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MiPNet12.23 FiberRespiration

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Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle.

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OROBOROS (2016-07-19) 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. »Versions

Methodological and conceptual features of highresolution respirometry 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 SUIT_NS(GM)01 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 Oxygraph-2k, 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, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.  Pathway: N, S, NS  HRR: Oxygraph-2k, O2k-Protocol 

O2k-Demo, O2k-Core, SUIT_NS(GM)01 

SUIT protocol

SUIT states: 1-3GM 4-5GMS 6S 7ROX

Step Respiratory state Pathway control Entry through electron transfer Complexx
1GM GM(L) N CI
2D GM(P) N CI
3c GM(P) N CI
4S GMS(P) NS CI&II
5U GMS(E) NS CI&II
6Rot S(E) S CII
7Ama 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 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.


  1. LEAK state with type N substrates, NL: Non-phosphorylating resting state with NADH-linked (type N) substrates glutamate&malate (GM; without adenalytes; CI-linked pathway to Q).
  2. OXPHOS capacity with type N substrates, NP: Respiratory capacity in the active coupled state (GM 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 (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 system (ETS) capacity with type NS substrates, NSE: Uncoupling by FCCP titration (avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ETS capacity by the phosphorylation system.
  6. ETS capacity with type S (CII) substrate, SE: ETS capacity with succinate, after blocking Complex I with rotenone.
  7. 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. - Open Access