Doerrier 2017 MiP2017 C1
The evaluation of mitochondrial damage during the transplantation process is crucial for evaluating different treatments which protect or rescue the tissue from an injury. Ischemia-reperfusion damage during the transplant process occurs mainly in three steps: (i) warm ischemia (WI), (ii) cold ischemia, (iii) reperfusion. WI is the time interval between a tissue remaining at body temperature after the blood supply is decreased or interrupted. Oxidative stress is considered as one of the main causes of injury in ischemia-reperfusion.
In the present study, we used the high-resolution O2k-FluoRespirometer (Oroboros, Innsbruck, Austria)  to investigate simultaneously mitochondrial respiration and hydrogen peroxide production (H2O2) in cardiac mitochondria isolated from C57BL/6N mice . H2O2 flux was measured in the O2k with the Amplex UltraRed assay. WI was induced by incubating the hearts in preservation buffer BIOPS for 1 h at 37 °C. Different substrate-uncoupler-inhibitor titration (SUIT) protocols were used for diagnostic OXPHOS analysis. Using inhibitors of the main mitochondrial H2O2 scavengers (dinitrochlorobenzene, DNCB for glutathione and auranofin; AF for thioredoxin reductase), we evaluated the total H2O2 production compared to net H2O2 production in the absence of these inhibitors . Fluxes were normalized for mt-protein.
OXPHOS capacities of N-, F-, FN-, FNS-, FNSGp-pathways and ET capacities of N-, NS-, FNS-, S-, SGp-, FNSGp-pathways were decreased, and CIV-activity was significantly reduced after 1 h WI. A significant injury of the mitochondrial outer membrane (mtOM) is consistent with ischemia-induced mt-permeability transition , which can explain a general respiratory defect. The use of flux control ratios (FCR) allowed the detection of a CI defect. In addition, application of a newly developed reference SUIT reference protocol RP2  revealed a specific defect of fatty acid β-oxidation (FAO, F-pathway) . H2O2 production increased after WI. H2O2 flux more than doubled after application of AF and DNCB inhibitors in the controls and after WI. The glutathione and thioredoxin antioxidant system did not protect mitochondria after WI from this increased H2O2 production when we evaluated the SUIT reference protocol RP1. However, antioxidant systems could have a role in WI during FAO. 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.
Labels: MiParea: Respiration
Stress:Ischemia-reperfusion Organism: Mouse Tissue;cell: Heart Preparation: Isolated mitochondria
Coupling state: LEAK, ET Pathway: F, N, S, CIV, NS HRR: Oxygraph-2k
- Doerrier C(1), Gnaiger E(1,2)
- Oroboros Instruments, Innsbruck, Austria
- Dept Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria. -firstname.lastname@example.org
- High-resolution respirometry
- 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.
- Aon MA, Stanley AS, Sivakumaran V, Kembro JM, O´Rourke B, Paolocci N, Cortassa S (2012) Glutathione/thioredoxin sytems modulate mitochondrial H2O2 emission: An experimental-computational study. J Gen Physiol 139:479-91.
- 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.