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Difference between revisions of "Doerrier 2019 MitoFit Preprint Arch EA"

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{{Publication
{{Publication
|title=Doerrier C, Gama-Perez P, Distefano G, Pesta D, Soendergaard SD, Chroeis KM, Gonzalez-Franquesa A, Goodpaster BH, Coen P, Larsen S, Gnaiger E, Garcia-Roves PM (2019) Inter-laboratory harmonization of respiratory protocols in permeabilized human muscle fibers. MitoFit Preprint Arch  [[doi:10.26124/mitofit:ea19.MiPSchool.0009]].
|title=Doerrier C, Gama-Perez P, Distefano G, Pesta D, Soendergaard SD, Chroeis KM, Gonzalez-Franquesa A, Goodpaster BH, Coen P, Larsen S, Gnaiger E, Garcia-Roves PM (2019) Inter-laboratory harmonization of respiratory protocols in permeabilized human muscle fibers. MitoFit Preprint Arch  [[doi:10.26124/mitofit:ea19.MiPSchool.0009]].
|info=[[File:MitoFit Preprint Arch pdf.png|left|160px|link= |MitoFit pdf]]  <big><big>'''[https:pdf Inter-laboratory harmonization of respiratory protocols in permeabilized human muscle fibers]'''</big></big>
|info=[[File:MitoFit Preprint Arch pdf.png|left|160px|link=https://www.mitoeagle.org/images/7/7c/Doerrier_2019_MitoFit_Preprint_Arch_doi_10.26214mitofitea19.MiPSchool.0009.pdf |MitoFit pdf]]  <big><big>'''[https://www.mitoeagle.org/images/7/7c/Doerrier_2019_MitoFit_Preprint_Arch_doi_10.26214mitofitea19.MiPSchool.0009.pdf Inter-laboratory harmonization of respiratory protocols in permeabilized human muscle fibers]'''</big></big>
|authors=Doerrier C, Gama-Perez P, Distefano G, Pesta D, Soendergaard SD, Chroeis KM, Gonzalez-Franquesa A, Goodpaster BH, Coen P, Larsen S, Gnaiger E, Garcia-Roves PM
|authors=Doerrier C, Gama-Perez P, Distefano G, Pesta D, Soendergaard SD, Chroeis KM, Gonzalez-Franquesa A, Goodpaster BH, Coen P, Larsen S, Gnaiger E, Garcia-Roves PM
|year=2019
|year=2019
|journal=MitoFit Preprint Arch
|journal=MitoFit Preprint Arch
|abstract=Version 1 ('''v1''') '''2019-07-04''' [http:.pdf doi:10.26124/mitofit:ea19.MiPSchool.0009]
|abstract=Version 1 ('''v1''') '''2019-07-04''' [https://www.mitoeagle.org/images/7/7c/Doerrier_2019_MitoFit_Preprint_Arch_doi_10.26214mitofitea19.MiPSchool.0009.pdf doi:10.26124/mitofit:ea19.MiPSchool.0009]
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoeagle.org/index.php/MitoEAGLE|COST Action MitoEAGLE]]
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoeagle.org/index.php/MitoEAGLE|COST Action MitoEAGLE]]
Permeabilized muscle fibers have been extensively used for the analysis of mitochondrial function in pathophysiology. Inter- and intra-laboratory comparisons of published studies in permeabilized muscle fibers are very difficult because different experimental procedures (e.g., sample preparation, substrate-uncoupler-inhibitor titration protocols, respiratory media, and oxygen regimes) have been employed. Oxygen dependence of mitochondrial respiration in permeabilized fibers (about 100-fold higher compared to small intact cells and isolated mitochondria) reveals the requirement of using hyperoxic incubation (oxygen levels between 400-250 µM) for respirometric studies [1]. However, different research groups have shown discrepancy in the oxygen dependence of permeabilized muscle fibers by using different experimental conditions (respiratory media and the presence/absence of the myosin II-specific inhibitor blebbistatin) [2-5].  
Permeabilized muscle fibers have been extensively used for the analysis of mitochondrial function in pathophysiology. Inter- and intra-laboratory comparisons of published studies in permeabilized muscle fibers are very difficult because different experimental procedures (e.g., sample preparation, substrate-uncoupler-inhibitor titration protocols, respiratory media, and oxygen regimes) have been employed. Oxygen dependence of mitochondrial respiration in permeabilized fibers (about 100-fold higher compared to small intact cells and isolated mitochondria) reveals the requirement of using hyperoxic incubation (oxygen levels between 400-250 µM) for respirometric studies [1]. However, different research groups have shown discrepancy in the oxygen dependence of permeabilized muscle fibers by using different experimental conditions (respiratory media and the presence/absence of the myosin II-specific inhibitor blebbistatin) [2-5].  

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Doerrier 2019 MitoFit Preprint Arch EA

Publications in the MiPMap
Doerrier C, Gama-Perez P, Distefano G, Pesta D, Soendergaard SD, Chroeis KM, Gonzalez-Franquesa A, Goodpaster BH, Coen P, Larsen S, Gnaiger E, Garcia-Roves PM (2019) Inter-laboratory harmonization of respiratory protocols in permeabilized human muscle fibers. MitoFit Preprint Arch doi:10.26124/mitofit:ea19.MiPSchool.0009.

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MitoFit pdf

Inter-laboratory harmonization of respiratory protocols in permeabilized human muscle fibers

Doerrier C, Gama-Perez P, Distefano G, Pesta D, Soendergaard SD, Chroeis KM, Gonzalez-Franquesa A, Goodpaster BH, Coen P, Larsen S, Gnaiger E, Garcia-Roves PM (2019) MitoFit Preprint Arch

Abstract: Version 1 (v1) 2019-07-04 doi:10.26124/mitofit:ea19.MiPSchool.0009

COST Action MitoEAGLE

Permeabilized muscle fibers have been extensively used for the analysis of mitochondrial function in pathophysiology. Inter- and intra-laboratory comparisons of published studies in permeabilized muscle fibers are very difficult because different experimental procedures (e.g., sample preparation, substrate-uncoupler-inhibitor titration protocols, respiratory media, and oxygen regimes) have been employed. Oxygen dependence of mitochondrial respiration in permeabilized fibers (about 100-fold higher compared to small intact cells and isolated mitochondria) reveals the requirement of using hyperoxic incubation (oxygen levels between 400-250 µM) for respirometric studies [1]. However, different research groups have shown discrepancy in the oxygen dependence of permeabilized muscle fibers by using different experimental conditions (respiratory media and the presence/absence of the myosin II-specific inhibitor blebbistatin) [2-5].

In the framework of the MitoEAGLE COST Action project, our main goals for the current study are: (1) address the lack of harmonization between research laboratories, (2) stablish the best experimental conditions, and (3) evaluate source(s) of experimental variability in studies with human permeabilized muscle fibers. For this purpose, we performed an inter-laboratory blinded test in human permeabilized fibers. Briefly, six groups from different countries (Austria, Denmark, Germany, Spain, and USA) measured simultaneously (at the same laboratory) mitochondrial respiration by high-resolution respirometry (Oroboros Instruments, Austria) in N=3 human biopsies (vastus lateralis) from the same volunteer. A total of 96 (32/day) permeabilized fiber preparations were included in this study. The follow conditions were compared in parallel: (1) fiber preparation; (2) respiration media MiR05-Kit and Buffer Z in the presence of blebbistatin (25 µM) versus carrier; (3) normoxia (200-100 µM) versus hyperoxia (450-250 µM). The substrate-uncoupler-inhibitor titration protocol applied in the present study is shown in the figure 1 [6].

Our preliminary data show similar results between the two main experimental conditions used in the literature (MiR05 hyperoxia without blebbistatin versus Buffer Z normoxia in the presence of blebbistatin) in NADH-OXPHOS capacity (Fig 2A). However, our results reflect differences (e.g. NADH&succinate-OXPHOS capacity) which must be evaluated carefully in order to identify the specific source(s) of these variations (Fig 2B). Our first analysis indicates that oxygen dependence seems to be the critical aspect triggering differences in both experimental buffers (Fig 2C, 2D). We ambition that this inter-laboratory study provides a robust experimental design with careful statistical data to harmonize results in permeabilized muscle fibers and to address the reproducibility crisis [7]. - Extended abstract

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

Template NextGen-O2k.jpg

Affiliations

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

  1. Oroboros Instruments, Innsbruck, Austria - [email protected]
  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, Denmark
  7. The Novo Nordisk Center Basic Metabolic Research, Section Integrative Physiology; Univ Copenhagen, Denmark
  8. D Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Med Univ Innsbruck, Austria

Results

Figures

Doerrier Figure1.jpg

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









Doerrier Figure2.jpg

Figure 2: Comparison of the main respiratory media and experimental conditions used for mitochondrial respiration studies in permeabilized fibers. Scatter dot plots illustrate the oxygen flux per mass in OXPHOS-state with (A) NADH-linked substrates (pyruvate and malate supporting N-pathway) and (B) NADH&succinate-linked substrates (pyruvate, malate, glutamate and succinate supporting NS-pathway) in human permeabilized fibers in MiR05-Kit at hyperoxic conditions (400-250 µM O2) in the absence of blebbistatin (black dot plots), and Buffer Z at normoxic conditions (200-100 µM O2) in the presence of 25 µM blebbistatin (green dot plots). Oxygen flux per mass in OXPHOS-state in NS-pathway at different oxygen regimes in Buffer Z in (C) the presence and (D) absence of blebbistatin (light green dot plots). Data are represented as the median with interquartile range (n=8 permeabilized fiber preparations per condition from N=3 human biopsies).










References

  1. Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25.
  2. Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS (2008) Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3:965-76.
  3. Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543:39-55.
  4. 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.
  5. 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. Abstract MitoFit Science Camp 2016.
  6. SUIT-008_O2_pfi_D014
  7. Baker M (2016) 1,500 scientists lift the lid on reproducibility. Survey sheds light on the ‘crisis’ rocking research. Nature 533:452–4.


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, O2k-Protocol