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

Abstract

Aim: 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. Methods: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.Results: 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. Conclusion: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.

Ischaemic preconditioning blunts exercise‐induced mitochondrial dysfunction, speeds oxygen uptake kinetics but does not alter severe‐intensity exercise capacity

“…Furthermore, variability of CS activity in different samples obtained from the same muscle biopsy might also be a contributory factor. The variability of resting high‐resolution respirometry data in this study is within that previously reported in the literature (Cardinale et al., 2018 ; Kuang et al., 2022 ); nevertheless, this remains an important consideration, and caution should be exercised when interpreting the results relating to CIL. Examination of the relative contributions of CIL to P and E, demonstrated in InvRCR and LCR, therefore provides a more appropriate representation of the potential for IPC to maintain mitochondrial coupling efficiency.…”

“…Correcting to CI+IIP as a marker of mitochondrial content might then be useful to adjust for baseline differences and to reduce the influence of variability in CS activity in different samples from the same muscle biopsy (Kuang et al, 2022). These data collectively corroborate the finding of greater CIL after exercise in SHAM, but not IPC, evidenced in the more established normalizations to wet mass and CS activity, again with no difference in the baselines (P = 0.36).…”

“…The CI+IIP is demonstrated to correlate strongly with mitochondrial content, determined by fractional area ( r = 0.81, P < 0.001; Larsen et al., 2012 ) and was found to be significantly lower at baseline in SHAM compared with IPC. Correcting to CI+IIP as a marker of mitochondrial content might then be useful to adjust for baseline differences and to reduce the influence of variability in CS activity in different samples from the same muscle biopsy (Kuang et al., 2022 ). These data collectively corroborate the finding of greater CIL after exercise in SHAM, but not IPC, evidenced in the more established normalizations to wet mass and CS activity, again with no difference in the baselines ( P = 0.36).…”

Increased mitochondrial surface area and cristae density in the skeletal muscle of strength athletes

“…It has been suggested that the increases in fibre cross-sectional area (fCSA) observed following resistance training may dilute, and therefore mask, the magnitude of mitochondrial volumetric adaptations (MacDougall et al ., 1979; Lüthi et al ., 1986; Parry et al ., 2020). Therefore, given that the use of biochemical assays to interrogate mitochondrial adaptations (Larsen et al ., 2012; Kuang et al ., 2022) are unable to account for difference in fCSA, the use of transmission electron microscopy (TEM) may better reveal the distinct structural mitochondrial characteristics of resistance-trained individuals, such as mitochondrial morphology (Castro-Sepulveda et al ., 2020) and cristae density (Nielsen et al ., 2017b), that are independent of fCSA.…”

Increased mitochondrial surface area and cristae density in the skeletal muscle of strength athletes

“…It has been suggested that the increases in fibre cross‐sectional area (fCSA) observed following resistance training may dilute, and therefore mask, the magnitude of mitochondrial volumetric adaptations (Lüthi et al., 1986; MacDougall et al., 1979; Parry et al., 2020). Therefore, given that the use of biochemical assays to interrogate mitochondrial adaptations (Kuang et al., 2022; Larsen et al., 2012) are unable to account for difference in fCSA, the use of transmission electron microscopy (TEM) may better reveal the distinct structural mitochondrial characteristics of resistance‐trained individuals, such as mitochondrial morphology (Castro‐Sepulveda et al., 2020) and cristae density (Nielsen, Gejl et al., 2017), that are independent of fCSA.…”