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Adult muscle stem cells (MuSCs) are known to replicate upon activation before differentiating and fusing to regenerate myofibers. It is unclear whether MuSC differentiation is intrinsically linked to cell division, which has implications for stem cell population maintenance. We use single-cell RNA-sequencing to identify transcriptionally diverse subpopulations of MuSCs after 5 days of a growth stimulus in adult muscle. Trajectory inference in combination with a novel mouse model for tracking MuSC-derived myonuclei and in vivo labeling of DNA replication revealed an MuSC population that exhibited division-independent differentiation and fusion. These findings demonstrate that in response to a growth stimulus in the presence of intact myofibers, MuSC division is not obligatory. [ABSTRACT FROM AUTHOR]

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]

A key determinant of geriatric frailty is sarcopenia, the age-associated loss of skeletal muscle mass and strength (1,2). Although the etiology of sarcopenia is unknown, the correlation during aging between the loss of activity of satellite cells, which are endogenous muscle stem cells, and impaired muscle regenerative capacity has led to the hypothesis that the loss of satellite cell activity is also a cause of sarcopenia (3,4). We tested this hypothesis in male sedentary mice by experimentally depleting satellite cells in young adult animals to a degree sufficient to impair regeneration throughout the rest of their lives. A detailed analysis of multiple muscles harvested at various time points during aging in different cohorts of these mice showed that the muscles were of normal size, despite low regenerative capacity, but did have increased fibrosis. These results suggest that lifelong reduction of satellite cells neither accelerated nor exacerbated sarcopenia and that satellite cells did not contribute to the maintenance of muscle size or fiber type composition during aging, but that their loss may contribute to age-related muscle fibrosis., Recent estimates indicate that up to one-third of elderly people suffer from frailty, characterized by a common set of symptoms including loss of muscle strength, increased fatigability, modest levels of [...]

Exercise promotes functional improvements in aged tissues, but the extent to which it simulates partial molecular reprogramming is unknown. Using transcriptome profiling from (1) a skeletal muscle‐specific in vivo Oct3/4, Klf4, Sox2 and Myc (OKSM) reprogramming‐factor expression murine model; (2) an in vivo inducible muscle‐specific Myc induction murine model; (3) a translatable high‐volume hypertrophic exercise training approach in aged mice; and (4) human exercise muscle biopsies, we collectively defined exercise‐induced genes that are common to partial reprogramming. Late‐life exercise training lowered murine DNA methylation age according to several contemporary muscle‐specific clocks. A comparison of the murine soleus transcriptome after late‐life exercise training to the soleus transcriptome after OKSM induction revealed an overlapping signature that included higher JunB and Sun1. Also, within this signature, downregulation of specific mitochondrial and muscle‐enriched genes was conserved in skeletal muscle of long‐term exercise‐trained humans; among these was muscle‐specific Abra/Stars. Myc is the OKSM factor most induced by exercise in muscle and was elevated following exercise training in aged mice. A pulse of MYC rewired the global soleus muscle methylome, and the transcriptome after a MYC pulse partially recapitulated OKSM induction. A common signature also emerged in the murine MYC‐controlled and exercise adaptation transcriptomes, including lower muscle‐specific Melusin and reactive oxygen species‐associated Romo1. With Myc, OKSM and exercise training in mice, as well habitual exercise in humans, the complex I accessory subunit Ndufb11 was lower; low Ndufb11 is linked to longevity in rodents. Collectively, exercise shares similarities with genetic in vivo partial reprogramming. Key points: Advances in the last decade related to cellular epigenetic reprogramming (e.g. DNA methylome remodelling) toward a pluripotent state via the Yamanaka transcription factors Oct3/4, Klf4, Sox2 and Myc (OKSM) provide a window into potential mechanisms for combatting the deleterious effects of cellular ageing.Using global gene expression analysis, we compared the effects of in vivo OKSM‐mediated partial reprogramming in skeletal muscle fibres of mice to the effects of late‐life murine exercise training in muscle.Myc is the Yamanaka factor most induced by exercise in skeletal muscle, and so we compared the MYC‐controlled transcriptome in muscle to Yamanaka factor‐mediated and exercise adaptation mRNA landscapes in mice and humans.A single pulse of MYC is sufficient to remodel the muscle methylome.We identify partial reprogramming‐associated genes that are innately altered by exercise training and conserved in humans, and propose that MYC contributes to some of these responses. [ABSTRACT FROM AUTHOR]

Background: This study evaluated the association of smoking with mitochondrial function in gastrocnemius muscle of people with peripheral artery disease (PAD). Methods: Participants were enrolled from Chicago, Illinois and consented to gastrocnemius biopsy. Mitochondrial oxidative capacity was measured in muscle with respirometry. Abundance of voltage-dependent anion channel (VDAC) (mitochondrial membrane abundance), peroxisome proliferator-activated receptor-γ coactivator (PGC-1α) (mitochondrial biogenesis), and electron transport chain complexes I–V were measured with Western blot. Results: Fourteen of 31 people with PAD (age 72.1 years, ABI 0.64) smoked cigarettes currently. Overall, there were no significant differences in mitochondrial oxidative capacity between PAD participants who currently smoked and those not currently smoking (complex I+II-mediated oxidative phosphorylation: 86.6 vs 78.3 pmolO2/s/mg, respectively [ p = 0.39]). Among participants with PAD, those who currently smoked had a higher abundance of PGC-1α (p < 0.01), VDAC (p = 0.022), complex I (p = 0.021), and complex III (p = 0.021) proteins compared to those not currently smoking. People with PAD who currently smoked had lower oxidative capacity per VDAC unit (complex I+II-mediated oxidative phosphorylation [137.4 vs 231.8 arbitrary units, p = 0.030]) compared to people with PAD not currently smoking. Among people without PAD, there were no significant differences in any mitochondrial measures between currently smoking (n = 5) and those not currently smoking (n = 63). Conclusions: Among people with PAD, cigarette smoking may stimulate mitochondrial biogenesis to compensate for reduced oxidative capacity per unit of mitochondrial membrane, resulting in no difference in overall mitochondrial oxidative capacity according to current smoking status among people with PAD. However, these results were cross-sectional and a longitudinal study is needed. [ABSTRACT FROM AUTHOR]

Evaluated the effectiveness of gaming to create an interactive, stimulating learning environment as a review format for the Dental Hygiene National Board examination. Students (n=28) participated in either the gaming or a lecture review format. The gaming group scored higher on the exam on eight of 12 topics as well as on the case-based learning section. (DB)

Importance: Patients with lower extremity peripheral artery disease (PAD) have reduced lower extremity perfusion, impaired lower extremity skeletal muscle function, and poor walking performance. Telmisartan (an angiotensin receptor blocker) has properties that reverse these abnormalities.Objective: To determine whether telmisartan improves 6-minute walk distance, compared with placebo, in patients with lower extremity PAD at 6-month follow-up.Design, Setting, and Participants: Double-blind, randomized clinical trial conducted at 2 US sites and involving 114 participants. Enrollment occurred between December 28, 2015, and November 9, 2021. Final follow-up occurred on May 6, 2022.Interventions: The trial randomized patients using a 2 × 2 factorial design to compare the effects of telmisartan plus supervised exercise vs telmisartan alone and supervised exercise alone and to compare telmisartan alone vs placebo. Participants with PAD were randomized to 1 of 4 groups: telmisartan plus exercise (n = 30), telmisartan plus attention control (n = 29), placebo plus exercise (n = 28), or placebo plus attention control (n = 27) for 6 months. The originally planned sample size was 240 participants. Due to slower than anticipated enrollment, the primary comparison was changed to the 2 combined telmisartan groups vs the 2 combined placebo groups and the target sample size was changed to 112 participants.Main Outcomes and Measures: The primary outcome was the 6-month change in 6-minute walk distance (minimum clinically important difference, 8-20 m). The secondary outcomes were maximal treadmill walking distance; Walking Impairment Questionnaire scores for distance, speed, and stair climbing; and the 36-Item Short-Form Health Survey physical functioning score. The results were adjusted for study site, baseline 6-minute walk distance, randomization to exercise vs attention control, sex, and history of heart failure at baseline.Results: Of the 114 randomized patients (mean age, 67.3 [SD, 9.9] years; 46 were women [40.4%]; and 81 were Black individuals [71.1%]), 105 (92%) completed 6-month follow-up. At 6-month follow-up, telmisartan did not significantly improve 6-minute walk distance (from a mean of 341.6 m to 343.0 m; within-group change: 1.32 m) compared with placebo (from a mean of 352.3 m to 364.8 m; within-group change: 12.5 m) and the adjusted between-group difference was -16.8 m (95% CI, -35.9 m to 2.2 m; P = .08). Compared with placebo, telmisartan did not significantly improve any of the 5 secondary outcomes. The most common serious adverse event was hospitalization for PAD (ie, lower extremity revascularization, amputation, or gangrene). Three participants (5.1%) in the telmisartan group and 2 participants (3.6%) in the placebo group were hospitalized for PAD.Conclusions and Relevance: Among patients with PAD, telmisartan did not improve 6-minute walk distance at 6-month follow-up compared with placebo. These results do not support telmisartan for improving walking performance in patients with PAD.Trial Registration: ClinicalTrials.gov Identifier: NCT02593110. [ABSTRACT FROM AUTHOR]

Adipose tissue macrophages are associated with insulin resistance and are linked to changes in the extracellular matrix. To better characterize adipose macrophages, the extracellular matrix, and adipocyte-macrophage interactions, gene expression from adipose tissue and the stromal vascular fraction was assessed for markers of inflammation and fibrosis, and macrophages from obese and lean subjects were counted and characterized immunohistochemically. Coculture experiments examined the effects of adipocyte-macrophage interaction. Collagen VI gene expression was associated with insulin sensitivity and CD68 (r = -0.56 and 0.60, P < 0.0001) and with other markers of inflammation and fibrosis. Compared with adipose tissue from lean subjects, adipose tissue from obese subjects contained increased areas of fibrosis, which correlated inversely with insulin sensitivity (r = -0.58, P < 0.02) and positively with macrophage number (r = 0.70, P < 0.01). Although macrophages in crownlike structures (CLS) were more abundant in obese adipose tissue, the majority of macrophages were associated with fibrosis and were not organized in CLS. Macrophages in CLS were predominantly M1, but most other macrophages, particularly those in fibrotic areas, were M2 and also expressed CD150, a marker of M2c macrophages. Coculture of THP-1 macrophages with adipocytes promoted the M2 phenotype, with a lower level of IL-1 expression and a higher ratio of IL-10 to IL-12. Transforming growth factor-[beta] (TGF-[beta]) was more abundant in M2 macrophages and was further increased by coculture with adipocytes. Downstream effectors of TGF-[beta], such as plasminogen activator inhibitor-l, collagen VI, and phosphorylated Smad, were increased in macrophages and adipocytes. Thus adipose tissue of insulin-resistant humans demonstrated increased fibrosis, M2 macrophage abundance, and TGF-[beta] activity. alternatively activated macrophages; inflammation; crownlike structures doi: 10.1152/ajpendo.00329.2010.

Obesity is characterized by adipose tissue expansion as well as macrophage infiltration of adipose tissue. This results in an increase in circulating inflammatory cytokines and nonesterified fatty acids, factors that cause skeletal muscle insulin resistance. Whether obesity also results in skeletal muscle inflammation is not known. In this study, we quantified macrophages immunohistochemically in vastus lateralis biopsies from eight obese and eight lean subjects. Our study demonstrates that macrophages infiltrate skeletal muscle in obesity, and we developed an in vitro system to study this mechanistically. Myoblasts were isolated from vastus lateralis biopsies and differentiated in culture. Coculture of differentiated human myotubes with macrophages in the presence of palmitic acid, to mimic an obese environment, revealed that macrophages in the presence of palmitic acid synergistically augment cytokine and chemokine expression in myotubes, decrease I[kappa]B-[alpha] protein expression, increase phosphorylated JNK, decrease phosphorylated Akt, and increase markers of muscle atrophy. These results suggest that macrophages alter the inflammatory state of muscle cells in an obese milieu, inhibiting insulin signaling. Thus in obesity both adipose tissue and skeletal muscle inflammation may contribute to insulin resistance. muscle cells; inflammation

Dramatic advances in understanding mechanisms of aging have recently been made in model systems. Interventions have been devised that successfully enhance survival. Major issues still in need of resolution include whether these interventions not only increase survival but also enhance function, delay frailty, and can be translated into clinical application. It seems there are basic biologic findings close to being ready for translation. However, a number of barriers exist to translating these findings into realistic clinical interventions. Steps and resources needed include measuring not only survival but also impact of interventions on age-related disability, frailty, and onset of disease in model systems; development of clinically relevant measures of disability, frailty, and disease for each animal model and genetically tractable animal models of frailty; training and career-long funding mechanisms for geriatricians in basic science research and for basic scientists in geriatric issues; translationally capable review and funding mechanisms; emphasis on studies of interventions that can be initiated in later life for preventing or reversing disability; genetic association studies in humans to identify new candidate genes and pathways that correlate with disability, frailty, and age-related disease onset as well as longevity; study of exposure to environmental agents or toxins early in life on survival, disability, frailty, and disease in later life. Key Words: Healthspan--Translation--Animal studies of aging.

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]

Changes in gene expression associated with skeletal muscle atrophy due to aging are distinct from those due to disuse, suggesting that the response of old muscle to inactivity may be altered. The goal of this study was to identify changes in muscle gene expression that may contribute to loss of adaptability of old muscle. Muscle atrophy was induced in young adult (6-mo) and old (32-mo) male Brown Norway/ F344 rats by 2 wk of hindlimb suspension (HS), and soleus muscles were analyzed by cDNA microarrays. Overall, similar changes in gene expression with HS were observed in young and old muscles for genes encoding proteins involved in protein folding (heat shock proteins), muscle structure, and contraction, extracellular matrix, and nucleic acid binding. More genes encoding transport and receptor proteins were differentially expressed in the soleus muscle from young rats, while in soleus muscle from old rats more genes that encoded ribosomal proteins were upregulated. The gene encoding the cold-shock protein RNA-binding motif protein-3 (RBM3) was induced most highly with HS in muscle from old rats, verified by real-time RT-PCR, while no difference with age was observed. The cold-inducible RNA-binding protein (Cirp) gene was also overexpressed with HS, whereas cold-shock protein Y-box-binding protein-1 was not. A time course analysis of RBM3 mRNA abundance during HS showed that upregulation occurred after apoptotic nuclei and markers of protein degradation increased. We conclude that a cold-shock response may be part of a compensatory mechanism in muscles undergoing atrophy to preserve remaining muscle mass and that RBM3 may be a therapeutic target to prevent muscle loss. aging; microarray; Cirp; atrophy

We report an age-dependent increase in nonimmunohematopoietic cells ([CD45.sup.neg]) in regenerating muscle characterized by high stem-cell antigen (Sca-1) expression. In aged regenerating muscle, only 14% of these [CD45.sup.neg][Sca-1.sup.pos] cells express MyoD, whereas 82% of [CD45.sup.neg][Sca-1.sup.pos] cells are [MyoD.sup.pos] in young adult muscle. In vitro, [CD45.sup.neg][MyoD.sup.neg][Sca-1.sup.pos] cells overexpress fibrosis-promoting genes, potentially controlled by Wnt2. The cells are proliferative, nonmyogenic, and nonadipogenic, and arise in clonally derived myoblast cultures from aged mice. [MyoD.sup.neg] [Sca-1.sup.pos] nonmyogenic cells also emerge in C2C12 myoblast cultures at late passage. Both in vitro and in vivo studies suggest that [MyoD.sup.neg][Sca-1.sup.pos] cells from aged muscle are more susceptible to apoptosis than myoblasts, which may contribute to depletion of the satellite cell pool. Thus, with age, a subset of myoblasts takes on an altered phenotype, which is marked by high Sca-1 expression. These cells do not participate in muscle regeneration, and instead may contribute to muscle fibrosis in aged muscle. Key Words: Sca-1--Skeletal muscle regeneration--Muscle stem cells--Fibrosis--Aging.

Skeletal muscle atrophy is associated with an increase in apoptosis, and we showed previously that endonuclease G (EndoG) is localized to nuclei following unloading. The goal of this study was to determine whether the onset of apoptosis in response to disuse was consistent with the hypothesis that EndoG is involved in myofiber nuclear loss. Atrophy was induced by hindlimb suspension for 12 h or 1, 2, 4 and 7 days in 6-mo-old rats. Soleus myofiber cross-sectional area decreased significantly by 2 days, whereas muscle mass and muscle-to-body mass ratio decreased by 4 and 7 days, respectively. By contrast, a significant increase in apoptosis, evidenced by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive nuclei, occurred as early as 12 h after suspension, preceding the elevation in muscle atrophy F-box gene expression. The early increase in apoptosis appeared to be specific to myofiber nuclei, whereas TUNEL-positive interstitial cells did not become significantly elevated until 2 days after suspension. Furthermore, TUNEL-positive myofiber nuclei colocalized with EndoG as early as 12 h after suspension, and no such localization was observed in interstitial cells. Although no significant change in total activated caspase-3, -7, or -12 protein abundance was apparent, activated caspase-3 was expressed in interstitial cells undergoing apoptosis, some of which were endothelial cells. These data indicate that apoptosis is an early, and therefore possibly causative, event in the process of muscle atrophy, and that EndoG nuclear translocation is specific for myofiber nuclear apoptosis, whereas interstitial cells may undergo apoptosis via a more classical, caspase-dependent pathway. caspase; muscle atrophy F-box; hindlimb suspension; endothelial cells; endonuclease G

Recent studies have implicated the mTOR-signaling pathway as a primary component for muscle growth in mammals. The purpose of this investigation was to examine signaling pathways for muscle protein synthesis after resistance exercise. Sprague-Dawley rats (male, 6 mo old) were assigned to either resistance exercise or control groups. Resistance exercise was accomplished in operantly conditioned animals using a specially designed flywheel apparatus. Rats performed two sessions of resistance exercise, separated by 48 h, each consisting of 2 sets of 25 repetitions. Sixteen hours after the second session, animals were killed, and soleus muscles were examined for rates of protein synthesis with and without insulin and/or rapamycin (mTOR inhibitor) and/or PD-098059 (PD; MEK kinase inhibitor). Results of this study demonstrated that rates of synthesis were higher (P < 0.05) with insulin after exercise compared with without insulin, or to control muscles, regardless of insulin. Rapamycin lowered (P < 0.05) rates of synthesis in controls, with or without insulin, and after exercise without insulin. However, insulin was able to overcome the inhibition of rapamycin after exercise (P < 0.05). PD had no effect on protein synthesis in control rats, but the addition of PD to exercised muscle resulted in lower (P < 0.05) rates of synthesis, and this inhibition was not rescued by insulin. Western blot analyses demonstrated that the inhibitors used in the present study were selective and effective for preventing activation of specific signaling proteins. Together, these results suggest that the insulin-facilitated increase of muscle protein synthesis after resistance exercise requires multiple signaling pathways. mammalian target of rapamycin: rapamycin; extracellular signal-related kinases; PD-098059

Hindlimb suspension (HS) results in rapid losses of muscle mass, which may in part be explained by attenuated rates of protein synthesis. Mammalian target of rapamycin (mTOR) regulates protein synthesis and has been implicated as a potential mediator of the muscle mass decrement with HS. This study examined the effect of resistance exercise, a muscle hypertrophy stimulant, on rates of protein synthesis after 4 days of HS in mature male Sprague-Dawley rats. Flywheel resistance exercise (2 sets x 25 repetitions) was conducted on days 2 and 4 of HS (HSRE). Sixteen hours after the last exercise bout, soleus muscles were assessed for in vitro rates of protein synthesis, with and without insulin (signaling agonist) and/or rapamycin (mTOR inhibitor). Results demonstrated that soleus mass was reduced (P < 0.05) with HS, but this loss of mass was not observed (P > 0.05) with HSRE. Muscle protein synthesis was diminished (P < 0.05) with HS, with or without insulin. HSRE also had reduced rates of synthesis without insulin; however, insulin administration yielded higher (P < 0.05) rates in HSRE compared with HS or control. Rapamycin diminished protein synthesis in all groups (P < 0.05), but insulin rescued synthesis rates in HS and HSRE to levels similar to insulin alone for each group, suggesting that alternate signaling pathways develop to increase protein synthesis with HS. These results demonstrate that the capacity for an augmented anabolic response to resistance exercise is maintained after 4 days of HS and is independent of a rapamycin-sensitive pathway. muscle atrophy; flywheel technology; phenylalanine; mammalian target of rapamycin; microgravity