Harmonization of experimental procedures to assess mitochondrial respiration in human permeabilized skeletal muscle fibers.

Pablo M Garcia-Roves, Carolina Doerrier, Pau Gama-Perez, Giovanna Distefano, Steen Larsen, Clara Prats, Marta Sales-Pardo, Alba Gonzalez-Franquesa, Dominik Pesta, Erich Gnaiger, Paul M Coen, Bret H Goodpaster, Stine D Soendergaard, Karoline Maise Chroeis, Roger Guimera

Journal: Free radical biology & medicine 2024;223():384-397

PMID: 39097206

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Abstract

AIM

High-resolution respirometry in human permeabilized muscle fibers is extensively used for analysis of mitochondrial adaptions to nutrition and exercise interventions, and is linked to athletic performance. However, the lack of standardization of experimental conditions limits quantitative inter- and intra-laboratory comparisons.

METHODS

In our study, an international team of investigators measured mitochondrial respiration of permeabilized muscle fibers obtained from three biopsies (vastus lateralis) from the same healthy volunteer to avoid inter-individual variability. High-resolution respirometry assays were performed together at the same laboratory to assess whether the heterogenity in published results are due to the effects of respiration media (MiR05 versus Z) with or without the myosin inhibitor blebbistatin at low- and high-oxygen regimes.

RESULTS

Our findings reveal significant differences between respiration media for OXPHOS and ETcapacities supported by NADH&succinate-linked substrates at different oxygen concentrations. Respiratory capacities were approximately 1.5-fold higher in MiR05 at high-oxygen regimes compared to medium Z near air saturation. The presence or absence of blebbistatin in human permeabilized muscle fiber preparations was without effect on oxygen flux.

CONCLUSION

Our study constitutes a basis to harmonize and establish optimum experimental conditions for respirometric studies of permeabilized human skeletal muscle fibers to improve reproducibility.

Address: Oroboros Instruments, Schöpfstrasse 18, 6020, Innsbruck, Austria. Electronic address: [email protected].; Dept Physiological Sciences, Univ Barcelona and Bellvitge Biomedical Research Inst, Spain. Electronic address: [email protected].; Inst Clinical Diabetology, German Diabetes Center, Leibniz Center Diabetes Research Heinrich-Heine Univ Düsseldorf, Germany; German Center Diabetes Research, Munich, Neuherberg, Germany; Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany; Center for Endocrinology, Diabetes and Preventive Medicine (CEDP), University Hospital Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany. Electronic address: [email protected].; Translational Research Institute AdventHealth, Orlando, FL, USA. Electronic address: [email protected].; Xlab, Dept Biomedical Sciences, Center Healthy Aging, Fac Health Sciences, Denmark. Electronic address: [email protected].; Xlab, Dept Biomedical Sciences, Center Healthy Aging, Fac Health Sciences, Denmark. Electronic address: [email protected].; The Novo Nordisk Center Basic Metabolic Research, Section Integrative Physiology, Univ Copenhagen, Denmark. Electronic address: [email protected].; Translational Research Institute AdventHealth, Orlando, FL, USA. Electronic address: [email protected].; Dept Biomedical Sciences, Center Healthy Aging, Fac Health Sciences, Denmark; The Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address: [email protected].; Dept of Chemical Engineering, Universitat Rovira I Virgili, Tarragona, Spain. Electronic address: [email protected].; Dept of Chemical Engineering, Universitat Rovira I Virgili, Tarragona, Spain; Institució Catalana de Recerca I Estudis Avançats (ICREA), Barcelona, Spain. Electronic address: [email protected].; Translational Research Institute AdventHealth, Orlando, FL, USA. Electronic address: [email protected].; Oroboros Instruments, Schöpfstrasse 18, 6020, Innsbruck, Austria. Electronic address: [email protected].; Xlab, Dept Biomedical Sciences, Center Healthy Aging, Fac Health Sciences, Denmark; Clinical Research Centre, Medical University of Bialystok, Bialystok, Poland. Electronic address: [email protected].; Dept Physiological Sciences, Univ Barcelona and Bellvitge Biomedical Research Inst, Spain. Electronic address: [email protected].

DOI: 10.1016/j.freeradbiomed.2024.07.039

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MeSH Terms: Humans, Muscle Fibers, Skeletal, Cell Respiration, Mitochondria, Muscle, Oxygen Consumption, Oxidative Phosphorylation, Male, Oxygen, Adult, Heterocyclic Compounds, 4 or More Rings, Healthy Volunteers