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The pathophysiology of muscle damage in peripheral artery disease (PAD) includes increased oxidant production and impaired antioxidant defenses. Epicatechin (EPI), a naturally occurring flavanol, has antioxidant properties that may mediate the beneficial effects of natural products such as cocoa. In a phase II randomized trial, a cocoa-flavanol-rich beverage significantly improved walking performance compared with a placebo in people with PAD. In the present work, the molecular mechanisms underlying the therapeutic effect of cocoa flavanols were investigated by analyzing baseline and follow-up muscle biopsies from participants. Increases in nuclear factor erythroid 2-related factor 2 (Nrf2) target antioxidants heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1) in the cocoa group were significantly associated with reduced accumulation of central nuclei, a myopathy indicator, in type II muscle fibers (P ¼ 0.017 and P ¼ 0.023, respectively). Protein levels of the mitochondrial respiratory complex III subunit, cytochrome b-c1 complex subunit 2 (UQCRC2), were significantly higher in the cocoa group than in the placebo group (P ¼ 0.032), and increases in UQCRC2 were significantly associated with increased levels of Nrf2 target antioxidants HO-1 and NQO1 (P ¼ 0.001 and P ¼ 0.035, respectively). Exposure of non-PAD human myotubes to ex vivo serum from patients with PAD reduced Nrf2 phosphorylation, an indicator of activation, increased hydrogen peroxide production and oxidative stress, and reduced mitochondrial respiration. Treatment of myotubes with EPI in the presence of serum from patients with PAD increased Nrf2 phosphorylation and protected against PAD serum-induced oxidative stress and mitochondrial dysfunction. Overall, these findings suggest that cocoa flavanols may enhance antioxidant capacity in PAD via Nrf2 activation. NEW & NOTEWORTHY The current study supports the hypothesis that in people with PAD, cocoa flavanols activate Nrf2, thereby increasing antioxidant protein levels, protecting against skeletal muscle damage, and increasing mitochondrial protein abundance. These results suggest that Nrf2 activation may be an important therapeutic target for improving walking performance in people with PAD. [ABSTRACT FROM AUTHOR]

In older Individuals, hypertrophy from progressive resistance training (PRT) is compromised In approximately one-third of participants In exercise trials. The objective of this study was to establish novel relationships between baseline muscle features and/or their PRT-induced change in vastus lateralis muscle biopsies with hypertrophy outcomes. Multiple linear regression analyses adjusted for sex were performed on phenotypic data from older adults (n = 48 participants, 70.8±4.5 yr) completing 14 wk of PRT. Results show that baseline muscle size associates with growth regardless of hypertrophy outcome measure [fiber cross-sectional area (fCSA), β = -0.76, Adj. P < 0.01; thigh muscle area by computed tomography (CT), β = -0.75, Adj. P < 0.01; dual-energy X-ray absorptiometry (DXA) thigh lean mass, β = -0.47, Adj. P < 0.05]. Furthermore, loosely packed collagen organization (CO, β = -0.44, Adj. P < 0.05) and abundance of CD11b+/CD206- immune cells (β = -0.36, Adj. P = 0.10) were negatively associated with whole muscle hypertrophy, with a significant sex interaction on the latter. In addition, a composite hypertrophy score generated using all three measures reinforces significant fiber level findings that changes in myonuclei (MN) (β = 0.67, Adj. P < 0.01), changes in immune cells (β = 0.48, Adj. P < 0.05; both CD11b+/CD206 + and CD11b+/CD206- cells), and capillary density (β = 0.56, Adj. P < 0.01) are significantly associated with growth. Exploratory single-cell RNA-sequencing of CD11b+ cells in muscle in response to resistance exercise showed that macrophages have a mixed phenotype. Collagen associations with macrophages may be an important aspect in muscle response heterogeneity. Detailed histological phenotyping of muscle combined with multiple measures of growth response to resistance training in older persons identify potential new mechanisms underlying response heterogeneity and possible sex differences. NEW & NOTEWORTHY Extensive analyses of muscle features associated with muscle size and resistance training response in older persons, including sex differences, and evaluation of multiple measures of hypertrophy are discussed. Collagen organization and CD11b-expressing immune cells offer potential new targets to augment growth response in older individuals. A hypertrophy composite score reveals that changes in immune cells, myonuclei, and capillary density are critically important for overall muscle growth while sc-RNAseq reveals evidence for macrophage heterogeneity. [ABSTRACT FROM AUTHOR]

The number of myonuclei within a muscle fiber is an important factor in muscle growth, but its regulation during muscle adaptation is not well understood. We aimed to elucidate the time course of myonuclear dynamics during endurance training, loaded and concentric resistance training, and nerve silencing-induced disuse atrophy with subsequent recovery. We modified tibialis anterior muscle activity in free-living rats with electrical stimulation from implantable pulse generators, or with implantable osmotic pumps delivering tetrodotoxin (TTX) to silence the motor nerve without transection. We used the updated, automated software MyoVision to measure fiber-type-specific responses in whole tibialis anterior cross sections (~8,000 fibers each). Seven days of continuous low-frequency stimulation (CLFS) reduced muscle mass (-12%), increased slower myosin isoforms and reduced IIX/IIB fibers (-32%), and substantially increased myonuclei especially in IIX/IIB fibers (55.5%). High-load resistance training (spillover) produced greater hypertrophy (~16%) in muscle mass and fiber cross-sectional area (CSA) than low-load resistance training (concentric, ~6%) and was associated with myonuclear addition in all fiber types (35%-46%). TTX-induced nerve silencing resulted in progressive loss in muscle mass, fiber CSA, and myonuclei per fiber cross section (-50.7%, -53.7%, and -40.7%, respectively, at 14 days). Myonuclear loss occurred in a fiber-type-independent manner, but subsequent recovery during voluntary habitual activity suggested that type IIX/IIB fibers contained more new myonuclei during recovery from severe atrophy. This study demonstrates the power and accuracy provided by the updated MyoVision software and introduces new models for studying myonuclear dynamics in training, detraining, retraining, repeated disuse, and recovery. [ABSTRACT FROM AUTHOR]

The skeletal muscle hypertrophic response to resistance exercise training (RT) is highly variable across individuals. The molecular underpinnings of this heterogeneity are unclear. This study investigated transcriptional networks linked to RT-induced muscle hypertrophy, classified as 1) predictive of hypertrophy, 2) responsive to RT independent of muscle hypertrophy, or 3) plastic with hypertrophy. Older adults (n = 31, 18 F/13 M, 70 ± 4 yr) underwent 14-wk RT (3 days/wk, alternating high-low-high intensity). Muscle hypertrophy was assessed by pre- to post-RT change in mid-thigh muscle cross-sectional area (CSA) [computed tomography (CT), primary outcome] and thigh lean mass [dual-energy X-ray absorptiometry (DXA), secondary outcome]. Transcriptome-wide poly-A RNA-seq was performed on vastus lateralis tissue collected pre- (n = 31) and post-RT (n = 22). Prediction networks (using only baseline RNA-seq) were identified by weighted gene correlation network analysis (WGCNA). To identify Plasticity networks, WGCNA change indices for paired samples were calculated and correlated to changes in muscle size outcomes. Pathway-level information extractor (PLIER) was applied to identify Response networks and link genes to biological annotation. Prediction networks (n = 6) confirmed transcripts previously connected to resistance/aerobic training adaptations in the MetaMEx database while revealing novel member genes that should fuel future research to understand the influence of baseline muscle gene expression on hypertrophy. Response networks (n = 6) indicated RT-induced increase in aerobic metabolism and reduced expression of genes associated with spliceosome biology and type-I myofibers. A single exploratory Plasticity network was identified. Findings support that interindividual differences in baseline gene expression may contribute more than RT-induced changes in gene networks to muscle hypertrophic response heterogeneity. Code/Data: https://github.com/kallavin/MASTERS_manuscript/tree/master. [ABSTRACT FROM AUTHOR]

As a consequence of military operations, many veterans suffer from penetrating wounds and long-term retention of military-grade heavy metal fragments. Fragments vary in size and location, and complete surgical removal may not be feasible or beneficial in all cases. Increasing evidence suggests retention of heavy metal fragments may have serious biological implications, including increased risks for malignant transformation. Previous studies assessed the tumorigenic effects of metal alloys in rats, demonstrating combinations of metals are sufficient to induce tumor formation after prolonged retention in skeletal muscle tissue. In this study, we analyzed transcriptional changes in skeletal muscle tissue in response to eight different military-relevant pure metals over 12 mo. We found that most transcriptional changes occur at 1 and 3 mo after metal pellets are embedded in skeletal muscle and these effects resolve at 6 and 12 mo. We also report significant immunogenic effects of nickel and cobalt and suppressive effects of lead and depleted uranium on gene expression. Overall, skeletal muscle exhibits a remarkable capacity to adapt to and recover from internalized metal fragments; however, the cellular response to chronic exposure may be restricted to the metal-tissue interface. These data suggest that unless affected regions are specifically captured by biopsy, it would be difficult to reliably detect changes in muscle gene expression that would be indicative of long-term adverse health outcomes. [ABSTRACT FROM AUTHOR]

The ability to genetically manipulate mice allows for gainand 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 crosssectional 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-paperthat- shows-testosterone-induced-skeletal-muscle-hypertrophydoes- not-need-muscle-stem-cells/). [ABSTRACT FROM AUTHOR]

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 weightedwheel- 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 fasttwitch 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Skeletal muscle is an exceptionally adaptive tissue that compromises 40% of mammalian body mass. Skeletal muscle functions in locomotion, but also plays important roles in thermogenesis and metabolic homeostasis. Thus characterizing the structural and functional properties of skeletal muscle is important in many facets of biomedical research, ranging from myopathies to rehabilitation sciences to exercise interventions aimed at improving quality of life in the face of chronic disease and aging. In this paper, we focus on automated quantification of three important morphological features of muscle: 1) muscle fiber-type composition; 2) muscle fiber-type-specific cross-sectional area, and 3) myonuclear content and location. We experimentally prove that the proposed automated image analysis approaches for fiber-type-specific assessments and automated myonuclei counting are fast, accurate, and reliable. [ABSTRACT FROM AUTHOR]