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Difference between revisions of "Sumbalova 2011 Abstract Bordeaux"

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(Created page with "{{Publication |title=Sumbalova Z, Harrison DK, Gradl P, Fasching M, Gnaiger E (2011) Mitochondrial membrane potential, coupling control, H<sub>2</sub>O<sub>2</sub> production, an...")
 
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{{Publication
{{Publication
|title=Sumbalova Z, Harrison DK, Gradl P, Fasching M, Gnaiger E (2011) Mitochondrial membrane potential, coupling control, H<sub>2</sub>O<sub>2</sub> production, and the upper limit of mitochondrial performance. Abstract Kagoshima.
|title=Sumbalova Z, Wiethüchter A, Fasching M, Gnaiger E (2011) Coupling control and substrate control of mitochondrial membrane potential and respiration in the mouse brain, and comparison with skeletal and cardiac muscle. Abstract Bordeaux.
|authors=Sumbalova Z, Harrison DK, Gradl P, Fasching M, Gnaiger E
|authors=Sumbalova Z, Wiethüchter A, Fasching M, Gnaiger E
|year=2011
|year=2011
|journal=Abstract
|journal=Abstract
|abstract=Electron gating through either Complex I (CI) or CII exerts an experimental limitation on OXPHOS capacity in mitochondrial preparations, artificially alters the production of reactive oxygen species (ROS), and restricts the driving force for generating the mitochondrial (mt) membrane potential. We applied physiological substrate cocktails to reconstitute tricarboxylic acid cycle function in mouse brain mitochondria to (i) support convergent CI+II-linked electron input into the [[Q-junction]] ([[Gnaiger 2009 IJBCB]]), (ii) quantify maximum capacities of oxidative phosphorylation ([[OXPHOS]]) and of the electron transfer system ([[ETS]]), and (iii) monitor simultaneously oxygen consumption (''J''<sub>O2</sub>) and mt-membrane potential (ΔΨ), and (iv) ''J''<sub>O2</sub> and hydrogen peroxide production (''J''<sub>H2O2</sub>). An inverse relationship between Δ''Ψ'' and ''J''<sub>O2</sub> and direct relation between Δ''Ψ'' and ''J''<sub>H2O2</sub> is well established when stimulating respiration by ADP and uncoupling. Applying CI- and/or CII-linked substrates, Δ''Ψ'' dropped by 20-25 mV as flux was increased by coupling control from the resting LEAK state to OXPHOS capacity (State 3), and JH2O2 decreased. Dissipation of Δ''Ψ'' by uncoupling (FCCP) was accompanied by a further stimulation of flux in the non-coupled ETS state (CI or CI+II substrates), comparable to human muscle mitochondria ([[Boushel_2007_Diabetologia]]; [[Pesta_2011_AJP]]). Opposite to this coupling paradigm of an inverse Δ''Ψ''/''J''<sub>O2</sub> relationship, both Δ''Ψ'' and ''J''<sub>O2</sub> increased significantly when the upper limit of OXPHOS capacity was obtained with convergent CI+II electron input (pyruvate +malate +glutamate +succinate). Despite the higher membrane potential supported by the CI+II substrate cocktail compared to CI-linked substrates, H<sub>2</sub>O<sub>2</sub> production remained unchanged in the active OXPHOS state of respiration, but CI+II electron supply increased JH2O2 further in the passive LEAK state of respiration. The upper limit of respiratory capacity and the scope of ROS signalling, therefore, are significantly higher under conditions of physiological substrate supply compared with conventional minimal substrate combinations (Contribution to ''[[MitoCom Tyrol]]'').
|abstract=Physiological substrate cocktails are required to reconstitute tricarboxylic acid cycle (TCA) function in mitochondrial (mt) preparations, to quantify maximum capacities of oxidative phosphorylation ([[OXPHOS]], ''P'') and of the electron transfer system ([[ETS]], ''E''). Functional differences between mitochondria from different tissues and species are largely masked when restricting flux artificially by applying either Complex I (CI) or Complex II (CII) linked substrates which do not support convergent CI+II-linked electron input into the [[Q-junction]] [1]. We applied and validated different protocols with substrate-uncoupler-inhibitor titrations ([[SUIT]]), monitoring simultaneously mt-membrane potential (TPP<sup>+</sup>) and respiration.
 
High-resolution respirometry was combined with an ion selective electrode system (TPP<sup>+</sup>; OROBOROS Oxygraph-2k MultiSensor system; [[MiR06]] at 37 °C) to measure respiration, ''J''<sub>O2</sub>, and mt-membrane potential, Δ''Ψ'', in three mouse brain preparations: isolated mitochondria, homogenate after 3 min centrifugation at 1300 g, and crude tissue homogenate. Coupling control and substrate control states [2] were established sequentially in SUIT protocols, and respiratory flux control patterns were compared between mouse brain and permeabilized fibres from skeletal muscle (gastrocnemius) and heart.
 
''J''<sub>O2</sub> in the [[LEAK}} state (''L''; no ADP) with CI-linked substrates (pyruvate +malate +glutamate) represented only 0.05 and 0.07 of OXPHOS capacity (saturating [ADP]) in the brain and skeletal muscle ([[RCR]]= 22 and 14), but 0.29 in heart (RCR=3.4). OXPHOS capacity with CI-linked substrates constituted only 0.77 of physiological OXPHOS capacity (CI+II substrate cocktail, pyruvate +malate +glutamate +succinate) in brain, compared to a CI/CI+II flux ratio of 0.87 and 0.68 in skeletal muscle and heart. OXPHOS capacity was strongly limited by the phosphorylation system in the brain, as revealed by the increase of ADP-stimulated respiration by uncoupling, with a corresponding ''[[P/E]]'' flux control ratio of 0.77. In contrast, the ''P/E'' ratio was 0.94 (close to the maximum value of 1.0) in skeletal muscle, despite of the similar ''[[L/P]]'' coupling control ratio, and 0.92 in heart.  Our results are in accordance with the investigation of Rossignol et al. [3] on tissue variation in the control of OXPHOS. While respiration is controlled mainly by the ETS in the heart and skeletal muscle, the phosphorylation system (ANT, ATP synthase and phosphate carrier) exerts significant control over respiration in the brain.
 
In mt-preparations of brain, Δ''Ψ'' dropped by 20-25 mV as flux was increased by coupling control from the resting LEAK state to OXPHOS capacity, and dissipation of Δ''Ψ'' by uncoupling (FCCP) was accompanied by a further stimulation of flux in the non-coupled state ''E''. Opposite to this coupling paradigm of an inverse Δ''Ψ''/''J''<sub>O2</sub> relationship, both Δ''Ψ'' and ''J''<sub>O2</sub> increased when flux was varied by substrate control. Under these conditions, Δ''Ψ'' increased with an increase of flux. The shift of ΔΨ was 4-6 mV both in the OXPHOS and LEAK state. The higher membrane potential supported by the CI+II substrate cocktail compared to CI-linked substrates requires re-investigations of ROS production and challenges the paradigm [4] that mitochondrial ROS plays a minor role during exercise and in active states of ATP turnover.
Our results challenge the simplistic State 3/State 4 paradigm of mitochondrial respiratory coupling control and inverse regulation of Δ''Ψ''. Substrate control is complementary to coupling control of mitochondrial respiration, as emphasized in the ingenious work of Chance and Williams [5].
 
Supported by FEMtech (NMVIT, Austria). Contribution to ''[[MitoCom Tyrol]]''.
 
1. Gnaiger E ed (2007) Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications, Innsbruck: 96 pp. - www.oroboros.at/index.php?mipnet-publications
 
2. [[Gnaiger_2009_IJBCB|Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int. J. Biochem. Cell Biol. 41: 1837–1845.]]
 
3. Rossignol R et al. (2000) Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases. Biochem. J. 347: 45-53.
 
4. St-Pierre J, Buckingham JA, Roebuck SJ, Brand MD (2002) Topology of superoxide production from different sites in the mitochondrial electron transport chain. J. Biol. Chem. 277: 44784–44790.
 
5. Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. III. The steady state. J. Biol. Chem. 217: 409-427.
 
|keywords=High-resolution respirometry, OXPHOS, mitochondrial membrane potential, ROS production, brain mitochondria, O2k-Fluorimeter
|keywords=High-resolution respirometry, OXPHOS, mitochondrial membrane potential, ROS production, brain mitochondria, O2k-Fluorimeter
|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS
|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS
}}
}}
{{Labeling
{{Labeling
|instruments=Oxygraph-2k, TPP, Spectrofluorimetry
|instruments=Oxygraph-2k, TPP
|injuries=RONS; Oxidative Stress
|organism=Mouse
|organism=Mouse
|tissues=Neurons; Brain
|tissues=Cardiac Muscle, Skeletal Muscle, Neurons; Brain
|preparations=Permeabilized Cell or Tissue; Homogenate
|preparations=Permeabilized Cell or Tissue; Homogenate
|topics=Respiration; OXPHOS; ETS Capacity
|topics=Respiration; OXPHOS; ETS Capacity
}}
}}

Revision as of 15:37, 23 September 2011

{{Publication |title=Sumbalova Z, Wiethüchter A, Fasching M, Gnaiger E (2011) Coupling control and substrate control of mitochondrial membrane potential and respiration in the mouse brain, and comparison with skeletal and cardiac muscle. Abstract Bordeaux. |authors=Sumbalova Z, Wiethüchter A, Fasching M, Gnaiger E |year=2011 |journal=Abstract |abstract=Physiological substrate cocktails are required to reconstitute tricarboxylic acid cycle (TCA) function in mitochondrial (mt) preparations, to quantify maximum capacities of oxidative phosphorylation (OXPHOS, P) and of the electron transfer system (ETS, E). Functional differences between mitochondria from different tissues and species are largely masked when restricting flux artificially by applying either Complex I (CI) or Complex II (CII) linked substrates which do not support convergent CI+II-linked electron input into the Q-junction [1]. We applied and validated different protocols with substrate-uncoupler-inhibitor titrations (SUIT), monitoring simultaneously mt-membrane potential (TPP+) and respiration.

High-resolution respirometry was combined with an ion selective electrode system (TPP+; OROBOROS Oxygraph-2k MultiSensor system; MiR06 at 37 °C) to measure respiration, JO2, and mt-membrane potential, ΔΨ, in three mouse brain preparations: isolated mitochondria, homogenate after 3 min centrifugation at 1300 g, and crude tissue homogenate. Coupling control and substrate control states [2] were established sequentially in SUIT protocols, and respiratory flux control patterns were compared between mouse brain and permeabilized fibres from skeletal muscle (gastrocnemius) and heart.

JO2 in the [[LEAK}} state (L; no ADP) with CI-linked substrates (pyruvate +malate +glutamate) represented only 0.05 and 0.07 of OXPHOS capacity (saturating [ADP]) in the brain and skeletal muscle (RCR= 22 and 14), but 0.29 in heart (RCR=3.4). OXPHOS capacity with CI-linked substrates constituted only 0.77 of physiological OXPHOS capacity (CI+II substrate cocktail, pyruvate +malate +glutamate +succinate) in brain, compared to a CI/CI+II flux ratio of 0.87 and 0.68 in skeletal muscle and heart. OXPHOS capacity was strongly limited by the phosphorylation system in the brain, as revealed by the increase of ADP-stimulated respiration by uncoupling, with a corresponding P/E flux control ratio of 0.77. In contrast, the P/E ratio was 0.94 (close to the maximum value of 1.0) in skeletal muscle, despite of the similar L/P coupling control ratio, and 0.92 in heart. Our results are in accordance with the investigation of Rossignol et al. [3] on tissue variation in the control of OXPHOS. While respiration is controlled mainly by the ETS in the heart and skeletal muscle, the phosphorylation system (ANT, ATP synthase and phosphate carrier) exerts significant control over respiration in the brain.

In mt-preparations of brain, ΔΨ dropped by 20-25 mV as flux was increased by coupling control from the resting LEAK state to OXPHOS capacity, and dissipation of ΔΨ by uncoupling (FCCP) was accompanied by a further stimulation of flux in the non-coupled state E. Opposite to this coupling paradigm of an inverse ΔΨ/JO2 relationship, both ΔΨ and JO2 increased when flux was varied by substrate control. Under these conditions, ΔΨ increased with an increase of flux. The shift of ΔΨ was 4-6 mV both in the OXPHOS and LEAK state. The higher membrane potential supported by the CI+II substrate cocktail compared to CI-linked substrates requires re-investigations of ROS production and challenges the paradigm [4] that mitochondrial ROS plays a minor role during exercise and in active states of ATP turnover. Our results challenge the simplistic State 3/State 4 paradigm of mitochondrial respiratory coupling control and inverse regulation of ΔΨ. Substrate control is complementary to coupling control of mitochondrial respiration, as emphasized in the ingenious work of Chance and Williams [5].

Supported by FEMtech (NMVIT, Austria). Contribution to MitoCom Tyrol.

1. Gnaiger E ed (2007) Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications, Innsbruck: 96 pp. - www.oroboros.at/index.php?mipnet-publications

2. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int. J. Biochem. Cell Biol. 41: 1837–1845.

3. Rossignol R et al. (2000) Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases. Biochem. J. 347: 45-53.

4. St-Pierre J, Buckingham JA, Roebuck SJ, Brand MD (2002) Topology of superoxide production from different sites in the mitochondrial electron transport chain. J. Biol. Chem. 277: 44784–44790.

5. Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. III. The steady state. J. Biol. Chem. 217: 409-427.

|keywords=High-resolution respirometry, OXPHOS, mitochondrial membrane potential, ROS production, brain mitochondria, O2k-Fluorimeter |mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS }}

Labels:


Organism: Mouse  Tissue;cell: Cardiac Muscle"Cardiac Muscle" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property., Skeletal Muscle"Skeletal Muscle" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property., Neurons; Brain"Neurons; Brain" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property.  Preparation: Permeabilized Cell or Tissue; Homogenate"Permeabilized Cell or Tissue; Homogenate" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

Regulation: Respiration; OXPHOS; ETS Capacity"Respiration; OXPHOS; ETS Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property. 


HRR: Oxygraph-2k, TPP 


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