Mitochondrial TOM70 in Hyperoxia-induced Acute Lung Injury

STUDY OBJECTIVE: Hyperoxia-induced acute lung injury (HALI) leads to respiratory failure and mitochondrial dysfunction is a critical mediator of HALI. We previously reported Toll-Like Receptor (TLR)4 prevents oxidant-induced ALI by maintaining mitochondrial bioenergetics in mammalian lung endothelium (Ec). Translocase of Outer Mitochondrial Membrane (TOM) have recently been identified to play important roles in mitochondrial quality control, primarily in yeast [1]. TOM70 is an essential subunit of TOM and mediates uptake of newly synthesized proteins to mitochondria as well has putative anti-oxidant and anti-apoptotic properties. TOM70 appeared to parallel many of the cytoprotective signaling properties of TLR4 [2-4]. Therefore, our hypothesis is TLR4 regulates TOM70 expression and TOM70 has mitochondrial and lung protective functions in HALI. METHODS: C57BL/6J mice (6-8 wks, both sex) were purchased from Jackson Laboratory, and Tlr4-/- mice were bred at Duke Animal facilities (IACUC A160-19-07) [5]. For hyperoxia exposure, mice were placed in a Plexiglas chamber with 5L/min of 100% oxygen continuous flow with food and water ad libitum. We previously reported 72 h of continuous hyperoxia as a time of maximal lung injury [6], which we used as a representative time point. Bodyweight and survival were monitored daily. Bronchoalveolar lavage (BAL) were collected and BAL protein were determined by a BCA assay. Protein and RNA were extracted from lung tissues and expression was determined by western blot and qPCR, respectively. Mouse lung Ec (MLEC) were isolated from lung tissues and treated with control or Tom70siRNA for 48 h. Statistical differences were analyzed by a Student-t test. RESULTS: Tlr4-/- MLEC decreased TOM70 and increased Parkin, PINK1, and MFN2 protein expression compared to WT. Tom70 siRNA effectively silenced Tom70 with >75% efficiency at 48 h after transfection. Tom70 siRNA-treated MLEC showed increased Mfn1, Mff, and Fis1mRNA expression, which are markers of mitochondrial fusion-fission. Hyperoxia-exposed mice showed marked weight loss and higher levels of BAL proteins compared to control, indicating increased permeability of the alveolar-capillary membrane. Moreover, TOM70 protein expression was significantly decreased by hyperoxia in both male and female mice. CONCLUSION: TLR4 deficiency decreased TOM70 expression and induced mitophagy and mitochondrial fusion, indicating that TOM70 is regulated by TLR4. Silencing TOM70 resulted in increased mitochondrial fusion and fission in MLEC, suggesting that TOM70 impacts mitochondrial quality control in mammalian, primary cells (MLEC) that are critical for lung airspace barrier function. Hyperoxia decreased TOM70 protein expression in mouse lungs, which may be a potential mechanism whereby hyperoxia leads to respiratory failure. Given that TOM70 maintains mitochondrial health, our data suggest that TOM70 may play a key role in the pathological mechanisms of HALI. 1. Liu et al. (2021) bioRxiv, 2. Zhang et al. (2019) Antioxid Redox Signal, 3. Zhang et al. (2016) Antioxid Redox Signal, 4. Takyar et al. (2015) FASEB J. 5. Kim et al. (2019) Aging Cell. 6. Zhang et al. (2021) AJP Lung Cell Mol Physiol.

Duke Scholars

Author Elias Coutavas Medicine, Pulmonary, Allergy, and Critical Care Medicine