What about hybrids? Supplemental methods for skeletal muscle fibre typing

High-intensity exercise relies heavily on muscle glycogen metabolism, with reductions in muscle glycogen content being linked to impaired exercise tolerance. There is little known about fibre type-specific subcellular glycogen utilization during high-intensity exercise. In a recent paper in The Journal of Physiology, Vigh-Larsen et al. (2022) sought to investigate fibre type-specific utilization of subcellular muscle glycogen in the vastus lateralis muscle during high-intensity intermittent exercise inmoderate to well-trained adults. Glycogen utilization was observed for slow-twitch (type I) and fast-twitch (type II) skeletal muscle fibres in three distinct subcellular regions, the intermyofibrillar (IMF), intramyofibrillar (Intra) and subsarcolemmal (SS) regions. Glycogen in the IMF region is found closest in proximity to the sarcoplasmic reticulum and mitochondria; Intra glycogen is interspersed within the contractile filaments; and SS glycogen is the most superficial, just beneath the surface of the membrane (Nielsen & Ortenblad, 2012). Relative distribution of subcellular glycogen depends on various factors, but it is understood that 75% of the cell’s glycogen storage is found in IMF region, whereas only 5–15% is in each of the Intra and SS regions (Nielsen & Ortenblad, 2012). Previous studies have identified fibre-type differences between IMF, Intra and SS glycogen content, and the differences appear to be dependent on the training status of muscle (Nielsen & Ortenblad, 2012). In the current study, moderately to well-trained participants were used, and subcellular glycogen depletion was quantified as changes in absolute subcellular volume relative to the geometric mean and 95% confidence intervals (Vigh-Larsen et al., 2022). Glycogen utilization between fibre types showed observable differences. Type I fibres exhibited homogeneous glycogen utilization among all three subfractions, across all three exercise periods (Vigh-Larsen et al., 2022). Conversely, type II fibres were seen to have uneven breakdown of muscle glycogen, favouring utilization in IMF and Intra regions at the onset of exercise (Vigh-Larsen et al., 2022). At the whole-muscle level, type II fibres demonstrated glycogen depletion nearly twice as high, which is consistent with historical research that demonstrates an elevation of glycogen metabolism in type II fibres during short term high-intensity exercise. The overall participant fibre type distribution was reported as 54 ± 6% type I, 45 ± 5% type IIa and 5 ± 4% type IIx (Vigh-Larsen et al., 2022). However, the authors noted a limitation of this study in the lack of distinction between type IIa and IIx fibres during fibre typing analysis. The decision was made to include type IIx fibres as type IIa fibres because of the low amount of type IIx fibres found in the biopsy samples (Vigh-Larsen et al., 2022). This low distribution of type IIx fibres is consistent with current understanding of fibre type distribution in humans, though it should be noted that the methods used in the current study did not detect hybrid skeletal muscle fibres. Hybrid fibres are classified as including two or more myosin isoforms simultaneously: myosin heavy chain (MyHC) I/IIa, IIa/IIx or I/IIa/IIx (Medler, 2019). These hybrids are a common component of normal muscle, and their prevalence varies greatly, though it is observed that humans can have up to 20–40% hybrid fibres in a given muscle (Medler, 2019). The hybrid fibres become less abundant as individuals become more highly trained, with endurance training prompting greater shifts towards MyHC I fibres, and resistance training prompting greater shifts towards MyHC IIa and IIx fibres (Medler, 2019). Vigh-Larsen and colleagues (2022) have advanced the understanding of glycogen utilization differences between type I and II fibres. Moving towards greater ecological validity, the inclusion of hybrid fibre analysis in the context of subcellular glycogen utilization will be a worthwhile direction for future research. Future follow-ups of this study may want to include supplemental methods of fibre typing via MyHC analysis to make hybrid fibre assessment possible. Vigh-Larsen et al. (2022) used transmission electron microscopy (TEM) to analyse image Z-disk widths of individual fibres, then visually decided whether fibres were to be placed in the category of type I or type II. Muscle specimens were prepared for TEM at×13,500 magnification. Prior to imaging, an ultramicrotome was used to cut ∼60 nm longitudinal sections from each specimen, yielding 10–12 fibres that were later contrasted with uranyl acetate and lead citrate. In total, 24 images were produced for each fibre sample: 12 of subsarcolemmal regions and 12 along both sides of the sarcolemma. Z-disk width was measured on each image, and fibres with>4 clearmeasurements were included in typing. Three fibres with the broadest Z-disk width were defined as type I and the threemost narrow as type II, while four intermediate fibres were discarded from the study (Vigh-Larsen et al., 2022). It could be assumed that these intermediate fibres were hybrid fibres, but this distinction is likely too simplistic. Alternatively, future studies may consider use of SDS-PAGE for single muscle fibre MyHC identification as this can identify fibres across all types including hybrids. This method is considered the ‘gold standard’ for fibre typing due to its time-effectiveness and ability to minimize misinterpretation of fibre type (Pandorf et al., 2010). A single fibre is isolated from a bundle then subjected to SDS-PAGE, separating the proteins by molecular mass. Samples then go through periods of staining and rinsing before revealing the intensity of colour of the stains. Finally, MyHC protein distribution is assessed via migration distance of the stains. More recently, Christiansen et al. (2019) have introduced the dot blotting method as the most novel approach for rapid skeletal muscle fibre type determination. A small amount of fibre (1–3 mm) is prepared and exposed to antibodies of MyHC I, IIa and IIx, yielding protein