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.
Highlights
The Bergstrom muscle biopsy technique has been an essential tool for the direct assessment of human skeletal muscle responses to a variety of physiological perturbations (Bergstrom, 1975)
41% of microbiopsy permeabilized muscle fiber bundles (PmFB) from −BLEB disintegrated into multiple pieces, many of which were so small that they could not be removed from the oxygraph chamber for imaging (Figure 1B) whereas most PmFB in +BLEB for either biopsy could be removed after the experiments
Using video capture in separate PmFB, we observed a progressive contraction in response to ADP in −BLEB from Bergstrom (Supplementary Video Figure 1) as previously reported (Perry et al, 2011, 2012) that was apparent in PmFB from microbiopsy (Supplementary Video Figure 3)
Summary
The Bergstrom muscle biopsy technique has been an essential tool for the direct assessment of human skeletal muscle responses to a variety of physiological perturbations (Bergstrom, 1975). Microbiopsies are often perceived as less invasive than Bergstrom given they obtain smaller samples and do not require an incision on the skin and fascia for the common procedure of sampling human vastus lateralis skeletal muscle. While these arguments have not been fully validated, interest in microbiopsies has increased for the assessment of skeletal muscle metabolism (Hayot et al, 2005; Jorge et al, 2011; Krause et al, 2012; Votion et al, 2012). Permeabilized muscle fiber bundles (PmFB) are commonly used to assess mitochondrial bioenergetics in skeletal muscle. Loss of PmFB integrity could impair respiratory assessments given it has previously been established that preservation of mitochondrial structure and morphology are critical for optimizing respiratory assessments in PmFB (Veksler et al, 1987; Kuznetsov et al, 2008; Picard et al, 2011)
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