title: Compartment-specific Control of Reactive Oxygen Species Scavenging by Antioxidant Pathway Enzymes.
abstract: Oxidative stress arises from an imbalance in the production and scavenging rates of reactive oxygen species (ROS) and is a key factor in the pathophysiology of cardiovascular disease and aging. The presence of parallel pathways and multiple intracellular compartments, each having its own ROS sources and antioxidant enzymes, complicates the determination of the most important regulatory nodes of the redox network. Here we quantified ROS dynamics within specific intracellular compartments in the cytosol and mitochondria and determined which scavenging enzymes exert the most control over antioxidant fluxes in H9c2 cardiac myoblasts. We used novel targeted viral gene transfer vectors expressing redox-sensitive GFP fused to sensor domains to measure H2O2 or oxidized glutathione. Using genetic manipulation in heart-derived H9c2 cells, we explored the contribution of specific antioxidant enzymes to ROS scavenging and glutathione redox potential within each intracellular compartment. Our findings reveal that antioxidant flux is strongly dependent on mitochondrial substrate catabolism, with availability of NADPH as a major rate-controlling step. Moreover, ROS scavenging by mitochondria significantly contributes to cytoplasmic ROS handling. The findings provide fundamental information about the control of ROS scavenging by the redox network and suggest novel interventions for circumventing oxidative stress in cardiac cells.
authors.affiliation: From the Division of Cardiology, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205.
authors.forename: Swati
authors.initials: S
authors.lastname: Dey
authors.affiliation: From the Division of Cardiology, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Sidor
authors.affiliation: From the Division of Cardiology, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205 bor@jhmi.edu.
authors.forename: Brian
authors.initials: B
authors.lastname: O'Rourke
mesh_terms: D000818:Animals; D002460:Cell Line; D016166:Free Radical Scavengers / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008928:Mitochondria / Q000201:enzymology*; D032386:Myoblasts, Cardiac / Q000201:enzymology*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats
chemical_list: D016166:Free Radical Scavengers; D006861:Hydrogen Peroxide
keywords: NADPH; antioxidant; cell metabolism; glutathione peroxidase; mitochondria; peroxiredoxin; reactive oxygen species (ROS); redox regulation; thioredoxin; thioredoxin reductase