Department of Biology, York University, Toronto, ON, Canada.
School of Kinesiology and Health Science, York University, Toronto, ON, Canada.
Cell Death Dis. 2023 Apr 7;14(4):252. doi: 10.1038/s41419-022-05484-3.
Iron overload is associated with various pathological changes which contribute to metabolic syndrome, many of which have been proposed to occur via damaging tissue through an excessive amount of reactive oxygen species (ROS) production. In this study, we established a model of iron overload in L6 skeletal muscle cells and observed that iron enhanced cytochrome c release from depolarized mitochondria, assayed by immunofluorescent colocalization of cytochrome c with Tom20 and the use of JC-1, respectively. This subsequently elevated apoptosis, determined via use of a caspase-3/7 activatable fluorescent probe and western blotting for cleaved caspase-3. Using CellROX deep red and mBBr, we observed that iron increased generation of reactive oxygen species (ROS), and that pretreatment with the superoxide dismutase mimetic MnTBAP reduced ROS production and attenuated iron-induced intrinsic apoptosis and cell death. Furthermore, using MitoSox Red we observed that iron enhanced mROS and the mitochondria-targeted anti-oxidant SKQ1 reduced iron-induced ROS generation and cell death. Western blotting for LC3-II and P62 levels as well as immunofluorescent detection of autophagy flux with LC3B and P62 co-localization indicated that iron acutely (2-8 h) activated and later (12-24 h) attenuated autophagic flux. We used autophagy-deficient cell models generated by overexpressing a dominant-negative Atg5 mutant or CRISPR-mediated ATG7 knock out to test the functional significance of autophagy and observed that autophagy-deficiency exacerbated iron-induced ROS production and apoptosis. In conclusion, our study showed that high iron levels promoted ROS production, blunted the self-protective autophagy response and led to cell death in L6 skeletal muscle cells.
铁过载与各种病理变化有关，这些变化导致代谢综合征，其中许多被认为是通过过量产生活性氧物种 (ROS) 对组织造成损害而发生的。在这项研究中，我们在 L6 骨骼肌细胞中建立了铁过载模型，观察到铁增强了去极化线粒体中细胞色素 c 的释放，分别通过免疫荧光共定位细胞色素 c 与 Tom20 和使用 JC-1 进行测定。这随后升高了细胞凋亡，通过 caspase-3/7 激活荧光探针和 cleaved caspase-3 的 western blot 进行测定。使用 CellROX deep red 和 mBBr，我们观察到铁增加了活性氧物种 (ROS) 的产生，而超氧化物歧化酶模拟物 MnTBAP 的预处理减少了 ROS 的产生，并减轻了铁诱导的内在凋亡和细胞死亡。此外，使用 MitoSox Red，我们观察到铁增强了 mROS，并且线粒体靶向抗氧化剂 SKQ1 减少了铁诱导的 ROS 产生和细胞死亡。LC3-II 和 P62 水平的 western blot 以及用 LC3B 和 P62 共定位进行的自噬流的免疫荧光检测表明，铁急性（2-8 小时）激活并随后（12-24 小时）减弱自噬流。我们使用通过过表达显性负性 Atg5 突变体或 CRISPR 介导的 ATG7 敲除产生的自噬缺陷细胞模型来测试自噬的功能意义，并观察到自噬缺陷加剧了铁诱导的 ROS 产生和细胞凋亡。总之，我们的研究表明，高铁水平促进了 ROS 的产生，减弱了自我保护的自噬反应，并导致 L6 骨骼肌细胞死亡。
