title: Increased mitochondrial prooxidant activity mediates up-regulation of Complex I S-glutathionylation via protein thiyl radical in the murine heart of eNOS(-/-).
abstract: In response to oxidative stress, mitochondrial Complex I is reversibly S-glutathionylated. We hypothesized that protein S-glutathionylation (PrSSG) of Complex I is mediated by a kinetic mechanism involving reactive protein thiyl radical (PrS(•)) and GSH in vivo. Previous studies have shown that in vitro S-glutathionylation of isolated Complex I at the 51 and 75-kDa subunits was detected under the conditions of (•)O2(-) production, and mass spectrometry confirmed that formation of Complex I PrS(•) mediates PrSSG. Exposure of myocytes to menadione resulted in enhanced Complex I PrSSG and PrS(•) (Kang et al., Free Radical Biol. Med.52:962-973; 2012). In this investigation, we tested our hypothesis in the murine heart of eNOS(-/-). The eNOS(-/-) mouse is known to be hypertensive and develops the pathological phenotype of progressive cardiac hypertrophy. The mitochondria isolated from the eNOS(-/-) myocardium exhibited a marked dysfunction with impaired state 3 respiration, a declining respiratory control index, and decreasing enzymatic activities of ETC components. Further biochemical analysis and EPR measurement indicated defective aconitase activity, a marked increase in (•)O2(-) generation activity, and a more oxidized physiological setting. These results suggest increasing prooxidant activity and subsequent oxidative stress in the mitochondria of the eNOS(-/-) murine heart. When Complex I from the mitochondria of the eNOS(-/-) murine heart was analyzed by immunospin trapping and probed with anti-GSH antibody, both PrS(•) and PrSSG of Complex I were significantly enhanced. Overexpression of SOD2 in the murine heart dramatically diminished the detected PrS(•), supporting the conclusion that mediation of Complex I PrSSG by oxidative stress-induced PrS(•) is a unique pathway for the redox regulation of mitochondrial function in vivo.
authors.affiliation: Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, OH 44272, USA.
authors.forename: Patrick T
authors.initials: PT
authors.lastname: Kang
authors.affiliation: Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, OH 44272, USA.
authors.forename: Chwen-Lih
authors.initials: CL
authors.lastname: Chen
authors.affiliation: Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, OH 44272, USA. Electronic address: ychen1@neomed.edu.
authors.forename: Yeong-Renn
authors.initials: YR
authors.lastname: Chen
mesh_terms: D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D005978:Glutathione / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008929:Mitochondria, Heart / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D017382:Reactive Oxygen Species; D013481:Superoxides; D052250:Nitric Oxide Synthase Type III; D005978:Glutathione
keywords: Complex I; Mitochondria; Oxidative stress; Protein thiyl radical; S-Glutathionylation; eNOS(−/−) murine heart