1. Real-time quantification of subcellular H 2 O 2 and glutathione redox potential in living cardiovascular tissues.
- Author
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Panieri E, Millia C, and Santoro MM
- Subjects
- 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 ultrastructure, Gene Expression, Glutaredoxins genetics, Glutaredoxins metabolism, Glutathione metabolism, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Hydrogen Peroxide metabolism, Microscopy, Fluorescence, Mitochondria metabolism, Mitochondria ultrastructure, Myocytes, Cardiac drug effects, Myocytes, Cardiac ultrastructure, Oxidation-Reduction, Pentose Phosphate Pathway drug effects, Recombinant Fusion Proteins genetics, Zebrafish, Endothelial Cells metabolism, Glutathione analysis, Hydrogen Peroxide analysis, Myocytes, Cardiac metabolism, Recombinant Fusion Proteins metabolism
- 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 (H
2 O2 ) 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
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