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Difference between revisions of "Laner 2014 Abstract MiP2014"

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(Created page with "{{Abstract |title=Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. |info=[[File:Krumschnabel Gerhard.jpg|150px|right|Krumsc...")
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{{Abstract
{{Abstract
|title=Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production.
|title=Cytochrome c flux control factor as a quality criterion in respiratory OXPHOS analysis in canine permeabilized fibres.
|info=[[File:Krumschnabel Gerhard.jpg|150px|right|Krumschnabel G]] [[Laner 2014 Mitochondr Physiol Network MiP2014|Mitochondr Physiol Network 19.13]] - [http://www.mitophysiology.org/index.php?mip2014 MiP2014]
|info=[[File:VG.jpg|150px|right|Laner V]] [[Laner 2014 Mitochondr Physiol Network MiP2014|Mitochondr Physiol Network 19.13]] - [http://www.mitophysiology.org/index.php?mip2014 MiP2014]
|authors=Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E
|authors=Laner V, Boushel RC, Hamilton KL, Miller BF, Williamson KK, Davis MS, Gnaiger E
|year=2014
|year=2014
|event=MiP2014
|event=MiP2014
|abstract=Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O<sub>2</sub><sup>β€’-</sup>) and particularly hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Since H<sub>2</sub>O<sub>2</sub> plays important roles in physiology and pathology, measurement of hydrogen peroxide has received considerable attention over many years. Here we describe how the well-established Amplex Red assay can be used to detect H2O2 production in combination with the simultaneous assessment of mitochondrial bioenergetics by high-resolution respirometry. Fundamental instrumental and methodological parameters were optimized for analysis of the effects of various substrate, uncoupler and inhibitor titrations (SUIT [1]) on respiration versus H<sub>2</sub>O<sub>2</sub> production. The sensitivity of the H<sub>2</sub>O<sub>2</sub> assay was strongly influenced by compounds present in different mitochondrial respiration media, which exerted significant effects on chemical background fluorescence changes. Near-linearity of the fluorescence signal was restricted to narrow ranges of accumulating resorufin concentrations independent of the nature of mitochondrial respiration media. Finally, we show an application example using mouse brain mitochondria as an experimental model for the simultaneous measurement of mitochondrial respiration and H2O2 production in SUIT protocols.
|abstract=Mitochondrial (mt) preparations (isolated mitochondria, permeabilized cells and tissues, tissue homogenates) provide a fundamental basis for comprehensive OXPHOS analysis for the study of substrate and coupling control of mitochondrial respiration [1]. Plasma membrane permeabilization with mechanical separation of muscle fibre bundles and chemical permeabilization with mild detergents may influence the integrity of the outer mt-membrane and thus induce partial release of cytochrome c (c). In mitochondria isolated from healthy skeletal muscle, CI&II-linked OXPHOS capacity decreases linearly with cytochrome c loss during isolation [2]. The cytochrome c effect is expressed as the flux control factor FCFc, which is the increase of OXPHOS capacity after addition of 10 Β΅M c normalized for c-stimulated respiration [1-3]. There is no consensus as to the threshold of FCFc applied as a quantitative exclusion criterion in permeabilized fibres obtained from healthy muscle tissue.
We aimed at establishing a reference method for the application of a cytochrome c threshold as exclusion criterion in mitochondrial OXPHOS analyses. Our study involved Alaskan sled dogs (N=6) studied 72 to 120 h after finishing a competitive 1,000 mile race in nine days. Permeabilized fibres (wet weight per chamber of 0.81-1.28 mg Β± 0.12 SD) were prepared from needle biopsies and immediately studied by high-resolution respirometry [4] using 12 chambers in parallel (OROBOROS Oxygraph-2k). Compared to human skeletal muscle fibres, the canine samples were more trexturally supple and sticky, requiring delicate fiber separation under light microscope, and disintegrating to various degrees during substrate-uncoupler-inhibitor titration (SUIT) protocols. This was reflected in variable and sometimes extremely high cytochrome c effects. However, there was no loss of CI- or CI&II-linked OXPHOS and ETS capacity with increasing FCFc (Figure 1). Apparently, the damage caused by mt-preparation even in cases with FCFc up to 0.25 could be rescued by addition of 10 Β΅M c and thus restore capacities comparable with samples of negligible FCFc. In contrast, multiple defects associated with increasing FCFc in human muscle fibres cannot be compensated fully by addition of cytochrome c [2,5]. Cytochrome c was applied early in the two SUIT protocols, in the CI-linked or CI&FAO-linked OXPHOS state. This allowed consistent analysis of subsequent respiratory states which were all supported by the externally added cytochrome c (Figure 1).
OXPHOS and ETS capacities with FAO- and CI&II-linked substrates were higher than in muscle from competitive horses and humans [5,6]. The present approach (Figure 1) allows evaluation of the FCFc threshold as a potential exclusion criterion in healthy controls.
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|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom
|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom
}}
}}
{{Labeling
{{Labeling
|area=Respiration
|area=Respiration
|organism=Mouse
|organism=Dog
|tissues=Nervous system
|topics=Cyt c
|preparations=Homogenate, Isolated Mitochondria
|substratestates=CI+II
|injuries=RONS; Oxidative Stress
|couplingstates=LEAK, OXPHOS, ETS
|substratestates=CI, CII, CI+II
|instruments=Oxygraph-2k, Fluorometry
|instruments=Oxygraph-2k, Fluorometry
|event=B4, Poster
|event=B4, Poster

Revision as of 09:14, 25 August 2014

Cytochrome c flux control factor as a quality criterion in respiratory OXPHOS analysis in canine permeabilized fibres.

Link:

Laner V

Mitochondr Physiol Network 19.13 - MiP2014

Laner V, Boushel RC, Hamilton KL, Miller BF, Williamson KK, Davis MS, Gnaiger E (2014)

Event: MiP2014

Mitochondrial (mt) preparations (isolated mitochondria, permeabilized cells and tissues, tissue homogenates) provide a fundamental basis for comprehensive OXPHOS analysis for the study of substrate and coupling control of mitochondrial respiration [1]. Plasma membrane permeabilization with mechanical separation of muscle fibre bundles and chemical permeabilization with mild detergents may influence the integrity of the outer mt-membrane and thus induce partial release of cytochrome c (c). In mitochondria isolated from healthy skeletal muscle, CI&II-linked OXPHOS capacity decreases linearly with cytochrome c loss during isolation [2]. The cytochrome c effect is expressed as the flux control factor FCFc, which is the increase of OXPHOS capacity after addition of 10 Β΅M c normalized for c-stimulated respiration [1-3]. There is no consensus as to the threshold of FCFc applied as a quantitative exclusion criterion in permeabilized fibres obtained from healthy muscle tissue. We aimed at establishing a reference method for the application of a cytochrome c threshold as exclusion criterion in mitochondrial OXPHOS analyses. Our study involved Alaskan sled dogs (N=6) studied 72 to 120 h after finishing a competitive 1,000 mile race in nine days. Permeabilized fibres (wet weight per chamber of 0.81-1.28 mg Β± 0.12 SD) were prepared from needle biopsies and immediately studied by high-resolution respirometry [4] using 12 chambers in parallel (OROBOROS Oxygraph-2k). Compared to human skeletal muscle fibres, the canine samples were more trexturally supple and sticky, requiring delicate fiber separation under light microscope, and disintegrating to various degrees during substrate-uncoupler-inhibitor titration (SUIT) protocols. This was reflected in variable and sometimes extremely high cytochrome c effects. However, there was no loss of CI- or CI&II-linked OXPHOS and ETS capacity with increasing FCFc (Figure 1). Apparently, the damage caused by mt-preparation even in cases with FCFc up to 0.25 could be rescued by addition of 10 Β΅M c and thus restore capacities comparable with samples of negligible FCFc. In contrast, multiple defects associated with increasing FCFc in human muscle fibres cannot be compensated fully by addition of cytochrome c [2,5]. Cytochrome c was applied early in the two SUIT protocols, in the CI-linked or CI&FAO-linked OXPHOS state. This allowed consistent analysis of subsequent respiratory states which were all supported by the externally added cytochrome c (Figure 1). OXPHOS and ETS capacities with FAO- and CI&II-linked substrates were higher than in muscle from competitive horses and humans [5,6]. The present approach (Figure 1) allows evaluation of the FCFc threshold as a potential exclusion criterion in healthy controls.


β€’ O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom


Labels: MiParea: Respiration 


Organism: Dog 


Regulation: Cyt c 


HRR: Oxygraph-2k, Fluorometry"Fluorometry" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.  Event: B4, Poster  MiP2014 

Affiliation

1-OROBOROS INSTRUMENTS, Innsbruck, Austria; 2-Present address: Pharmacobioch Lab 3rd Dep Intern Medicine, Comenius Univ, Bratislava, Medical Fac; 3-Daniel Swarovski Research Lab, Mitochondrial Physiol, Dep Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck. – [email protected]

References and acknowledgements

Supported by K-Regio project MitoCom Tyrol.

  1. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.