Department of Cellular and Integrative Physiology, School of Medicine, University of Texas Health Science Center at San Antonio , San Antonio, Texas.
Department of Biomedical Engineering, University of Texas at San Antonio , San Antonio, Texas.
Am J Physiol Cell Physiol. 2018 Jul 1;315(1):C28-C43. doi: 10.1152/ajpcell.00230.2017. Epub 2018 Feb 28.
Mitofilin is an inner membrane protein that has been defined as a mitochondria-shaping protein in controlling and maintaining mitochondrial cristae structure and remodeling. We determined the role of mitofilin in cell survival by investigating the mechanism underlying mitofilin knockdown-induced cell death by apoptosis. Cultured H9c2 myoblasts and HEK 293 cells were treated with mitofilin siRNA or scrambled siRNA for 24 h. Cell death (apoptosis), caspase 3 activity and cell cycle phases were assessed by flow cytometry, while cytochrome c release and intracellular ATP production were measured by ELISA. Mitofilin, apoptosis-inducing factor (AIF) and poly(ADP-ribose) polymerase (PARP) expression were measured by Western blot analysis and calpain activity was assessed using a calpain activity kit. Mitochondrial images were taken using electron microscopy. We found that mitofilin knockdown increases apoptosis mainly via activation of the AIF-PARP pathway leading to nuclear fragmentation that is correlated with S phase arrest of the cell cycle. Knockdown of mitofilin also led to mitochondrial swelling and damage of cristae that is associated with the increase in reactive oxygen species production and mitochondrial calpain activity, as well as a marked decrease in intracellular ATP production and mitochondrial membrane potential. Together, these results indicate that mitofilin knockdown by siRNA increases calpain activity that presumably leads to mitochondrial structural degradation resulting in a critical reduction of mitochondrial function that is responsible for the increase in cell death by apoptosis via an AIF-PARP mechanism and associated with nuclear fragmentation, and S phase arrest of the cell cycle.
肌球蛋白是一种内膜蛋白，被定义为一种控制和维持线粒体嵴结构和重塑的线粒体成形蛋白。我们通过研究肌球蛋白敲低诱导细胞凋亡的细胞死亡机制来确定肌球蛋白在细胞存活中的作用。用肌球蛋白 siRNA 或乱序 siRNA 处理培养的 H9c2 成肌细胞和 HEK 293 细胞 24 h。通过流式细胞术评估细胞死亡（凋亡）、caspase 3 活性和细胞周期相，通过 ELISA 测量细胞色素 c 释放和细胞内 ATP 产生。通过 Western blot 分析测量肌球蛋白、凋亡诱导因子（AIF）和多聚（ADP-核糖）聚合酶（PARP）的表达，并用钙蛋白酶活性试剂盒评估钙蛋白酶活性。使用电子显微镜拍摄线粒体图像。我们发现肌球蛋白敲低主要通过激活 AIF-PARP 途径增加凋亡，导致核片段化，与细胞周期 S 期停滞相关。肌球蛋白的敲低还导致线粒体肿胀和嵴损伤，与活性氧产生和线粒体钙蛋白酶活性增加以及细胞内 ATP 产生和线粒体膜电位明显减少相关。总之，这些结果表明，siRNA 介导的肌球蛋白敲低增加钙蛋白酶活性，可能导致线粒体结构降解，从而导致线粒体功能严重降低，这是通过 AIF-PARP 机制导致细胞凋亡增加和核片段化以及细胞周期 S 期停滞的原因。
