Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins. Issue 3 (March 2018)

• Record Type: Journal Article
• Authors: Ma, Cui; Beyer, Andreas M.; Durand, Matthew; Clough, Anne V.; Zhu, Daling; Norwood Toro, Laura; Terashvili, Maia; Ebben, Johnathan D.; Hill, R. Blake; Audi, Said H.; Medhora, Meetha; Jacobs, Elizabeth R.
• Is Part Of: Arteriosclerosis, thrombosis, and vascular biology. Volume 38:Issue 3(2018)
• Publication Date: 2018-03
• Subjects: cell survival -- endothelial cells -- hyperoxia -- mitochondria -- reactive oxygen species; Arteriosclerosis -- Periodicals; Thrombosis -- Periodicals; Blood-vessels -- Pathophysiology -- Periodicals; Electronic journals; 616.13
• DOI: 10.1161/ATVBAHA.117.310605
• Languages: English
• ISSNs: 1079-5642

Abstract

Abstract : Objective—: We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia. Approach and Results—: Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related protein 1) was overexpressed or silenced to test the role of this protein in cell survival or transwell resistance. Hyperoxia increased fragmentation of PEC mitochondria in a time-dependent manner through 48 hours of exposure. Hyperoxic PECs exhibited increased phosphorylation of Drp-1 (serine 616), decreases in Mfn1 (mitofusion protein 1), but increases in OPA-1 (optic atrophy 1). Pro-autophagy proteins p62 (LC3 adapter–binding protein SQSTM1/p62), PINK-1 (PTEN-induced putative kinase 1), and LC3B (microtubule-associated protein 1A/1B-light chain 3) were increased. Returning cells to normoxia for 24 hours reversed the increased mt-fragmentation and changes in expression of pro-fission proteins. Hyperoxia-induced changes in mitochondrial structure or cell survival were mitigated by antioxidants mito-TEMPOL, Drp-1 silencing, or inhibition or protection by the mitochondrial endonuclease ENDO III. Hyperoxia induced oxidation and mitochondrial depolarization and impaired transwell resistance. Decrease in resistance was mitigated by mito-TEMPOL or ENDO III and reproduced by overexpression of Drp-1. Conclusions—: Because hyperoxia evoked mt-fragmentation, cell survival and transwell resistance are prevented by ENDO III and mito-TEMPOL and Drp-1 silencing, and these data link hyperoxia-induced mt-DNA damage, Drp-1 expression, mt-fragmentation, and PEC dysfunction. Abstract : Supplemental Digital Content is available in the text.