|Lemieux H, Votion DM, Gnaiger E (2007) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23.|
Abstract: O2k-Protocol for Mitochondrial Respiration in Permeabilized Fibres: Needle Biopsies from Horse Skeletal Muscle
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS Oxygraph- 2k (O2k). The experiment demonstrates manual titrations applied to study respiratory capacity in permeabilized fibres. Application of the DatLab 4 software is shown for on-line data analysis MiPNet12.09 The following guideline describes the experimental protocol and includes a short discussion of results. The experiments was carried out by participants of an O2k-Course on HRR in December 2007 (IOC44; Schroecken, Austria).
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.
- LEAK state, L: Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N).
- OXPHOS capacity with Complex I substrates, P: Respiratory capacity in the active coupled state (with ADP, GMD).
- 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).
- OXPHOS capacity with Complex I+II substrates, P: Respiratory stimulation by convergent electron flow from Complexes I+II at the Q-junction, in the coupled state after further addition of succinate, as an estimate of OXPHOS capacity (Gnaiger 2009).
- Electron transfer system (ETS) capacity with CI+II substrates, E: 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.
- ETS capacity with CII substrate, E: ETS 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).
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.
HRR: Oxygraph-2k, Protocol"Protocol" 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.
- Version 1: 2007
- Version 6: 2011-12-11