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Difference between revisions of "Doerrier 2016b Abstract Mito Xmas Meeting Innsbruck"

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{{Abstract
{{Abstract
|title=Searching specific targets of ischemic damage of cardiac mitochondria using O2k-Fluorometry.
|title=Searching for specific targets of ischemic damage of cardiac mitochondria using [[O2k-Fluorometry]].
 
|authors=Doerrier C, Gnaiger E
|authors=Doerrier C, Gnaiger E
|year=2016
|year=2016
|event=Mito Xmas Meeting 2016 Innsbruck AT
|event=Mito Xmas Meeting 2016 Innsbruck AT
|abstract=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.
|abstract=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) [1] 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 [2].
In the present work we used high-resolution respirometry (O2k-Fluorometer; Oroboros , Innsbruck, Austria) [1] to investigate simultaneously respiration and hydrogen peroxide production (H<sub>2</sub>O<sub>2</sub>) of mitochondria isolated from the hearts of C57BL/6 mice. By using inhibitors of the main mitochondrial H<sub>2</sub>O<sub>2</sub> scavengers (DNCB for glutathione and AF for thioredoxin) we evaluated the total H<sub>2</sub>O<sub>2</sub> production compared to net H<sub>2</sub>O<sub>2</sub> production in the absence of these inhibitors [2].  
OXPHOS and ETS 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 [3], which can explain a general respiratory defect. In addition, application of a newly developed substrate-uncoupler-inhibitor titration (SUIT) protocol [4] revealed a specific defect of fatty acid β-oxidation (FAO) [5] 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.


|mipnetlab=AT Innsbruck OROBOROS, ES Granada Acuna-Castroviejo D, AT Innsbruck Gnaiger E, AT Innsbruck MitoFit
[[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 [3], which can explain a general respiratory defect. In addition, application of a newly developed substrate-uncoupler-inhibitor titration (SUIT) protocol [4] revealed a specific defect of fatty acid β-oxidation (FAO) [5] H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> production. Taken together, standardized respiratory SUIT protocols combined with SOPs in the fluorometric assay of H<sub>2</sub>O<sub>2</sub> production offer a sensitive diagnostic tool for comprehensive OXPHOS analysis.
}}
|mipnetlab=AT Innsbruck Oroboros, AT Innsbruck MitoFit
{{Labeling
|event=Poster
}}
}}
== Affiliations ==
== Affiliations ==
Line 18: Line 14:
:::: Doerrier C(1), Gnaiger E(1,2)
:::: Doerrier C(1), Gnaiger E(1,2)


::::# OROBOROS INSTRUMENTS, Innsbruck, Austria  
::::# Oroboros Instruments, Innsbruck, Austria  
::::# D.Swarovski Research Lab, Dept.Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria.
::::# D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria


==Reference==
==Reference==
Line 27: Line 23:
::::# 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.
::::# 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.
::::# 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.
{{Labeling
|area=Respiration
|injuries=Ischemia-reperfusion
|organism=Mouse
|tissues=Heart
|preparations=Isolated mitochondria
|couplingstates=LEAK, OXPHOS, ET
|pathways=F, N, S, Gp, CIV, ROX
|instruments=Oxygraph-2k, O2k-Fluorometer
|event=B2, Oral
|additional=Warm ischemia, AmR, RP1, RP2, AF, DNCB
}}

Latest revision as of 17:41, 10 January 2022

Searching for specific targets of ischemic damage of cardiac mitochondria using O2k-Fluorometry.

Link:

Doerrier C, Gnaiger E (2016)

Event: Mito Xmas Meeting 2016 Innsbruck AT

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) [1] 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 [2].

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 [3], which can explain a general respiratory defect. In addition, application of a newly developed substrate-uncoupler-inhibitor titration (SUIT) protocol [4] revealed a specific defect of fatty acid β-oxidation (FAO) [5] 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.


O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck MitoFit


Affiliations

Doerrier C(1), Gnaiger E(1,2)
  1. Oroboros Instruments, Innsbruck, Austria
  2. D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria

Reference

  1. 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.
  2. 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(6):479-91.
  3. 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.
  4. 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.
  5. 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