Assessing mitochondrial respiration in permeabilized fibres and biomarkers for mitochondrial content in human skeletal muscle.

Jujiao Kuang, Nicholas J Saner, Javier Botella, Matthew J-C Lee, Cesare Granata, Zhenhuan Wang, Xu Yan, Jia Li, Amanda J Genders, David J Bishop

Acta physiologica (Oxford, England) 2022 Feb

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Assessments of mitochondrial respiration and mitochondrial content are common in skeletal muscle research and exercise science. However, many sources of technical and biological variation render these analyses susceptible to error. This study aimed to better quantify the reliability of different experimental designs and/or techniques so as to assist researchers to obtain more reliable data. We examined the repeatability of maximal mitochondrial oxidative phosphorylation in permeabilized muscle fibres via high-resolution respirometry, and citrate synthase activity (a biomarker for mitochondrial content) in a microplate with spectrophotometery. For mitochondrial respiration using permeabilized skeletal muscle fibres, the variability was reduced using three chambers and removing outliers compared to two chambers (CV reduced from 12.7% to 11.0%), and the minimal change that can be detected with 10 participants reduced from 17% to 13% according to modelling. For citrate synthase activity, the within-plate CV (3.5%) increased when the assay was repeated after 4 hours (CV = 10.2%) and 4 weeks (CV = 30.5%). The readings were correlated, but significantly different after 4 hours and 4 weeks. This research provides evidence for important technical considerations when measuring mitochondrial respiration and content using citrate synthase activity as a biomarker. When assessing mitochondrial respiration in human skeletal muscle, the technical variability of high-resolution respirometry can be reduced by increasing technical repeats and excluding outliers, practices which are not currently common. When analysing citrate synthase activity, our results highlight the importance of analysing all samples from the same study at the same time. © 2022 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Citation

Jujiao Kuang, Nicholas J Saner, Javier Botella, Matthew J-C Lee, Cesare Granata, Zhenhuan Wang, Xu Yan, Jia Li, Amanda J Genders, David J Bishop. Assessing mitochondrial respiration in permeabilized fibres and biomarkers for mitochondrial content in human skeletal muscle. Acta physiologica (Oxford, England). 2022 Feb;234(2):e13772