Methodological considerations when assessing mitochondrial respiration and biomarkers for mitochondrial content in human skeletal muscle

Abstract

Background: The assessment of mitochondrial respiration and mitochondrial content are two common measurements in the fields of skeletal muscle research and exercise science. However, to verify the validity of the observed changes in both mitochondrial respiration and mitochondrial content following an intervention such as exercise training, it is important to determine the reliability and reproducibility of the experimental design and/or techniques employed. We examined the repeatability of widely used methodologies for assessing mitochondrial respiration and mitochondrial content, respectively; the measurement of maximal mitochondrial oxidative phosphorylation in permeabilized muscle fibres using high-resolution respirometry, and the measurement of citrate synthase activity as a biomarker for mitochondrial content in a microplate with spectrophotometer. Result: For mitochondrial respiration, the coefficient of variation for repeated measurements using muscle sampled from same biopsy decreased from 12.7% to 11% when measured in triplicate with outliers excluded, rather than in duplicate. The coefficient of variation was 9.7% for repeated muscle biopsies sampled across two separated days. For measurements of citrate synthase activity, the coefficient of variation was 3.5% of three technical repeats on the same plate, 10.2% for duplicate analyses using the same muscle lysate when conducted in the same day, and 30.5% when conducted four weeks apart. Conclusion: We have provided evidence for important technical considerations when measuring mitochondrial respiration with human skeletal muscle: 1) the relatively large technical variability can be reduced by increasing technical repeats and excluding outliers; 2) the biological variability and absolute mitochondrial respiration value of the participants should be considered when estimating the required sample size; 3) a new threshold of 15% for the increase in respiration rate after the addition of cytochrome c test for testing mitochondrial outer membrane integrity. When analysing citrate synthase activity, our evidence suggests it is important to consider the following: 1) all samples from the same study should be homogenized and measured at the same time using the same batch of freshly made chemical reagents; 2) biological variability should be considered when detecting small change in mitochondrial content; 3) the relative change should be used to compare the outcomes from different studies.

Short-term step reduction reduces citrate synthase activity without altering skeletal muscle markers of oxidative metabolism or insulin-mediated signaling in young males

“…The average enzyme activity across three replicates was taken forward for analysis. The within-plate coefficient variation of the three technical replicates was 3.51 ± 2.51% and within the assay’s acceptable range, as previously reported ( 39 ).…”

Acute resistance exercise training does not augment mitochondrial remodelling in master athletes or untrained older adults

“…CS activity was calculated using the extinction coefficient (CS ε = 13.6). Measurement CV% within-plate was 6.6 ± 1.7% for three technical repeats ( Kuang et al, 2021 ).…”

No effect of five days of bed rest or short‐term resistance exercise prehabilitation on markers of skeletal muscle mitochondrial content and dynamics in older adults

“…CS activity was calculated using the extinction coefficient (CS ε = 13.6). Measurement CV% within‐plate was 7.01 ± 0.9% for three technical repeats (Kuang et al, 2021).…”