Difference between revisions of "MiPNet12.01 Suppl T-issue"

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MiPNet12.01 Suppl T-issue

MitoPathways at the Q-junction: mouse skeletal muscle fibers.

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Oroboros (2018-11-18) Mitochondr Physiol Network

Abstract: Oroboros (2018) MitoPathways at the Q-junction: mouse skeletal muscle fibers. Mitochondr Physiol Network 12.01(03): Suppl T-issue.

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• O2k-Network Lab: AT_Innsbruck_Oroboros

Labels: MiParea: Respiration

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

Coupling state: LEAK, ROUTINE, ET Pathway: N, S, NS, ROX HRR: Oxygraph-2k, O2k-Protocol

MitoPathways, O2k-Demo, O2k-Core

@@ Line 1: / Line 1: @@
+{{OROBOROS header page name}}
 {{Publication
-|title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols contents]] MitoPathways at the Q-junction.
-|info=[[Gnaiger 2014 MitoPathways]]
+|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] MitoPathways at the Q-junction: mouse skeletal muscle fibers.
-|authors=OROBOROS
+|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/2/2d/MiPNet12.01_Suppl_T-issue.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.01_Suppl_T-issue.pdf Versions]
-|year=2014-07
+|authors=Oroboros
+|year=2018-11-18
 |journal=Mitochondr Physiol Network
-|abstract=[[File:MiPNet12.01 SupplementaryT-issue.jpg|left|500px|Supplementary T-issue: MitoPathways at the Q-junction]] '''OROBOROS (2014) MitoPathways at the Q-junction. Mitochondr Physiol Network 12.01(02): Supplementary T-issue.'''
+|abstract= Oroboros (2018) MitoPathways at the Q-junction: mouse skeletal muscle fibers. Mitochondr Physiol Network 12.01(03): Suppl T-issue.
+{{MiPNet pdf page linking to MitoPedia}}
-[[High-resolution respirometry]] with a [[SUIT protocol]]<sup>1</sup> for [[OXPHOS]] analysis<sup>2</sup> is presented as supplementary '''''T-issue''''' ([[OROBOROS]] T-shirt).
+|mipnetlab=AT_Innsbruck_Oroboros
-[[Pyruvate]]+[[glutamate]]+[[malate]] (PGM) were used in combination to induce C<sub>I</sub>-linked [[LEAK respiration]] in permeabilized mouse skeletal muscle ([[MiPNet12.14 IOC39  |IOC39]]; Fig. O2).<sup>3,4</sup> Saturating [[ADP]] (D; 2.5 mM final concentration) stimulated respiration to the level of [[OXPHOS capacity]] (''P'' state), with a small effect of 10 µM [[cytochrome c]] (''c''), expressed as the [[Cytochrome c control factor |cytochrome ''c'' control factor]] (''FCF<sub>c</sub>''<0.5; indicating integrity of the outer mt-membrane). Addition of [[succinate]] (S) stimulated respiration by convergent e-input through the [[Q-junction]]. C<sub>I+II</sub> OXPHOS capacity was not stimulated further by [[uncoupler]] titration (U). Therefore, the capacity of the [[phosphorylation system]] matched the [[ETS capacity]] (''E'' state). At ''E=P'' the [[E-P coupling control factor |''E-P'' coupling control factor]] is zero, indicating that there is no ETS excess capacity over ''P'', in striking contrast to human skeletal and cardiac muscle mitochondria.<sup>1,5,6</sup> Inhibition of C<sub>I</sub> by [[rotenone]] (Rot) inhibited respiration to the level of C<sub>II</sub>-linked ETS capacity, which was higher than C<sub>I</sub>-linked respiratory capacity (''E=P''). C<sub>I+II</sub>-linked respiratory capacity was higher than respiration with any single e-input substrate state, indicating an additive effect at the Q-junction. However, since C<sub>I+II</sub> < C<sub>I</sub> + C<sub>II</sub>, the additive effect was incomplete, which indicates that any electron channelling through [[Respiratory complexes |supercomplexes]] to C<sub>IV</sub> was incomplete. Addition of [[azide]] (Azd) inhibited respiration to the level of [[residual oxygen consumption]] (ROX).
-|mipnetlab=AT_Innsbruck_OROBOROS
 }}
 {{Labeling
@@ Line 17: / Line 18: @@
 |tissues=Skeletal muscle
 |preparations=Permeabilized tissue
-|couplingstates=LEAK, ROUTINE, ETS
+|couplingstates=LEAK, ROUTINE, ET
-|substratestates=CI, CII, CI+II, ROX
+|pathways=N, S, NS, ROX
-|instruments=Oxygraph-2k, Protocol
+|instruments=Oxygraph-2k, O2k-Protocol
 |additional=MitoPathways, O2k-Demo, O2k-Core
 }}
-<ref> 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. [[Pesta 2012 Methods Mol Biol |»Bioblast Access]] </ref>
-<ref> Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 3rd ed. Mitochondr Physiol Network 19.12. OROBOROS MiPNet Publications, Innsbruck: 64 pp. [[Gnaiger 2014 MitoPathways |»Open Access]] </ref>
-<ref> OROBOROS IOC39. International course on high-resolution respirometry. Schroecken 13-17 April 2007. Mitochondr Physiol Network 12.14: 1-8. [[MiPNet12.14 IOC39 |»Open Access]] - O2k-Demo experiment 2007-04-14 A-03 carried out by [[Lemieux H |Hélène Lemieux]] at [[MiPNet12.14 IOC39  |IOC39]], Schröcken. </ref>
-<ref> OROBOROS (2014) Oxygraph-2k manual titrations: SUIT protocols with mitochondrial preparations. Mitochondr Physiol Network 09.12(11): 1. [[MiPNet09.12 O2k-Titrations |»Open Access]] </ref>
-<ref> Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845. [[Gnaiger 2009 Int J Biochem Cell Biol |»PMID: 19467914]] </ref>
-<ref> Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43: 1729–38. [[Lemieux 2011 Int J Biochem Cell Biol |»Bioblast Access]] </ref>
-== Limitations of the SUIT protocol ==
-=== Maximum OXPHOS and ETS capacity ===
-Evaluation of maximum respiratory capacities requires titration of further substrates activating additional [[respiratory complexes]] at the Q-junction ([[Electron-transferring flavoprotein complex |C<sub>ETF</sub>]] and [[glycerophosphate dehydrogenase complex |C<sub>GpDH</sub>]]).
-=== Malate concentration ===
-The [[malate]] concentration was 2 mM, to saturate C<sub>I</sub>-linked respiration. However, at 2 mM malate, the fumarate concentration is increased to a level which inhibits succinate dehydrogenase. Then C<sub>I+II</sub>- and C<sub>II</sub>-linked respiratory capacities are underestimated. A malate concentration of 0.5 mM, which saturates C<sub>I</sub>-linked respiration and inhibits C<sub>II</sub>-linked respiration to a lesser extent, represents and improved standard.
-»[[Talk:Malate |Optimum malate concentration in SUIT protocols]]
-=== ROX correction ===
-The fact that ROX was higher in the C<sub>I+II</sub> substrate state compared to C<sub>I</sub>-linked LEAK respiration indicates that ROX is partially controlled by the substrate state. Therefore, a single measurement of ROX cannot be applied for correction of total oxygen consumption in the different substrate states. Total respiration, therefore, represents apparent coupling states ''L''´, ''P''´ and ''E''´ (Fig. 1). ROX correction is possible in the present experiment only for C<sub>I+II</sub>- and C<sub>II</sub>-linked respiration. [[Azide]] inhibits not only C<sub>IV</sub> but other heme-based oxidases and peroxidases, and therefore may interfere with ROX beyond blocking respiratory electron transfer. Based on this argument, a combination of C<sub>II</sub>- and C<sub>III</sub>-inhibitors (malonic acid, antimycin A, myxothiazol) may yield more consistent results, although any ROS scavenged by cytochrome ''c'' may in the absence of a C<sub>IV</sub>-inhibitor result in respiratory oxygen consumption through C<sub>IV</sub>.
-== References ==
-<references/>
-:» Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]]