|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.|
Abstract: Version 1 (v1) 2019-07-04 doi:10.26124/mitofit:ea19.MiPSchool.0009
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 ) reveals the requirement of using hyperoxic incubation conditions to avoid oxygen limitation of respiratory capacity . 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 , (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  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 .
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
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
Preprints, MitoEAGLE, Vastus lateralis, Blebbistatin
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)
- Oroboros Instruments, Innsbruck, Austria - [email protected]
- Dept Physiological Sciences, Univ Barcelona and Bellvitge Biomedical Research Inst, Spain
- Translational Research Inst Metabolism Diabetes, Florida Hospital, Orlando, FL, USA
- Inst Clinical Diabetology, German Diabetes Center, Leibniz Center Diabetes Research Heinrich-Heine Univ Düsseldorf
- German Center Diabetes Research, Munich, Neuherberg; Germany
- Dept Biomedical Sciences, Center Healthy Aging, Fac Health Sciences, 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
- This preprint is based upon work from COST Action CA15203 MitoEAGLE, supported by COST (European Cooperation in Science and Technology).
Figure 1. Substrate-uncoupler-inhibitor titration protocol (SUIT-008 O2 pfi D014). 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: 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).
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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