77 Microtubule Network Disturbance is Likely the Target of Oxidative-Stress-Induced Mitophagy Perturbation

Abstract Introduction Oxidative stress is considered one of the key mechanisms in burn injury-induced organ dysfunctions. The roles of oxidative stress in autophagy/mitophagy dysfunction have recently been discussed in basic biology, but its molecular target remained somewhat elusive. In this study, we have examined the effect of oxidative stress on the formation of microtubules (MTs), which, in normal circumstances, serves as an essential guide rail for auto/mitophagosome vesicle trafficking and thus a key component of auto/mitophagy pathway. Methods By stably transfecting auto/mitophagy markers in C2C12 myocyte, we have established a few new muscle cell lines, expressing mCherry-tubulin, GFP-LC3, and/or tfLC3. To monitor mitochondria and lysosomes, the cells were stained by MitoTracker DeepRed and LysoTracker Blue. The cells were cultured with or without oxidative stress by hydorgen peroxide (H2O2). The response of mitophagy was monitored by stimulating the cells with CCCP. To confirm the mitophagy turnover blockade under oxidative stress, Western Blotting was performed against mitochondria-associated LC3. Results Normal cells showed mitophagy-induced MT network formation. All the mitophagosomes and lysosomes traveled along the MTs. Cells under the oxidative stress, however, showed completely abolished MT network formation, and the vesicle motion was markedly inhibited. Observation of tfLC3-expressing cells demonstrated the inhibition of mitophagosome maturation. Western Blotting against mitochondria-LC3 showed the blockade of mitophagy flux under oxidative stress. Conclusions MT network disturbance is likely the target of oxidative stress. The disturbed maturation of mitophagosome under oxidative stress is consistent with the notion that MTs are essential for vesicle fusion and thus for the turnover of mitophagy. Applicability of Research to Practice Oxidative stress-induced MT disturbance can be the pivotal key event in the critical illness-related mitochondrial dysfunction in skeletal muscles, and needs further investigation. Amelioration of the perturbed MTs may serve a novel therapeutic target in the future.