Skeletal Muscle Maintenance Research Articles

Stem Cell Aging in Skeletal Muscle Regeneration and Disease.

Skeletal muscle comprises 30–40% of the weight of a healthy human body and is required for voluntary movements in humans. Mature skeletal muscle is formed by multinuclear cells, which are called myofibers. Formation of myofibers depends on the proliferation, differentiation, and fusion of muscle progenitor cells during development and after injury. Muscle progenitor cells are derived from muscle satellite (stem) cells (MuSCs), which reside on the surface of the myofiber but beneath the basement membrane. MuSCs play a central role in postnatal maintenance, growth, repair, and regeneration of skeletal muscle. In sedentary adult muscle, MuSCs are mitotically quiescent, but are promptly activated in response to muscle injury. Physiological and chronological aging induces MuSC aging, leading to an impaired regenerative capability. Importantly, in pathological situations, repetitive muscle injury induces early impairment of MuSCs due to stem cell aging and leads to early impairment of regeneration ability. In this review, we discuss (1) the role of MuSCs in muscle regeneration, (2) stem cell aging under physiological and pathological conditions, and (3) prospects related to clinical applications of controlling MuSCs.

The Autophagy Regulatory Molecule CSRP3 Interacts with LC3 and Protects Against Muscular Dystrophy.

CSRP3/MLP (cysteine-rich protein 3/muscle Lim protein), a member of the cysteine-rich protein family, is a muscle-specific LIM-only factor specifically expressed in skeletal muscle. CSRP3 is critical in maintaining the structure and function of normal muscle. To investigate the mechanism of disease in CSRP3 myopathy, we performed siRNA-mediated CSRP3 knockdown in chicken primary myoblasts. CSRP3 silencing resulted in the down-regulation of the expression of myogenic genes and the up-regulation of atrophy-related gene expressions. We found that CSRP3 interacted with LC3 protein to promote the formation of autophagosomes during autophagy. CSRP3-silencing impaired myoblast autophagy, as evidenced by inhibited autophagy-related ATG5 and ATG7 mRNA expression levels, and inhibited LC3II and Beclin-1 protein accumulation. In addition, impaired autophagy in CSRP3-silenced cells resulted in increased sensitivity to apoptosis cell death. CSRP3-silenced cells also showed increased caspase-3 and caspase-9 cleavage. Moreover, apoptosis induced by CSRP3 silencing was alleviated after autophagy activation. Together, these results indicate that CSRP3 promotes the correct formation of autophagosomes through its interaction with LC3 protein, which has an important role in skeletal muscle remodeling and maintenance.

Isolation, Culture, and Differentiation of Primary Myoblasts Derived from Muscle Satellite Cells

The skeletal muscle is key for body mobility and motor performance, but aging and diseases often lead to progressive loss of muscle mass due to wasting or degeneration of muscle cells. Muscle satellite cells (MuSCs) represent a population of tissue stem cells residing in the skeletal muscles and are responsible for homeostatic maintenance and regeneration of skeletal muscles. Growth, injury, and degenerative signals activate MuSCs, which then proliferate (proliferating MuSCs are called myoblasts), differentiate and fuse with existing multinuclear muscle cells (myofibers) to mediate muscle growth and repair. Here, we describe a protocol for isolating MuSCs from skeletal muscles of mice for in vitro analysis. In addition, we provide a detailed protocol on how to culture and differentiate primary myoblasts into myotubes and an immunofluorescent staining procedure to characterize the cells. These methods are essential for modeling regenerative myogenesis in vitro to understand the dynamics, function and molecular regulation of MuSCs.

Isolation of muscle stem cells from rat skeletal muscles.

Muscle stem cells (MuSCs) are involved in homeostatic maintenance of skeletal muscle and play a central role in muscle regeneration in response to injury. Thus, understanding MuSC autonomous properties is of fundamental importance for studies of muscle degenerative diseases and muscle plasticity. Rat, as an animal model, has been widely used in the skeletal muscle field, however rat MuSC isolation through fluorescence-activated cell sorting has never been described. This work validates a protocol for effective MuSC isolation from rat skeletal muscles. Tibialis anterior was harvested from female rats and digested for isolation of MuSCs. Three protocols, employing different cell surface markers (CD106, CD56, and CD29), were compared for their ability to isolate a highly enriched MuSC population. Cells isolated using only CD106 as a positive marker showed high expression of Pax7, ability to progress through myogenic lineage while in culture, and complete differentiation in serum-deprived conditions. The protocol was further validated in gastrocnemius, diaphragm, and the individual components of the pelvic floor muscle complex (coccygeus, iliocaudalis, and pubocaudalis), proving to be reproducible. CD106 is an efficient marker for reliable isolation of MuSCs from a variety of rat skeletal muscles.

Mitophagy Regulation in Skeletal Muscle: Effect of Endurance Exercise and Age

Mitochondria are essential energy-providing organelles that are required in the maintenance of healthy skeletal muscle. As such, the removal of damaged mitochondria, through mitophagy, is necessary to maintain mitochondrial quality. In aging muscle, mitochondrial content and function are often found to be reduced compared to young individuals. This occurs despite the fact that measures of mitophagy are elevated, suggesting that mitophagy is insufficiently high to remove all of the dysfunctional organelles in aging muscle. Recent evidence has shown that acute exercise promotes mitophagic signaling, leading to organelle degradation. This exercise-induced signaling is attenuated in aging muscle, suggesting that aging muscle loses its capacity for mitochondrial turnover in response to exercise. This contributes to the reduction in muscle health in elderly individuals. Chronic exercise training improves mitochondrial content and function, even in aging muscle, leading to reduced mitophagy signaling. Thus, exercise training should be prescribed for both young and elderly populations to promote the maintenance of a healthy mitochondrial pool, through the stimulation of both organelle biogenesis and mitophagy.

MicroRNAs facilitate skeletal muscle maintenance and metabolic suppression in hibernating brown bears.

Hibernating brown bears, Ursus arctos, undergo extended periods of inactivity and yet these large hibernators are resilient to muscle disuse atrophy. Physiological characteristics associated with atrophy resistance in bear muscle have been examined (e.g., muscle mechanics, neural activity) but roles for molecular signaling/regulatory mechanisms in the resistance to muscle wasting in bears still require investigation. Using quantitative reverse transcription PCR (RT-qPCR), the present study characterized the responses of 36 microRNAs linked with development, metabolism, and regeneration of skeletal muscle, in the vastus lateralis of brown bears comparing winter hibernating and summer active animals. Relative levels of mRNA of selected genes (mef2a, pax7, id2, prkaa1, and mstn) implicated upstream and downstream of the microRNAs were examined. Results indicated that hibernation elicited a myogenic microRNA, or "myomiR", response via MEF2A-mediated signaling. Upregulation of MEF2A-controlled miR-1 and miR-206 and respective downregulation of pax7 and id2 mRNA are suggestive of responses that promote skeletal muscle maintenance. Increased levels of metabolic microRNAs, such as miR-27, miR-29, and miR-33, may facilitate metabolic suppression during hibernation via mechanisms that decrease glucose uptake and fatty acid oxidation. This study identified myomiR-mediated mechanisms for the promotion of muscle regeneration, suppression of ubiquitin ligases, and resistance to muscle atrophy during hibernation mediated by observed increases in miR-206, miR-221, miR-31, miR-23a, and miR-29b. This was further supported by the downregulation of myomiRs associated with a muscle injury and inflammation (miR-199a and miR-223) during hibernation. The present study provides evidence of myomiR-mediated signaling pathways that are activated during hibernation to maintain skeletal muscle functionality in brown bears.

The emergence of protein arginine methyltransferases in skeletal muscle and metabolic disease

Protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the methylation of arginine residues on target proteins and thus alter the stability, localization, or activity of the substrate. In doing so, PRMTs mediate a variety of intracellular functions that are essential for survival. Additionally, PRMT dysregulation is involved in a number of the most prevalent health disorders, including cancer and neurodegenerative and cardiovascular diseases, as well as in the aging process. Investigations of PRMT biology in skeletal muscle cells began in 2002, and since then these enzymes have emerged as regulators of skeletal muscle phenotype determination, maintenance, and remodeling. Specifically, more recent in vivo studies have revealed that PRMTs impact multiple aspects of skeletal muscle biology, including satellite cell function and phenotypic plasticity in response to exercise and disuse. Skeletal muscle plays critically important roles in regulating whole body metabolism, and recent investigations have also begun elucidating PRMT expression and function under conditions of metabolic dysfunction. The goals of this review are to 1) summarize the literature on PRMT biology in skeletal muscle with a particular emphasis on the in vivo evidence and 2) survey PRMTs in metabolic disorders, namely, obesity and type 2 diabetes mellitus. We also identify notable knowledge gaps therein and present opportunities to further expand our understanding of these enzymes so critical to health and disease.

Cross-talk between TGF-β and PDGFRα signaling pathways regulates the fate of stromal fibro-adipogenic progenitors.

Fibro-adipogenic progenitors (FAPs) are tissue-resident mesenchymal stromal cells (MSCs) required for proper skeletal muscle development, regeneration and maintenance. However, FAPs are also responsible for fibro-fatty scar deposition following chronic damage. We aimed to investigate the role of functional cross-talk between TGF-β and PDGFRα signaling pathways in the fate of FAPs. Here, we show that the number of FAPs correlates with TGF-β levels and with extracellular matrix deposition during regeneration and repair. Interestingly, the expression of PDGFRα changed dynamically in the fibroblast lineage after injury. Furthermore, PDGFRα-dependent immediate early gene expression changed during regeneration and repair. We also found that TGF-β signaling reduces PDGFRα expression in FAPs, mouse dermal fibroblasts and in two related mesenchymal cell lines. Moreover, TGF-β promotes myofibroblast differentiation of FAPs but inhibits their adipogenicity. Accordingly, TGF-β impairs the expression of PDGFRα-dependent immediate early genes in a TGFBR1-dependent manner. Finally, pharmacological inhibition of PDGFRα activity with AG1296 impaired TGF-β-induced extracellular matrix remodeling, Smad2 signaling, myofibroblast differentiation and migration of MSCs. Thus, our work establishes a functional cross-talk between TGF-β and PDGFRα signaling pathways that is involved in regulating the biology of FAPs and/or MSCs.This article has an associated First Person interview with the first author of the paper.

I.8Reversible formation of TDP-43 assemblies during skeletal muscle regeneration

Skeletal muscle maintenance and repair is dependent on the resident adult muscle stem cell. Following an injury and in diseased muscle, stem cells are engaged to replace or repair damaged tissue, which requires that the stem cells exit quiescence, expand, fuse and rebuild the contractile apparatus. Severe, progressive neuromuscular diseases manifest a constant injury, possibly disrupting the timing of regeneration, and coupled with attempts to continuously regenerate, may contribute to loss of muscle tissue and function. Proteostasis is disrupted in muscle during neuromuscular disease progression and often accompanied by the accumulation of irreversible protein aggregates in the cytoplasm, which are considered cytotoxic. Tar DNA binding Protein 43 (TDP-43), is a common component of these cytotoxic cytoplasmic cellular aggregates, even though TDP-43 is often not mutated. Unexpectedly, we observed cytoplasmic TDP-43 in amyloid assemblies or myo-granules during muscle regeneration following an induced muscle injury in normal mice. Myo-granules appear following an induced injury and dissipate during muscle regeneration. In regenerating muscle, TDP-43 is associated with transcripts encoding sarcomeric structural proteins as well as vasolin containing protein. We propose that cytoplasmic TDP-43-containing assemblies are not cytotoxic but are required for muscle formation, providing protection, transport and localized translation for TDP-43-associated transcripts. The relationship of cytoplasmic TDP-43-containing aggregates in diseased skeletal muscle to myo-granules in normal muscle is not known, however, myo-granules can seed the formation of insoluble amyloid fibrils. Thus, the formation of insoluble cytoplasmic aggregates occurring in progressive neuromuscular diseases may arise from altered myo-granule composition or from aberrant formation or clearance of myo-granules during continuous regeneration occurring in diseased muscle. Determining the relationship between cytotoxic TDP-43-containing cytoplasmic aggregates and myo-granules is essential to better understand disease progression and for the development of new therapies aimed at eliminating or preventing the accumulation of insoluble cytoplasmic aggregates arising during the progression of neuromuscular diseases.

Giving combined medium-chain fatty acids and glucose protects against cancer-associated skeletal muscle atrophy.

Skeletal muscle volume is associated with prognosis of cancer patients. Maintenance of skeletal muscle is an essential concern in cancer treatment. In nutritional intervention, it is important to focus on differences in metabolism between tumor and skeletal muscle. We examined the influence of oral intake of glucose (0%, 10%, 50%) and 2% medium‐chain fatty acid (lauric acid, LAA, C12:0) on tumor growth and skeletal muscle atrophy in mouse peritoneal metastasis models using CT26 mouse colon cancer cells and HT29 human colon cancer cells. After 2 weeks of experimental breeding, skeletal muscle and tumor were removed and analyzed. Glucose intake contributed to prevention of skeletal muscle atrophy in a sugar concentration‐dependent way and also promoted tumor growth. LAA ingestion elevated the level of skeletal muscle protein and suppressed tumor growth by inducing tumor‐selective oxidative stress production. When a combination of glucose and LAA was ingested, skeletal muscle mass increased and tumor growth was suppressed. Our results confirmed that although glucose is an important nutrient for the prevention of skeletal muscle atrophy, it may also foster tumor growth. However, the ingestion of LAA inhibited tumor growth, and its combination with glucose promoted skeletal muscle integrity and function, without stimulating tumor growth. These findings suggest novel strategies for the prevention of skeletal muscle atrophy.

Walk the Line: The Role of Ubiquitin in Regulating Transcription in Myocytes.

The ubiquitin-proteasome offers novel targets for potential therapies with their specific activities and tissue localization. Recently, the expansion of our understanding of how ubiquitin ligases (E3s) specifically regulate transcription has demonstrated their roles in skeletal muscle, complementing their roles in protein quality control and protein degradation. This review focuses on skeletal muscle E3s that regulate transcription factors critical to myogenesis and the maintenance of skeletal muscle wasting diseases.

Limited Oxidative Stress Favors Resistance to Skeletal Muscle Atrophy in Hibernating Brown Bears (Ursus Arctos).

Oxidative stress, which is believed to promote muscle atrophy, has been reported to occur in a few hibernators. However, hibernating bears exhibit efficient energy savings and muscle protein sparing, despite long-term physical inactivity and fasting. We hypothesized that the regulation of the oxidant/antioxidant balance and oxidative stress could favor skeletal muscle maintenance in hibernating brown bears. We showed that increased expressions of cold-inducible proteins CIRBP and RBM3 could favor muscle mass maintenance and alleviate oxidative stress during hibernation. Downregulation of the subunits of the mitochondrial electron transfer chain complexes I, II, and III, and antioxidant enzymes, possibly due to the reduced mitochondrial content, indicated a possible reduction of the production of reactive oxygen species in the hibernating muscle. Concomitantly, the upregulation of cytosolic antioxidant systems, under the control of the transcription factor NRF2, and the maintenance of the GSH/GSSG ratio suggested that bear skeletal muscle is not under a significant oxidative insult during hibernation. Accordingly, lower levels of oxidative damage were recorded in hibernating bear skeletal muscles. These results identify mechanisms by which limited oxidative stress may underlie the resistance to skeletal muscle atrophy in hibernating brown bears. They may constitute therapeutic targets for the treatment of human muscle atrophy.

Interleukin 4 Moderately Affects Competence of Pluripotent Stem Cells for Myogenic Conversion.

Pluripotent stem cells convert into skeletal muscle tissue during teratoma formation or chimeric animal development. Thus, they are characterized by naive myogenic potential. Numerous attempts have been made to develop protocols enabling efficient and safe conversion of pluripotent stem cells into functional myogenic cells in vitro. Despite significant progress in the field, generation of myogenic cells from pluripotent stem cells is still challenging—i.e., currently available methods require genetic modifications, animal-derived reagents, or are long lasting—and, therefore, should be further improved. In the current study, we investigated the influence of interleukin 4, a factor regulating inter alia migration and fusion of myogenic cells and necessary for proper skeletal muscle development and maintenance, on pluripotent stem cells. We assessed the impact of interleukin 4 on proliferation, selected gene expression, and ability to fuse in case of both undifferentiated and differentiating mouse embryonic stem cells. Our results revealed that interleukin 4 slightly improves fusion of pluripotent stem cells with myoblasts leading to the formation of hybrid myotubes. Moreover, it increases the level of early myogenic genes such as Mesogenin1, Pax3, and Pax7 in differentiating embryonic stem cells. Thus, interleukin 4 moderately enhances competence of mouse pluripotent stem cells for myogenic conversion.

Exercise-Induced Mitohormesis for the Maintenance of Skeletal Muscle and Healthspan Extension.

Oxidative damage is one mechanism linking aging with chronic diseases including the progressive loss of skeletal muscle mass and function called sarcopenia. Thus, mitigating oxidative damage is a potential avenue to prevent or delay the onset of chronic disease and/or extend healthspan. Mitochondrial hormesis (mitohormesis) occurs when acute exposure to stress stimulates adaptive mitochondrial responses that improve mitochondrial function and resistance to stress. For example, an acute oxidative stress via mitochondrial superoxide production stimulates the activation of endogenous antioxidant gene transcription regulated by the redox sensitive transcription factor Nrf2, resulting in an adaptive hormetic response. In addition, acute stresses such as aerobic exercise stimulate the expansion of skeletal muscle mitochondria (i.e., mitochondrial biogenesis), constituting a mitohormetic response that protects from sarcopenia through a variety of mechanisms. This review summarized the effects of age-related declines in mitochondrial and redox homeostasis on skeletal muscle protein homeostasis and highlights the mitohormetic mechanisms by which aerobic exercise mitigates these age-related declines and maintains function. We discussed the potential efficacy of targeting the Nrf2 signaling pathway, which partially mediates adaptation to aerobic exercise, to restore mitochondrial and skeletal muscle function. Finally, we highlight knowledge gaps related to improving redox signaling and make recommendations for future research.

Roles of Wnt7a in embryo development, tissue homeostasis, and human diseases.

Human Wnt family comprises 19 proteins which are critical to embryo development and tissue homeostasis. Binding to different frizzled (FZD) receptor, Wnt7a initiates both β-catenin dependent pathway, and β-catenin independent pathways such as PI3K/Akt, RAC/JNK, and extracellular signal-regulated kinase 5/peroxisome proliferator-activated receptor-γ. In the embryo, Wnt7a plays a crucial role in cerebral cortex development, synapse formation, and central nervous system vasculature formation and maintenance. Wnt7a is also involved in the development of limb and female reproductive system. Wnt7a mutation leads to human limb malformations and animal female reproductive system defects. Wnt7a is implicated in homeostasis maintenance of skeletal muscle, cartilage, cornea and hair follicle, and Wnt7a treatment may be potentially applied in skeletal muscle dystrophy, corneal damage, wound repair, and hair follicle regeneration. Wnt7a plays dual roles in human tumors. Wnt7a is downregulated in lung cancers, functioning as a tumor suppressor, however, it is upregulated in several other malignancies such as ovarian cancer, breast cancer, and glioma, acting as a tumor promoter. Moreover, Wnt7a overexpression is associated with inflammation and fibrosis, but its roles need to be further investigated.

Mesenchymal Stromal Cells Are Required for Regeneration and Homeostatic Maintenance of Skeletal Muscle

The necessity of mesenchymal stromal cells, called fibroadipogenic progenitors (FAPs), in skeletal muscle regeneration and maintenance remains unestablished. We report the generation of a PDGFRαCreER knockin mouse model that provides a specific means of labeling and targeting FAPs. Depletion of FAPs using Cre-dependent diphtheria toxin expression results in loss of expansion of muscle stem cells (MuSCs) and CD45+ hematopoietic cells after injury and impaired skeletal muscle regeneration. Furthermore, FAP-depleted mice under homeostatic conditions exhibit muscle atrophy and loss of MuSCs, revealing that FAPs are required for the maintenance of both skeletal muscle and the MuSC pool. We also report that local tamoxifen metabolite delivery to target CreER activity in a single muscle, removing potentially confounding systemic effects of ablating PDGFRα+ cells distantly, also causes muscle atrophy. These data establish a critical role of FAPs in skeletal muscle regeneration and maintenance.

Human Muscle Protein Synthesis Rates after Intake of Hydrolyzed Porcine-Derived and Cows' Milk Whey Proteins-A Randomized Controlled Trial.

Background: Whey protein has been shown to be one of the best proteins to stimulate muscle protein synthesis rate (MPS), but other high quality proteins, e.g., animal/porcine-derived, could have similar effects. Objective: To investigate the effects of hydrolyzed porcine proteins from blood (HPB) and muscle (HPM), in comparison to hydrolyzed whey protein (HW), on MPS after intake of 15 g alone or 30 g protein as part of a mixed meal. We hypothesized that the postprandial MPS would be similar for porcine proteins and whey protein. Design: Eighteen men (mean ± SD age: 24 ± 1 year; BMI: 21.7 ± 0.4 kg/m2) participated in the randomized, double-blind, three-way cross-over study. Subjects consumed the three test products (HPB, HPM and HW) in a random order in two servings at each test day. Serving 1 consisted of a drink with 15 g protein and serving 2 of a drink with 30 g protein together with a mixed meal. A flood-primed continuous infusion of (ring-13C6) phenylalanine was performed and muscle biopsies, blood and urine samples were collected for determination of MPS, muscle free leucine, plasma amino acid concentrations and urea excretion. Results: There were no statistical differences between the MPS measured after consuming 15 g protein alone or 30 g with a mixed meal (p = 0.53) of HPB (0.048 ± 0.007 vs. 0.049 ± 0.008%/h, resp.), HPM (0.063 ± 0.011 vs. 0.062 ± 0.011 %/h, resp.) and HW (0.058 ± 0.007 vs. 0.071 ± 0.013%/h, resp.). However, the impact of protein type on MPS reached statistical tendency (HPB vs. HPM (p = 0.093) and HPB vs. HW (p = 0.067)) with no difference between HPM and HW (p = 0.88). Plasma leucine, branched-chain, essential and total amino acids were generally higher for HPB and HW than HPM (p < 0.01), which reflected their content in the proteins. Muscle-free leucine was higher for HPB than HW and HPM (p < 0.05). Conclusion: Hydrolyzed porcine proteins from blood and muscle resulted in an MPS similar to that of HW, although with a trend for porcine blood proteins to be inferior to muscle proteins and whey. Consequently, these porcine-derived muscle proteins can be used similarly to whey protein to support maintenance of skeletal muscle as part of supplements and ingredients in foods.

Massage Protects against Disuse Atrophy in Young Adult Rats

Maintenance of skeletal muscle (SkM) mass is essential for maintaining mobility and metabolic health. SkM unloading or disuse, such as extended bed rest or limb immobilization, leads to rapid SkM wasting. Interventions to prevent disuse atrophy have had limited success. We recently found that applying massage, in the form of cyclic compressive loading (CCL), during the reloading period after hindlimb unloading, results in a greater increase in SkM protein synthesis (MPS) and SkM fiber cross‐sectional area (CSA) compared to reloading alone in 10 month old male rats. The purpose of the current investigation was to determine if CCL has similar anabolic effects when applied during normal weight bearing (WB versus WBM) as an anabolic stimulus, or during hindlimb suspension (HS versus HSM) to prevent muscle loss. We hypothesized that CCL applied during WB or HS would enhance (WBM) or protect against SkM fiber CSA loss (HSM), respectively, by increasing ribosome biogenesis and MPS. Adult (10 month) male F344/Brown Norway rats underwent either HS (n=8) or WB (n=8) for 7 days. In the treatment groups, CCL was applied directly to the gastrocnemius muscle at a load of 4.5 N and 0.5 Hz for 30 min. every other day for a total of 4 bouts during HSM (n=9) or WBM (n=9). WBM had no significant differences (NSD) in any anabolic parameters assessed compared to WB. CSA was significantly lower in HS vs WB (2373.5±267.6 vs 3796.7±80.2 mm2), with HSM significantly minimizing the loss compared to HS (2737.4±64.0 vs 2373.5±267.6 mm2). We used D2O and modeling for non‐steady state conditions to measure myofibrillar protein synthesis (Ksyn) and breakdown (Kdeg), and ribosomal biogenesis (rRNA Ksyn) and breakdown (rRNA Kdeg). HS and HSM had significant decreases in myofibrillar Ksyn vs WB (13.5±0.63 and 22.8±2.1 vs 35.5±2.3 mg/day), with HSM being significantly greater than HS. Myofibrillar Kdeg was significantly elevated in HS and HSM vs WB (.030±.001 and .030±.001 vs .018±.001 1/t), with NSD between HS and HSM. Further, in HSM only, the contralateral non‐massaged gastrocnemius also had significantly greater myofibrillar Ksyn and CSA (22.5±1.8 mg/day and 2713.5±96.5 mm2) vs HS. There were NSD in rRNA Ksyn between any group, which was despite a significant decrease in total RNA in HS and HSM vs WB (520.8±25.3 and 545.2±35.9 vs 600.5±42.0 ng/mg muscle), with NSD between HS and HSM. However, rRNA Kdeg was significantly elevated in HS and HSM vs WB (.045±.000 and .040±.000 vs .006±.000 1/t), with HSM being significantly reduced vs HS. In summary, massage in weight bearing rats did not affect any anabolic parameter assessed. In contrast, there were strong and significant effects of massage during HS (HSM) by minimizing the loss of CSA through enhancing myofibrillar protein synthesis and protecting against ribosomal breakdown vs HS alone. These findings support the highly translatable potential of CCL to protect against SkM loss during disuse by increasing MPS in either the massaged or contralateral non‐massaged limb.Support or Funding InformationThis research was supported by NIH grant R01AT009268 and R21AG042699.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High-Frequency Stimulation on Skeletal Muscle Maintenance in Female Cachectic Mice.

The purpose of this study was to determine whether high-frequency electric stimulation (HFES) could attenuate muscle mass loss during the progression of cancer cachexia in female tumor-bearing mice. Female wild-type (WT) and Apc (Min) mice (16-18 wk old) performed either repeated bouts or a single bout of HFES (10 sets of 6 repetitions, ~22 min), which eccentrically contracts the tibialis anterior (TA) muscle. TA myofiber size, oxidative capacity, anabolic signaling, and catabolic signaling were examined. Min had reduced TA muscle mass and type IIa and type IIb fiber sizes compared with WT. HFES increased the muscle weight and the mean cross-sectional area of type IIa and type IIb fibers in WT and Min mice. HFES increased mTOR signaling and myofibrillar protein synthesis and attenuated cachexia-induced AMPK activity. HFES attenuated the cachexia-associated decrease in skeletal muscle oxidative capacity. HFES in female mice can activate muscle protein synthesis through mTOR signaling and repeated bouts of contraction can attenuate cancer-induced muscle mass loss.

PUFA supplementation may confer enhanced metabolic capacity in stimulated skeletal muscle cells

Skeletal muscle is an energetically demanding tissue that catabolizes a variety of different substrates, a phenomenon that is largely activity‐dependent. Lipids are an energetically‐rich macromolecule and, in this light, are advantageous for skeletal muscle performance and maintenance. It has long been established that lipids have dichotomous lasting effects on the body that appear to be, at least in part, species‐specific. While lipid‐related pathologies are largely associated with species such as saturated fatty acids, diacylglycerols, and ceramides, other lipid species, such as various unsaturated fatty acids, confer adaptive changes and may actually protect against diseases. Lipids have previously been linked to increased aerobic capacity in skeletal muscle, though the role of fatty acid quality in this context (e.g., degree of saturation) is less clear. In the present study, we used C2C12 cells, an immortalized line of mouse skeletal muscle cells, to better understand how polyunsaturated fatty acids (PUFAs), in combination with exercise, affect metabolism and protein concentration in skeletal muscle. Following standard proliferation protocols, mature myotubes were differentiated for 7 days in either control media or media supplemented with a 5% addition of linoleic acid (PUFA); additionally, to stimulate contractile activity, a subset of cell cohorts from each media group were exposed to caffeine for 20 minutes per day during differentiation days 4–7. All cells were harvested for protein on differentiation day seven. Protein concentration across cohorts was measured using a Bradford protein assay. Metabolic changes were characterized by running functional enzyme assays for lactate dehydrogenase (LDH) and citrate synthase (CS) to compare anaerobic and aerobic changes, respectively. Preliminary data show that non‐exercised cohorts have higher protein concentration when supplemented PUFA, and that exercise, both with and without PUFA supplementation, decreases protein concentration as compared to respective non‐exercised cohorts. Regarding enzyme activity, PUFA supplementation appears to decrease LDH activity in non‐exercised cells, while PUFA supplementation appears to increase LDH activity in exercised cells; CS activity also appears to change in PUFA supplemented cohorts. Collectively, these preliminary data suggest activity‐induced adaptive changes in skeletal muscle metabolism are contingent upon specific substrate availability, whereby PUFA availability may advantageously prime skeletal muscle for a higher overall metabolic capacity.Support or Funding InformationMcNair Scholars Program and The College of Saint ScholasticaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.