Analysis of skeletal muscle cross sections is an important experimental technique in muscle biology. Many aspects of immunohistochemistry and fluorescence microscopy can now be automated, but most image quantification techniques still require extensive human input, slowing progress and introducing the possibility of user bias. MyoVision is a new software package that was developed to overcome these limitations. The software improves upon previously reported automatic techniques and analyzes images without requiring significant human input and correction. When compared with data derived by manual quantification, MyoVision achieves an accuracy of ≥94% for basic measurements such as fiber number, fiber type distribution, fiber cross-sectional area, and myonuclear number. Scientists can download the software free from www.MyoVision.org and use it to automate the analysis of their own experimental data. This will improve the efficiency and consistency of the analysis of muscle cross sections and help to reduce the burden of routine image quantification in muscle biology. NEW & NOTEWORTHY Scientists currently analyze images of immunofluorescently labeled skeletal muscle using time-consuming techniques that require sustained human supervision. As well as being inefficient, these techniques can increase variability in studies that quantify morphological adaptations of skeletal muscle at the cellular level. MyoVision is new software that overcomes these limitations by performing high-content analysis of muscle cross sections with minimal manual input. It is open source and freely available.

BackgroundPax7+ satellite cells are required for skeletal muscle fiber growth during post-natal development in mice. Satellite cell-mediated myonuclear accretion also appears to persist into early adulthood. Given the important role of satellite cells during muscle development, we hypothesized that the necessity of satellite cells for adaptation to an imposed hypertrophic stimulus depends on maturational age.MethodsPax7CreER-R26RDTA mice were treated for 5 days with vehicle (satellite cell-replete, SC+) or tamoxifen (satellite cell-depleted, SC-) at 2 months (young) and 4 months (mature) of age. Following a 2-week washout, mice were subjected to sham surgery or 10 day synergist ablation overload of the plantaris (n = 6–9 per group). The surgical approach minimized regeneration, de novo fiber formation, and fiber splitting while promoting muscle fiber growth. Satellite cell density (Pax7+ cells/fiber), embryonic myosin heavy chain expression (eMyHC), and muscle fiber cross sectional area (CSA) were evaluated via immunohistochemistry. Myonuclei (myonuclei/100 mm) were counted on isolated single muscle fibers.ResultsTamoxifen treatment depleted satellite cells by ≥90% and prevented myonuclear accretion with overload in young and mature mice (p < 0.05). Satellite cells did not recover in SC- mice after overload. Average muscle fiber CSA increased ~20% in young SC+ (p = 0.07), mature SC+ (p < 0.05), and mature SC- mice (p < 0.05). In contrast, muscle fiber hypertrophy was prevented in young SC- mice. Muscle fiber number increased only in mature mice after overload (p < 0.05), and eMyHC expression was variable, specifically in mature SC+ mice.ConclusionsReliance on satellite cells for overload-induced hypertrophy is dependent on maturational age, and global responses to overload differ in young versus mature mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-017-0132-z) contains supplementary material, which is available to authorized users.

Myonuclei gained during exercise-induced skeletal muscle hypertrophy may be long-lasting and could facilitate future muscle adaptability after deconditioning, a concept colloquially termed “muscle memory.” The evidence for this is limited, mostly due to the lack of a murine exercise-training paradigm that is nonsurgical and reversible. To address this limitation, we developed a novel progressive weighted-wheel-running (PoWeR) model of murine exercise training to test whether myonuclei gained during exercise persist after detraining. We hypothesized that myonuclei acquired during training-induced hypertrophy would remain following loss of muscle mass with detraining. Singly housed female C57BL/6J mice performed 8 wk of PoWeR, while another group performed 8 wk of PoWeR followed by 12 wk of detraining. Age-matched sedentary cage-dwelling mice served as untrained controls. Eight weeks of PoWeR yielded significant plantaris muscle fiber hypertrophy, a shift to a more oxidative phenotype, and greater myonuclear density than untrained mice. After 12 wk of detraining, the plantaris muscle returned to an untrained phenotype with fewer myonuclei. A finding of fewer myonuclei simultaneously with plantaris deconditioning argues against a muscle memory mechanism mediated by elevated myonuclear density in primarily fast-twitch muscle. PoWeR is a novel, practical, and easy-to-deploy approach for eliciting robust hypertrophy in mice, and our findings can inform future research on the mechanisms underlying skeletal muscle adaptive potential and muscle memory.

Background In the context of mass regulation, 'muscle memory' can be defined as long-lasting cellular adaptations to hypertrophic exercise training that persist during detraining-induced atrophy and may facilitate future adaptation. The cellular basis of muscle memory is not clearly defined but may be related to myonuclear number and/or epigenetic changes within muscle fibres. Methods Utilizing progressive weighted wheel running (PoWeR), a novel murine exercise training model, we explored myonuclear dynamics and skeletal muscle miRNA levels with training and detraining utilizing immunohistochemistry, single fibre myonuclear analysis, and quantitative analysis of miRNAs. We also used a genetically inducible mouse model of fluorescent myonuclear labelling to study myonuclear adaptations early during exercise. Results In the soleus, oxidative type 2a fibres were larger after 2 months of PoWeR (P ¼ 0.02), but muscle fibre size and myonuclear number did not return to untrained levels after 6 months of detraining. Soleus type 1 fibres were not larger after PoWeR but had significantly more myonuclei, as well as central nuclei (P < 0.0001), the latter from satellite cell-derived or resident myonuclei, appearing early during training and remaining with detraining. In the gastrocnemius muscle, oxidative type 2a fibres of the deep region were larger and contained more myonuclei after PoWeR (P < 0.003), both of which returned to untrained levels after detraining. In the gastrocnemius and plantaris, two muscles where myonuclear number was comparable with untrained levels after 6 months of detraining, myonuclei were significantly elongated with detraining (P < 0.0001). In the gastrocnemius, miR-1 was lower with training and remained lower after detraining (P < 0.002). Conclusions This study found that (i) myonuclei gained during hypertrophy are lost with detraining across muscles, even in oxidative fibres; (ii) complete reversal of muscle adaptations, including myonuclear number, to untrained levels occurs within 6 months in the plantaris and gastrocnemius; (iii) the murine soleus is resistant to detraining; (iv) myonuclear accretion occurs early with wheel running and can be uncoupled from muscle fibre hypertrophy; (v) resident (non-satellite cell-derived) myonuclei can adopt a central location; (vi) myonuclei change shape with training and detraining; and (vii) miR-1 levels may reflect a memory of previous adaptation that facilitates future growth.

Recent loss-of-function studies show that satellite cell depletion does not promote sarcopenia or unloading-induced atrophy, and does not prevent regrowth. Although overload-induced muscle fiber hypertrophy is normally associated with satellite cell-mediated myonuclear accretion, hypertrophic adaptation proceeds in the absence of satellite cells in fully grown adult mice, but not in young growing mice. Emerging evidence also indicates that satellite cells play an important role in remodeling the extracellular matrix during hypertrophy.

Over the last 30+ years, it has become axiomatic that performing aerobic exercise within the same training program as resistance exercise (termed concurrent exercise training) interferes with the hypertrophic adaptations associated with resistance exercise training. However, a close examination of the literature reveals that the interference effect of concurrent exercise training on muscle growth in humans is not as compelling as previously thought. Moreover, recent studies show that, under certain conditions, concurrent exercise may augment resistance exercise-induced hypertrophy in healthy human skeletal muscle. The purpose of this article is to outline the contrary evidence for an acute and chronic interference effect of concurrent exercise on skeletal muscle growth in humans and provide practical literature-based recommendations for maximizing hypertrophy when training concurrently.

Satellite cell-mediated myonuclear accretion is thought to be required for skeletal muscle fiber hypertrophy, and even drive hypertrophy by preceding growth. Recent studies in humans and rodents provide evidence that challenge this axiom. Specifically, Type 2 muscle fibers reliably demonstrate a substantial capacity to hypertrophy in the absence of myonuclear accretion, challenging the notion of a tightly regulated myonuclear domain (i.e., area that each myonucleus transcriptionally governs). In fact, a “myonuclear domain ceiling”, or upper limit of transcriptional output per nucleus to support hypertrophy, has yet to be identified. Satellite cells respond to muscle damage, and also play an important role in extracellular matrix remodeling during loading-induced hypertrophy. We postulate that robust satellite cell activation and proliferation in response to mechanical loading is largely for these purposes. Future work will aim to elucidate the mechanisms by which Type 2 fibers can hypertrophy without additional myonuclei, the extent to which Type 1 fibers can grow without myonuclear accretion, and whether a true myonuclear domain ceiling exists.

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