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"Version-201-20(-27-27'v1-27-27')-20-27-27'2019-2D07-2D04-27-27'-20-5Bhttps:-2F-2Fwww.mitoeagle.org-2Fimages-2F7-2F7c-2FDoerrier_2019_MitoFit_Preprint_Arch_doi_10.26214mitofitea19.MiPSchool.0009.pdf-20doi:10.26124-2Fmitofit:ea19.MiPSchool.0009-5D-0A-5B-5BImage:MITOEAGLE-2Dlogo.jpg-7Cleft-7C100px-7Clink=http:-2F-2Fwww.mitoeagle.org-2Findex.php-2FMitoEAGLE-7CCOST-20Action-20MitoEAGLE-5D-5D-0APermeabilized-20muscle-20fibers-20are-20extensively-20used-20for-20analysis-20of-20mitochondrial-20function-20in-20exercise-20and-20pathophysiological-20studies.-20Inter-2D-20and-20intra-2Dlaboratory-20comparisons-20of-20published-20results-20on-20permeabilized-20muscle-20fibers-20are-20difficult-20due-20to-20application-20of-20different-20experimental-20procedures,-20including-20sample-20preparation,-20substrate-2Duncoupler-2Dinhibitor-20titrations-20(SUIT),-20respiratory-20media,-20and-20oxygen-20regimes.-20Oxygen-20dependence-20of-20mitochondrial-20respiration-20in-20permeabilized-20fibers-20(about-20100-2Dfold-20higher-20p50-20compared-20to-20small-20living-20cells-20and-20isolated-20mitochondria-20-5B1-5D)-20reveals-20the-20requirement-20of-20using-20hyperoxic-20incubation-20conditions-20to-20avoid-20oxygen-20limitation-20of-20respiratory-20capacity-20-5B2-5D.-20However,-20controversial-20results-20on-20the-20oxygen-20dependence-20of-20permeabilized-20muscle-20fibers-20have-20been-20reported-20by-20different-20research-20groups-20using-20different-20respiration-20media-20in-20the-20presence-20or-20absence-20of-20the-20myosin-20II-2Dspecific-20inhibitor-20blebbistatin-20-5B3,4-5D.-20-0A-0AIn-20the-20framework-20of-20COST-20Action-20MitoEAGLE,-20our-20main-20goals-20for-20the-20current-20study-20of-20permeabilized-20human-20muscle-20fibers-20are:-20(1)-20a-20comparison-20of-20protocols-20used-20in-20different-20research-20laboratories,-20(2)-20harmonization-20of-20results-20to-20address-20the-20reproducibility-20crisis-20-5B5-5D,-20(3)-20evaluation-20of-20optimum-20experimental-20conditions,-20and-20(4)-20analysis-20of-20the-20causes-20of-20experimental-20variability.-0A-0AWe-20performed-20a-20blinded-20test-20with-20human-20permeabilized-20skeletal-20fibers.-20Six-20groups-20from-20Austria,-20Denmark,-20Germany,-20Spain,-20and-20USA-20measured-20simultaneously-20in-20the-20same-20laboratory-20mitochondrial-20respiration-20using-20high-2Dresolution-20respirometry-20(O2k-3B-20Oroboros-20Instruments,-20Austria)-20in-20three-20human-20biopsies-20(vastus-20lateralis)-20from-20the-20same-20healthy-20volunteer-20sampled-20on-20three-20consecutive-20days.-20A-20total-20of-2096-20(32-2Fday)-20permeabilized-20fiber-20preparations-20were-20assayed.-20The-20wet-20mass-20of-20permeabilized-20fibers-20ranged-20from-200.38-20to-202.83-20mg-20per-20chamber.-20Protocols-20were-20compared-20at-20several-20levels:-20(1)-20permeabilized-20fiber-20preparation-3B-20(2)-20respiration-20media-20MiR05-2DKit-20and-20Buffer-20Z-20in-20the-20presence-2Fabsence-20of-20blebbistatin-20(25-20µM),-20covering-20the-20most-20frequently-20used-20experimental-20conditions-20in-20the-20literature-3B-20(3)-20‘normoxia’-20(200-2D100-20µM)-20versus-20hyperoxia-20(450-2D250-20µM).-20The-20SUIT-2D008-20protocol-20-5B6-5D-20was-20applied-20in-20all-20assays.-20Results-20were-20excluded-20from-20analysis-20if-20the-20cytochrome-20c-20flux-20control-20factor,-20-27-27FCF-3Csub-3Ec-3C-2Fsub-3E-27-27-20=-20(-27-27I-27-27-3Csub-3EO2,-27-27c-27-27PM-3C-2Fsub-3E-2D-27-27I-27-27-3Csub-3EO2,PM-3C-2Fsub-3E)-2F-27-27I-27-27-3Csub-3EO2,-27-27c-27-27PM-3C-2Fsub-3E,-20exceeded-200.1-20in-20the-20OXPHOS-2Dstate-20(Fig.-201-3B-20steps-202D-20and-202c).-20For-20abbreviations-20see-20Figure-201-20and-20Gnaiger-20et-20al-202019-20-5B7-5D.-0A-0ANS-2DOXPHOS-20capacity-20was-20oxygen-2Dlimited-20under-20‘normoxic’-20compared-20to-20hyperoxic-20conditions-20in-20both-20media-20(Figure-202A-2DD).-20Blebbistatin-20did-20not-20prevent-20the-20decrease-20of-20respiration-20in-20the-20‘normoxic’-20regime-20(Figure-202A-20and-202C),-20and-20exerted-20minor-20effects-20on-20oxygen-20flux-20in-20both-20media-20(Figure-202E-2DF).-20These-20results-20indicate-20that-20oxygen-20dependence-20is-20critical-20and-20independent-20of-20experimental-20buffers-20and-20blebbistatin-20(Figure-202A-2DD).-20Comparing-20respiratory-20capacity-20in-20both-20media-20under-20hyperoxic-20conditions,-20oxygen-20flux-20per-20mass-20was-20higher-20in-20MiR05-2DKit-20than-20in-20Buffer-20Z-20(Figure-202E-2DF).-20Evaluation-20of-20these-20trends-20will-20be-20completed-20based-20on-20an-20in-2Ddepth-20statistical-20analysis.-20Our-20inter-2Dlaboratory-20study-20provides-20a-20basis-20to-20harmonize-20published-20results-20on-20permeabilized-20human-20skeletal-20muscle-20fibers-20and-20establishes-20guidelines-20for-20selecting-20optimum-20experimental-20conditions.-20-2D-20-27-27Extended-20abstract-27-27" 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