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Doerrier 2019 MitoFit Preprint Arch EA
Additional label Preprints  +
Coupling states LEAK  + , OXPHOS  + , ET  +
Has abstract Version 1 ('''v1''') '''2019-07-04''' [htt
Version 1 ('''v1''') '''2019-07-04''' [ doi:10.26124/mitofit:ea19.MiPSchool.0009] [[Image:MITOEAGLE-logo.jpg|left|100px|link=|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, ''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''
mental conditions. - ''Extended abstract''  +
Has editor [[Gnaiger E]] & [[Beno M]] & [[Gnaiger C]]  +
Has info [[File:MitoFit Preprint Arch pdf.png|left|
[[File:MitoFit Preprint Arch pdf.png|left|160px|link= |MitoFit pdf]] <big><big>'''[ Inter-laboratory harmonization of respiratory protocols in permeabilized human muscle fibers]'''</big></big>
muscle fibers]'''</big></big>  +
Has title Doerrier C, Gama-Perez P, Distefano G, Pes
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]].
oi:10.26124/mitofit:ea19.MiPSchool.0009]].  +
Instrument and method Oxygraph-2k  +
Mammal and model Human  +
MiP area Respiration  + , Instruments;methods  +
Pathways N  + , S  + , NS  + , ROX  +
Preparation Permeabilized tissue  +
Tissue and cell Skeletal muscle  +
Was published by MiPNetLab 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 +
Was published in journal MitoFit Preprint Arch +
Was published in year 2019  +
Was written by Doerrier C + , Gama Perez P + , Distefano G + , Pesta D + , Soendergaard SD + , Chroeis KM + , Gonzalez-Franquesa A + , Goodpaster BH + , Coen PM + , Larsen S + , Gnaiger E + , Garcia-Roves PM +
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Doerrier 2019 MitoFit Preprint Arch EA  +
Categories MitoFit , Publications , Category:NextGen-O2k
Modification date
"Modification date" is a predefined property that corresponds to the date of the last modification of a subject and is provided by Semantic MediaWiki.
13:34:03, 11 October 2019  +
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