Reliable determination of training‐induced alterations in muscle fiber composition in human skeletal muscle using quantitative polymerase chain reaction

Abstract

Determination of muscle fiber composition in human skeletal muscle biopsies is often performed using immunohistochemistry, a method that tends to be both time consuming, technically challenging, and complicated by limited availability of tissue. Here, we introduce quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)-based Gene-family profiling (GeneFam) of myosin heavy chain (MyHC) mRNA expression as a high-throughput, sensitive, and reliable alternative. We show that GeneFam and immunohistochemistry result in similar disclosures of alterations in muscle fiber composition in biopsies from musculus vastus lateralis and musculus biceps brachii of previously untrained young women after 12 weeks of progressive strength training. The adaptations were evident as (a) consistent increases in MyHC2A abundance; (b) consistent decreases in MyHC2X abundance; and (c) consistently stable MyHC1 abundance, and were not found using traditional reference gene-based qRT-PCR analyses. Furthermore, muscle fiber composition found using each of the two approaches was correlated with each other (r = 0.50, 0.74, and 0.78 for MyHC1, A, and X, respectively), suggesting that GeneFam may be suitable for ranking of individual muscle phenotype, particularly for MyHC2 fibers. In summary, GeneFam of MyHC mRNA resulted in reliable assessment of alterations in muscle fiber composition in skeletal muscle of previously untrained women after 12 weeks of strength training.

Irisin and FNDC5: effects of 12-week strength training, and relations to muscle phenotype and body mass composition in untrained women

“…The efficacy of the training program with respect to muscle fiber conversion was comparable to those seen in other studies (Staron et al 1994;Adams et al 1993), evident as stable MyHC1 abundances and increased MyHC2A abundances, as discussed in Ellefsen et al (2014). No effect of training was observed on whole-body FM (Table 1), with individual responses varying from -14 % to +15 %.…”

“…The latter was recently shown to be accompanied by increased abundances of MyHC2A (Ellefsen et al 2014). Furthermore, we found FNDC5 expression to be closely correlated with proportions of aerobic muscle fibers in untrained (pre) but not in trained (post) muscle, suggesting that strength training has an intricate effect on irisin biology.…”

“…This approach provides a normalization-free alternative to reference gene-based normalization by treating individual genes as constituting fractions of collective Gene-family expression, thereby increasing the physiological relevance of mRNA data. The approach seems particularly well-suited for MyHC mRNA-based fiber-typing in skeletal muscle tissue (Ellefsen and Stensløkken 2010), for which it was recently validated by our group (Ellefsen et al 2014). Notably, this validation study was performed on the very same biopsy material that were analysed in the current study.…”

“…Formalin-fixed muscle biopsies were paraffin-embedded and sectioned, whereupon transverse, serial sections were labelled for MyHC1 (A4.840), MyHC2A (EPR5280) and MyHC2X (6H1), as previously described (Ellefsen et al 2014). For images showing representative labeling, see Fig.…”

“…Unfortunately, of the eighteen subject for which we had complete sets of biopsies for RNA-analysis from VL and BB (pre and post), we only had sixteen complete sets of biopsies for immunohistochemical analysis. Of these, we were only able to retrieve complete sets of MyHC-labeled serial tissue sections (1+2A+2X) for eleven and twelve subjects in VL and BB, respectively, as reported in Ellefsen et al (Ellefsen et al 2014). This was due to an issue with the performance of the 6H1-antibody, possibly linked to the combined fragility of the antibody and high levels of intramuscular fatty acids, distorting tissue preparation.…”