Doerrier 2016b Abstract Mito Xmas Meeting Innsbruck
|Searching for specific targets of ischemic damage of cardiac mitochondria using O2k-Fluorometry.|
Ischemia-reperfusion damage during the transplant process occurs mainly in three steps: (i) warm ischemia (WI), (ii) cold ischemia, (iii) reperfusion. WI is defined as the time interval between a tissue remaining at body temperature after blood supply has been reduced or interrupted. Oxidative stress is considered to be one of the main causes of injury during ischemia-reperfusion. In the present work we used high-resolution respirometry (O2k-Fluorometer; Oroboros , Innsbruck, Austria)  to investigate simultaneously respiration and hydrogen peroxide production (H2O2) of mitochondria isolated from the hearts of C57BL/6 mice. By using inhibitors of the main mitochondrial H2O2 scavengers (DNCB for glutathione and AF for thioredoxin) we evaluated the total H2O2 production compared to net H2O2 production in the absence of these inhibitors .
OXPHOS and Electron transfer pathway respiratory capacities of isolated mitochondria (normalized per mg mt-protein) were decreased after 1-h WI of the excised heart. A significant injury of the outer mt-membrane is consistent with ischemia-induced mt-permeability transition , which can explain a general respiratory defect. In addition, application of a newly developed substrate-uncoupler-inhibitor titration (SUIT) protocol  revealed a specific defect of fatty acid β-oxidation (FAO)  H2O2 flux based on the Amplex red assay more than doubled after application of AF and DNCB inhibitors to the controls. The glutathione and thioredoxin antioxidant system did not protect mitochondria after WI from this increased H2O2 production. Taken together, standardized respiratory SUIT protocols combined with SOPs in the fluorometric assay of H2O2 production offer a sensitive diagnostic tool for comprehensive OXPHOS analysis.
- Doerrier C(1), Gnaiger E(1,2)
- Oroboros Instruments, Innsbruck, Austria
- D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria
- Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38.
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- Borutaite V, Toleikis A, Brown GC (2013) In the eye of the storm: mitochondrial damage during heart and brain ischaemia. FEBS J 280:4999-5014.
- Doerrier C, Sumbalova Z, Krumschnabel G, Hiller E, Gnaiger E (2016) SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. 21.06 Mitochondr Physiol Network.
- Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43:1729–38.
Labels: MiParea: Respiration
Stress:Ischemia-reperfusion Organism: Mouse Tissue;cell: Heart Preparation: Isolated mitochondria
Coupling state: LEAK, OXPHOS, ET Pathway: F, N, S, Gp, CIV, ROX HRR: Oxygraph-2k, O2k-Fluorometer Event: B2, Oral Warm ischemia, AmR, RP1, RP2, AF, DNCB