Mitochondrial Bioenergetics and Fiber Type Assessments in Microbiopsy vs. Bergstrom Percutaneous Sampling of Human Skeletal Muscle

Abstract

Microbiopsies of human skeletal muscle are increasingly adopted by physiologists for a variety of experimental assays given the reduced invasiveness of this procedure compared to the classic Bergstrom percutaneous biopsy technique. However, a recent report demonstrated lower mitochondrial respiration in saponin-permeabilized muscle fiber bundles (PmFB) prepared from microbiopsies vs. Bergstrom biopsies. We hypothesized that ADP-induced contraction (rigor) of smaller length microbiopsy PmFB causes a greater reduction in maximal respiration vs. Bergstrom, such that respiration could be increased by a myosin II ATPase-inhibitor (Blebbistatin; BLEB). Eleven males and females each received a 2 mm diameter percutaneous microbiopsy and a 5 mm diameter Bergstrom percutaneous biopsy in opposite legs. Glutamate/malate (5/0.5 mM)—supported respiration in microbiopsy PmFB was lower than Bergstrom at submaximal concentrations of ADP. 5 μM BLEB reduced this impairment such that there were no differences relative to Bergstrom ± BLEB. Surprisingly, pyruvate (5 mM)-supported respiration was not different between either biopsy technique ±BLEB, whereas BLEB increased succinate-supported respiration in Bergstrom only. H2O2 emission was lower in microbiopsy PmFB compared to Bergstrom PmFB in the presence of BLEB. Microbiopsies contained fewer type I fibers (37 vs. 47%) and more type IIX fibers (20 vs. 8%) compared to Bergstrom possibly due to sampling site depth and/or longitudinal location. These findings suggest that smaller diameter percutaneous biopsies yield lower glutamate-supported mitochondrial respiratory kinetics which is increased by preventing ADP-induced rigor with myosin inhibition. Microbiopsies of human skeletal muscle can be utilized for assessing mitochondrial respiratory kinetics in PmFB when assay conditions are supplemented with BLEB, but fiber type differences with this method should be considered.

Impairments in left ventricular mitochondrial bioenergetics precede overt cardiac dysfunction and remodelling in Duchenne muscular dystrophy

“…, ; Hughes et al . ). Each protocol was initiated with a starting [O 2 ] of approximately 350 μM and was completed before the oxygraph chamber [O 2 ] reached 150 μM as done previously (Perry et al .…”

“…Mitochondrial respiration. High-resolution O 2 consumption measurements were conducted in 2 ml of respiration medium (MiR05) using the Oroboros Oxygraph-2k (Oroboros Instruments, Corp., Innsbruck, Austria) with stirring at 750 rpm at 37°C (Perry et al 2011(Perry et al , 2013Pesta & Gnaiger, 2012;Hughes et al 2015;Ydfors et al 2016). Respiration medium contained 20 mM creatine (Cr) to saturate mitochondrial creatine kinase (mtCK) or no Cr to prevent the activation of mtCK (Anderson et al 2007;Perry et al 2011).…”

Repeatability of exercise‐induced changes in mRNA expression and technical considerations for qPCR analysis in human skeletal muscle

“…Biopsies were obtained from the lateral portion of the vastus lateralis (right leg) by percutaneous needle biopsy technique (Hughes et al., ) using a 14‐gauge Medax Biofeather micro‐biopsy disposable needle (San Possidonio (MO), Italy) while the participant lay in a supine position. The skin was punctured under local anaesthesia (2% xylocaine with adrenaline) using a 12‐gauge cannula inserted 4 cm deep to guide the biopsy needle to a final depth of 8 cm into the muscle.…”

Measuring biological aging in humans: A quest

“…In the scheme, we show only three cycles of epigenetic adaptation, at any point in time; the epigenetic landscape results from the sum of hundreds or even thousands of adaptive cycles that occur throughout life; and some more relevant than others. Importantly, very little is known about how environmental stresses are sensed and encoded into epigenetic changes the quantification of characterization of morphological changes using microscopy imaging techniques (Coggan, 1995;Hughes et al, 2015). Many of these indexes have been associated with aging and increased risk of chronic conditions (Consolini et al, 2017;Gonzalez-Freire et al, 2018).…”

“…P 31 MRS is noninvasive and reliable but is too expensive for large population studies, and this method only measures global skeletal muscle oxidative phosphorylation, which depends not only on the intrinsic mitochondrial function but also on the capacity of circulatory and microcirculatory system to deliver to mitochondria adequate amount of oxygen and oxidative substrates. Muscle biopsies are invasive but safe and allow for a variety of measurements—including direct mitochondrial respiration—as well as a wide range of biochemical assays that target different components of the energetic and biogenesis machinery, and the quantification of characterization of morphological changes using microscopy imaging techniques (Coggan, ; Hughes et al, ). Many of these indexes have been associated with aging and increased risk of chronic conditions (Consolini et al, ; Gonzalez‐Freire et al, ).…”