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Roberts et al. have provided an insightful counterpoint to our review article on the utility of the synergist ablation model. The purpose of this review is to provide some further dialogue regarding the strengths and weaknesses of the synergist ablation model. Specifically, we highlight that the robustness of the model overshadows surgical limitations. We also compare the transcriptomic responses to synergist ablation in mice and resistance exercise in humans to identify common pathways. We conclude that "cell growth is cell growth" and that the mechanisms available to cells to accumulate biomass and increase in size are similar across cell types and independent of the rate of growth.

In cell biology, ribosomal RNA (rRNA) 2' O -methyl (2'- O -Me) is the most prevalent posttranscriptional chemical modification contributing to ribosome heterogeneity. The modification involves a family of small nucleolar RNAs (snoRNAs) and is specified by box C/D snoRNAs (SNORDs). Given the importance of ribosome biogenesis for skeletal muscle growth, we asked if rRNA 2'- O -Me in nascent ribosomes synthesized in response to a growth stimulus is an unrecognized mode of ribosome heterogeneity in muscle. To determine the pattern and dynamics of 2'- O -Me rRNA, we used a sequencing-based profiling method called RiboMeth-seq (RMS). We applied this method to tissue-derived rRNA of skeletal muscle and rRNA specifically from the muscle fiber using an inducible myofiber-specific RiboTag mouse in sedentary and mechanically overloaded conditions. These analyses were complemented by myonuclear-specific small RNA sequencing to profile SNORDs and link the rRNA epitranscriptome to known regulatory elements generated within the muscle fiber. We demonstrate for the first time that mechanical overload of skeletal muscle 1 ) induces decreased 2'- O -Me at a subset of skeletal muscle rRNA and 2 ) alters the SNORD profile in isolated myonuclei. These findings point to a transient diversification of the ribosome pool via 2'- O -Me during growth and adaptation in skeletal muscle. These findings suggest changes in ribosome heterogeneity at the 2'- O -Me level during muscle hypertrophy and lay the foundation for studies investigating the functional implications of these newly identified "growth-induced" ribosomes. NEW & NOTEWORTHY Ribosomal RNAs (rRNAs) are posttranscriptionally modified by 2' O -methyl (2'- O -Me). This study applied RiboMeth-seq (RMS) to detect changes in 2'- O -Me levels during skeletal muscle hypertrophy, uncovering transient diversification of the ribosome pool in skeletal muscle fibers. This work implies a role for ribosome heterogeneity in skeletal muscle growth and adaptation.

Disuse-induced muscle atrophy is a common clinical problem observed mainly in older adults, intensive care units patients, or astronauts. Previous studies presented biological sex divergence in progression of disuse-induced atrophy along with differential changes in molecular mechanisms possibly underlying muscle atrophy. The aim of this study was to perform transcriptomic profiling of male and female mice during the onset and progression of unloading disuse-induced atrophy. Male and female mice underwent hindlimb unloading (HU) for 24, 48, 72, and 168 h ( n = 8/group). Muscles were weighed for each cohort and gastrocnemius was used for RNA-sequencing analysis. Females exhibited muscle loss as early as 24 h of HU, whereas males after 168 h of HU. In males, pathways related to proteasome degradation were upregulated throughout 168 h of HU, whereas in females these pathways were upregulated up to 72 h of HU. Lcn2 , a gene contributing to regulation of myogenesis, was upregulated by 6.46- to 19.86-fold across all time points in females only. A reverse expression of Fosb , a gene related to muscle degeneration, was observed between males (4.27-fold up) and females (4.57-fold down) at 24-h HU. Mitochondrial pathways related to tricarboxylic acid (TCA) cycle were highly downregulated at 168 h of HU in males, whereas in females this downregulation was less pronounced. Collagen-related pathways were consistently downregulated throughout 168 h of HU only in females, suggesting a potential biological sex-specific protective mechanism against disuse-induced fibrosis. In conclusion, females may have protection against HU-induced skeletal muscle mitochondrial degeneration and fibrosis through transcriptional mechanisms, although they may be more vulnerable to HU-induced muscle wasting compared with males. NEW & NOTEWORTHY Herein, we have assessed the transcriptomic response across biological sexes during the onset and progression of unloading disuse-induced atrophy in mice. We have demonstrated an inverse expression of Fosb between males and females, as well as differentially timed patterns of expressing atrophy-related pathways between sexes that are concomitant to the accelerated atrophy in females. We also identified in females signs of mechanisms to combat disuse-induced mitochondrial degeneration and fibrosis.

A gradual decline in skeletal muscle mass and function is closely tied to increased mortality and disease risk during organismal aging. Exercise training is the most effective way to enhance muscle health, but the adaptive response to exercise as well as muscle repair potential is blunted in older individuals. Numerous mechanisms contribute to the loss of muscle mass and plasticity as aging progresses. An emerging body of recent evidence implicates an accumulation of senescent ("zombie") cells in muscle as a contributing factor to the aging phenotype. Senescent cells cannot divide but can release inflammatory factors and create an unfavorable environment for homeostasis and adaptation. On balance, some evidence indicates that cells with senescent characteristics can be beneficial for the muscle adaptive process, specifically at younger ages. Emerging evidence also suggests that multinuclear muscle fibers could become senescent. In this review, we summarize current literature on the prevalence of senescent cells in skeletal muscle and highlight the consequences of senescent cell removal on muscle mass, function, and adaptability. We examine key limitations in the field of senescence specifically in skeletal muscle and identify areas of research that require future investigation. NEW & NOTEWORTHY There is evidence to suggest that senescent "zombie" cells may or may not accrue in aging skeletal muscle. When muscle is perturbed regardless of age, senescent-like cells do appear, and the benefits of removing them could be age-dependent. More work is needed to determine the magnitude of accumulation and source of senescent cells in muscle. Regardless, pharmacological senolytic treatment of aged muscle is beneficial for adaptation.

MicroRNAs (miRs) control stem cell biology and fate. Ubiquitously expressed and conserved miR-16 was the first miR implicated in tumorigenesis. miR-16 is low in muscle during developmental hypertrophy and regeneration. It is enriched in proliferating myogenic progenitor cells but is repressed during differentiation. The induction of miR-16 blocks myoblast differentiation and myotube formation, whereas knockdown enhances these processes. Despite a central role for miR-16 in myogenic cell biology, how it mediates its potent effects is incompletely defined. In this investigation, global transcriptomic and proteomic analyses after miR-16 knockdown in proliferating C2C12 myoblasts revealed how miR-16 influences myogenic cell fate. Eighteen hours after miR-16 inhibition, ribosomal protein gene expression levels were higher relative to control myoblasts and p53 pathway-related gene abundance was lower. At the protein level at this same time point, miR-16 knockdown globally upregulated tricarboxylic acid (TCA) cycle proteins while downregulating RNA metabolism-related proteins. miR-16 inhibition induced specific proteins associated with myogenic differentiation such as ACTA2, EEF1A2, and OPA1. We extend prior work in hypertrophic muscle tissue and show that miR-16 is lower in mechanically overloaded muscle in vivo. Our data collectively point to how miR-16 is implicated in aspects of myogenic cell differentiation. A deeper understanding of the role of miR-16 in myogenic cells has consequences for muscle developmental growth, exercise-induced hypertrophy, and regenerative repair after injury, all of which involve myogenic progenitors.

Multinuclear muscle fibers are the most voluminous cells in skeletal muscle and the primary drivers of growth in response to loading. Outside the muscle fiber, however, is a diversity of mononuclear cell types that reside in the extracellular matrix (ECM). These muscle-resident cells are exercise-responsive and produce the scaffolding for successful myofibrillar growth. Without proper remodeling and maintenance of this ECM scaffolding, the ability to mount an appropriate response to resistance training in adult muscles is severely hindered. Complex cellular choreography takes place in muscles following a loading stimulus. These interactions have been recently revealed by single-cell explorations into muscle adaptation with loading. The intricate ballet of ECM remodeling involves collagen production from fibrogenic cells and ECM modifying signals initiated by satellite cells, immune cells, and the muscle fibers themselves. The acellular collagen-rich ECM is also a mechanical signal-transducer and rich repository of growth factors that may directly influence muscle fiber hypertrophy once liberated. Collectively, high levels of collagen expression, deposition, and turnover characterize a well-trained muscle phenotype. The purpose of this review is to highlight the most recent evidence for how the ECM and its cellular components affect loading-induced muscle hypertrophy. We also address how the muscle fiber may directly take part in ECM remodeling, and whether ECM dynamics are rate limiting for muscle fiber growth.

Muscle fibers are syncytial postmitotic cells that can acquire exogenous nuclei from resident muscle stem cells, called satellite cells. Myonuclei are added to muscle fibers by satellite cells during conditions such as load-induced hypertrophy. It is difficult to dissect the molecular contributions of resident versus satellite cell-derived myonuclei during adaptation due to the complexity of labeling distinct nuclear populations in multinuclear cells without label transference between nuclei. To sidestep this barrier, we used a genetic mouse model where myonuclear DNA can be specifically and stably labeled via nonconstitutive H2B-GFP at any point in the lifespan. Resident myonuclei (Mn) were GFP-tagged in vivo before 8 wk of progressive weighted wheel running (PoWeR) in adult mice (>4-mo-old). Resident + satellite cell-derived myonuclei (Mn+SC Mn) were labeled at the end of PoWeR in a separate cohort. Following myonuclear isolation, promoter DNA methylation profiles acquired with low-input reduced representation bisulfite sequencing (RRBS) were compared to deduce epigenetic contributions of satellite cell-derived myonuclei during adaptation. Resident myonuclear DNA has hypomethylated promoters in genes related to protein turnover, whereas the addition of satellite cell-derived myonuclei shifts myonuclear methylation profiles to favor transcription factor regulation and cell-cell signaling. By comparing myonucleus-specific methylation profiling to previously published single-nucleus transcriptional analysis in the absence (Mn) versus the presence of satellite cells (Mn+SC Mn) with PoWeR, we provide evidence that satellite cell-derived myonuclei may preferentially supply specific ribosomal proteins to growing myofibers and retain an epigenetic "memory" of prior stem cell identity. These data offer insights on distinct epigenetic myonuclear characteristics and contributions during adult muscle growth.

The ability to genetically manipulate mice allows for gain- and loss-of-function in vivo, making them an ideal model for elucidating mechanisms of skeletal muscle mass regulation. Combining genetic models with mechanical muscle loading enables identification of specific factors involved in the hypertrophic response as well as the ability to test the requirement of those factors for adaptation, thereby informing performance and therapeutic interventions. Until recently, approaches for inducing mechanically mediated muscle hypertrophy (i.e., resistance-training analogs) have been limited and considered "nontranslatable" to humans. This mini-review outlines recent translational advances in loading-mediated strategies for inducing muscle hypertrophy in mice, and highlights the advantages and disadvantages of each method. The skeletal muscle field is poised for new breakthroughs in understanding mechanisms regulating load-induced muscle growth given the numerous murine tools that have very recently been described.

To date, studies that have aimed to investigate the role of satellite cells during adult skeletal muscle adaptation and hypertrophy have utilized a nontranslational stimulus and/or have been performed over a relatively short time frame. Although it has been shown that satellite cell depletion throughout adulthood does not drive skeletal muscle loss in sedentary mice, it remains unknown how satellite cells participate in skeletal muscle adaptation to long-term physical activity. The current study was designed to determine whether reduced satellite cell content throughout adulthood would influence the transcriptome-wide response to physical activity and diminish the adaptive response of skeletal muscle. We administered vehicle or tamoxifen to adult Pax7-diphtheria toxin A (DTA) mice to deplete satellite cells and assigned them to sedentary or wheel-running conditions for 13 mo. Satellite cell depletion throughout adulthood reduced balance and coordination, overall running volume, and the size of muscle proprioceptors (spindle fibers). Furthermore, satellite cell participation was necessary for optimal muscle fiber hypertrophy but not adaptations in fiber type distribution in response to lifelong physical activity. Transcriptome-wide analysis of the plantaris and soleus revealed that satellite cell function is muscle type specific; satellite cell-dependent myonuclear accretion was apparent in oxidative muscles, whereas initiation of G protein-coupled receptor (GPCR) signaling in the glycolytic plantaris may require satellite cells to induce optimal adaptations to long-term physical activity. These findings suggest that satellite cells play a role in preserving physical function during aging and influence muscle adaptation during sustained periods of physical activity.

Skeletal muscle myosin heavy chain (MyHC) fiber type composition is a critical determinant of overall muscle function and health. Various approaches interrogate fiber type at the single cell, but the two most commonly utilized are single-muscle fiber sodium dodecyl sulfate-polyacrylamide gel electrophoresis (smfSDS-PAGE) and fluorescent immunohistochemistry (IHC). Although smfSDS-PAGE is generally considered the "gold standard," IHC is more commonly used because of its time-effectiveness and relative ease. Unfortunately, there is lingering inconsistency on how best to accurately and quickly determine fiber type via IHC and an overall misunderstanding regarding pure fiber type proportions, specifically the abundance of fibers exclusively expressing highly glycolytic MyHC IIX in humans. We therefore 1 ) present information and data showing the low abundance of pure MyHC IIX muscle fibers in healthy human skeletal muscle and 2 ) leverage this information to provide straightforward protocols that are informed by human biology and employ inexpensive, easily attainable antibodies for the accurate determination of fiber type.

It is postulated that testosterone-induced skeletal muscle hypertrophy is driven by myonuclear accretion as the result of satellite cell fusion. To directly test this hypothesis, we utilized the Pax7-DTA mouse model to deplete satellite cells in skeletal muscle followed by testosterone administration. Pax7-DTA mice (6 mo of age) were treated for 5 days with either vehicle [satellite cell replete (SC+)] or tamoxifen [satellite cell depleted (SC-)]. Following a washout period, a testosterone propionate or sham pellet was implanted for 21 days. Testosterone administration caused a significant increase in muscle fiber cross-sectional area in SC+ and SC- mice in both oxidative (soleus) and glycolytic (plantaris and extensor digitorum longus) muscles. In SC+ mice treated with testosterone, there was a significant increase in both satellite cell abundance and myonuclei that was completely absent in testosterone-treated SC- mice. These findings provide direct evidence that testosterone-induced muscle fiber hypertrophy does not require an increase in satellite cell abundance or myonuclear accretion.Listen to a podcast about this Rapid Report with senior author E. E. Dupont-Versteegden (https://ajpcell.podbean.com/e/podcast-on-paper-that-shows-testosterone-induced-skeletal-muscle-hypertrophy-does-not-need-muscle-stem-cells/).

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.

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.

We examined single muscle fiber contractile function of the oldest-old (3F/2M, 89 ± 1 yr old) enrolled in The Health, Aging, and Body Composition Study (The Health ABC Study). Vastus lateralis muscle biopsies were obtained and single muscle fiber function was determined (n = 105) prior to myosin heavy chain (MHC) isoform identification with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-sectional area of MHC I muscle fibers (5,576 ± 333 μm 2 ; n = 58) was 21% larger (P < 0.05) than MHC IIa fibers (4,518 ± 386 μm 2 ; n = 47). Normalized power (an indicator of muscle fiber quality incorporating size, strength, and speed) of MHC I and IIa muscle fibers was 2.3 ± 0.1 and 17.4 ± 0.8 W/l, respectively. Compared with previous research from our lab using identical procedures, MHC I normalized power was 28% higher than healthy 20 yr olds and similar to younger octogenarians (∼80 yr old). Normalized power of MHC IIa fibers was 63% greater than 20 yr olds and 39% greater than younger octogenarians. These comparative data suggest that power output per unit size (i.e., muscle quality) of remaining muscle fibers improves with age, a phenomenon more pronounced in MHC IIa fibers. Age-related single muscle fiber quality improvements may be a compensatory mechanism to help offset decrements in whole muscle function.