Real-time quantification of subcellular H 2 O 2 and glutathione redox potential in living cardiovascular tissues

Free Radic Biol Med. 2017 Aug;109:189-200. doi: 10.1016/j.freeradbiomed.2017.02.022. Epub 2017 Feb 10.

Abstract

Detecting and measuring the dynamic redox events that occur in vivo is a prerequisite for understanding the impact of oxidants and redox events in normal and pathological conditions. These aspects are particularly relevant in cardiovascular tissues wherein alterations of the redox balance are associated with stroke, aging, and pharmacological intervention. An ambiguous aspect of redox biology is how redox events occur in subcellular organelles including mitochondria, and nuclei. Genetically-encoded Rogfp2 fluorescent probes have become powerful tools for real-time detection of redox events. These probes detect hydrogen peroxide (H2O2) levels and glutathione redox potential (EGSH), both with high spatiotemporal resolution. By generating novel transgenic (Tg) zebrafish lines that express compartment-specific Rogfp2-Orp1 and Grx1-Rogfp2 sensors we analyzed cytosolic, mitochondrial, and the nuclear redox state of endothelial cells and cardiomyocytes of living zebrafish embryos. We provide evidence for the usefulness of these Tg lines for pharmacological compounds screening by addressing the blocking of pentose phosphate pathways (PPP) and glutathione synthesis, thus altering subcellular redox state in vivo. Rogfp2-based transgenic zebrafish lines represent valuable tools to characterize the impact of redox changes in living tissues and offer new opportunities for studying metabolic driven antioxidant response in biomedical research.

Keywords: Cardiomyocytes; Endothelial cells; Redox metabolism; Rogfp2 probes; Subcellular redox biology; Zebrafish model.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 6-Aminonicotinamide / pharmacology
  • Animals
  • Animals, Genetically Modified
  • Buthionine Sulfoximine / pharmacology
  • Cardiovascular System / cytology
  • Cardiovascular System / drug effects
  • Cardiovascular System / metabolism
  • Cell Nucleus / metabolism
  • Cell Nucleus / ultrastructure
  • Cytosol / metabolism
  • Cytosol / ultrastructure
  • Embryo, Nonmammalian
  • Embryonic Development / genetics
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism*
  • Endothelial Cells / ultrastructure
  • Gene Expression
  • Glutaredoxins / genetics
  • Glutaredoxins / metabolism
  • Glutathione / analysis*
  • Glutathione / metabolism
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Hydrogen Peroxide / analysis*
  • Hydrogen Peroxide / metabolism
  • Microscopy, Fluorescence
  • Mitochondria / metabolism
  • Mitochondria / ultrastructure
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / ultrastructure
  • Oxidation-Reduction
  • Pentose Phosphate Pathway / drug effects
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Zebrafish

Substances

  • Glutaredoxins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • 6-Aminonicotinamide
  • Buthionine Sulfoximine
  • Hydrogen Peroxide
  • Glutathione