Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

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

From Bioblast
Line 1: Line 1:
{{Publication
{{Publication
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] MitoPathways at the Q-junction: mouse skeletal muscle fibres.
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] MitoPathways at the Q-junction: mouse skeletal muscle fibres.
|info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/2/2d/MiPNet12.01_Suppl_T-issue.pdf |Bioblast pdf]]
|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 Versions]
|authors=Oroboros
|authors=Oroboros
|year=2014-07-20
|year=2014-07-20

Revision as of 09:47, 11 January 2018

Publications in the MiPMap
O2k-Protocols contents
MitoPathways at the Q-junction: mouse skeletal muscle fibres.

» Bioblast pdf »Versions

Oroboros (2014-07-20) Mitochondr Physiol Network

Abstract:

Oroboros (2014) MitoPathways at the Q-junction: mouse skeletal muscle fibres. Mitochondr Physiol Network 12.01(02): Suppl T-issue. » Versions

High-resolution respirometry with a SUIT protocol1 for OXPHOS analysis2 is presented as supplementary T-issue (Oroboros T-shirt).

Pyruvate&glutamate&malate (PGM) were used in combination to induce CI-linked LEAK respiration in permeabilized mouse skeletal muscle (IOC39; Fig. O2).3,4 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 (FCFc<0.01; indicating integrity of the outer mt-membrane). Without correction for residual oxygen consumption (ROX), the biochemical coupling efficiency, (P-L)/P, was 0.68 (RCR=3.1). Addition of succinate (S) stimulated respiration by convergent e-input through the Q-junction. The corresponding succinate control factor was (CI&II-CI)/CI&II=0.47, i.e. succinate increased respiration by 47%. CI&II-linked OXPHOS capacity was not stimulated further by uncoupler titration (U). Therefore, the capacity of the phosphorylation system matched the ET-capacity (E state). At E=P the Excess E-P capacity is zero, in striking contrast to human skeletal and cardiac muscle mitochondria.1,5,6 Inhibition of CI by rotenone (Rot) inhibited respiration to the level of CII-linked ET-capacity. The corresponding CI-control factor is (CI&II-CII)/CI&II=0.25. CII- was higher than CI-linked respiratory capacity (E=P). CI&II-linked respiratory capacity was higher than respiration with any single e-input substrate state, indicating an additive effect at the Q-junction. However, since CI&II < CI + CII, the additive effect was incomplete, which indicates that any electron channelling through supercomplexes to CIV was incomplete. Addition of azide (Azd) inhibited respiration to the level of residual oxygen consumption (ROX). ROX was 0.18 of CI&II-linked ET-capacity.


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 

[1] [2] [3] [4] [5] [6]

Limitations of the SUIT protocol

Maximum OXPHOS and ET-capacity

Evaluation of maximum respiratory capacities requires titration of further substrates activating additional respiratory complexes at the Q-junction (CETF and CGpDH<).

Malate concentration

The malate concentration was 2 mM, to saturate CI-linked respiration. However, at 2 mM malate, the fumarate concentration is increased to a level which inhibits succinate dehydrogenase. Then CI&II- and CII-linked respiratory capacities are underestimated. A malate concentration of 0.5 mM, which saturates CI-linked respiration and inhibits CII-linked respiration to a lesser extent, represents and improved standard.
» Optimum malate concentration in SUIT protocols

ROX correction

The fact that ROX was higher in the CI&II substrate state compared to CI-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 CI&II- and CII-linked respiration. Azide inhibits not only CIV 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 CII- and CIII-inhibitors (malonic acid, antimycin A, myxothiazol) may yield more consistent results, although any ROS scavenged by cytochrome c may in the absence of a CIV-inhibitor result in respiratory oxygen consumption through CIV.

References

  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. »Bioblast link
  2. Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. Oroboros MiPNet Publications, Innsbruck:80 pp. »Bioblast link
  3. Oroboros IOC39. International course on high-resolution respirometry. Schroecken 13-17 April 2007. Mitochondr Physiol Network 12.14: 1-8. »Bioblast link - O2k-Demo experiment 2007-04-14 A-03 carried out by Hélène Lemieux at IOC39, Schröcken.
  4. Oroboros (2014) Oxygraph-2k manual titrations: SUIT protocols with mitochondrial preparations. Mitochondr Physiol Network 09.12(11): 1. »Bioblast link
  5. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845. »Bioblast link
  6. 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. »Bioblast link
» Product: Oroboros O2k, Oroboros O2k-Catalogue