Abstract

An increase in cardiomyocyte apoptosis is the main contributor to the observed high morbidity of cardiac disease during aging. Mitochondria play important roles in cardiac apoptosis, and dynamin-related protein 1 (Drp1) is the critical factor that participates in mitochondrial fission and induces mitophagy to maintain mitochondria quality. However, whether Drp1 is involved in the increase of apoptosis in aging heart remains unclear. The purpose of this study was to determine whether Drp1 participates in inducing the apoptosis through regulating mitophagy in aging myocardium. To explore the effect of mitophagy and apoptosis in aging heart, we detected the expression of COX IV and the co-localization of COX IV and LC3 II, which reflect mitophagy, and measured adenosine triphosphate and reactive oxygen species contents, which reflect mitochondrial injury. Cell apoptosis was detected by measuring the activity of caspase-3 and the expression of cleaved caspase-3 and further confirmed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The results showed an increase in apoptosis and a decrease in mitophagy in aging cardiomyocytes, and apoptosis was ameliorated after the induction of mitophagy by carbonyl cyanide m-chlorophenyl hydrazone (a mitophagy activator) in D-galactose (D-gal)-induced senescence H9c2 cells. To clarify the role of Drp1 in apoptosis, we knocked down Drp1 by transfecting si-Drp1, or overexpressed Drp1 in senescent cells, and then detected mitophagy, mitochondrial injury, and apoptosis. The data showed that downregulated Drp1 induces mitochondrial damage and apoptosis. In addition, to explore the regulatory relationship between Drp1 and phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy, we detected the expressions of PINK1 and Parkin after the overexpression of Drp1 in the D-gal group cells and found that Drp1-mediated mitophagy inhibited the PINK1/Parkin pathway in senescent cells. Our results demonstrated that insufficient Drp1 induces cardiomyocyte apoptosis by inhibiting mitophagy, and Drp1 affects the PINK1/Parkin pathway of mitophagy in the aging heart.

中文翻译：

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动力相关蛋白1相关线粒体自噬减少诱导衰老心脏心肌细胞凋亡

 抽象的

心肌细胞凋亡的增加是衰老期间心脏病高发病率的主要原因。线粒体在心脏细胞凋亡中发挥重要作用，动力相关蛋白1（Drp1）是参与线粒体裂变并诱导线粒体自噬以维持线粒体质量的关键因子。然而，Drp1是否参与衰老心脏细胞凋亡的增加尚不清楚。本研究的目的是确定Drp1是否通过调节衰老心肌中的线粒体自噬来参与诱导细胞凋亡。为了探讨线粒体自噬和细胞凋亡对衰老心脏的影响，我们检测了反映线粒体自噬的COX IV的表达以及COX IV和LC3 II的共定位，并测量了反映线粒体损伤的三磷酸腺苷和活性氧含量。通过测量caspase-3的活性和裂解的caspase-3的表达来检测细胞凋亡，并通过末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记（TUNEL）测定进一步证实细胞凋亡。结果显示，衰老心肌细胞凋亡增加，线粒体自噬减少，在D-半乳糖（D-gal）诱导的衰老H9c2中，羰基氰化物间氯苯腙（线粒体自噬激活剂）诱导线粒体自噬后，细胞凋亡得到改善细胞。为了阐明Drp1在细胞凋亡中的作用，我们通过转染si-Drp1敲低Drp1，或在衰老细胞中过表达Drp1，然后检测线粒体自噬、线粒体损伤和细胞凋亡。数据显示，下调的 Drp1 会诱导线粒体损伤和细胞凋亡。 此外，为了探讨Drp1与磷酸酶和张力蛋白同源物（PTEN）诱导的推定激酶1（PINK1）/Parkin介导的线粒体自噬之间的调节关系，我们检测了D-gal中Drp1过表达后PINK1和Parkin的表达组细胞，发现 Drp1 介导的线粒体自噬抑制衰老细胞中的 PINK1/Parkin 通路。我们的结果表明，Drp1不足会通过抑制线粒体自噬来诱导心肌细胞凋亡，并且Drp1会影响衰老心脏中线粒体自噬的PINK1/Parkin通路。