Mitochondrial respiration variability and simulations in human skeletal muscle: The Gene SMART study

Abstract

Sarah Voisin and Nir Eynon are Co-senior authorship.Abbreviations: ATP, adenosine triphosphate; CI, complex I, electron input through CI; CV, coefficient of variation; CI P , oxidative phosphorylation (OXPHOS) capacity (P) through CI; ETS, electron transport system; CI+CII, convergent electron input through CI and CII CI+CII E , capacity (E) through CI+CII; CI+CII P , complex II (CII) linked respiration; E, ETS capacity; ETS, measurement of electron transport system; FCCP, p-trifluoromethoxyphenylhydrazone; Gene SMART, genes and skeletal muscle adaptive response to training; Inv-RCR, inverse of respiratory control ratio (CIL/CI+II P ); L, leak respiration; LCR, leak control ratio (CIL/CI+II E ); OXPHOS, oxidative phosphorylation; P, oxphos capacity; PCR, phosphorylation control ratio (CI+II P / CI+II E ); ROX, residual oxygen consumption; SCR, substrate control ratio at constant P (CIP/CI+II P ); TCA, tricarboxylic acid; TE M , technical error of measurement. AbstractMitochondrial respiration using the oxygraph-2k respirometer (Oroboros) is widely used to estimate mitochondrial capacity in human skeletal muscle. Here, we measured mitochondrial respiration variability, in a relatively large sample, and for the first time, using statistical simulations, we provide the sample size required to detect meaningful respiration changes following lifestyle intervention. Muscle biopsies were taken from healthy, young men from the Gene SMART cohort, at multiple time points. We utilized samples for each measurement with two technical repeats using two respirometer chambers (n = 160 pairs of same muscle after removal of low-quality samples). We measured the Technical Error of measurement (TE M ) and the coefficient of variation (CV) for each mitochondrial complex. There was a high correlation between measurements from the two chambers (R > 0.7 P < .001) for all complexes, but the TE M was large (7.9-27 pmol s −1 mg −1 ; complex dependent), and the CV was >15% for all complexes. We performed statistical simulations of a range of effect sizes at 80% power and found that 75 participants (with duplicate measurements) are required to detect a 6% change in mitochondrial respiration after an intervention, while for interventions with 11% effect size, ~24 participants are sufficient.The high variability in respiration suggests that the typical sample sizes in exercise studies may not be sufficient to capture exercise-induced changes. K E Y W O R D Sexercise, intervention, mitochondria, OXPHOS, oxygen consumption | 2979 JACQUES Et Al.

Contextualizing the biological relevance of standardized high‐resolution respirometry to assess mitochondrial function in permeabilized human skeletal muscle

“…The intra‐individual CV (duplicate or more measures obtained from the same skeletal muscle biopsy) for OXPHOS p is 14.8% (n = 89). This within‐participant variation agrees with previous reports of 15.2% (n = 25) 89 and 15.3% (n = 68) 90 …”

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

“…When analyzing the same muscle sample in duplicate, we identified a greater CV (12.7%) and TEM (12.8 pmol•s -1 •mg -1 or 15.4%); however, our observed variability is in a similar range to, or slightly lower than, previous studies [19,32,33]. Sahl et al…”

“…("Values Within 4× CV") vs 18.1% ("All Three Values")). Using statistical simulations [19], we then calculated that 10 and 20 samples measured in triplicate (using all values for calculation) could determine 20% and 13% difference in maximal mitochondrial respiration with 80% power, respectively. After excluding outliers ("Values Within 4×…”

“…Consequently, the use of permeabilized muscle fibres with highresolution respirometry has become the most common method to measure mitochondrial respiration in human skeletal muscle [6,17,18]. However, despite its widespread use in human exercise training studies ( [5,[19][20][21][22][23][24][25][26][27][28][29][30][31], and several studies have been conducted to assess factors that might cause technical and/or biological variation [32][33][34], uncertainty remains regarding the reliability of this method. As such, additional research is needed to establish the reliability of mitochondrial respiration measures using permeabilized muscle fibres with high-resolution respirometry.…”

The Molecular Adaptive Responses of Skeletal Muscle to High-Intensity Exercise/Training and Hypoxia

“…A separate study also showed similar findings in mitochondrial respiratory function; however, a modest but significant increase in CS activity was observed after four weeks of HIIT, suggesting an increase in mitochondrial content [42]. The failure to detect significant changes in mitochondrial function could be due to technical variations [43] or genetic factors [32,33,[44][45][46][47].…”