Reliability of maximal mitochondrial oxidative phosphorylation in permeabilized fibers from thevastus lateralisemploying high-resolution respirometry

Abstract

The purpose was to assess the impact of various factors on methodological errors associated with measurement of maximal oxidative phosphorylation (OXPHOS) in human skeletal muscle determined by high‐resolution respirometry in saponin‐permeabilized fibers. Biopsies were collected from 25 men to assess differences in OXPHOS between two muscle bundles and to assess the correlation between OXPHOS and the wet weight of the muscle bundle. Biopsies from left and right thighs of another five subjects were collected on two occasions to compare limbs and time‐points. A single muscle specimen was used to assess effects of the anesthetic carbocaine and the influence of technician. The difference in OXPHOS between two fiber‐bundles from the same biopsy exhibited a standard error of measurement (SEM) of 10.5 pmol · s−1 · mg−1 and a coefficient of variation (CV) of 15.2%. The differences between left and right thighs and between two different time‐points had SEMs of 9.4 and 15.2 pmol · s−1 · mg−1 and CVs of 23.9% and 33.1%, respectively. The average (±SD) values obtained by two technicians monitoring different bundles of fibers from the same biopsy were 31.3 ± 7.1 and 26.3 ± 8.1 pmol · s−1 · mg−1. The time that elapsed after collection of the biopsy (up to a least 5 h in preservation medium), wet weight of the bundle (from 0.5 to 4.5 mg) and presence of an anesthetic did not influence OXPHOS. The major source of variation in OXPHOS measurements is the sample preparation. The thigh involved, time‐point of collection, size of fiber bundles, and time that elapsed after biopsy had minor or no effect.

Muscle mass and inspired oxygen influence oxygen extraction at maximal exercise: Role of mitochondrial oxygen affinity

“…Permeabilized fibre preparation was conducted as published elsewhere . Briefly, a second portion of the muscle biopsy (~5 mg wet weight) was immediately transferred into ice‐cold relaxing medium (BIOPS) containing 10 mmol L −1 Ca 2+ /EGTA buffer, 20 mmol·L −1 imidazole, 50 mmol·L −1 K + ‐4‐morpholinoethanesulfonic acid (Mes), 0.5 mmol·L −1 dithiothreitol, 6.56 mmol·L −1 MgCl 2 , 5.77 mmol·L −1 ATP and 15 mmol·L −1 phosphocreatine at pH 7.1.…”

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

“…17 Mitochondrial respiration values, as well as improvements in mitochondrial respiration following exercise training are also quite variable between studies. 3,10,14,16,18,19 For example, Irving et al 18 observed a ~1.5-fold change in mitochondrial respiration after moderate intensity endurance training (n = 34), while Robach et al 20 did not observe any change after a similar intervention (n = 17). Mitochondrial respiration capacity in human exercise intervention studies is not exclusive to skeletal muscle samples.…”

“…2 Mitochondrial respiration measured by maximal oxygen consumption in skeletal muscle fibers, is currently the primary way of assessing mitochondrial capacity. 3,4 The most common method in human skeletal muscle uses fibers permeabilized by saponin. 5,6 Using a combination of different substrates, this technique is able to mimic the processes (ie, TCA cycle and ETS) occurring within the mitochondrion.…”

“…17 In the only study to date investigating the reliability of mitochondrial respiration measurement, 3 the TE M between two fiber bundles from the same biopsy was 10.5 pmol s −1 mg −1 in the maximal oxidative phosphorylation (OXPHOS) and the coefficient of variations (CVs) were worryingly high (15.2% between two fiber bundles, 23.9% between legs, and 33.1% at different time points). 3 More studies are required to confirm whether such variability is consistently high, but, more importantly, this technical variability needs to be put into perspective with typical mitochondrial respiration changes following interventions (eg, exercise training interventions). The qualifiers "high" and "low" variability only make sense when compared with the magnitude of the intervention-induced changes, which will determine how likely we are to detect those changes.…”

High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle

“…When isolated mitochondria are evaluated, large amounts of tissue are needed [13][14][15][16]. The preparation of permeabilised muscle fibres is manually demanding and results can be operator-dependent [17,18].…”