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Laner 2014 Abstract MiP2014

From Bioblast
Cytochrome c flux control factor as a quality criterion in respiratory OXPHOS analysis in canine permeabilized fibres.

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Laner V

Mitochondr Physiol Network 19.13 - MiP2014

Laner V, Boushel RC, Hamilton KL, Miller BF, Williamson KK, Davis MS, Gnaiger E (2014)

Event: MiP2014

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 [1]. Plasma membrane permeabilization with mechanical separation of muscle fibre 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 [2]. The cytochrome c effect is expressed as the flux control factor FCFc, which is the increase of OXPHOS capacity after addition of 10 Β΅M 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 fibres 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 fibres (wet weight per chamber of 0.81-1.28 mg Β± 0.12 SD) were prepared from needle biopsies and immediately studied by high-resolution respirometry [4] using 12 chambers in parallel (OROBOROS Oxygraph-2k). Compared to human skeletal muscle fibres, 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 ETS 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 Β΅M c and thus restore capacities comparable with samples of negligible FCFc. In contrast, multiple defects associated with increasing FCFc in human muscle fibres 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 ETS 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.


β€’ O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom


Labels: MiParea: Respiration 


Organism: Dog 


Regulation: Cyt c 


HRR: Oxygraph-2k, Fluorometry"Fluorometry" 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.  Event: B4, Poster  MiP2014 

Affiliation

1-OROBOROS INSTRUMENTS, Innsbruck, Austria; 2-Present address: Pharmacobioch Lab 3rd Dep Intern Medicine, Comenius Univ, Bratislava, Medical Fac; 3-Daniel Swarovski Research Lab, Mitochondrial Physiol, Dep Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck. – [email protected]

References and acknowledgements

Supported by K-Regio project MitoCom Tyrol.

  1. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.