心肌细胞。Western印迹和共聚焦显微镜分析表明,TFAM的CRISPR-Cas9敲除显著增加蛋白酶Calpain1、MMP9、调节因子Serca2a和NFAT4蛋白的表达。CRISPR敲除HL-1心肌细胞中的TFAM可上调降解因子,导致心肌细胞不稳定。过氧化氢氧化应激降低TFAM表达,增加钙蛋白酶1、MMP9和NFAT4蛋白表达。TFAM过度表达使氧化应激下病理性肥大因子NFAT4正常化。

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