bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2021‒06‒20
thirty-one papers selected by
Anna Vainshtein
Craft Science Inc.

  1. Cell Death Dis. 2021 Jun 12. 12(6): 611
      Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer-/- mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45+ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.
  2. Bone. 2021 Jun 07. pii: S8756-3282(21)00191-5. [Epub ahead of print]151 116029
      Osteoporosis commonly affects the elderly and is associated with significant morbidity and mortality. Loss of bone mineral density induces muscle atrophy and increases fracture risk. However, muscle lipid content and droplet size are increased by aging and mobility impairments, inversely correlated with muscle function, and a cause of reduced motor function. Teriparatide, the synthetic form of human parathyroid hormone (PTH) 1-34, has been widely used to treat osteoporosis. Although PTH positively affects muscle differentiation in vitro, the precise function and mechanisms of muscle mass and power preservation are still poorly understood, especially in vivo. In this study, we investigated the effect of PTH on skeletal muscle atrophy and dysfunction using an ovariectomized murine model. Eight-week-old female C57BL/6J mice were ovariectomized or sham-operated. Within each surgical group, the mice were divided into PTH injection or control subgroups. Motor function was evaluated based on grip strength, treadmill running, and lactic acid concentration. PTH receptor was expressed in skeletal muscle cells and myoblasts. PTH inhibited ovariectomy-induced bone loss but not uterine atrophy or increased body weight; PTH not only abolished ovariectomy-induced reduction in grip strength and maximum running speed, but also significantly reduced the ovariectomy-induced increase in lactic acid concentration (compared with that observed in the vehicle control). PTH also abrogated the ovariectomy-induced reduction in the oxidative capacity of muscle fibers, their cross-sectional area, and intramyocellular lipid content, and induced cell proliferation, cell migration, and muscle differentiation, while reducing lipid secretion by C2C12 myoblasts via the Wnt/β-catenin pathway. PTH significantly ameliorated muscle weakness and attenuated exercise-induced lactate levels in ovariectomized mice. Our in vitro study demonstrated that PTH/Wnt signaling regulated the proliferation, migration, and differentiation of myoblasts and also reduced lipid secretion in myoblasts. Thus, PTH could regulate several aspects of muscle function and physiology, and may represent a novel therapeutic strategy for patients with osteoporosis.
    Keywords:  Intramyocellular lipids; Ovariectomized murine model; Parathyroid hormone; Skeletal muscle; Wnt signal
  3. Sci Rep. 2021 Jun 18. 11(1): 12904
      The process of myogenesis which operates during skeletal muscle regeneration involves the activation of muscle stem cells, the so-called satellite cells. These then give rise to proliferating progenitors, the myoblasts which subsequently exit the cell cycle and differentiate into committed precursors, the myocytes. Ultimately, the fusion of myocytes leads to myofiber formation. Here we reveal a role for the transcriptional co-regulator nTRIP6, the nuclear isoform of the LIM-domain protein TRIP6, in the temporal control of myogenesis. In an in vitro model of myogenesis, the expression of nTRIP6 is transiently up-regulated at the transition between proliferation and differentiation, whereas that of the cytosolic isoform TRIP6 is not altered. Selectively blocking nTRIP6 function results in accelerated early differentiation followed by deregulated late differentiation and fusion. Thus, the transient increase in nTRIP6 expression appears to prevent premature differentiation. Accordingly, knocking out the Trip6 gene in satellite cells leads to deregulated skeletal muscle regeneration dynamics in the mouse. Thus, dynamic changes in nTRIP6 expression contributes to the temporal control of myogenesis.
  4. FEBS J. 2021 Jun 18.
      The characterization of fibro/adipogenic progenitor cells (FAPs) in the skeletal muscle has contributed to modify the monocentric view of muscle regeneration beyond muscle satellite cells (MuSCs). Now, we are aware that each population of the muscle niche plays a critical role in modulating homeostasis and regeneration. In the healthy muscle, FAPs contribute to maintain tissue homeostasis and assist MuSCs to cope with limited insults. Here, FAPs sense and integrate niche signals that keep in check their differentiation potential. The disruption of these niche cues leads to FAP differentiation into adipocytes and fibroblasts, both detrimental hallmarks of a large variety of muscle wasting diseases. FAP biology is still in its infancy and current efforts are focused on the understanding of the molecular circuits governing their double-edged behavior. The present review offers a detailed overview of the pathways and metabolic routes that can be modulated to halt and redirect their fibro/adipogenic potential while favoring their supportive role in muscle regeneration. Finally, we discuss on how single-cell technologies have contributed to resolve FAP transitional states with distinctive roles in muscle regeneration and myopathies.
    Keywords:  Duchenne Muscular Dystrophy; Fibro/adipogenic progenitors (FAPs); adipogenesis; fibrosis; muscle metabolism; muscle regeneration; muscle stem cells (MuSCs); muscle wasting; signaling pathways; single-cell
  5. J Muscle Res Cell Motil. 2021 Jun 17.
      SUMOylation is one of the post-translational modifications that involves the covalent attachment of the small ubiquitin-like modifier (SUMO) to the substrate. SUMOylation regulates multiple biological processes, including myoblast proliferation, differentiation, and apoptosis. 2-D08 is a synthetically available flavone, which acts as a potent cell-permeable SUMOylation inhibitor. Its mechanism of action involves preventing the transfer of SUMO from the E2 thioester to the substrate without influencing SUMO-activating enzyme E1 (SAE-1/2) or E2 Ubc9-SUMO thioester formation. However, both the effects and mechanisms of 2-D08 on C2C12 myoblast cells remain unclear. In the present study, we found that treatment with 2-D08 inhibits C2C12 cell proliferation and differentiation. We confirmed that 2-D08 significantly hampers the viability of C2C12 cells. Additionally, it inhibited myogenic differentiation, decreasing myosin heavy chain (MHC), MyoD, and myogenin expression. Furthermore, we confirmed that 2-D08-mediated anti-myogenic effects impair myoblast differentiation and myotube formation, reducing the number of MHC-positive C2C12 cells. In addition, we found that 2-D08 induces the activation of ErK1/2 and the degradation of MyoD and myogenin in C2C12 cells. Taken together, these results indicated that 2-D08 treatment results in the deregulated proliferation and differentiation of myoblasts. However, further research is needed to investigate the long-term effects of 2-D08 on skeletal muscles.
    Keywords:  2-D08; C2C12; Differentiation; Myogenesis; SUMOylation; Skeletal Muscle
  6. JCI Insight. 2021 Jun 15. pii: 149381. [Epub ahead of print]
      Neurogenic muscle atrophy is the loss of skeletal muscle mass and function that occurs with nerve injury and in denervating diseases such as amyotrophic lateral sclerosis. Aside from prompt restoration of innervation and exercise where feasible, there are currently no effective strategies for maintaining skeletal muscle mass in the setting of denervation. We conducted a longitudinal analysis of gene expression changes occurring in atrophying skeletal muscle, and identified Gadd45a as a gene that shows one of the earliest and most sustained increases in expression in skeletal muscle after denervation. We evaluated the role of this induction using genetic mouse models and found that mice lacking GADD45A show accelerated and exacerbated neurogenic muscle atrophy, as well as loss of fiber type identity. Our genetic analyses demonstrate that, rather than directly contributing to muscle atrophy as proposed in earlier studies, GADD45A induction likely represents a protective negative feedback response to denervation. Establishing the downstream effectors that mediate this protective effect and the pathways they participate in may yield new opportunities to modify the course of muscle atrophy.
    Keywords:  Muscle Biology; Neuromuscular disease; Neuroscience; Skeletal muscle
  7. Front Mol Biosci. 2021 ;8 685362
      In cancer patients, chemotherapeutic medication induces aberrant ROS (reactive oxygen species) accumulation in skeletal muscles, resulting in myofiber degradation, muscle weakness, and even cachexia, which further leads to poor therapeutic outcomes. Acting as an antioxidant, taurine is extensively used to accelerate postexercise muscle recovery in athletes. The antioxidant effects of taurine have been shown in mature myotubes and myofibers but not yet in myoblasts, the myotube precursor. The proliferation and differentiation ability of myoblasts play a very important role in myofiber repair and regeneration, which is usually impaired during chemotherapeutics in cancer patients as well. Here, we explored the effects of taurine supplementation on C2C12 myoblasts exposed to cisplatin-induced ROS. We found that cisplatin treatment led to dramatically decreased cell viability; accumulated ROS level; down-regulated expressions of MyoD1 (myoblast determination protein 1), myogenin, and MHC (myosin heavy chain); and impaired myotube differentiation in myoblasts. Significantly, taurine supplementation protected myoblasts against cisplatin-induced cell viability decrease, promoted cellular ROS clearance, and, most importantly, preserved the expressions of MyoD1, myogenin, and MHC as well as myotube differentiation ability. We further conducted NMR-based metabolomic analysis to clarify the underlying molecular mechanisms. We identified 14 characteristic metabolites primarily responsible for the discrimination of metabolic profiles between cisplatin-treated cells and normal counterparts, including increased levels of BCAAs (branched-chain amino acids: leucine and isoleucine), alanine, glycine, threonine, glucose, ADP (adenosine diphosphate), phenylalanine, and PC (O-phosphocholine), and decreased levels of lysine, β-alanine, choline, GPC (sn-glycero-3-phosphocholine), and myo-inositol. Evidently, taurine supplementation partially reversed the changing trends of several metabolites (isoleucine, threonine, glycine, PC, β-alanine, lysine, and myo-inositol). Furthermore, taurine supplementation promoted the proliferation and myotube differentiation of myoblasts by alleviating cellular catabolism, facilitating GSH (reduced glutathione) biosynthesis, improving glucose utilization and TCA (tricarboxylic acid) cycle anaplerosis, and stabilizing cellular membranes. Our results demonstrated the protective effects of taurine on cisplatin-impaired myoblasts and elucidated the mechanistic rationale for the use of taurine to ameliorate muscle toxicity in clinical chemotherapy cancer patients.
    Keywords:  C2C12 myoblasts; NMR-based metabolomics; ROS; cisplatin; myotube differentiation; taurine
  8. Mol Metab. 2021 Jun 10. pii: S2212-8778(21)00116-2. [Epub ahead of print] 101271
      OBJECTIVE: NAD+ is a co-factor and substrate for enzymes maintaining energy homeostasis. Nicotinamide phosphoribosyltransferase (NAMPT) controls NAD+ synthesis, and in skeletal muscle, NAD+ is important for muscle integrity. However, the underlying molecular mechanisms by which NAD+ synthesis affects muscle health remain poorly understood. Thus, the objective of the current study was to delineate the role of NAMPT-mediated NAD+ biosynthesis in skeletal muscle development and function.METHODS: To determine the role of Nampt in muscle development and function, we generated skeletal muscle-specific Nampt KO (SMNKO) mice. We performed a comprehensive phenotypic characterization of the SMNKO mice including metabolic measurements, histological examinations, and RNA sequencing analyses of skeletal muscle from SMNKO mice and WT littermates.
    RESULTS: SMNKO mice are smaller, with phenotypic changes in skeletal muscle, including reduced fiber area and increased number of centralized nuclei. The majority of SMNKO mice die prematurely. Transcriptomic analysis identified that the gene encoding the mitochondrial permeability transition pore (mPTP) regulator Cyclophilin D (Ppif) is upregulated in skeletal muscle of SMNKO mice from 2 weeks of age, with associated increased sensitivity of mitochondria to Ca2+-stimulated mPTP opening. Treatment of SMNKO mice with the Cyclophilin D inhibitor, Cyclosporine A, increased membrane integrity, decreased the number of centralized nuclei, and increased survival.
    CONCLUSION: Our study demonstrates that NAMPT is crucial for maintaining cellular Ca2+ homeostasis and skeletal muscle development, which is vital for juvenile survival.
    Keywords:  Cyclophilin D; NAD(+); mitochondrial permeability transition pore (mPTP); myopathy; nicotinamide riboside; sarcopenia
  9. J Cell Sci. 2021 Jun 15. pii: jcs258349. [Epub ahead of print]134(12):
      Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of both upper and lower motor neurons (MNs). The main clinical features of ALS are motor function impairment, progressive muscle weakness, muscle atrophy and, ultimately, paralysis. Intrinsic skeletal muscle deterioration plays a crucial role in the disease and contributes to ALS progression. Currently, there are no effective treatments for ALS, highlighting the need to obtain a deeper understanding of the molecular events underlying degeneration of both MNs and muscle tissue, with the aim of developing successful therapies. Muscle tissue is enriched in a group of microRNAs called myomiRs, which are effective regulators of muscle homeostasis, plasticity and myogenesis in both physiological and pathological conditions. After providing an overview of ALS pathophysiology, with a focus on the role of skeletal muscle, we review the current literature on myomiR network dysregulation as a contributing factor to myogenic perturbations and muscle atrophy in ALS. We argue that, in view of their critical regulatory function at the interface between MNs and skeletal muscle fiber, myomiRs are worthy of further investigation as potential molecular targets of therapeutic strategies to improve ALS symptoms and counteract disease progression.
    Keywords:  Amyotrophic lateral sclerosis; Muscle atrophy; Muscle homeostasis; MyomiRs
  10. Semin Cell Dev Biol. 2021 Jun 14. pii: S1084-9521(21)00140-3. [Epub ahead of print]
      The adult skeletal muscle fully regenerates after injury thanks to the properties of muscle stem cells that follow the adult myogenic program to replace damaged myofibers. Muscle regeneration also relies upon the coordinated actions of several other cell types, among which immune cells. Leukocytes infiltrate the damaged muscle soon after injury and support the regeneration process in a variety of ways, from the activation of muscle stem cells to the maturation of newly formed myofibers. Leukocytes also interact with other cell types such as fibroadipogenic precursors and endothelial cells. This review presents the interactions that leukocytes develop with the cells present in their vicinity and the impact they have on skeletal muscle regeneration.
    Keywords:  Macrophages; Resolution of inflammation; Skeletal muscle regeneration
  11. Front Pharmacol. 2021 ;12 599393
      NLRP3 inflammasome has been implicated in impaired post-injury muscle healing and in muscle atrophy. Histamine receptors play an important role in inflammation, but the role of histamine H3 receptor (H3R) in myocyte regeneration and in the regulation of NLRP3 inflammasome is not known. We studied the effects of H3R signaling on C2C12 myocyte viability, apoptosis, and tumor necrosis factor alpha (TNFα)-induced NLRP3 inflammasome activation during striated myogenic differentiation at three time points (days 0, 3, and 6). Expression of Nlrp3, interleukin-1β (IL-1β), and myogenesis markers were determined. TNFα reduced overall viability of C2C12 cells, and exposure to TNFα induced apoptosis of cells at D6. Activation of H3R had no effect on viability or apoptosis, whereas inhibition of H3R increased TNFα-induced apoptosis. Stimulation of C2C12 cells with TNFα increased Nlrp3 mRNA expression at D3 and D6. Moreover, TNFα reduced the expression of myogenesis markers MyoD1, Myogenin, and Myosin-2 at D3 and D6. H3R attenuated TNFα-induced expression of Nlrp3 and further inhibited the myogenesis marker expression; while H3R -blockage enhanced the proinflammatory effects of TNFα and increased the myogenesis marker expression. TNFα-induced secretion of mature IL-1β was dependent on the activation of the NLRP3 inflammasome, as shown by the reduced secretion of mature IL-1β upon treatment of the cells with the small molecule inhibitor of the NLRP3 inflammasome (MCC950). The activation of H3R reduced TNFα-induced IL-1β secretion, while the H3R blockage had an opposite effect. In conclusion, the modulation of H3R activity regulates the effects of TNFα on C2C12 myocyte differentiation and TNFα-induced activation of NLRP3 inflammasome. Thus, H3R signaling may represent a novel target for limiting postinjury muscle inflammation and muscle atrophy.
    Keywords:  C2C12 myocyte; IL-1β; NLRP3 inflammasome; TNFα; histamine H3 receptor; inflammation; myogenesis
  12. Cell Biochem Funct. 2021 Jun 15.
      Energetically inefficient inter-organ substrate shuttles are proposed contributors to cachexia-related weight loss. Here, we examined glycolytic pathway metabolites, enzyme activity and transport proteins in skeletal muscle, liver and tumours of mice with cachexia-related weight loss induced by colon-26 cancer cells. Skeletal muscle of cachexic mice had increased [L-lactate]/[pyruvate], LDH activity and lactate transporter MCT1. Cachexic livers also showed increased MCT1. This is consistent with the proposal that the rate of muscle-derived lactate shuttling to liver for use in gluconeogenesis is increased, that is, an increased Cori cycle flux in weight-losing cachexic mice. A second shuttle between liver and tumour may also contribute to disrupted energy balance and weight loss. We found increased high-affinity glucose transporter GLUT1 in tumours, suggesting active glucose uptake, tumour MCT1 detection and decreased intratumour [L-lactate]/[pyruvate], implying increased lactate efflux and/or intratumour lactate oxidation. Last, high [L-lactate]/[pyruvate] and MCT1 in cachexic muscle provides a potential muscle-derived lactate supply for the tumour (a 'reverse Warburg effect'), supporting tumour growth and consequent cachexia. Our findings suggest several substrate shuttles among liver, skeletal muscle and tumour contribute to metabolic disruption and weight loss. Therapies that aim to normalize dysregulated substrate shuttling among energy-regulating tissues may alleviate unintended weight loss in cancer cachexia. SIGNIFICANCE OF THE STUDY: Cachexia is a serious complication of cancer characterized by severe weight loss, muscle atrophy and frailty. Cachexia occurs in roughly half of all cancer patients, and in up to 80% of patients with advanced disease. Cachexia independently worsens patient prognosis, lowers treatment efficacy, increases hospitalization cost and length of stay, and accounts for 20-30% of cancer-related deaths. There are no effective treatments. Our findings suggest several substrate shuttles among liver, skeletal muscle and tumour contribute to metabolic disruption and weight loss in cancer cachexia. Identifying therapies that normalize dysregulated substrate shuttling among energy-regulating tissues may protect against cachexia-related weight loss.
    Keywords:  Colon-26; Cori cycle; Warburg effect; energy metabolism; glucose transporter; lactate; monocarboxylate transporter
  13. Front Cell Dev Biol. 2021 ;9 670435
      The unfolded protein response (UPR) plays important roles in various cells that have a high demand for protein folding, which are involved in the process of cell differentiation and development. Here, we separately knocked down the three sensors of the UPR in myoblasts and found that PERK knockdown led to a marked transformation in myoblasts from a fusiform to a rounded morphology, which suggests that PERK is required for early myoblast differentiation. Interestingly, knocking down PERK induced reprogramming of C2C12 myoblasts into stem-like cells by altering the miRNA networks associated with differentiation and stemness maintenance, and the PERK-ATF4 signaling pathway transactivated muscle differentiation-associated miRNAs in the early stage of myoblast differentiation. Furthermore, we identified Ppp1cc as a direct target gene of miR-128 regulated by the PERK signaling pathway and showed that its repression is critical for a feedback loop that regulates the activity of UPR-associated signaling pathways, leading to cell migration, cell fusion, endoplasmic reticulum expansion, and myotube formation during myoblast differentiation. Subsequently, we found that the RNA-binding protein ARPP21, encoded by the host gene of miR-128-2, antagonized miR-128 activity by competing with it to bind to the 3' untranslated region (UTR) of Ppp1cc to maintain the balance of the differentiation state. Together, these results reveal the crucial role of PERK signaling in myoblast maintenance and differentiation and identify the mechanism underlying the role of UPR signaling as a major regulator of miRNA networks during early differentiation of myoblasts.
    Keywords:  C2C12 (mouse skeletal myoblasts); PERK signaling; differentiation; microRNA network; myoblasts
  14. Nutr Metab (Lond). 2021 Jun 12. 18(1): 61
      BACKGROUND: Previous work in HEK-293 cells demonstrated the importance of amino acid-induced mTORC1 translocation to the lysosomal surface for stimulating mTORC1 kinase activity and protein synthesis. This study tested the conservation of this amino acid sensing mechanism in human skeletal muscle by treating subjects with chloroquine-a lysosomotropic agent that induces in vitro and in vivo lysosome dysfunction.METHODS: mTORC1 signaling and muscle protein synthesis (MPS) were determined in vivo in a randomized controlled trial of 14 subjects (10 M, 4 F; 26 ± 4 year) that ingested 10 g of essential amino acids (EAA) after receiving 750 mg of chloroquine (CHQ, n = 7) or serving as controls (CON, n = 7; no chloroquine). Additionally, differentiated C2C12 cells were used to assess mTORC1 signaling and myotube protein synthesis (MyPS) in the presence and absence of leucine and the lysosomotropic agent chloroquine.
    RESULTS: mTORC1, S6K1, 4E-BP1 and rpS6 phosphorylation increased in both CON and CHQ 1 h post EAA ingestion (P < 0.05). MPS increased similarly in both groups (CON, P = 0.06; CHQ, P < 0.05). In contrast, in C2C12 cells, 1 mM leucine increased mTORC1 and S6K1 phosphorylation (P < 0.05), which was inhibited by 2 mg/ml chloroquine. Chloroquine (2 mg/ml) was sufficient to disrupt mTORC1 signaling, and MyPS.
    CONCLUSIONS: Chloroquine did not inhibit amino acid-induced activation of mTORC1 signaling and skeletal MPS in humans as it does in C2C12 muscle cells. Therefore, different in vivo experimental approaches are required for confirming the precise role of the lysosome and amino acid sensing in human skeletal muscle. Trial registration NCT00891696. Registered 29 April 2009.
    Keywords:  Amino acid sensing; Chloroquine; Muscle protein turnover; mTOR signaling
  15. J Physiol. 2021 Jun 18.
      KEY POINTS: Maximal endurance performance is greater in the early daytime Timed exercise differentially alters the muscle transcriptome and (phospho)-proteome Early daytime exercise triggers energy provisioning and tissue regeneration Early nighttime exercise activates stress-related and catabolic pathways Scheduled training has limited effects on the muscle and liver circadian clocks ABSTRACT: Timed physical activity might potentiate the health benefits of training. The underlying signaling events triggered by exercise at different times of the day are, however, poorly understood. Here, we found that time-dependent variations in maximal treadmill exercise capacity of naïve mice were associated with energy stores, mostly hepatic glycogen levels. Importantly, running at different times of the day resulted in a vastly different activation of signaling pathways, e.g., related to stress response, vesicular trafficking, repair, and regeneration. Second, voluntary wheel running at the opposite phase of the dark, feeding period surprisingly revealed minimal Zeitgeber (i.e., phase-shifting) effect of training on the muscle clock. This integrated study provides important insights into the circadian regulation of endurance performance and the control of the circadian clock by exercise. In future studies, these results could contribute to better understand circadian aspects of training design in athletes and the application of chrono-exercise-based interventions in patients. Geraldine Maier completed her PhD in Biomedical Research in 2020 at the Biozentrum, Univerity of Basel (Switzerland). Julien Delezie completed his PhD in Neurobiology in 2012 at the Univerity of Strasbourg (France). Both are interested in exercise and circadian biology and, in particular, how the circadian clock promotes skeletal muscle and overall health. This article is protected by copyright. All rights reserved.
    Keywords:  Zeitgeber; circadian clock; energy homeostasis; exercise; metabolism; proteomics; skeletal muscle; transcriptomics
  16. Diabetologia. 2021 Jun 16.
      AIMS/HYPOTHESIS: Increased levels of branched-chain amino acids (BCAAs) are associated with type 2 diabetes pathogenesis. However, most metabolomic studies are limited to an analysis of plasma metabolites under fasting conditions, rather than the dynamic shift in response to a metabolic challenge. Moreover, metabolomic profiles of peripheral tissues involved in glucose homeostasis are scarce and the transcriptomic regulation of genes involved in BCAA catabolism is partially unknown. This study aimed to identify differences in circulating and skeletal muscle BCAA levels in response to an OGTT in individuals with normal glucose tolerance (NGT) or type 2 diabetes. Additionally, transcription factors involved in the regulation of the BCAA gene set were identified.METHODS: Plasma and vastus lateralis muscle biopsies were obtained from individuals with NGT or type 2 diabetes before and after an OGTT. Plasma and quadriceps muscles were harvested from skeletal muscle-specific Ppargc1a knockout and transgenic mice. BCAA-related metabolites and genes were assessed by LC-MS/MS and quantitative RT-PCR, respectively. Small interfering RNA and adenovirus-mediated overexpression techniques were used in primary human skeletal muscle cells to study the role of PPARGC1A and ESRRA in the expression of the BCAA gene set. Radiolabelled leucine was used to analyse the impact of oestrogen-related receptor α (ERRα) knockdown on leucine oxidation.
    RESULTS: Impairments in BCAA catabolism in people with type 2 diabetes under fasting conditions were exacerbated after a glucose load. Branched-chain keto acids were reduced 37-56% after an OGTT in the NGT group, whereas no changes were detected in individuals with type 2 diabetes. These changes were concomitant with a stronger correlation with glucose homeostasis biomarkers and downregulated expression of branched-chain amino acid transaminase 2, branched-chain keto acid dehydrogenase complex subunits and 69% of downstream BCAA-related genes in skeletal muscle. In primary human myotubes overexpressing peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, encoded by PPARGC1A), 61% of the analysed BCAA genes were upregulated, while 67% were downregulated in the quadriceps of skeletal muscle-specific Ppargc1a knockout mice. ESRRA (encoding ERRα) silencing completely abrogated the PGC-1α-induced upregulation of BCAA-related genes in primary human myotubes.
    CONCLUSIONS/INTERPRETATION: Metabolic inflexibility in type 2 diabetes impacts BCAA homeostasis and attenuates the decrease in circulating and skeletal muscle BCAA-related metabolites after a glucose challenge. Transcriptional regulation of BCAA genes in primary human myotubes via PGC-1α is ERRα-dependent.
    Keywords:  Branched-chain amino acid; Oestrogen-related receptor α; Oral glucose tolerance test; Peroxisome proliferator-activated receptor γ coactivator 1-α; Skeletal muscle; Type 2 diabetes
  17. Front Cell Dev Biol. 2021 ;9 656604
      Skeletal muscle protein synthesis is a highly complex process, influenced by nutritional status, mechanical stimuli, repair programs, hormones, and growth factors. The molecular aspects of protein synthesis are centered around the mTORC1 complex. However, the intricacies of mTORC1 regulation, both up and downstream, have expanded overtime. Moreover, the plastic nature of skeletal muscle makes it a unique tissue, having to coordinate between temporal changes in myofiber metabolism and hypertrophy/atrophy stimuli within a tissue with considerable protein content. Skeletal muscle manages the push and pull between anabolic and catabolic pathways through key regulatory proteins to promote energy production in times of nutrient deprivation or activate anabolic pathways in times of nutrient availability and anabolic stimuli. Branched-chain amino acids (BCAAs) can be used for both energy production and signaling to induce protein synthesis. The metabolism of BCAAs occur in tandem with energetic and anabolic processes, converging at several points along their respective pathways. The fate of intramuscular BCAAs adds another layer of regulation, which has consequences to promote or inhibit muscle fiber protein anabolism. This review will outline the general mechanisms of muscle protein synthesis and describe how metabolic pathways can regulate this process. Lastly, we will discuss how BCAA availability and demand coordinate with synthesis mechanisms and identify key factors involved in intramuscular BCAA trafficking.
    Keywords:  AMPK (5′-AMP activated kinase); BCKD; branch chain amino acids; branched-chain α-ketoacid dehydrogenase; mammalian target of rapamycin; protein synthesis; skeletal muscle
  18. J Biol Chem. 2021 Jun 12. pii: S0021-9258(21)00674-8. [Epub ahead of print] 100874
      In skeletal muscle tissue, an intriguing mechanical coupling exists between two ion channels from different membranes: the L-type voltage-gated calcium channel (CaV1.1), located in the plasma membrane, and Ryanodine Receptor 1 (RyR1) located in the sarcoplasmic reticulum membrane. Excitable cells rely on Cavs to initiate Ca2+ entry in response to action potentials. RyRs can amplify this signal by releasing Ca2+ from internal stores. Whereas this process can be mediated through Ca2+ as a messenger, an overwhelming amount of evidence suggests that RyR1 has recruited CaV1.1 directly as its voltage sensor. The exact mechanisms that underlie this coupling have been enigmatic, but a recent wave of reports have illuminated the coupling protein STAC3 as a critical player. Without STAC3, the mechanical coupling between Cav1.1 and RyR1 is lost, and muscles fail to contract. Various sequence variants of this protein have been linked to congenital myopathy. Other STAC isoforms are expressed in the brain and may serve as regulators of L-type CaVs. Despite the short length of STACs, several points of contacts have been proposed between them and CaVs. However, it is currently unclear whether STAC3 also forms direct interactions with RyR1, and whether this modulates RyR1 function. In this review, we discuss the 3D architecture of STAC proteins, the biochemical evidence for their interactions, the relevance of these connections for functional modulation, and their involvement in myopathy.
    Keywords:  adaptor protein; allosteric regulation; calcium channel; excitation-contraction coupling; ryanodine receptor; skeletal muscle; structure-function
  19. Sci Rep. 2021 Jun 10. 11(1): 12301
      Oxidative and glycolytic muscle fibers differ in their ultrastructure, metabolism, and responses to physiological stimuli and pathological insults. We examined whether these fibers respond differentially to exogenous anabolic androgenic steroids (AASs) by comparing morphological and histological changes between the oxidative anterior latissimus dorsi (ALD) and glycolytic pectoralis major (PM) fibers in adult avian muscles. Adult female White Leghorn chickens (Gallus gallus) were randomly divided into five groups: a vehicle control and four mesterolone treatment groups (4, 8, 12, and 16 mg/kg). Mesterolone was administered orally every three days for four weeks. Immunocytochemical techniques and morphometric analyses were employed to measure the changes in muscle weight, fiber size, satellite cell (SC) composition, and number of myonuclei. Mesterolone increased both body and muscle weights and induced hypertrophy in glycolytic PM fibers but not in oxidative ALD fibers. Mesterolone induced SC proliferation in both muscles; however, the myonuclear accretion was noticeable only in the PM muscle. In both muscles, the collective changes maintained a constant myonuclear domain size and the changes were dose independent. In conclusion, mesterolone induced distinct dose-independent effects in avian oxidative and glycolytic skeletal muscle fibers; these findings might be clinically valuable in the treatment of age-related sarcopenia.
  20. Front Physiol. 2021 ;12 670381
      Background: The cause of sarcopenia has been observed over decades by clinical trials, which, however, are still insufficient to systematically unravel the enigma of how resistance exercise mediates skeletal muscle mass. Materials and Methods: Here, we proposed a minimal regulatory network and developed a dynamic model to rigorously investigate the mechanism of sarcopenia. Our model is consisted of eight ordinary differential equations and incorporates linear and Hill-function terms to describe positive and negative feedbacks between protein species, respectively. Results: A total of 720 samples with 10 scaled intensities were included in simulations, which revealed the expression level of AKT (maximum around 3.9-fold) and mTOR (maximum around 5.5-fold) at 3, 6, and 24 h at high intensity, and non-monotonic relation (ranging from 1.2-fold to 1.7-fold) between the graded intensities and skeletal muscle mass. Furthermore, continuous dynamics (within 24 h) of AKT, mTOR, and other proteins were obtained accordingly, and we also predicted the delaying effect with the median of maximized muscle mass shifting from 1.8-fold to 4.6-fold during a 4-fold increase of delay coefficient. Conclusion: The de novo modeling framework sheds light on the interdisciplinary methodology integrating computational approaches with experimental results, which facilitates the deeper understandings of exercise training and sarcopenia.
    Keywords:  gut microbiota; mathematical model; protein synthesis; resistance training; sarcopenia
  21. Mol Cell Endocrinol. 2021 Jun 11. pii: S0303-7207(21)00208-2. [Epub ahead of print] 111364
      Capmatinib (CAP) has been used to treat metastatic non-small lung cancer (NSCL) and suppress inflammation. It causes hypoglycemia in NSCL patients. Therefore, it is expected that CAP improves inflammation-mediated insulin resistance due to its anti-inflammatory effect. However, the impacts of CAP on insulin signaling in skeletal muscle cells have not yet been fully elucidated. Herein, we investigated the effect of CAP on insulin resistance in palmitate-treated C2C12 myocytes and explored the related molecular mechanisms. We found that treatment of C2C12 myocytes with CAP reversed palmitate-induced impairment of insulin signaling and glucose uptake. CAP treatment ameliorated phosphorylation of inflammatory markers, including NFκB and IκB, in palmitate-treated C2C12 myocytes. Further, it augmented PPARδ expression and suppressed palmitate-induced p38 phosphorylation in a dose-dependent manner. siRNA-mediated suppression of PPARδ abolished the effects of CAP on palmitate-induced insulin resistance and inflammation as well as p38 phosphorylation. Therefore, it has been shown that CAP treatment ameliorates insulin resistance in palmitate-treated C2C12 myocytes via PPARδ/p38 signaling-mediated suppression of inflammation. These results may represent a novel therapeutic approach that could halt insulin resistance and type 2 diabetes.
    Keywords:  Capmatinib; PPARδ; inflammation; insulin resistance; myocyte; p38
  22. Am J Physiol Renal Physiol. 2021 06 14.
      Preclinical models of chronic kidney disease (CKD) are critical to investigate the mechanisms of disease and to evaluate novel therapeutics aimed to treat CKD-associated pathologies. The objective of the present study was to compare the adenine diet and 5/6 nephrectomy (5/6 Nx) models in mice. Male and female 10-week-old C57BL/6J mice (N=5-9/sex/group) were randomly allocated to CKD groups (0.2-0.15% adenine-supplemented diet or 5/6 Nx surgery) or corresponding control groups (casein diet or sham surgery). Glomerular filtration rate was reduced to a similar level in adenine and 5/6 Nx mice (adenine male: 81.1 ± 41.9 µL/min vs. 5/6 Nx male: 160 ± 80.9 µL/min, P=0.5875; adenine female: 112.9 ± 32.4 µL/min vs. 5/6 Nx female: 107.0 ± 45.7 µL/min, P=0.9995). Serum metabolomics analysis indicated that established uremic toxins were robustly elevated in both CKD models, although some differences were observed between CKD models (i.e. p-Cresol sulfate). Dysregulated phosphate homeostasis was observed in the adenine model only, whereas calcium homeostasis was disturbed in male mice with both models. Muscle mass and myofiber cross-sectional area of extensor digitorum longus and soleus muscles were ~18-24% smaller in male CKD mice regardless of model, but were not different in female CKD mice (P>0.05). Skeletal muscle mitochondrial respiratory function was significantly decreased (19-24%) in CKD mice in both models and sexes. These findings demonstrate that adenine diet and 5/6 Nx models of CKD have similar levels of renal dysfunction and skeletal myopathy, but had less mortality (P<0.05 for both sexes) compared with the 5/6 Nx model.
    Keywords:  atrophy; cachexia; metabolism; mitochondria; uremia
  23. J Appl Physiol (1985). 2021 06 17.
      Chronic obesity and insulin resistance are considered to inhibit contraction-induced muscle hypertrophy, through impairment of mTORC1 and muscle protein synthesis (MPS). A high-fat diet is known to rapidly induce obesity and insulin resistance within a month. However, the influence of a short-term high-fat diet on the response of mTORC1 activation and MPS to acute resistance exercise (RE) is unclear. Thus, the purpose of this study was to investigate the effect of a short-term high-fat diet on the response of mTORC1 activation and MPS to acute RE. Male Sprague-Dawley rats were randomly assigned to groups and fed a normal diet (ND), high-fat diet (HFD 4wk), or pair feed (PF 4wk) for 4 weeks. After dietary habituation, acute RE was performed on the gastrocnemius muscle via percutaneous electrical stimulation. The results showed that 4 weeks of a high fat-diet induced intramuscular lipid accumulation and insulin resistance, without affecting basal mTORC1 activity or MPS. The response of RE-induced mTORC1 activation and MPS was not altered by a high-fat diet. On the other hand, analysis of each fiber type demonstrated that response of MPS to an acute RE was disappeared specifically in type I and IIa fiber. These results indicate that a short-term high-fat diet causes anabolic resistance to acute RE, depending on the fiber type.
    Keywords:  high-fat diet; muscle protein synthesis; resistance exercise; mTOR
  24. Biochem J. 2021 Jun 15. pii: BCJ20210264. [Epub ahead of print]
      Reductions in mitochondrial function have been proposed to cause insulin resistance, however the possibility that impairments in insulin signaling negatively affects mitochondrial bioenergetics has received little attention. Therefore, we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a key process linked to oxidative phosphorylation and redox balance, and if this phenomenon would be lost following high-fat diet (HFD)-induced insulin resistance. Insulin acutely (60 minutes post I.P.) increased submaximal (100-1000 μM ADP) mitochondrial respiration ~2-fold without altering maximal (>1000 μM ADP) respiration, suggesting insulin rapidly improves mitochondrial bioenergetics. The consumption of HFD impaired submaximal ADP-supported respiration ~50%, however, despite the induction of insulin resistance, the ability of acute insulin to stimulate ADP sensitivity and increase submaximal respiration persisted. While these data suggest that insulin mitigates HFD-induced impairments in mitochondrial bioenergetics, the presence of a high intracellular lipid environment reflective of an HFD (i.e. presence of palmitoyl-CoA) completely prevented the beneficial effects of insulin. Altogether, these data show that while insulin rapidly stimulates mitochondrial bioenergetics through an improvement in ADP sensitivity, this phenomenon is possibly lost following HFD due to the presence of intracellular lipids.
    Keywords:  insulin resistance; mitochondria; muscle metabolism; obesity; skeletal muscle
  25. PLoS One. 2021 ;16(6): e0253269
      AMP-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor. Activation of AMPK leads to a number of metabolic benefits, including improved mitochondrial function in skeletal muscle and lowering of serum glucose levels in type-2 diabetes models. However, direct activation of AMPK leads to cardiac enlargement, and an alternative strategy that activates AMPK without affecting the heart is needed. Inhibition of phosphodiesterase 4 (PDE4), which is poorly expressed in the human heart, activates AMPK in other tissues. In a screen to identify novel PDE4 inhibitors, we discovered compound CBU91, which is 5-10 fold more potent than rolipram, the best characterized PDE4 inhibitor. CBU91, like rolipram, is able to activate AMPK and Sirt1 and increase mitochondrial function in myotubes. These findings suggest that activation of AMPK in myotubes is a general property of PDE4 inhibition and that PDE4 inhibition may activate AMPK in metabolically relevant tissues without affecting the heart.
  26. Sci Rep. 2021 Jun 15. 11(1): 12572
      Ischemia reperfusion (IR) injury plays a pivotal role in many diseases and leads to collateral damage during surgical interventions. While most studies focus on alleviating its severity in the context of brain, liver, kidney, and cardiac tissue, research as regards to skeletal muscle has not been conducted to the same extent. In the past, myostatin (MSTN), primarily known for supressing muscle growth, has been implicated in inflammatory circuits, and research provided promising results for cardiac IR injury mitigation by inhibiting MSTN cell surface receptor ACVR2B. This generated the question if interrupting MSTN signaling could temper IR injury in skeletal muscle. Examining human specimens from free myocutaneous flap transfer demonstrated increased MSTN signaling and tissue damage in terms of apoptotic activity, cell death, tissue edema, and lipid peroxidation. In subsequent in vivo MstnLn/Ln IR injury models, we identified potential mechanisms linking MSTN deficiency to protective effects, among others, inhibition of p38 MAPK signaling and SERCA2a modulation. Furthermore, transcriptional profiling revealed a putative involvement of NK cells. Collectively, this work establishes a protective role of MSTN deficiency in skeletal muscle IR injury.
  27. High Alt Med Biol. 2021 Jun 17.
      Slivka, Dustin, Charles Dumke, Walter Hailes, and Brent Ruby. Impact of hypoxic exercise recovery on skeletal muscle glycogen and gene expression. High Alt Med Biol 00:000-000, 2021. Background: The impact of altitude during recovery from exercise is largely unknown. The purpose of this study was to determine the acute gene response and muscle glycogen re-synthesis after exercise when exposed to simulated high altitude during recovery. Materials and Methods: Twelve male participants (age, 25 ± 2 years; height, 178 ± 7 cm; weight, 78.8 ± 7.8 kg; VO2peak, 4.25 ± 0.59 l/min; Wpeak 307 ± 44 W; and body fat, 13.1% ± 1.2%) completed two trials (random order), which consisted of cycling for 90 minutes in laboratory conditions and then recovering for 6 hours in laboratory conditions (975 m; normoxia) or at a high simulated altitude (5,000 m; hypoxia). Results: Skeletal muscle biopsies from the vastus lateralis were obtained before exercise, after exercise, and 6 hours after exercise for the measurement of metabolic gene expression and muscle glycogen. Muscle glycogen decreased with exercise (61% ± 13%, p < 0.05) and increased with recovery (78% ± 35%, p < 0.05) with no difference between trials (p > 0.05). Hypoxia-inducible factor (HIF)-1α, HIF-2α, optic atrophy gene 1 (OPA-1), mitofusin 2 (MFN-2), and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) gene expression were suppressed after altitude exposure (p < 0.05), while mitochondrial fission 1 protein (FIS-1), phosphofructokinase (PFK), Cytochrome c oxidase (COX), and hexokinase (HK) were unaffected by altitude exposure (p > 0.05). Conclusions: High-altitude exposure during recovery from exercise inhibits gene expression associated with mitochondrial development without affecting muscle glycogen re-synthesis.
    Keywords:  HIF-1; PGC-1α; altitude; biopsy; environmental; hypoxia; mRNA
  28. Elife. 2021 Jun 14. pii: e68211. [Epub ahead of print]10
      Time-resolved X-ray diffraction from isolated fast-twitch muscles of the mouse was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by complete recovery of the folded motor conformation on the filament backbone but incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscle.
    Keywords:  molecular biophysics; mouse; physics of living systems; structural biology
  29. Cell Death Dis. 2021 Jun 16. 12(7): 625
      Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.
  30. Sci Rep. 2021 Jun 10. 11(1): 12317
      Aging is associated with widespread physiological changes, including skeletal muscle weakening, neuron system degeneration, hair loss, and skin wrinkling. Previous studies have identified numerous molecular biomarkers involved in these changes, but their regulatory mechanisms and functional repercussions remain elusive. In this study, we conducted next-generation sequencing of DNA methylation and RNA sequencing of blood samples from 51 healthy adults between 20 and 74 years of age and identified aging-related epigenetic and transcriptomic biomarkers. We also identified candidate molecular targets that can reversely regulate the transcriptomic biomarkers of aging by reconstructing a gene regulatory network model and performing signal flow analysis. For validation, we screened public experimental data including gene expression profiles in response to thousands of chemical perturbagens. Despite insufficient data on the binding targets of perturbagens and their modes of action, curcumin, which reversely regulated the biomarkers in the experimental dataset, was found to bind and inhibit JUN, which was identified as a candidate target via signal flow analysis. Collectively, our results demonstrate the utility of a network model for integrative analysis of omics data, which can help elucidate inter-omics regulatory mechanisms and develop therapeutic strategies against aging.
  31. Sci Rep. 2021 Jun 15. 11(1): 12598
      Facioscapulohumeral muscular dystrophy (FSHD) is a debilitating muscle disease that currently does not have an effective cure or therapy. The abnormal reactivation of DUX4, an embryonic gene that is epigenetically silenced in somatic tissues, is causal to FSHD. Disease-specific reactivation of DUX4 has two common characteristics, the presence of a non-canonical polyadenylation sequence within exon 3 of DUX4 that stabilizes pathogenic transcripts, and the loss of repressive chromatin modifications at D4Z4, the macrosatellite repeat which encodes DUX4. We used CRISPR/Cas9 to silence DUX4 using two independent approaches. We deleted the DUX4 pathogenic polyadenylation signal, which resulted in downregulation of pathogenic DUX4-fl transcripts. In another approach, we transcriptionally repressed DUX4 by seeding heterochromatin using the dCas9-KRAB platform within exon 3. These feasibility of targeting DUX4 experiments were initially tested in a non-myogenic carcinoma cell line that we have previously characterized. Subsequently, in an immortalized patient myoblast cell line, we demonstrated that targeting DUX4 by either approach led to substantial downregulation of not only pathogenic DUX4 transcripts, but also a subset of its target genes that are known biomarkers of FSHD. These findings offer proof-of-concept of the effect of silencing the polyadenylation sequence on pathogenic DUX4 expression.