Difference between revisions of "Laner 2015 Abstract MiPschool Greenville 2015"

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Comparative mitochondrial physiology: OXPHOS and ETS capacity in permeabilized fibres of canine superathletes.

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Laner V, Boushel RC, Hamilton KL, Miller BF, Williamson KK, Davis MS, Gnaiger E (2015)

Event: MiPschool Greenville 2015

Comparative mitochondrial physiology strongly relies on quantitative data sets for comparison of OXPHOS capacities and respiratory control patterns between species and tissues. Combination and interpretation of a wide variety of studies requires standardization of respiratory protocols, implementation of quality control criteria, and consistency of normalization. Previously, we described a reference method for the application of a cytochrome c threshold as exclusion criterion in mitochondrial OXPHOS analyses [1]. Alaskan sled dogs (N=6) were studied 72 to 120 h after finishing a competitive 1,000 mile race within less than nine days. Permeabilized fibres (0.81-1.28 mg ± 0.12 SD wet weight per assay) were prepared from needle biopsies and immediately studied by high-resolution respirometry [2] using 12 chambers in parallel (OROBOROS Oxygraph-2k). Compared to human skeletal muscle fibres, the canine samples were more delicate to handle, highly sticky and appeared to be fragile, disintegrating to various degrees during substrate-uncoupler-inhibitor titration (SUIT) protocols in mt-respiration medium MiR06Cr. Two substrate-uncoupler-inhibitor titration protocols were applied (Fig. 1). SUIT1 emphasized substrate control with fatty acid oxidation (FAO) versus carbohydrate oxidation capacity, whereas the focus of SUIT2 was on coupling control with CI-linked substrates. Both protocols were designed to provide a common reference state of CI&II-linked ETS capacity, in comparison to separate Complex I- and Complex II-linked substrate states (CI versus CII).

CI&II-linked ETS capacity was 262±41 pmol∙s^-1∙mg^-1 Ww independent of the presence or absence of 0.2 mM octanoyl carnitine (FAO). This is the highest value so far reported for mammalian skeletal muscle. Top human endurance athletes have a CI&II-linked ETS capacity approaching 200 pmol∙^-11∙mg^-1 Ww [3], compared to 153±19 pmol∙s^-1∙mg^-1 Ww in competitive racing horses [4].

• O2k-Network Lab: AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E, CA Montreal Bergdahl A, SE Stockholm Boushel RC, US OK Stillwater Davis MS, AT Innsbruck MitoCom

Labels: MiParea: Respiration, Instruments;methods, Comparative MiP;environmental MiP, Exercise physiology;nutrition;life style

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

Regulation: Cyt c Coupling state: 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. Event: Oral

Affiliation

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; 5-D Swarovski Research Lab, Dep Visceral Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. – [email protected]

Figures

Figure 1. Coupling/substrate control diagrams. Coupling states: LEAK, L; OXPHOS, P; ETS or electron transfer system capacity, E. Substrate states differ in SUIT1 and SUIT2. Octanoylcarnitine, Oct 0.2 mM; malate, M 0.5 mM (OctM: FAO); pyruvate; P 5 mM; glutamate, G 10 mM (PGM: CI); succinate, S 10 mM (CI&II); rotenone, Rot 0.5 µM (CII); residual oxygen consumption, ROX with malonate, 5 mM, and antimycin A, 2.5 µM.

References and Acknowledgement

Laner V, Boushel RC, Hamilton KL, Miller BF, Williamson KK, Davis MS, Gnaiger E (2014) Cytochrome c flux control factor as a quality criterion in respiratory OXPHOS analysis in canine permeabilized fibres. Mitochondr Physiol Network 19.13:63-4.
Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres 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.

Supported by K-Regio project MitoFit.

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 fibres 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.

@@ Line 1: / Line 1: @@
 {{Abstract
-|title=Cytochrome c flux control factor as a quality criterion in respiratory OXPHOS analysis in canine permeabilized fibres.
+|title=Comparative mitochondrial physiology: OXPHOS and ETS capacity in permeabilized fibres of canine superathletes.
 |authors=Laner V, Boushel RC, Hamilton KL, Miller BF, Williamson KK, Davis MS, Gnaiger E
 |year=2015
 |event=MiPschool Greenville 2015
-|abstract=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 ''FCF<sub>c</sub>'', 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 ''FCF<sub>c</sub>'' applied as a quantitative exclusion criterion in permeabilized fibres obtained from healthy muscle tissue.
+|abstract=Comparative mitochondrial physiology strongly relies on quantitative data sets for comparison of OXPHOS capacities and respiratory control patterns between species and tissues. Combination and interpretation of a wide variety of studies requires standardization of respiratory protocols, implementation of quality control criteria, and consistency of normalization. Previously, we described a reference method for the application of a cytochrome c threshold as exclusion criterion in mitochondrial OXPHOS analyses [1]. Alaskan sled dogs (N=6) were studied 72 to 120 h after finishing a competitive 1,000 mile race within less than nine days. Permeabilized fibres (0.81-1.28 mg ± 0.12 SD wet weight per assay) were prepared from needle biopsies and immediately studied by high-resolution respirometry [2] using 12 chambers in parallel (OROBOROS Oxygraph-2k). Compared to human skeletal muscle fibres, the canine samples were more delicate to handle, highly sticky and appeared to be fragile, disintegrating to various degrees during substrate-uncoupler-inhibitor titration (SUIT) protocols in mt-respiration medium MiR06Cr. Two substrate-uncoupler-inhibitor titration protocols were applied (Fig. 1). SUIT1 emphasized substrate control with fatty acid oxidation (FAO) versus carbohydrate oxidation capacity, whereas the focus of SUIT2 was on coupling control with CI-linked substrates. Both protocols were designed to provide a common reference state of CI&II-linked ETS capacity, in comparison to separate Complex I- and Complex II-linked substrate states (CI versus CII).
-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 ± 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 texturally 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 ETS capacity with increasing ''FCF<sub>c</sub>'' (Figure 1). Apparently, the damage caused by mt-preparation even in cases with ''FCF<sub>c</sub>'' up to 0.25 could be rescued by addition of 10 µM ''c'' and thus restore capacities comparable with samples of negligible ''FCF<sub>c</sub>''. In contrast, multiple defects associated with increasing ''FCF<sub>c</sub>'' 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).
+CI&II-linked ETS capacity was 262±41 pmol∙s<sup>-1</sup>∙mg<sup>-1</sup> Ww independent of the presence or absence of 0.2 mM octanoyl carnitine (FAO). This is the highest value so far reported for mammalian skeletal muscle. Top human endurance athletes have a CI&II-linked ETS capacity approaching 200 pmol∙<sup>-1</sup>1∙mg<sup>-1</sup> Ww [3], compared to 153±19 pmol∙s<sup>-1</sup>∙mg<sup>-1</sup> Ww in competitive racing horses [4].
-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 ''FCF<sub>c</sub>'' threshold as a potential exclusion criterion in healthy controls.
 |mipnetlab=AT Innsbruck OROBOROS, AT Innsbruck Gnaiger E, CA Montreal Bergdahl A, SE Stockholm Boushel RC, US OK Stillwater Davis MS, AT Innsbruck MitoCom
 }}
 {{Labeling
-|area=Respiration, Exercise physiology;nutrition;life style
+|area=Respiration, Instruments;methods, Comparative MiP;environmental MiP, Exercise physiology;nutrition;life style
 |organism=Dog
 |tissues=Skeletal muscle
@@ Line 19: / Line 17: @@
 |couplingstates=OXPHOS, ETS
 |substratestates=CI, CII, ETF, CI&II
-|instruments=Oxygraph-2k
+|instruments=Oxygraph-2k, Protocol
 |event=Oral
 }}
 == Affiliation ==
--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 protected]
+-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; 5-D Swarovski Research Lab, Dep Visceral Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. – [email protected]
 == Figures ==
-[[Image:MiP2014_Laner_Figure.jpg|left|400px]] Figure 1. Independence of O2 flux (ETS capacity in the presence of cytochrome ''c'') of the cytochrome ''c'' control factor,
+[[Image:MiPschool2015Greenville Laner Figure.jpg|left|500px]] Figure 1. Coupling/substrate control diagrams. Coupling states: LEAK, L; OXPHOS, P; ETS or electron transfer system capacity, E. Substrate states differ in '''SUIT1''' and  '''SUIT2'''. Octanoylcarnitine, Oct 0.2 mM; malate, M 0.5 mM (OctM: FAO); pyruvate; P 5 mM;  glutamate, G 10 mM (PGM: CI); succinate, S 10 mM (CI&II); rotenone, Rot 0.5 µM (CII); residual oxygen consumption, ROX with malonate, 5 mM, and antimycin A, 2.5 µM.
-''FCF<sub>c</sub>'' = (''J''<sub>CHO''c''</sub>-''J''<sub>CHO</sub>)/''J''<sub>CHO''c''</sub>
-ETS capacity was 238±64 pmol∙s<sup>-1</sup>∙mg<sup>-1</sup> ''W''<sub>w</sub> 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 Acknowledgement ==
+#Laner V, Boushel RC, Hamilton KL, Miller BF, Williamson KK, Davis MS, Gnaiger E (2014) Cytochrome c flux control factor as a quality criterion in respiratory OXPHOS analysis in canine permeabilized fibres. Mitochondr Physiol Network 19.13:63-4.
+#Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres 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.
+Supported by K-Regio project MitoFit.
 == References and acknowledgements ==
@@ Line 53: / Line 42: @@
 # 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.
-A preliminary version of this abstract was presented at MiP2014.