73 Establishment of New Cellular Model Supportive of Burn-Induced Perturbation in Mitophagy Response

Autophagy is considered an indispensable mechanism for cellular adaptation to various stresses. Mitophagy, or autophagic degradation of mitochondria sequesters damaged mitochondria and functions as the essential quality control (QC) system of this organelle. When mitophagy is disturbed, damaged mitochondria are not cleared and will produce superoxide. Our previous mouse study documented a compromised mitophagy response in burn injury (BI), suggestive of poor QC of mitochondria in severe BI. The detailed mechanisms have not been investigated, due to the lack of feasible cell culture model to investigate this phenomenon. We have established a C2C12 myocyte cell line stably expressing markers for auto/mitophagy, by transfecting (1) GFP-LC3 for monitoring auto/mitophagosomes, (2) mCherry-tubulin for studying the formation of microtubule (MT), and/or (3) tandem-fluorescent LC3 (tfLC3) to document the maturation/prematuration status. The cells were also stained by LysoTracker Blue and MitoTracker DeepRed for monitoring lysosomes and mitochondria, respectively. By incubating the cells with day 3 BI or sham-burn serum harvested from rats (30%, systemic BI), cells were primed with simulated BI-stress or the control treatment. Twelve hours later, mitophagy was invoked by incubating the cells with CCCP, and the response of mitophagy was monitored by time-lapse observation under confocal microscopy. The obtained findings were confirmed by Western Blotting. In control C2C12 cells, MTs were formed upon induction of mitophagy, and the mitophagosomes and lysosomes travelled along the MT. In the BI-serum treated group, however, the cells showed disturbed formation of MT, and the vesicle trafficking of mitophagosomes and lysosomes was markedly perturbed. Accordingly, the maturation of mitophagy was perturbed in BI group. Western Blotting experiment showed reduced flux of mitophagy in BI group, confirming the microscopy data. We have established a feasible cell culture experimental model to study mitophagy responses in skeletal muscles to study the effect of BI. MT formation and mitophagosome trafficking were both perturbed in BI group, suggesting that MT disturbance can be one of the important molecular event affected by BI, and involved in the poor adaptation of organs in BI subjects. We have identified MT network disturbance as the potential molecular target for future therapeutic intervention aimed at ameliorating disturbed auto/mitophagy response. Our newly established cell culture model will be a feasible tool to study the detailed mechanism of BI-induced muscle dysfunction, and to test the drug efficacy.