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

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|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]
 
|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 are extensively used for analysis of mitochondrial function in exercise and pathophysiological studies. Inter- and intra-laboratory comparisons of published results on permeabilized muscle fibers are difficult due to application of different experimental procedures, including sample preparation, substrate-uncoupler-inhibitor titrations (SUIT), respiratory media, and oxygen regimes. Oxygen dependence of mitochondrial respiration in permeabilized fibers (about 100-fold higher p50 compared to small living cells and isolated mitochondria [1]) reveals the requirement of using hyperoxic incubation conditions to avoid oxygen limitation of respiratory capacity [2]. However, controversial results on the oxygen dependence of permeabilized muscle fibers have been reported by different research groups using different respiration media in the presence or absence of the myosin II-specific inhibitor blebbistatin [3,4].  
  
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].  
+
In the framework of COST Action MitoEAGLE, our main goals for the current study of permeabilized human muscle fibers are: (1) a comparison of protocols used in different research laboratories, (2) harmonization of results to address the reproducibility crisis [5], (3) evaluation of optimum experimental conditions, and (4) analysis of the causes of experimental variability.
  
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''
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We performed a blinded test with human permeabilized skeletal fibers. Six groups from Austria, Denmark, Germany, Spain, and USA measured simultaneously in the same laboratory mitochondrial respiration using high-resolution respirometry (O2k; Oroboros Instruments, Austria) in three human biopsies (vastus lateralis) from the same healthy volunteer sampled on three consecutive days. A total of 96 (32/day) permeabilized fiber preparations were assayed. The wet mass of permeabilized fibers ranged from 0.38 to 2.83 mg per chamber. Protocols were compared at several levels: (1) permeabilized fiber preparation; (2) respiration media MiR05-Kit and Buffer Z in the presence/absence of blebbistatin (25 µM), covering the most frequently used experimental conditions in the literature; (3) ‘normoxia’ (200-100 µM) versus hyperoxia (450-250 µM). The SUIT-008 protocol [6] was applied in all assays. Results were excluded from analysis if the cytochrome c flux control factor, ''FCF<sub>c</sub>'' = (''I''<sub>O2,''c''PM</sub>-''I''<sub>O2,PM</sub>)/''I''<sub>O2,''c''PM</sub>, exceeded 0.1 in the OXPHOS-state (Fig. 1; steps 2D and 2c). For abbreviations see Figure 1 and Gnaiger et al 2019 [7].
 +
 
 +
NS-OXPHOS capacity was oxygen-limited under ‘normoxic’ compared to hyperoxic conditions in both media (Figure 2A-D). Blebbistatin did not prevent the decrease of respiration in the ‘normoxic’ regime (Figure 2A and 2C), and exerted minor effects on oxygen flux in both media (Figure 2E-F). These results indicate that oxygen dependence is critical and independent of experimental buffers and blebbistatin (Figure 2A-D). Comparing respiratory capacity in both media under hyperoxic conditions, oxygen flux per mass was higher in MiR05-Kit than in Buffer Z (Figure 2E-F). Evaluation of these trends will be completed based on an in-depth statistical analysis. Our inter-laboratory study provides a basis to harmonize published results on permeabilized human skeletal muscle fibers and establishes guidelines for selecting optimum experimental conditions. - ''Extended abstract''
 
|keywords=
 
|keywords=
 
|editor=[[Gnaiger E]] & [[Beno M]] & [[Gnaiger C]]
 
|editor=[[Gnaiger E]] & [[Beno M]] & [[Gnaiger C]]
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::::# The Novo Nordisk Center Basic Metabolic Research, Section Integrative Physiology; Univ Copenhagen, Denmark
 
::::# The Novo Nordisk Center Basic Metabolic Research, Section Integrative Physiology; Univ Copenhagen, Denmark
 
::::# D Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Med Univ Innsbruck, Austria
 
::::# D Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Med Univ Innsbruck, Austria
 
== Results ==
 
  
  
 
== Figures ==
 
== Figures ==
[[File:Doerrier_Figure1.jpg|left|400 px]] 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).
+
[[File:Doerrier_Figure1.jpg|left|400 px]] '''Figure 1. Substrate-uncoupler-inhibitor titration protocol (SUIT-008 O2 pfi D014).''''''Bold text''' Sequential titrations and respiratory states. '''1PM''': NADH-pathway (N-pathway) in the presence of 5 mM pyruvate and 2 mM malate in the N-LEAK state. '''2D''''''Bold text''': saturating ADP (N-OXPHOS state). '''2c''': 10 µM cytochrome c for evaluating the integrity of the outer mitochondrial membrane. '''3G''': 10 mM glutamate as an additional NADH-linked substrate (N-OXPHOS state). '''4S''': 10 mM succinate (NS-OXPHOS capacity). '''5U''': uncoupler titrations to evaluate the electron transfer- (ET-) capacity (NS-ET capacity). '''6Rot''': inhibition of CI by rotenone (S-ET capacity). '''7Ama''': inhibition of CIII by antimycin A (residual oxygen consumption, ''Rox'').
 
 
  
  
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[[File:Doerrier_Figure2.jpg|left|400 px]] 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).
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[[File:Doerrier_Figure2.jpg|left|400 px]] '''Figure 2. The effect of oxygen concentration and blebbistatin on mitochondrial respiration of permeabilized human skeletal muscle fibers in MiR05-Kit (A, B) and Buffer Z (C, D).''' Mass-specific NS-OXPHOS capacity (based on wet mass) supported by pyruvate, malate, glutamate and succinate. ('''E, F''') Comparison of the two media at hyperoxia in the presence and absence of blebbistatin. A biopsy was taken on three consecutive days from the same person. Scatter plots and median with interquartile range show results from individual chambers (''n'' = 8 to 10) with muscle fibers obtained from the three biopsies.
  
  
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== References ==
 
== References ==
::::#Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25.
+
::::# Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25.
::::#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.
+
::::# Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543:39-55.
::::#Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543:39-55.
+
::::# 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.
::::#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.
+
::::# 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.
::::#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.
+
::::# Baker M (2016) 1,500 scientists lift the lid on reproducibility. Survey sheds light on the ‘crisis’ rocking research. Nature 533:452–4.
::::#[[SUIT-008_O2_pfi_D014]]
+
::::# [[SUIT-008_O2_pfi_D014]]
::::#Baker M (2016) 1,500 scientists lift the lid on reproducibility. Survey sheds light on the ‘crisis’ rocking research. Nature 533:452–4.
+
::::# Gnaiger E, Aasander Frostner E, Abdul Karim N, Abumrad NA, Acuna-Castroviejo D, Adiele RC, et al (2019) Mitochondrial respiratory states and rates. MitoFit Preprint Arch doi:10.26124/mitofit:190001.v4.
  
 
{{Labeling
 
{{Labeling
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|couplingstates=LEAK, OXPHOS, ET
 
|couplingstates=LEAK, OXPHOS, ET
 
|pathways=N, S, NS, ROX
 
|pathways=N, S, NS, ROX
|instruments=Oxygraph-2k, O2k-Protocol
+
|instruments=Oxygraph-2k
 
}}
 
}}

Revision as of 13:42, 5 July 2019


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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.
»
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 are extensively used for analysis of mitochondrial function in exercise and pathophysiological studies. Inter- and intra-laboratory comparisons of published results on permeabilized muscle fibers are difficult due to application of different experimental procedures, including sample preparation, substrate-uncoupler-inhibitor titrations (SUIT), respiratory media, and oxygen regimes. Oxygen dependence of mitochondrial respiration in permeabilized fibers (about 100-fold higher p50 compared to small living cells and isolated mitochondria [1]) reveals the requirement of using hyperoxic incubation conditions to avoid oxygen limitation of respiratory capacity [2]. However, controversial results on the oxygen dependence of permeabilized muscle fibers have been reported by different research groups using different respiration media in the presence or absence of the myosin II-specific inhibitor blebbistatin [3,4].

In the framework of COST Action MitoEAGLE, our main goals for the current study of permeabilized human muscle fibers are: (1) a comparison of protocols used in different research laboratories, (2) harmonization of results to address the reproducibility crisis [5], (3) evaluation of optimum experimental conditions, and (4) analysis of the causes of experimental variability.

We performed a blinded test with human permeabilized skeletal fibers. Six groups from Austria, Denmark, Germany, Spain, and USA measured simultaneously in the same laboratory mitochondrial respiration using high-resolution respirometry (O2k; Oroboros Instruments, Austria) in three human biopsies (vastus lateralis) from the same healthy volunteer sampled on three consecutive days. A total of 96 (32/day) permeabilized fiber preparations were assayed. The wet mass of permeabilized fibers ranged from 0.38 to 2.83 mg per chamber. Protocols were compared at several levels: (1) permeabilized fiber preparation; (2) respiration media MiR05-Kit and Buffer Z in the presence/absence of blebbistatin (25 µM), covering the most frequently used experimental conditions in the literature; (3) ‘normoxia’ (200-100 µM) versus hyperoxia (450-250 µM). The SUIT-008 protocol [6] was applied in all assays. Results were excluded from analysis if the cytochrome c flux control factor, FCFc = (IO2,cPM-IO2,PM)/IO2,cPM, exceeded 0.1 in the OXPHOS-state (Fig. 1; steps 2D and 2c). For abbreviations see Figure 1 and Gnaiger et al 2019 [7].

NS-OXPHOS capacity was oxygen-limited under ‘normoxic’ compared to hyperoxic conditions in both media (Figure 2A-D). Blebbistatin did not prevent the decrease of respiration in the ‘normoxic’ regime (Figure 2A and 2C), and exerted minor effects on oxygen flux in both media (Figure 2E-F). These results indicate that oxygen dependence is critical and independent of experimental buffers and blebbistatin (Figure 2A-D). Comparing respiratory capacity in both media under hyperoxic conditions, oxygen flux per mass was higher in MiR05-Kit than in Buffer Z (Figure 2E-F). Evaluation of these trends will be completed based on an in-depth statistical analysis. Our inter-laboratory study provides a basis to harmonize published results on permeabilized human skeletal muscle fibers and establishes guidelines for selecting optimum experimental conditions. - 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 - carolina.doerrier@oroboros.at
  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


Figures

Doerrier Figure1.jpg
Figure 1. Substrate-uncoupler-inhibitor titration protocol (SUIT-008 O2 pfi D014).'Bold text Sequential titrations and respiratory states. 1PM: NADH-pathway (N-pathway) in the presence of 5 mM pyruvate and 2 mM malate in the N-LEAK state. 2D'Bold text: saturating ADP (N-OXPHOS state). 2c: 10 µM cytochrome c for evaluating the integrity of the outer mitochondrial membrane. 3G: 10 mM glutamate as an additional NADH-linked substrate (N-OXPHOS state). 4S: 10 mM succinate (NS-OXPHOS capacity). 5U: uncoupler titrations to evaluate the electron transfer- (ET-) capacity (NS-ET capacity). 6Rot: inhibition of CI by rotenone (S-ET capacity). 7Ama: inhibition of CIII by antimycin A (residual oxygen consumption, Rox).








Doerrier Figure2.jpg
Figure 2. The effect of oxygen concentration and blebbistatin on mitochondrial respiration of permeabilized human skeletal muscle fibers in MiR05-Kit (A, B) and Buffer Z (C, D). Mass-specific NS-OXPHOS capacity (based on wet mass) supported by pyruvate, malate, glutamate and succinate. (E, F) Comparison of the two media at hyperoxia in the presence and absence of blebbistatin. A biopsy was taken on three consecutive days from the same person. Scatter plots and median with interquartile range show results from individual chambers (n = 8 to 10) with muscle fibers obtained from the three 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. 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. 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.
  5. Baker M (2016) 1,500 scientists lift the lid on reproducibility. Survey sheds light on the ‘crisis’ rocking research. Nature 533:452–4.
  6. SUIT-008_O2_pfi_D014
  7. Gnaiger E, Aasander Frostner E, Abdul Karim N, Abumrad NA, Acuna-Castroviejo D, Adiele RC, et al (2019) Mitochondrial respiratory states and rates. MitoFit Preprint Arch doi:10.26124/mitofit:190001.v4.


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