Laner 2014 Abstract MiP2014
|Cytochrome c flux control factor as a quality criterion in respiratory OXPHOS analysis in canine permeabilized fibers.|
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 . Plasma membrane permeabilization with mechanical separation of muscle fiber 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 . The cytochrome c effect is expressed as the flux control factor FCFc, which is the increase of OXPHOS capacity after addition of 10 µM 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 fibers 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 fibers (wet weight per chamber of 0.81-1.28 mg ± 0.12 SD) were prepared from needle biopsies and immediately studied by high-resolution respirometry  using 12 chambers in parallel (Oroboros Oxygraph-2k). Compared to human skeletal muscle fibers, 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 ET 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 µM c and thus restore capacities comparable with samples of negligible FCFc. In contrast, multiple defects associated with increasing FCFc in human muscle fibers 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 ET 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.
• O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck Oroboros, CA Vancouver Boushel RC, US OK Stillwater Davis MS, US CO Fort Collins Miller BF, US CO Fort Collins Hamilton K, US OK Oklahoma City Miller BF
Labels: MiParea: Respiration
Organism: Dog Tissue;cell: Skeletal muscle Preparation: Permeabilized tissue
Regulation: Cyt c Coupling state: LEAK, ROUTINE, OXPHOS, ET Pathway: N, NS HRR: Oxygraph-2k Event: C2, Poster MiP2014
1-Oroboros Instruments, Innsbruck, Austria; 2-The Swedish School Sports Health Sc, Lindigovagen, Sweden; 3-College Health Human Sc, Colorado State Univ., Fort Collins, CO, US; 4-Land O’Lakes Purina Feed, St Louis, MO, US, 5Comparative Exercise Physiol Lab, Center Veterinary Health Sc, Oklahoma State Univ, Stillwater, OK, US; 5D Swarovski Research Lab, Dep Visceral Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria – email@example.com
Figure 1. Independence of O2 flux (ET capacity in the presence of cytochrome c) of the cytochrome c control factor,
FCFc = (JCHOc-JCHO)/JCHOc
ET capacity was 238±64 pmol∙s-1∙mg-1 Ww independent of the CHO substrate combination supporting CI&II-linked electron flow in the presence or absence of 0.2 mM octanoyl carnitine (FAO).
References and acknowledgements
Supported by K-Regio project MitoCom.
- Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. Oroboros MiPNet Publications, Innsbruck: 72 pp.
- Rasmussen HN, Rasmussen UF (1997) Small scale preparation of skeletal muscle mitochondria, criteria of integrity, and assays with reference to tissue function. Mol Cell Biochem 174: 55-60.
- Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am J Physiol Heart Circ Physiol 286: H1633–41.
- Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810: 25-58.
- Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-45.
- 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.