Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins

Authors

Cui Ma, Harbin Medical University, Daqing, China
Andreas M. Beyer, Medical College of Wisconsin, MilwaukeeFollow
Matthew J. Durand, Medical College of WisconsinFollow
Anne V. Clough, Marquette UniversityFollow
Daling Zhu, Harbin Medical University, Daqing, China
Laura Norwood Toro, Medical College of Wisconsin, Milwaukee
Maia Terashvili, Medical College of Wisconsin, Milwaukee
Johnathan D. Ebben, Medical College of Wisconsin, Milwaukee
R. Blake Hill, Medical College of Wisconsin, Milwaukee
Said H. Audi, Marquette UniversityFollow
Meetha Medhora, Medical College of Wisconsin
Elizabeth R. Jacobs, Medical College of WisconsinFollow

Document Type

Article

Language

eng

Publication Date

2-1-2018

Publisher

American Heart Association

Source Publication

Arteriosclerosis, Thrombosis, and Vascular Biology

Source ISSN

1079-5642

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.

Comments

Accepted version. Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 38 (February 1, 2018): 622-635. DOI. © 2018 American Heart Association, Inc. Used with permission.

Recommended Citation

Ma, Cui; Beyer, Andreas M.; Durand, Matthew J.; Clough, Anne V.; Zhu, Daling; Toro, Laura Norwood; Terashvili, Maia; Ebben, Johnathan D.; Hill, R. Blake; Audi, Said H.; Medhora, Meetha; and Jacobs, Elizabeth R., "Hyperoxia Causes Mitochondrial Fragmentation in Pulmonary Endothelial Cells by Increasing Expression of Pro-Fission Proteins" (2018). Biomedical Engineering Faculty Research and Publications. 501.
https://epublications.marquette.edu/bioengin_fac/501