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Difference between revisions of "MiPNet12.23 FiberRespiration"

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{{OROBOROS header page name}}
{{Publication
{{Publication
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]]Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle.
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|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/4/49/MiPNet12.23_FibreRespiration.pdf|Bioblast pdf]]
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]]Mitochondrial respiration in permeabilized fibers: Needle biopsies from horse skeletal muscle.
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/4/49/MiPNet12.23_FibreRespiration.pdf|Bioblast pdf]] Β»[http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions]
|authors=Oroboros
|authors=Oroboros
|year=2016-07-19
|year=2019-01-10
|journal=Mitochondr Physiol Network
|journal=Mitochondr Physiol Network
|abstract='''Doerrier C, Lemieux H, Votion DM, Gnaiger E (2007-16) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(08 in prep):1-8.''' Β»[http://www.bioblast.at/index.php/File:MiPNet12.23_FibreRespiration.pdf Versions]
|abstract=Lemieux H, Votion DM, Doerrier C, Gnaiger E (2019) Mitochondrial respiration in permeabilized fibers: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(08):1-4.
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:Β» O2k-Protocols:[[SUIT-011| SUIT-011]]
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the Oroboros O2k. The experiment demonstrates [http://bioblast.at/images/e/ef/MiPNet09.12_O2k-Titrations.pdf manual titrations] applied to study respiratory capacity in permeabilized fibres. Application of the [[DatLab]] software is shown for real-time data analysis (compare [[MiPNet12.09]]). The following guideline describes the [[1GM;2D;3S;4U;5Rot-]] SUIT protocol and includes a short discussion of results (see [[Votion 2012 PLoS One]]). The experiment was carried out by participants of an O2k-Workshop on HRR in December 2007 ([[MiPNet12.24 IOC44 |IOC44]]; Schroecken, Austria).
:Β» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]
:Β» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]
|mipnetlab=AT_Innsbruck_Oroboros, BE_Liege_Votion DM
|mipnetlab=AT_Innsbruck_Oroboros, BE_Liege_Votion DM
}}
}}
{{Labeling
|area=Respiration, Instruments;methods
|organism=Horse
|tissues=Skeletal muscle
|preparations=Permeabilized tissue
|topics=Coupling efficiency;uncoupling, Cyt c
|couplingstates=LEAK, OXPHOS, ETS
|pathways=N, S, NS
|instruments=Oxygraph-2k, O2k-Protocol
|additional=O2k-Demo, O2k-Core, 1GM;2D;3S;4U;5Rot-
}}
== SUIT protocol ==
[[File:1GM;2D;2c;3S;4U;5Rot;6Ama.jpg |right|300px|link=1GM;2D;3S;4U;5Rot-]]
****: SUIT protocol: [[1GM;2D;3S;4U;5Rot-]]
'''SUIT states:''' 1-2[[GM]] 3-4[[GMS]] 5[[S]] 6[[ROX]]
{| class="wikitable" border="1"
|-
! Step
! Respiratory state
! Pathway control
! Entry through electron transfer Complexx
|-
| 1GM
| [[GM]](L)
| N
| CI
|-
| 2D
| [[GM]](P)
| N
| CI
|-
| 2c
| [[GM]](P)
| N
| CI
|-
| 3S
| [[GMS]](P)
| NS
| CI<small>&</small>II
|-
| 4U
| [[GMS]](E)
| NS
| CI<small>&</small>II
|-
| 5Rot
| [[S]](E)
| S
| CII
|-
| 6Ama
| ROX
|}
[[Image:MiPNet12.23.jpg|center|800px]]
Figure 1. Oxygen concentration ([ΞΌM] blue line) and oxygen flux per mg wet weight of muscle ([pmolβˆ™s<sup>-1</sup>βˆ™mg<sup>-1</sup>] red lines) in O2k chamber B, in permeabilized fibres from horse skeletal muscle with the standard titration protocol.
A multiple substrate-uncoupler-inhibitor titration protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The experiment was aimed at an assessment of a sequence of titrations, inducing stepwise defined respiratory states.
:::#<span style="color:red"> '''LEAK state with type N substrates, N''<sub>L</sub>'':'''</span> Non-phosphorylating resting state with NADH-linked (type N) substrates glutamate&malate (GM; without adenalytes; CI-linked pathway to Q).
:::#<span style="color:green">'''OXPHOS capacity with type N substrates, N''<sub>P</sub>'':'''</span> Respiratory capacity in the active coupled state (GM with ADP).
:::#<span style="color:black">'''Cytochrome ''c'' test for quality control:'''</span> Further addition of cytochrome ''c'' yields a test for integrity of the outer mitochondrial membrane (loss of cytochrome ''c'' would be indicated by a stimulation of respiration).
:::#<span style="color:green">'''OXPHOS capacity with type NS substrates (CI<small>&</small>II-linked pathway to Q), NS''<sub>P</sub>'':'''</span> Respiratory stimulation by convergent electron flow through Complexes I<small>&</small>II at the Q-junction, in the coupled state after further addition of succinate (S), as an estimate of OXPHOS capacity with reconstitution of the TCA cycle ([[Gnaiger 2009 Int J Biochem Cell Biol]]).
:::#<span style="color:blue">'''Electron transfer system (ET-pathway) capacity with type NS substrates, NS''<sub>E</sub>'':'''</span> Uncoupling by FCCP titration (avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ET capacity by the phosphorylation system.
:::#<span style="color:blue">'''ET capacity with type S (CII) substrate, S''<sub>E</sub>'':'''</span> ET capacity with succinate, after blocking Complex I with rotenone.
:::#'''Residual oxygen consumption (ROX)''' due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (inhibitor of Complex III).
== Reference ==
::::* Votion DM, Gnaiger E, Lemieux H, Mouithys-Mickalad A, Serteyn D (2012) Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle. PLoS One 7: e34890. - [[Votion 2012 PLoS One |Open Access]]

Latest revision as of 10:34, 27 November 2019

                



MiPNet12.23 FiberRespiration

Publications in the MiPMap
O2k-Protocols contents
Mitochondrial respiration in permeabilized fibers: Needle biopsies from horse skeletal muscle.

Β» Bioblast pdf Β»Versions

Oroboros (2019-01-10) Mitochondr Physiol Network

Abstract: Lemieux H, Votion DM, Doerrier C, Gnaiger E (2019) Mitochondrial respiration in permeabilized fibers: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(08):1-4.

Β» O2k-Protocols: SUIT-011
Β» Product: Oroboros O2k, O2k-Catalogue


β€’ O2k-Network Lab: AT_Innsbruck_Oroboros, BE_Liege_Votion DM