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Difference between revisions of "MiPNet12.23 FiberRespiration"

From Bioblast
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:::: A substrate-uncoupler-inhibitor titration (SUIT) protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The protocol aims at an assessment of physiologically relevant maxumum respiratory capacity and coupling/pathway control.
:::: A substrate-uncoupler-inhibitor titration (SUIT) protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The protocol aims at an assessment of physiologically relevant maxumum respiratory capacity and coupling/pathway control.


:::#<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{L}; without adenalytes; CI-linked pathway to Q).
:::#<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<sub>''L''</sub>; 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{P} with ADP).
:::#<span style="color:green">'''OXPHOS capacity with type N substrates, N''<sub>P</sub>'':'''</span> Respiratory capacity in the active coupled state (GM<sub>''P''</sub> 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: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 (GMS{P}; 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:green">'''OXPHOS capacity with type NS substrates (GMS<sub>''P''</sub>; 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-pathway (ET-pathway) capacity with type NS substrates, NS''<sub>E</sub>'':'''</span> Uncoupling by FCCP titration (GMS{E}; 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">'''Electron transfer-pathway (ET-pathway) capacity with type NS substrates, NS''<sub>E</sub>'':'''</span> Uncoupling by FCCP titration (GMS<sub>''E''</sub>; 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 CI with rotenone (S{E}); .
:::#<span style="color:blue">'''ET capacity with type S (CII) substrate, S''<sub>E</sub>'':'''</span> ET capacity with succinate, after blocking CI with rotenone); .
:::#'''Residual oxygen consumption (ROX)''' due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (inhibitor of CIII).
:::#'''Residual oxygen consumption (ROX)''' due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (inhibitor of CIII).



Revision as of 11:16, 10 January 2019

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

» Bioblast pdf »Versions

Oroboros (2016-07-19) Mitochondr Physiol Network

Abstract: Lemieux H, Votion DM, Doerrier C, Gnaiger E (2007-19) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23(08 in prep):1-4. Methodological and conceptual features of High-Resolution Respirometry (HRR) are illustrated in an experiment with permeabilized fibres in the Oroboros O2k. The experiment demonstrates 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 (IOC44; Schroecken, Austria).

» Product: Oroboros O2k, O2k-Catalogue


O2k-Network Lab: AT_Innsbruck_Oroboros, BE_Liege_Votion DM


Labels: MiParea: Respiration, Instruments;methods 


Organism: Horse  Tissue;cell: Skeletal muscle  Preparation: Permeabilized tissue 

Regulation: Coupling efficiency;uncoupling, Cyt c  Coupling state: LEAK, OXPHOS, ET  Pathway: N, S, NS  HRR: Oxygraph-2k, O2k-Protocol 

O2k-Demo, O2k-Core, 1GM;2D;3S;4U;5Rot- 

SUIT protocol

1GM;2D;2c;3S;4U;5Rot;6Ama.png

SUIT states: 1-2GM 3-4GMS 5S 6ROX

Step Respiratory state Pathway control Q-junction entry through ET-Complex
1GM GML N CI
2D GMP N CI
2c GMP N CI
3S GMSP NS CI&II
4U GMSE NS CI&II
5Rot SE S CII
6Ama ROX
MiPNet12.23.jpg

Figure 1. Oxygen concentration ([μM] blue line) and oxygen flux per mg wet weight of muscle ([pmol∙s-1∙mg-1] red lines) in O2k chamber B, in permeabilized fibres from horse skeletal muscle with the standard titration protocol.

A substrate-uncoupler-inhibitor titration (SUIT) protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The protocol aims at an assessment of physiologically relevant maxumum respiratory capacity and coupling/pathway control.
  1. LEAK state with type N substrates, NL: Non-phosphorylating resting state with NADH-linked (type N) substrates glutamate&malate (GML; without adenalytes; CI-linked pathway to Q).
  2. OXPHOS capacity with type N substrates, NP: Respiratory capacity in the active coupled state (GMP with ADP).
  3. Cytochrome c test for quality control: 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).
  4. OXPHOS capacity with type NS substrates (GMSP; CI&II-linked pathway to Q), NSP: Respiratory stimulation by convergent electron flow through Complexes I&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).
  5. Electron transfer-pathway (ET-pathway) capacity with type NS substrates, NSE: Uncoupling by FCCP titration (GMSE; avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ET capacity by the phosphorylation system.
  6. ET capacity with type S (CII) substrate, SE: ET capacity with succinate, after blocking CI with rotenone); .
  7. Residual oxygen consumption (ROX) due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (inhibitor of CIII).

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. - »Bioblast link«