Kevin A. Murach

Methodological issues limit interpretation of negative effects of satellite cell depletion on adult muscle hypertrophy

We are writing regarding the recent publication in Development entitled ‘Satellite cell depletion prevents fiber hypertrophy in skeletal muscle’ ([Egner et al., 2016][1]). Egner et al. claim to have ‘essentially repeated’ our work ([McCarthy et al., 2011][2]); however, we think that

Cycle training modulates satellite cell and transcriptional responses to a bout of resistance exercise

This investigation evaluated whether moderate‐intensity cycle ergometer training affects satellite cell and molecular responses to acute maximal concentric/eccentric resistance exercise in middle‐aged women. Baseline and 72 h postresistance exercise vastus lateralis biopsies were obtained from seven healthy middle‐aged women (56 ± 5 years, BMI 26 ± 1, VO2max 27 ± 4) before and after 12 weeks of cycle training. Myosin heavy chain (MyHC) I‐ and II‐associated satellite cell density and cross‐sectional area was determined via immunohistochemistry. Expression of 93 genes representative of the muscle‐remodeling environment was also measured via NanoString. Overall fiber size increased ~20% with cycle training (P = 0.052). MyHC I satellite cell density increased 29% in response to acute resistance exercise before endurance training and 50% with endurance training (P < 0.05). Following endurance training, MyHC I satellite cell density decreased by 13% in response to acute resistance exercise (acute resistance × training interaction, P < 0.05). Genes with an interaction effect tracked with satellite cell behavior, increasing in the untrained state and decreasing in the endurance trained state in response to resistance exercise. Similar satellite cell and gene expression response patterns indicate coordinated regulation of the muscle environment to promote adaptation. Moderate‐intensity endurance cycle training modulates the response to acute resistance exercise, potentially conditioning the muscle for more intense concentric/eccentric activity. These results suggest that cycle training is an effective endurance exercise modality for promoting growth in middle‐aged women, who are susceptible to muscle mass loss with progressing age.

Starring or Supporting Role? Satellite Cells and Skeletal Muscle Fiber Size Regulation

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.

MicroRNAs, heart failure, and aging: potential interactions with skeletal muscle

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting mRNAs for degradation or translational repression. MiRNAs can be expressed tissue specifically and are altered in response to various physiological conditions. It has recently been shown that miRNAs are released into the circulation, potentially for the purpose of communicating with distant tissues. This manuscript discusses miRNA alterations in cardiac muscle and the circulation during heart failure, a prevalent and costly public health issue. A potential mechanism for how skeletal muscle maladaptations during heart failure could be mediated by myocardium-derived miRNAs released to the circulation is presented. An overview of miRNA alterations in skeletal muscle during the ubiquitous process of aging and perspectives on miRNA interactions during heart failure are also provided.

Depletion of Pax7+ satellite cells does not affect diaphragm adaptations to running in young or aged mice

Satellite cell depletion does not affect diaphragm adaptations to voluntary wheel running in young or aged mice. Satellite cell depletion early in life (4 months of age) has minimal effect on diaphragm phenotype by old age (24 months). Prolonged satellite cell depletion in the diaphragm does not result in excessive extracellular matrix accumulation, in contrast to what has been reported in hind limb muscles. Up‐regulation of Pax3 mRNA+ cells after satellite cell depletion in young and aged mice suggests that Pax3+ cells may compensate for a loss of Pax7+ satellite cells in the diaphragm. Future investigations should focus on the role of Pax3+ cells in the diaphragm during adaptation to exercise and ageing.

MyoVision: software for automated high-content analysis of skeletal muscle immunohistochemistry

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.

Myonuclear Domain Flexibility Challenges Rigid Assumptions on Satellite Cell Contribution to Skeletal Muscle Fiber Hypertrophy

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.

A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression

BackgroundThe tetracycline-responsive system (Tet-ON/OFF) has proven to be a valuable tool for manipulating gene expression in an inducible, temporal, and tissue-specific manner. The purpose of this study was to create and characterize a new transgenic mouse strain utilizing the human skeletal muscle α-actin (HSA) promoter to drive skeletal muscle-specific expression of the reverse tetracycline transactivator (rtTA) gene which we have designated as the HSA-rtTA mouse.MethodsTo confirm the HSA-rtTA mouse was capable of driving skeletal muscle-specific expression, we crossed the HSA-rtTA mouse with the tetracycline-responsive histone H2B-green fluorescent protein (H2B-GFP) transgenic mouse in order to label myonuclei.ResultsReverse transcription-PCR confirmed skeletal muscle-specific expression of rtTA mRNA, while single-fiber analysis showed highly effective GFP labeling of myonuclei in both fast- and slow-twitch skeletal muscles. Pax7 immunohistochemistry of skeletal muscle cross-sections revealed no appreciable GFP expression in satellite cells.ConclusionsThe HSA-rtTA transgenic mouse allows for robust, specific, and inducible gene expression across muscles of different fiber types. The HSA-rtTA mouse provides a powerful tool to manipulate gene expression in skeletal muscle.

Delineating the effects of aerobic training versus aerobic capacity on satellite cell behaviour in humans

Since the initial characterization of muscle stem cells (satellite cells) in 1961, significant time and energy has been dedicated to uncovering their function in skeletal muscle. Over 50 years later, researchers are still elucidating the role(s) satellite cells play in exercise adaptation, ageing, health and disease. Satellite cells are indisputably necessary for regeneration (i.e. successful recovery from injury), but their requirement as myonuclear donors during adult skeletal muscle hypertrophy has been challenged using an inducible satellite cell knockout mouse (McCarthy et al. 2011). Depletion of satellite cells prior to endurance training in adult mice does not affect fibre type transitioning or muscle aerobic adaptations, but does affect coordination and running performance by disrupting intrafusal fibre (i.e. muscle spindle) morphology and activity (Jackson et al. 2015). Satellite cells play a homeostatic role by contributing to muscle fibres (Keefe et al. 2015) and regulating fibrosis (Fry et al. 2015) throughout the lifespan in sedentary mice, but do not appear to mediate sarcopenia with ageing (Fry et al. 2015; Keefe et al. 2015). Transgenic mouse models that permit post-developmental manipulation of satellite cells in conjunction with rodent muscle training techniques provide invaluable information on satellite cell biology and behaviour, but the question of translatability to humans is always pertinent. To that point, a multitude of human research indicates that satellite cell activity and density increases profoundly in response to varying environmental stimuli (e.g. exercise), strongly signifying an active role in the muscle adaptive process. Most recently, how satellite cells interact with secreted factors as well as their influence on muscle fibre type-specific adaptations to exercise (e.g. slow versus fast twitch) has emerged as an area of inquiry in humans. In a recent issue of The Journal of Physiology, Hoedt and colleagues (Hoedt et al. 2016) sought to manipulate satellite cell behaviour in the vastus lateralis of healthy young men via two distinct but seemingly complementary strategies: chronic administration of a substance known to enhance aerobic exercise performance and a 10 week high-intensity interval (80–100% aerobic capacity) and continuous (70% aerobic capacity) cycling exercise training programme. The authors hypothesized that supplementation with an erythropoiesis-stimulating agent (ESA, darbepoetin-α, or synthetic erythropoietin that reportedly binds erythropoietin receptors) would enhance satellite cell myogenic commitment (i.e. MyoD+ indicating commitment to a myogenic/proliferative programme) and fibre type-specific density to an extent similar to that of aerobic training. In addition, the authors evaluated whether ESA treatment combined with aerobic exercise training resulted in a synergistic effect on satellite cell behaviour. Prior to this investigation, the evidence for whether erythropoietin could directly interact with satellite cells in humans was questionable based on methodological limitations. To address this, the investigators successfully employed an advanced cell sorting technique (fluorescent activated cell sorting, or FACS), rarely utilized in human muscle research, to isolate and better characterize satellite cells at the transcriptional level. The result from FACS revealed that purified satellite cells express receptors that can interact with erythropoietin, thereby forming a strong basis for the proposed hypotheses. The major findings of this investigation were: (1) ESA treatment and endurance training both increased satellite cell myogenic commitment, (2) there was no synergistic effect on satellite cell myogenic commitment between ESA treatment and endurance training, and (3) endurance training, regardless of ESA treatment, increased fast twitch-specific satellite cell density. A number of secreted substances, such as inflammatory-related cytokines and trophic factors, are known to modulate satellite cell behaviour. Although serum erythropoietin/darbepoetin-α levels were not significantly elevated after 10 weeks of treatment, Hoedt et al. provide compelling evidence that erythropoietin (a hormone with both therapeutic and performance-enhancing potential) can increase the myogenic commitment of satellite cells. Interestingly, neither ESA treatment nor aerobic training resulted in increased myonuclei per fibre for any fibre type, suggesting an enhanced proliferative state did not ultimately translate to myonuclear accretion. Increasing the number of proliferating satellite cells seemingly serves a different purpose in human skeletal muscle remodelling. Moreover, increased whole body aerobic capacity (via ESA treatment) in the absence of exercise training was not sufficient to affect satellite cell density. It can be concluded that the mechanical loading, energetic perturbation, and/or overall systemic environment during exercise is a more potent stimulator of satellite cell accumulation than aerobic fitness level per se in humans. Increased fast twitch-specific satellite cell density with aerobic training reported by Hoedt et al. (2016) is in agreement with one study (Verney et al. 2008), but contrasts with two recent investigations (Joanisse et al. 2013; Fry et al. 2014). In elderly men (70–76 years), Verney et al. found that 14 weeks of interval cycling (75–95% heart rate maximum) increased fast twitch-specific satellite cell density. This occurred in conjunction with fast twitch-specific hypertrophy, but global myonuclear density did not increase (Verney et al. 2008). In a diverse group of subjects (men and women, 26–68 years), Fry et al. reported hypertrophy of all fibre types after 12 weeks of continuous submaximal cycle training (70% heart rate reserve), but satellite cell and myonuclear density only increased in slow twitch fibres (Fry et al. 2014). In response to 6 weeks of high-intensity interval cycling ( 90% heart rate maximum), Joanisse et al. reported that satellite cell density increased in co-expressing slow/fast ‘hybrid’ fibres in the absence of hypertrophy in young untrained women (Joanisse et al. 2013). However, similar to Hoedt et al., Joanisse et al. report a low average abundance of hybrid fibres before and after aerobic training (1–5%);