Doerrier 2018 MiP2018c

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Carolina Doerrier
Inter-laboratory study on human permeabilized myofibres: effects of fibre preparation, respiration buffer, and blebbistatin on mitochondrial respiratory capacity and oxygen limitation.

Link: MiP2018

Doerrier C, Garcia-Roves PM, Distefano G, Pesta D, Gama-Perez P, Soendergaard SD, Chroeis KM, Gonzalez-Franquesa A, Coen P, Goodpaster BH, Larsen S, Gnaiger E (2018)

Event: MiP2018

COST Action MitoEAGLE

For many years, muscle biopsies have been applied for investigating mitochondrial (mt) function in health and disease. Compared to isolated mitochondria (imt), permeabilized fibres (pfi) show strongly decreased affinities for ADP [1] and oxygen [2] due to diffusion restrictions. ADP kinetics of myofibre respiration is modulated by creatine [1], and oxygen diffusion limitation is overcome by inhibition of myosin-ATPase [3]. However, diverse experimental procedures (e.g., sample preparation, protocols, media, and oxygen regimes) render inter- and intra-laboratory comparisons difficult and limit data reproducibility. Controversial results on mt-respiration within and between groups may be related to the effect exerted by respiration media on the oxygen dependence of fibre respiration [2-6]. In the framework of the COST MitoEAGLE project (WG2, muscle), an inter-laboratory blinded test was performed simultaneously in the same laboratory (Copenhagen, Denmark) by six groups from Austria, Denmark, Germany, Spain, and USA. N=3 human biopsies (vastus lateralis) from the same volunteer were employed for preparing n=96 pfi preparations (32/day). mt-Respiration of pfi was evaluated by High-Resolution Respirometry (HRR) comparing in parallel (n=16 pfi) the following conditions: (1) fiber preparation; (2) respiration media MiR05-Kit and Buffer Z in the presence of the myosin II ATPase inhibitor blebbistatin (25 µM) versus carrier; (3) ‘normoxia’ (200-100 µM) versus hyperoxia (450-250 µM. NADH (N-), NADH&succinate (NS-), and succinate (S-) pathways were studied as described in the substrate-uncoupler-inhibitor titration (SUIT) protocol SUIT-008 O2 pfi D014 (Fig. 1) [7]. Results of this study will be presented during the MitoEAGLE meeting and next steps will be discussed. Inter-laboratory studies, ring tests, more robust experimental design, and care in statistical data analysis are considered as the most effective approach to address the reproducibility crisis [8].


Bioblast editor: Plangger M O2k-Network Lab: AT Innsbruck Oroboros, ES Barcelona Garcia-Roves PM, US FL Orlando Goodpaster BH, DE Duesseldorf Roden M, DK Copenhagen Dela F, AT Innsbruck Gnaiger E, DK Copenhagen Larsen S


Labels: MiParea: Respiration, Instruments;methods 


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


Coupling state: LEAK, OXPHOS, ET  Pathway: N, S, NS, ROX  HRR: Oxygraph-2k  Event: Oral  MitoEAGLE 

Affiliations

Doerrier C(1), Garcia-Roves PM(2), Distefano G(3), Pesta D(4,5), Gama-Perez P(2), Soendergaard SD(6), Chroeis KM(6), Gonzalez-Franquesa A(7), Coen P(3), Goodpaster BH(3), Larsen S(6), Gnaiger E(1,8)

  1. Oroboros Instruments, Innsbruck, Austria
  2. Dept Physiological Sciences, Univ Barcelona and Bellvitge Biomedical Research Inst, Spain
  3. Translational Research Inst Metabolism Diabetes, Florida Hospital, Orlando, FL, USA
  4. Inst Clinical Diabetology, German Diabetes Center, Leibniz Center Diabetes Research Heinrich-Heine Univ Düsseldorf
  5. German Center Diabetes Research, Munich, Neuherberg; Germany
  6. Dept Biomedical Sciences, Center Healthy Aging, Fac Health Sciences
  7. The Novo Nordisk Center Basic Metabolic Research, Section Integrative Physiology; Univ Copenhagen, Denmark
  8. Dept Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria. - carolina.doerrier@oroboros.at

Support and author contributions

Contribution to COST Action CA15203 MitoEAGLE, supported by COST (European Cooperation in Science and Technology), and K-Regio project MitoFit (CD and EG).
The study was designed and co-ordinated by DC, DG, CP, PD, GRPM, GBH, LS, and EG. Experiments were carried out by DC, GRPM, DG, GPP, PD, SSD, GFA, CKM and LS. Data analysis was performed by DC, GRPM, GPP and PD. CD and EG wrote the abstract. All authors contributed to the abstract.

Figures

Doerrier c Figure MiP2018.jpg
Figure 1. SUIT-008 O2 pfi D014 protocol. 25 µM blebbistatin or carrier was added to Buffer Z and MiR05-Kit. NADH-pathway (N-pathway) in LEAK (1PM) and OXPHOS (2D) states was studied in the presence of 5 mM glutamate and 2 mM malate (LEAK state) and saturating ADP with MgCl2 (OXPHOS state). The addition of 10 µM cytochrome c was employed for evaluation of the outer mitochondrial membrane integrity (2c). 10 mM glutamate was added as an additional N-pathway substrate in the OXPHOS state (3G). Combined NS-OXPHOS capacity was evaluated after adding 10 mM succinate (4S). ET-state was assessed after uncoupler titrations for NS-ET (5U) and S-ET capacity after inhibition of CI by rotenone (6Rot). Finally, CIII was inhibited by antimycin A to obtain residual oxygen consumption (7Ama).










References

  1. Saks VA, Belikova YO, Kuznetsov AV (1991) In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP. Biochim Biophys Acta 1074:302-11.
  2. Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543:39-55.
  3. Perry CG, Kane DA, Lin CT, Kozy R, Cathey BL, Lark DS, Kane CL, Brophy PM, Gavin TP, Anderson EJ, Neufer PD (2011) Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle. Biochem J 437:215-22.
  4. Doerrier C, Distefano G, Coen P, Goodpaster B, Gnaiger E (2017) Upper limit of OXPHOS capacity in permeabilized myofibers: effect of oxygen, ADP and blebbistatin in MiR05Cr and Buffer Z. MiP2017. - Doerrier 2017 MiP2017 WG2
  5. Larsen S (2017) The effect on respiratory capacity using different respiration buffers. MiP2017. - Larsen 2017 MiP2017 WG2
  6. Bezuidenhout N, Doerrier C, Droescher S, Ojuka E, Gnaiger E (2016) Comparison of oxygen dependence of respiration in permeabilized mouse skeletal muscle fibers in two respiration media, MiR06Cr and Buffer Z containing Ctl, Cr and Blebbistatin. MitoFit Science Camp 2016. - Bezuidenhout 2016a Abstract MitoFit Science Camp 2016
  7. Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840. - SUIT-008 O2 pfi D014
  8. Baker M (2016) 1,500 scientists lift the lid on reproducibility. Survey sheds light on the ‘crisis’ rocking research. Nature 533:452–4.