Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells
Abstract 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...
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| Format: | Article |
| Language: | English |
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Nature Publishing Group
2023-04-01
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| Series: | Cell Death and Disease |
| Online Access: | https://doi.org/10.1038/s41419-022-05484-3 |
| _version_ | 1827970277447827456 |
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| author | Hye Kyoung Sung Mayoorey Murugathasan Ali A. Abdul-Sater Gary Sweeney |
| author_facet | Hye Kyoung Sung Mayoorey Murugathasan Ali A. Abdul-Sater Gary Sweeney |
| author_sort | Hye Kyoung Sung |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-09T18:50:22Z |
| format | Article |
| id | doaj.art-34cd5f36473b450697fad0cf69e7f97d |
| institution | Directory Open Access Journal |
| issn | 2041-4889 |
| language | English |
| last_indexed | 2024-04-09T18:50:22Z |
| publishDate | 2023-04-01 |
| publisher | Nature Publishing Group |
| record_format | Article |
| series | Cell Death and Disease |
| spelling | doaj.art-34cd5f36473b450697fad0cf69e7f97d2023-04-09T11:28:15ZengNature Publishing GroupCell Death and Disease2041-48892023-04-0114411310.1038/s41419-022-05484-3Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cellsHye Kyoung Sung0Mayoorey Murugathasan1Ali A. Abdul-Sater2Gary Sweeney3Department of Biology, York UniversitySchool of Kinesiology and Health Science, York UniversitySchool of Kinesiology and Health Science, York UniversityDepartment of Biology, York UniversityAbstract 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.https://doi.org/10.1038/s41419-022-05484-3 |
| spellingShingle | Hye Kyoung Sung Mayoorey Murugathasan Ali A. Abdul-Sater Gary Sweeney Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells Cell Death and Disease |
| title | Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells |
| title_full | Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells |
| title_fullStr | Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells |
| title_full_unstemmed | Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells |
| title_short | Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells |
| title_sort | autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells |
| url | https://doi.org/10.1038/s41419-022-05484-3 |
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