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Difference between revisions of "Doerrier 2017 MiP2017 C1"

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{{Abstract
{{Abstract
|title=[[Image:DoerrierC.JPG|left|90px|Carolina Anneliese Doerrier]] Effects of warm ischemia on cardiac mitochondria to detect specific targets by High-Resolution FluoRespirometry (HRFR)
|title=[[Image:DoerrierC.JPG|left|90px|Carolina Anneliese Doerrier]] Effects of warm ischemia on cardiac mitochondria to detect specific targets by high-resolution respirometry (HRR)
|info=[[MiP2017]]
|info=[[MiP2017]]
|authors=Doerrier C, Gnaiger E
|authors=Doerrier C, Gnaiger E
|year=2017
|year=2017
|event=MiP2017
|event=MiP2017
|abstract=[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]] The evaluation of the 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 remains at body temperature after blood supply decreased or has been interrupted. It was reported that oxidative stress is considered to be one of the main causes of injury during ischemia-reperfusion.  
|abstract=[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]] 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.  


Therefore, in the present work, we used High-Resolution FluoRespirometry (HRFR; Oroboros, Innsbruck, Austria) [1] to investigate simultaneously mitochondrial respiration and hydrogen peroxide production (H<sub>2</sub>O<sub>2</sub>) of mouse cardiac isolated mitochondria (from C57BL/6N mice) [2]. WI was induced by incubating the hearts in the preservation buffer BIOPS during 1 hour at 37 °C. Different substrate-uncoupler-inhibitor titration (SUIT) protocols were used to detect specific targets where mitochondria display any defect. By using inhibitors of the main mitochondrial H<sub>2</sub>O<sub>2</sub> scavengers (dinitrochlorobenzene, DNCB for glutathione and auranofin; AF for thioredoxin reductase) 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 [3].  
In the present study, we used the high-resolution O2k-FluoRespirometer (Oroboros, Innsbruck, Austria) [1] to investigate simultaneously mitochondrial respiration and hydrogen peroxide production (H<sub>2</sub>O<sub>2</sub>) in cardiac mitochondria isolated from C57BL/6N mice [2]. H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> scavengers (dinitrochlorobenzene, DNCB for glutathione and auranofin; AF for thioredoxin reductase), 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 [3]. Fluxes were normalized for mt-protein.


OXPHOS capacity evaluated in N-, F(N)-, FN-, FNS-, FNSGp-pathways and ET capacity with N-, NS-, FNS-, S-, SGp-, FNSGp-linked substrates were decreased after 1 hour WI of the excised heart (oxygen consumption was normalized per mg mt-protein). Moreover, CIV respirometric assay displayed a significant reduction of mitochondrial respiration in ET state after 1 hour of WI. A significant injury of the outer mt-membrane is consistent with ischemia-induced mt-permeability transition (MPT) [4], which can explain a general respiratory defect. The use of flux control ratios (FCR) allowed the detection of a CI defect during WI. In addition, application of a newly developed reference SUIT reference protocol RP2 [5] revealed a specific defect of fatty acid β-oxidation (FAO) [6]. H<sub>2</sub>O<sub>2</sub> production increases in the WI model. H<sub>2</sub>O<sub>2</sub> flux based on the Amplex UltraRed assay more than doubled after application of AF and DNCB inhibitors in the controls and after WI, reflecting that the use of the antioxidant inhibitors shows higher H<sub>2</sub>O<sub>2</sub> production which is not accounted in H<sub>2</sub>O<sub>2</sub> measurements. The glutathione and thioredoxin antioxidant system did not protect mitochondria after WI from this increased H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> production offer a sensitive diagnostic tool for comprehensive OXPHOS analysis.
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 [4], 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 [5] revealed a specific defect of fatty acid β-oxidation (FAO, F-pathway) [6]. H<sub>2</sub>O<sub>2</sub> production increased after WI. H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> production offer a sensitive diagnostic tool for comprehensive OXPHOS analysis.
|editor=[[Kandolf G]]
|editor=[[Kandolf G]]
|mipnetlab=AT Innsbruck Oroboros, AT Innsbruck Gnaiger E
|mipnetlab=AT Innsbruck Oroboros
}}
{{Labeling
|area=Respiration
|injuries=Ischemia-reperfusion
|organism=Mouse
|tissues=Heart
|preparations=Isolated mitochondria
|couplingstates=LEAK, ET-pathway
|pathways=F, N, S, CIV, NS
|instruments=Oxygraph-2k
|additional=Amplex UltraRed
}}
}}
== Affiliations ==
== Affiliations ==
Line 31: Line 20:


== References ==
== References ==
::::#[[High-Resolution_FluoRespirometry]]
::::#[[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.
::::#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 H<sub>2</sub>O<sub>2</sub> emission: An experimental-computational study. J Gen Physiol 139:479-91.  
::::#Aon MA, Stanley AS, Sivakumaran V, Kembro JM, O´Rourke B, Paolocci N, Cortassa S (2012) Glutathione/thioredoxin sytems modulate mitochondrial H<sub>2</sub>O<sub>2</sub> emission: An experimental-computational study. J Gen Physiol 139:479-91.  
Line 37: Line 26:
::::#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, ET
|pathways=F, N, S, CIV, NS
|instruments=Oxygraph-2k
|additional=AmR,
}}

Latest revision as of 17:48, 10 January 2022

Carolina Anneliese Doerrier
Effects of warm ischemia on cardiac mitochondria to detect specific targets by high-resolution respirometry (HRR)

Link: MiP2017

Doerrier C, Gnaiger E (2017)

Event: MiP2017

COST Action MITOEAGLE

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) [1] to investigate simultaneously mitochondrial respiration and hydrogen peroxide production (H2O2) in cardiac mitochondria isolated from C57BL/6N mice [2]. 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 [3]. 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 [4], 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 [5] revealed a specific defect of fatty acid β-oxidation (FAO, F-pathway) [6]. 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.


Bioblast editor: Kandolf G O2k-Network Lab: AT Innsbruck Oroboros


Affiliations

Doerrier C(1), Gnaiger E(1,2)
  1. Oroboros Instruments, Innsbruck, Austria
  2. Dept Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria. -carolina.doerrier@oroboros.at

References

  1. High-resolution respirometry
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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, ET  Pathway: F, N, S, CIV, NS  HRR: Oxygraph-2k 

AmR