Inhibition of complex I of the electron transport chain causes O₂⁻·-mediated mitochondrial outgrowth

TLDR

Oxidative stress, arising when reactive oxygen species production exceeds detoxification, is implicated in degenerative diseases, aging, and cancer, and cells often respond by altering mitochondrial morphology. The study aimed to determine how complex I inhibition–induced mitochondrial superoxide production affects mitochondrial morphology in human skin fibroblasts, using rotenone and the mitochondria‑targeted antioxidant mitoquinone. Human skin fibroblasts were chronically treated with 100 nM rotenone or 10 nM mitoquinone for 72 h, and mitochondrial morphology and superoxide production were quantified by video‑rate confocal microscopy and hydroethidine fluorescence. Rotenone increased mitochondrial length and branching, doubled lipid peroxidation and superoxide production, while mitoquinone prevented the morphological changes and lipid peroxidation without affecting superoxide levels, demonstrating that complex I inhibition–induced superoxide drives mitochondrial outgrowth that mitoquinone can block downstream.

Abstract

Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial O 2 − · production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial O 2 − · production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIPY 581/591 . Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by O 2 − · to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a twofold increase in the rate of O 2 − · production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial O 2 − · production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this O 2 − · to prevent alterations in mitochondrial morphology.