Cui Ma1, Andreas M Beyer1, Matthew Durand1, Anne V Clough1, Daling Zhu1, Laura Norwood Toro1, Maia Terashvili1, Johnathan D Ebben1, R Blake Hill1, Said H Audi1, Meetha Medhora1, Elizabeth R Jacobs2. 1. From the College of Medical Laboratory Science and Technology, Harbin Medical University, Daqing, China (C.M., D.Z., M.M., E.R.J.); Department of Medicine (C.M., A.M.B., A.C., L.N., J.E., M.M., E.R.J.), Department of Physical Medicine and Rehabilitation (M.D.), Department of Physiology (A.M.B., M.M., E.R.J.), Department of Biochemistry (B.H.), Department of Radiation Oncology (M.M.), Department of Biophysics (N.H.), and Cardiovascular Center (M.T., C.M., A.B., M.D., M.M., E.R.J.), Medical College of Wisconsin, Milwaukee; Research Service, Zablocki Veterans Affairs Medical Center, Milwaukee (A.C., S.H.A., M.M., E.R.J.); Department of Biomedical Engineering, Marquette University, Milwaukee (S.H.A.); and Department of Mathematics, Statistics and Computer Science, Marquette University, Milwaukee (A.C.). 2. From the College of Medical Laboratory Science and Technology, Harbin Medical University, Daqing, China (C.M., D.Z., M.M., E.R.J.); Department of Medicine (C.M., A.M.B., A.C., L.N., J.E., M.M., E.R.J.), Department of Physical Medicine and Rehabilitation (M.D.), Department of Physiology (A.M.B., M.M., E.R.J.), Department of Biochemistry (B.H.), Department of Radiation Oncology (M.M.), Department of Biophysics (N.H.), and Cardiovascular Center (M.T., C.M., A.B., M.D., M.M., E.R.J.), Medical College of Wisconsin, Milwaukee; Research Service, Zablocki Veterans Affairs Medical Center, Milwaukee (A.C., S.H.A., M.M., E.R.J.); Department of Biomedical Engineering, Marquette University, Milwaukee (S.H.A.); and Department of Mathematics, Statistics and Computer Science, Marquette University, Milwaukee (A.C.). ejacobs@mcw.edu.
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.
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.
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