bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2021‒05‒23
fifty-five papers selected by
Anna Vainshtein
Craft Science Inc.

  1. Development. 2020 Jan 01. pii: dev.194027. [Epub ahead of print]
      Formation of skeletal muscle is among the most striking examples of cellular plasticity in animal tissue development, where muscle progenitor cells are reprogrammed by epithelial-mesenchymal transition (EMT) to produce multinucleated myofibers. The regulation of EMT in muscle formation remains poorly understood. Here, we demonstrate that the long noncoding RNA (lncRNA) Meg3 regulates EMT in myoblast differentiation and skeletal muscle regeneration. Chronic inhibition of Meg3 in C2C12 myoblasts induced EMT, and suppressed cell state transitions required for differentiation. Furthermore, adenoviral Meg3 knockdown compromised muscle regeneration, which was accompanied by abnormal mesenchymal gene expression and interstitial cell proliferation. Transcriptomic and pathway analyses of Meg3-depleted C2C12 myoblasts and injured skeletal muscle revealed a significant dysregulation of EMT-related genes, and identified TGFβ as a key upstream regulator. Importantly, inhibition of TGFβR1 and its downstream effectors, and the EMT transcription factor Snai2, restored many aspects of myogenic differentiation in Meg3-depleted myoblasts in vitro. We further demonstrate that reduction of Meg3-dependent Ezh2 activity results in epigenetic alterations associated with TGFβ activation. Thus, Meg3 regulates myoblast identity to maintain proper cell state for progression into differentiation.
    Keywords:  Cell identity; Epithelial-mesenchymal transition; Long noncoding RNA; Meg3; Muscle differentiation; Regeneration; TGFβ
  2. Biol Open. 2020 Jan 01. pii: bio.053918. [Epub ahead of print]
      In response to injury, skeletal muscle stem cells (MuSCs) undergo myogenesis where they become activated, proliferate rapidly, differentiate and undergo fusion to form multinucleated myotubes. Dramatic changes in cell size, shape, metabolism and motility occur during myogenesis which cause cellular stress and alter proteostasis. The molecular chaperone heat shock protein 70 (HSP70) maintains proteostasis by regulating protein biosynthesis and folding, facilitating transport of polypeptides across intracellular membranes and preventing stress-induced protein unfolding/aggregation. Although HSP70 overexpression can exert beneficial effects in skeletal muscle diseases and enhance skeletal muscle repair after injury, its effect on myogenesis has not been investigated. Plasmid-mediated overexpression of HSP70 did not affect the rate of C2C12 proliferation or differentiation, but the median number of myonuclei per myotube and median myotube width in differentiated C2C12 myotubes were increased with HSP70 overexpression. These findings reveal that increased HSP70 expression can promote myoblast fusion, identifying a mechanism for its therapeutic potential to enhance muscle repair after injury.
    Keywords:  C2C12; Fusion; Heat shock protein 70; Myogenesis; Skeletal muscle
  3. Cell Death Dis. 2021 May 19. 12(6): 514
      Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.
  4. Front Physiol. 2021 ;12 642908
      Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease characterized by progressive muscle weakness and wasting. Stimulation of AMP-activated protein kinase (AMPK) has been demonstrated to increase muscle function and protect muscle against damage in dystrophic mice. Metformin is a widely used anti-hyperglycemic drug and has been shown to be an indirect activator of AMPK. Based on these findings, we sought to determine the effects of metformin on neuromuscular deficits in mdx murine model of DMD. In this study, we found metformin treatment increased muscle strength accompanied by elevated twitch and tetanic force of tibialis anterior (TA) muscle in mdx mice. Immunofluorescence and electron microscopy analysis of metformin-treated mdx muscles revealed an improvement in muscle fiber membrane integrity. Electrophysiological studies showed the amplitude of miniature endplate potentials (mEPP) was increased in treated mice, indicating metformin also improved neuromuscular transmission of the mdx mice. Analysis of mRNA and protein levels from muscles of treated mice showed an upregulation of AMPK phosphorylation and dystrophin-glycoprotein complex protein expression. In conclusion, metformin can indeed improve muscle function and diminish neuromuscular deficits in mdx mice, suggesting its potential use as a therapeutic drug in DMD patients.
    Keywords:  AMP-activated protein kinase; Duchenne muscular dystrophy; metformin; neuromuscular junction; skeletal muscle
  5. Dis Model Mech. 2020 Jan 01. pii: dmm.044354. [Epub ahead of print]
      Skeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies (CNM), that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive, muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role of BIN1 on intracellular organization in addition to membrane remodeling. Whereas restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle-specific exon delayed muscle regeneration. Thus, BIN1 ubiquitous function is necessary for muscle development and function while its muscle-specific isoforms fine-tune muscle regeneration in adulthood, supporting that BIN1 centronuclear myopathy with congenital onset are due to developmental defects while later onset may be due to regeneration defects.
    Keywords:  Animal model; BAR domain; Centronuclear myopathy; Dynamin; Myoblast fusion; Myotonic dystrophy; Myotubular myopathy; SH3 domain; Triad; XLMTM
  6. Development. 2020 Jan 01. pii: dev.194480. [Epub ahead of print]
      Translational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent satellite cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2α (P-eIF2α). Pharmacological inhibition of the eIF2α phosphatase with the small molecule sal003 maintains P-eIF2α and permits the expansion of satellite cells ex vivo. Paradoxically, P-eIF2α also increases the translation of specific mRNAs, which is mediated by P-eIF2α dependent readthrough of inhibitory upstream open reading frames (uORFs). Here, we ask whether P-eIF2α dependent mRNA translation enables expansion of satellite cells. Using transcriptomic and proteomic analyses, we show a number of genes associated with the assembly of the spindle pole to be upregulated at the level of protein, without corresponding change in mRNA levels, in satellite cells expanded in the presence of sal003. We show that uORFs in the 5'UTR of mRNA for the mitotic spindle stability gene Tacc3 direct P-eIF2α dependent translation. Satellite cells deficient for TACC3 exhibit defects in expansion, self-renewal and regeneration of skeletal muscle.
    Keywords:  Muscle stem cell; Satellite cell; Skeletal muscle regeneration; TACC3
  7. Development. 2020 Jan 01. pii: dev.187344. [Epub ahead of print]
      Satellite cells (SC) are muscle stem cells which can regenerate adult muscles upon injury. Most SC originate from PAX7-positive myogenic precursors set aside during development. While myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced Pluripotent Stem (iPS) cell reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPS-derived PAX7-positive myogenic precursors. We show that the PAX7-positive cells generated in culture can produce myofibers and self-renew in vitro and in vivo. Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPS cells, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.
    Keywords:  Human development; PAX7; Pluripotent stem cell; Satellite cell; Skeletal muscle
  8. Development. 2020 Jan 01. pii: dev.185975. [Epub ahead of print]
      Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the Sine oculis homeobox related Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior (TA) muscle. Mutant stem cells form hypotrophic myofibers that are not innervated but retain the ability to self-renew.
    Keywords:  Homing; Pax7; Six1; Six4; Stem cells
  9. Mol Ther Methods Clin Dev. 2021 Jun 11. 21 559-573
      Although receptor-activator of nuclear factor κB (RANK), its ligand RANKL, and osteoprotegerin (OPG), which are members of the tumor necrosis factor (TNF) superfamily, were first discovered in bone cells, they are also expressed in other cells, including skeletal muscle. We previously showed that the RANK/RANKL/OPG pathway is involved in the physiopathology of Duchenne muscular dystrophy and that a mouse full-length OPG-Fc (mFL-OPG-Fc) treatment is superior to muscle-specific RANK deletion in protecting dystrophic muscles. Although mFL-OPG-Fc has a beneficial effect in the context of muscular dystrophy, the function of human FL-OPG-Fc (hFL-OPG-Fc) during muscle repair is not yet known. In the present study, we investigated the impacts of an hFL-OPG-Fc treatment following the intramuscular injection of cardiotoxin (CTX). We show that a 7-day hFL-OPG-Fc treatment improved force production of soleus muscle. hFL-OPG-Fc also improved soleus muscle integrity and regeneration by increasing satellite cell density and fiber cross-sectional area, attenuating neutrophil inflammatory cell infiltration at 3 and 7 days post-CTX injury, increasing the anti-inflammatory M2 macrophages 7 days post-CTX injury. hFL-OPG-Fc treatment also favored M2 over M1 macrophage phenotypic polarization in vitro. We show for the first time that hFL-OPG-Fc improved myotube maturation and fusion in vitro and reduced cytotoxicity and cell apoptosis. These findings demonstrate that hFL-OPG-Fc has therapeutic potential for muscle diseases in which repair and regeneration are impaired.
    Keywords:  Osteoprotegerin; RANK/RANKL/OPG; human full-length OPG-Fc; muscle injury; muscle repair; protein-Fc therapy
  10. Biochemistry (Mosc). 2021 05;86(5): 597-610
      Skeletal muscles comprise more than a third of human body mass and critically contribute to regulation of body metabolism. Chronic inactivity reduces metabolic activity and functional capacity of muscles, leading to metabolic and other disorders, reduced life quality and duration. Cellular models based on progenitor cells isolated from human muscle biopsies and then differentiated into mature fibers in vitro can be used to solve a wide range of experimental tasks. The review discusses the aspects of myogenesis dynamics and regulation, which might be important in the development of an adequate cell model. The main function of skeletal muscle is contraction; therefore, electrical stimulation is important for both successful completion of myogenesis and in vitro modeling of major processes induced in the skeletal muscle by acute or regular physical exercise. The review analyzes the drawbacks of such cellular model and possibilities for its optimization, as well as the prospects for its further application to address fundamental aspects of muscle physiology and biochemistry and explore cellular and molecular mechanisms of metabolic diseases.
    Keywords:  electrical stimulation; gene expression; metabolism; myogenesis; physical exercise; satellite cells; skeletal muscle
  11. Neuropathol Appl Neurobiol. 2021 May 17.
      Utrophin is an autosomal paralogue of dystrophin, a protein whose deficit causes Duchenne and Becker muscular dystrophies (DMD/BMD). Utrophin is naturally overexpressed at the sarcolemma of mature dystrophin-deficient fibres in DMD and BMD patients as well as in the mdx Duchenne mouse model. Dystrophin and utrophin can co-localize in human fetal muscle, in the dystrophin-competent fibres from DMD/BMD carriers, and revertant fibre clusters in biopsies from DMD patients. These findings suggest that utrophin overexpression could act as a surrogate, compensating for the lack of dystrophin, and, as such, it could be used in combination with dystrophin restoration therapies. Different strategies to overexpress utrophin are currently under investigation. In recent years, many compounds have been reported to modulate utrophin expression efficiently in preclinical studies and ameliorate the dystrophic phenotype in animal models of the disease. In this manuscript, we discuss the current knowledge on utrophin protein and the different mechanisms that modulate its expression in skeletal muscle. We also include a comprehensive review of compounds proposed as utrophin regulators and, as such, potential therapeutic candidates for these muscular dystrophies.
    Keywords:  Becker muscular dystrophy; Duchenne muscular dystrophy; Dystrophin; Utrophin; biglycan; ezutromid; therapy
  12. J Neural Transm (Vienna). 2021 May 17.
      Spinal muscular atrophy (SMA) is a heritable, autosomal recessive neuromuscular disorder characterized by a loss of the survival of motor neurons (SMN) protein, which leads to degeneration of lower motor neurons, and muscle atrophy. Despite SMA being nosographically classified as a motor neuron disease, recent advances indicate that peripheral alterations at the level of the neuromuscular junction (NMJ), involving the muscle, and axons of the sensory-motor system, occur early, and may even precede motor neuron loss. In the present study, we used a mouse model of slow progressive (type III) SMA, whereby the absence of the mouse SMN protein is compensated by the expression of two human genes (heterozygous SMN1A2G, and SMN2). This leads to late disease onset and prolonged survival, which allows for dissecting slow degenerative steps operating early in SMA pathogenesis. In this purely morphological study carried out at transmission electron microscopy, we extend the examination of motor neurons and proximal axons towards peripheral components, including distal axons, muscle fibers, and also muscle spindles. We document remarkable ultrastructural alterations being consistent with early peripheral denervation in SMA, which may shift the ultimate anatomical target in neuromuscular disease from the spinal cord towards the muscle. This concerns mostly mitochondrial alterations within distal axons and muscle, which are quantified here through ultrastructural morphometry. The present study is expected to provide a deeper knowledge of early pathogenic mechanisms in SMA.
    Keywords:  Mitochondria; Muscle denervation; Muscle spindle; Neuromuscular disease; SMN; Transmission electron microscopy
  13. Mech Ageing Dev. 2021 May 18. pii: S0047-6374(21)00082-8. [Epub ahead of print] 111510
      Progressive loss of muscle mass and function due to muscle fiber atrophy and loss in the elderly and chronically ill is now defined as sarcopenia. It is a major contributor to loss of independence, disability, need of long-term care as well as overall mortality. Sarcopenia is a heterogenous disease and underlying mechanisms are not completely understood. Here, we newly identified and used Tmem158, alongside Cdkn1a, as relevant senescence and denervation markers (SDMs), associated with muscle fiber atrophy. Subsequent application of laser capture microdissection (LCM) and RNA analyses revealed age- and disease-associated differences in gene expression and alternative splicing patterns in a rodent sarcopenia model. Of note, genes exhibiting such differential alternative splicing (DAS) are mainly involved in the contractile function of the muscle. Many of these splicing events are also found in a mouse model for myotonic dystrophy type 1 (DM1), underscoring the premature aging phenotype of this disease. We propose to add differential alternative splicing to the hallmarks of aging.
    Keywords:  Cellular senescence; Differential alternative splicing; Laser Capture Microdissection; Myotonic dystrophy type 1; Sarcopenia
  14. J Trace Elem Med Biol. 2021 May 13. pii: S0946-672X(21)00074-2. [Epub ahead of print]67 126784
      BACKGROUND: Zinc transporters are thought to facilitate the mobilization of zinc (Zn) and the role of Zn as a signaling mediator during cellular events. Little is known about the response of Zn movement and zinc transporters during muscle proliferation and differentiation processes after damage.METHODS: After rats were subjected to one 90-min session of downhill running to cause muscle damage, the gastrocnemius muscles were harvested to assess the expression of zinc transporters SLC39A/ZIP7, ZIP8, ZIP14 and myogenic regulatory factors at the 0 h, 6 h, 12 h, 1 d, 2 d, 3 d, 1 w and 2 w time points after exercise.
    RESULTS: SLC39A/ZIP7, ZIP8 and ZIP14 had translocated to different compartments of the cell following damage, and they exhibited differential expression profiles after eccentric exercise. The results regarding the myogenetic regulators showed that nf-κb was upregulated 2 d after exercise, and STAT3 and Akt1 mRNA levels were mostly expressed 2 w after exercise. The upregulation of phosphatidylinositol 3-kinase, catalytic subunit gamma (pik3cg), erk1 and erk2 mostly occurred at the early stage (6 h or 12 h) after exercise. In addition, we found that zip7, zip8 and zip14 expression was moderately correlated with certain markers of muscle regeneration.
    CONCLUSION: The zinc transporters SLC39A/ZIP7, ZIP8 and ZIP14 have differential expression profiles upon eccentric exercise, and they might regulate muscle proliferation or differentiation processes through different cellular pathways after exercise-induced muscle damage.
    Keywords:  Myogenesis; Skeletal muscle damage; Zinc; Zinc transporter
  15. Skelet Muscle. 2021 May 17. 11(1): 13
      BACKGROUND: Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type.METHODS: We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers.
    RESULTS: Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types.
    CONCLUSION: This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.
    Keywords:  Fiber; Single cell RNA-sequencing; Skeletal muscle; Twitch
  16. Nat Commun. 2021 05 17. 12(1): 2860
      Bone regenerates by activation of tissue resident stem/progenitor cells, formation of a fibrous callus followed by deposition of cartilage and bone matrices. Here, we show that mesenchymal progenitors residing in skeletal muscle adjacent to bone mediate the initial fibrotic response to bone injury and also participate in cartilage and bone formation. Combined lineage and single-cell RNA sequencing analyses reveal that skeletal muscle mesenchymal progenitors adopt a fibrogenic fate before they engage in chondrogenesis after fracture. In polytrauma, where bone and skeletal muscle are injured, skeletal muscle mesenchymal progenitors exhibit altered fibrogenesis and chondrogenesis. This leads to impaired bone healing, which is due to accumulation of fibrotic tissue originating from skeletal muscle and can be corrected by the anti-fibrotic agent Imatinib. These results elucidate the central role of skeletal muscle in bone regeneration and provide evidence that skeletal muscle can be targeted to prevent persistent callus fibrosis and improve bone healing after musculoskeletal trauma.
  17. J Cachexia Sarcopenia Muscle. 2021 May 18.
      BACKGROUND: Eighty per cent of United States advanced cancer patients faces a worsened prognosis due to cancer-associated cachexia. Inflammation is one driver of muscle atrophy in cachexia, and skeletal muscle-resident immune cells could be a source of inflammation. This study explores the efficacy of cancer activated skeletal muscle-resident mast cells as a biomarker and mediator of cachexia.METHODS: Individual gene markers for immune cells were assessed in a publicly available colon carcinoma cohort of normal (n = 3), moderate cachexia (n = 3), and severe cachexia (n = 4) mice. Lewis lung carcinoma (LL/2) cells induced cachexia in C57BL/6 mice, and a combination of toluidine blue staining, immunofluorescence, quantitative polymerase chain reaction, and western blots measured innate immune cell expression in hind limb muscles. In vitro measurements included C2C12 myotube diameter before and after treatment with media from primary murine mast cells activated with LL/2 conditioned media. To assess translational potential in human samples, innate immune cell signatures were assessed for correlation with skeletal muscle atrophy and apoptosis, dietary excess, and cachexia signatures in normal skeletal muscle tissue. Gene set enrichment analysis was performed with innate immune cell signatures in publicly available cohorts for upper gastrointestinal (GI) cancer and pancreatic ductal adenocarcinoma (PDAC) patients (accession: GSE34111 and GSE130563, respectively).
    RESULTS: Individual innate immunity genes (TPSAB1 and CD68) showed significant increases in severe cachexia (weight loss > 15%) mice in a C26 cohort (GSE24112). Induction of cachexia in C57BL/6 mice with LL/2 subcutaneous injection significantly increased the number of activated skeletal muscle-resident degranulating mast cells. Murine mast cells activated with LL/2 conditioned media decreased C2C12 myotube diameter (P ≤ 0.05). Normal human skeletal muscle showed significant positive correlations between innate immune cell signatures and muscle apoptosis and atrophy, dietary excess, and cachexia signatures. The mast cell signature was up-regulated (positive normalized enrichment score and false discovery rate ≤ 0.1) in upper GI cachectic patients (n = 12) compared with control (n = 6), as well as in cachectic PDAC patients (n = 17) compared with control patients (n = 16).
    CONCLUSIONS: Activated skeletal muscle-resident mast cells are enriched in cachectic muscles, suggesting skeletal-muscle resident mast cells may serve as a biomarker and mediator for cachexia development to improve patient diagnosis and prognosis.
    Keywords:  Cancer-associated cachexia; Degranulation; Innate immunity; Mast cells; Skeletal muscle
  18. Exp Physiol. 2021 May 16.
      NEW FINDING: What is the central question of this study? Eccentric contraction exercises cause damage to muscle fibers, induce inflammatory responses; the exacerbation of this process can induce deposition of fibrous connective tissue, leading to decreased muscle function. The aim of this study is to examine the role of Angiotensin-(1-7) in this context. What is the main finding and its importance? Our results show that oral treatment with Angiotensin-(1-7) decreases muscle damage induced by eccentric exercise, reducing inflammation and fibrosis in the gastrocnemius and soleus muscle. This study shows potential effect of Ang-(1-7) for the prevention of muscle injuries induced by physical exercise.ABSTRACT: Eccentric contraction exercises cause damage to the muscle fibers and induce inflammatory reaction. The protective effect of Angiotensin-(1-7) [Ang-(1-7)] in skeletal muscle has led us to examine the role that this peptide plays in modifying processes associated with inflammation and fibrogenesis induced by eccentric exercise. In this study, we sought to investigate the effects of the oral administration of Ang-(1-7) included in hydroxypropyl β-cyclodextrin (HPβ-CD) in preventing and treating muscle damage following downhill running. Male Wistar rats were divided into three groups: control (untreated and not exercised) (n = 10), treated/exercised HPβ-CD Ang-(1-7) (n = 40), and treated/exercised HPβ-CD (n = 40). Exercised groups were subjected to a single eccentric contraction exercise session on a treadmill inclined to -13° and a constant speed of 20 m.min-1, for 60 min. Oral administration with HPβ-CD Ang-(1-7) and HPβ-CD was performed three hours before the exercise protocol and daily as a single dose, until day of euthanasia. Samples were collected at 04/12/24/48 and 72 hours after the exercise session. The animals treated with the Ang-(1-7) showed lower levels of CK, lower levels of TNF-α in soleus muscle and increased levels of IL-10 cytokines. The inflammatory cells and deposition of fibrous connective tissue were lower in the group treated with Ang-(1-7) in soleus and gastrocnemius muscles. The results of this study show that treatment with an oral formulation of Ang-(1-7) plays a fundamental role in the process of muscle injury repair induced by eccentric exercise. This article is protected by copyright. All rights reserved.
    Keywords:  Eccentric exercise; angiotensin-(1-7); fibrosis; inflammation; muscle damage
  19. J Exp Biol. 2020 Jan 01. pii: jeb.224410. [Epub ahead of print]
      Most of what we know about whole muscle behaviour comes from experiments on single fibres or small muscles that are scaled up in size without considering the effects of the additional muscle mass. Previous modelling studies have shown that tissue inertia acts to slow the rate of force development and maximum velocity of muscle during shortening contractions and decreases the work and power per cycle during cyclic contractions; however, these results have not yet been confirmed by experiments on living tissue. Therefore, in this study we conducted in situ work-loop experiments on rat plantaris muscle to determine the effects of increasing the mass of muscle on mechanical work during cyclic contractions. We additionally simulated these experimental contractions using a mass-enhanced Hill-type model to validate our previous modelling work. We found that greater added mass resulted in lower mechanical work per cycle relative to the unloaded trials in which no mass was added to the muscle (p=0.041 for both 85% and 123% increases in muscle mass). We additionally found that greater strain resulted in lower work per cycle relative to unloaded trials at the same strain to control for length change and velocity effects on the work output, possibly due to greater accelerations of the muscle mass at higher strains. These results confirm that tissue mass reduces muscle mechanical work at larger muscle sizes, and that this effect is likely amplified for lower activations.
    Keywords:  Cyclic contractions; Inertia; Mechanical work; Muscle mass; Muscle mechanics; Skeletal muscle
  20. Nat Commun. 2021 05 17. 12(1): 2887
      Obesity is a major risk factor underlying the development of metabolic disease and a growing public health concern globally. Strategies to promote skeletal muscle metabolism can be effective to limit the progression of metabolic disease. Here, we demonstrate that the levels of the Hippo pathway transcriptional co-activator YAP are decreased in muscle biopsies from obese, insulin-resistant humans and mice. Targeted disruption of Yap in adult skeletal muscle resulted in incomplete oxidation of fatty acids and lipotoxicity. Integrated 'omics analysis from isolated adult muscle nuclei revealed that Yap regulates a transcriptional profile associated with metabolic substrate utilisation. In line with these findings, increasing Yap abundance in the striated muscle of obese (db/db) mice enhanced energy expenditure and attenuated adiposity. Our results demonstrate a vital role for Yap as a mediator of skeletal muscle metabolism. Strategies to enhance Yap activity in skeletal muscle warrant consideration as part of comprehensive approaches to treat metabolic disease.
  21. Physiol Rep. 2021 May;9(9): e14869
      Myoglobin is an important regulator of muscle and whole-body metabolism and exercise capacity. Caffeine, an activator of the calcium and cyclic AMP (cAMP)/protein kinase A (PKA) pathway, enhances glucose uptake, fat oxidation, and mitochondrial biogenesis in skeletal muscle cells. However, no study has shown that caffeine increases the endogenous expression of myoglobin in muscle cells. Further, the molecular mechanism underlying the regulation of myoglobin expression remains unclear. Therefore, our aim was to investigate whether caffeine and activators of the calcium signaling and cAMP/PKA pathway increase the expression of myoglobin in L6 myotubes and whether the pathway mediates caffeine-induced myoglobin expression. Caffeine increased myoglobin expression and activated the cAMP/PKA pathway in L6 muscle cells. Additionally, a cAMP analog significantly increased myoglobin expression, whereas a ryanodine receptor agonist showed no significant effect. Finally, PKA inhibition significantly suppressed caffeine-induced myoglobin expression in L6 myotubes. These results suggest that caffeine increases myoglobin expression via the cAMP/PKA pathway in skeletal muscle cells.
    Keywords:  caffeine; cyclic AMP; myoglobin; protein kinase A; skeletal muscle
  22. PLoS One. 2021 ;16(5): e0251921
      The skeletal muscle mass has been shown to be affected by catecholamines, such as epinephrine (Epi), norepinephrine (NE), and isoproterenol (ISO). On the other hand, lipopolysaccharide (LPS), one of the causative substances of sepsis, induces muscle wasting via toll-like receptors expressed in skeletal muscle. Although catecholamines are frequently administered to critically ill patients, it is still incompletely understood how these drugs affect skeletal muscle during critical illness, including sepsis. Herein, we examined the direct effects of catecholamines on LPS-induced skeletal muscle wasting using the C2C12 myoblast cell line. Muscle wasting induced by catecholamines and/or LPS was analyzed by the use of the differentiated C2C12 myotubes, and its underlying mechanism was explored by immunoblotting analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and the TransAM kit for p-65 NF-κB. Epi augmented myosin heavy chain (MHC) protein loss and reduction of the myotube diameter induced by LPS. LPS induced C/EBPδ protein, Atrogin-1 and inteleukin-6 (IL-6), and these responses were potentiated by Epi. An IL-6 inhibitor, LMT28, suppressed the potentiating effect of Epi on the LPS-induced responses. NF-κB activity was induced by LPS, but was not affected by Epi and recombinant IL-6, and the NF-κB inhibitor, Bay 11-7082, abolished Atrogin-1 mRNA expression induced by LPS with or without Epi. NE and ISO also potentiated LPS-induced IL-6 and Atroign-1 mRNA expression. Carvedilol, a nonselective β-adrenergic receptor antagonist, suppressed the facilitating effects of Epi on the Atrogin-1 mRNA induction by LPS, and abolished the effects of Epi on the MHC protein loss in the presence of LPS. It was concluded that Epi activates the β-adrenergic receptors in C2C12 myotubes and the IL-6-STAT3 pathway, leading to the augmentation of LPS-induced activation of the NF-κB- C/EBPδ-Atrogin-1 pathway and to the exacerbation of myotube wasting.
  23. J Cell Biol. 2021 Jul 05. pii: e202101057. [Epub ahead of print]220(7):
      Circadian rhythms regulate cell proliferation and differentiation, but circadian control of tissue regeneration remains elusive at the molecular level. Here, we show that proper myoblast differentiation and muscle regeneration are regulated by the circadian master regulators Per1 and Per2. Depletion of Per1 or Per2 suppressed myoblast differentiation in vitro and muscle regeneration in vivo, demonstrating their nonredundant functions. Both Per1 and Per2 were required for the activation of Igf2, an autocrine promoter of myoblast differentiation, accompanied by Per-dependent recruitment of RNA polymerase II, dynamic histone modifications at the Igf2 promoter and enhancer, and the promoter-enhancer interaction. This circadian epigenetic priming created a preferred time window for initiating myoblast differentiation. Consistently, muscle regeneration was faster if initiated at night, when Per1, Per2, and Igf2 were highly expressed compared with morning. This study reveals the circadian timing as a significant factor for effective muscle cell differentiation and regeneration.
  24. Nat Commun. 2021 05 19. 12(1): 2951
      The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG's glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies.
  25. Am J Physiol Endocrinol Metab. 2021 05 17.
      Obesity is accompanied by numerous systemic and tissue-specific derangements, including systemic inflammation, insulin resistance, and mitochondrial abnormalities in skeletal muscle. Despite growing recognition that adipose tissue dysfunction plays a role in obesity-related disorders, the relationship between adipose tissue inflammation and other pathological features of obesity is not well-understood. We assessed macrophage populations and measured the expression of inflammatory cytokines in abdominal adipose tissue biopsies in 39 non-diabetic adults across a range of body mass indexes (BMI 20.5-45.8 kg/m2). Skeletal muscle biopsies were used to evaluate mitochondrial respiratory capacity, ATP production capacity, coupling, and reactive oxygen species production. Insulin sensitivity (SI) and beta cell responsivity were determined from test meal postprandial glucose, insulin, c-peptide, and triglyceride kinetics. We examined the relationships between adipose tissue inflammatory markers, systemic inflammatory markers, SI, and skeletal muscle mitochondrial physiology. BMI was associated with increased adipose tissue and systemic inflammation, reduced SI, and reduced skeletal muscle mitochondrial oxidative capacity. Adipose-resident macrophage numbers were positively associated with circulating inflammatory markers, including tumor necrosis factor-α (TNFα) and C-reactive protein (CRP). Local adipose tissue inflammation and circulating concentrations of TNFα and CRP were negatively associated with SI, and circulating concentrations of TNFα and CRP were also negatively associated with skeletal muscle oxidative capacity. These results demonstrate that obese humans exhibit increased adipose tissue inflammation concurrently with increased systemic inflammation, reduced insulin sensitivity, and reduced muscle oxidative capacity, and suggest that adipose tissue and systemic inflammation may drive obesity-associated metabolic derangements.
    Keywords:  adipose tissue; inflammation; macrophages; mitochondria; obesity
  26. Gene. 2021 May 16. pii: S0378-1119(21)00319-X. [Epub ahead of print] 145725
      Ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic protein found in the nuclei and sarcomeres of cardiac and skeletal muscles, with a proposed role in linking myofibrilar stress and transcriptional regulation. Rapid upregulation of its expression in response to both physiological and pathological stress supports the involvement of ANKRD1 in muscle tissue adaptation and remodeling. However, the exact role of ANKRD1 remains poorly understood. To begin to investigate its function at higher resolution, we have generated and characterized a TgBAC(ankrd1a:EGFP) zebrafish line. This reporter line displays transgene expression in slow skeletal muscle fibers during development and exercise responsiveness in adult cardiac muscle. To better understand the role of Ankrd1a in pathological conditions in adult zebrafish, we assessed ankrd1a expression after cardiac ventricle cryoinjury and observed localized upregulation in cardiomyocytes in the border zone. We show that this expression in injured hearts is recapitulated by the TgBAC(ankrd1a:EGFP) reporter. Our results identify novel expression domains of ankrd1a and suggest an important role for Ankrd1a in the early stress response and regeneration of cardiac tissue. This new reporter line will help decipher the role of Ankrd1a in striated muscle stress response, including after cardiac injury.
    Keywords:  cryoinjury; exercise; reporter line; slow muscle fibers
  27. J Cell Sci. 2020 Jan 01. pii: jcs.243162. [Epub ahead of print]
      Striated muscle is a highly specialized collection of tissues with contractile properties varying according to functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-dependent adaptation. Functional adaptation requires molecular adaptation, partially provided by miRNA-mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-206 expression increases in both physiologic (including female sex and endurance exercise) and pathologic conditions (muscular dystrophy and adrenergic agonism) that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-206 knockout mice displays a faster phenotype than wild-type mice. Moreover, left ventricles of male miR-206 knockout mice have a faster myosin profile accompanied by dilation and systolic dysfunction. Thus, miR-206 appears necessary to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms.
    Keywords:  Heart; MiR-206; MiRNA; Sexual Dimorphism; Skeletal Muscle
  28. Development. 2020 Jan 01. pii: dev.190868. [Epub ahead of print]
      The Myostatin/Activin branch of the TGFβ superfamily acts as a negative regulator of vertebrate skeletal muscle size, in part, through downregulation of insulin/IGF-1 signaling. Surprisingly, recent studies in Drosophila indicate that motoneuron derived Activin signaling acts as a positive regulator of muscle size. Here we demonstrate that Drosophila Activin signaling promotes growth of the muscle cells along all three axes; width, thickness and length. Activin signaling positively regulates the InR/dTORC1 pathway and the level of Mhc, an essential sarcomeric protein, via increased Pdk1 and Akt1 expression. Enhancing InR/dTORC1 signaling in the muscle of Activin pathway mutants restores Mhc levels close to wild-type, but only increases muscle width. In contrast, hyperactivation of the Activin pathway in muscles increases overall larval body and muscle fiber length even when Mhc levels were lowered by suppression of dTORC1. Together, these results indicate that the Drosophila Activin pathway regulates larval muscle geometry and body size via promoting InR/dTORC1-dependent Mhc production and the differential assembly of sarcomeric components into either pre-existing or new sarcomeric units depending on the balance of InR/dTORC1 and Activin signals.
    Keywords:  Activin; Drosophila; Insulin; Muscle
  29. Physiol Rep. 2021 May;9(9): e14856
      Functional interaction between the selective water channel AQP4 and several ion channels, such as TRPV4, NKCC1, and Na+ /K+ -ATPase, closely participate to regulate osmotic homeostasis. In the skeletal muscles, the decrease in APQ4 expression due to denervation was followed by the restoration of AQP4 expression during reinnervation. These findings raised the possibility that innervation status is an essential factor to regulate AQP4 expression in the skeletal muscles. This study investigated this hypothesis using disuse muscle atrophy model with innervation. Adult female Fischer 344 rats (8 weeks of age) were randomly assigned to either control (C) or cast immobilization (IM) groups (n = 6 per group). Two weeks after cast immobilization, the tibialis anterior muscles of each group were removed and the expression levels of some target proteins were quantified by western blot analysis. The expression level of AQP4 significantly decreased at 2 weeks post-immobilization (p < 0.05). Moreover, the expression levels of TRPV4, NKCC1, and Na+ /K+ -ATPase significantly decreased at 2 weeks post-immobilization (p < 0.05). This study suggested that innervation status is not always a key regulatory factor to maintain the expression of AQP4 in the skeletal muscles. Moreover, the transport of water and ions by AQP4 may be changed during immobilization-induced muscle atrophy.
    Keywords:  AQP4; disuse muscle atrophy; immobilization; skeletal muscle
  30. Acta Cir Bras. 2021 ;pii: S0102-86502021000400202. [Epub ahead of print]36(4): e360403
      PURPOSE: To study effects of Rehmannia glutinosa polysaccharides (RGP) on bone tissue structure and skeletal muscle atrophy in rats with disuse.METHODS: A rat model of disuse osteoporosis combined with muscle atrophy was established by removing the bilateral ovaries of rats and fixing their hind limbs for a long time. Forty SD rats were administered intragastrically for 12 weeks. The bone histomorphometry parameters and the level of oxidative stress were measured. In addition, the changes of muscle atrophy F-box (MAFbx), muscle RING-finger protein-1 (MuRF1), forkhead box O1 (FOXO1) mRNA expression in skeletal muscle of rats were observed.
    RESULTS: RGP significantly increased the percentage of fluorescence perimeter and bone mineralization deposition rate of the second lumbar vertebrae of rats. It also significantly increased the wet weight ratio and muscle fiber cross-sectional area of the gastrocnemius muscle of rats. At the same time, RGP significantly increased the levels of super oxide dismutase (SOD) and catalase (CAT) in the skeletal muscle of rats, and reduced the content of malondialdehyde (MDA). Rehmannia glutinosa polysaccharides also significantly reduced the expression levels of FOXO1, MAFbx and MuRF1 mRNA in rat skeletal muscle.
    CONCLUSIONS: RGP could improve the bone structure of osteoporotic rats. It could also improve muscle that atrophy may be related to the inhibition of FOXO1-mediated ubiquitin-proteasome pathway.
  31. J Exp Biol. 2020 Jan 01. pii: jeb.215020. [Epub ahead of print]
      The periodic striation pattern in skeletal muscle reflects the length of the basic contractile unit: the sarcomere. More than half a century ago, Gordon, Huxley and Julian provided strong support for the 'sliding filament' theory that allowed prediction of the active isometric force as a function of sarcomere length (SL). Although the theory was established using single muscle fibers, the sarcomere force-length (FL) relationship has been extrapolated to whole muscles in an attempt to unravel in-vivo muscle function. However, these extrapolations were frequently associated with non-trivial assumptions, such as muscle length changes corresponding linearly to SL changes. However, the sarcomere force-length relationship in whole muscles may not behave as theoretically predicted due to the structural complexity of muscles, the force transmission, pressures and shear forces developed inside muscles, and the great SL non-uniformity recently observed in whole muscles. Here, we determined the in-situ sarcomere FL relationship in a whole muscle preparation by simultaneously measuring muscle force and individual SLs in an intact muscle-tendon unit (MTU) using state-of-the-art multi-photon excitation microscopy. We found that despite great SL non-uniformity, the mean value of SLs measured from a minute volume of the mid-belly, equivalent to about 5 x 10-6 % of the total muscle volume, agrees well with the theoretically predicted FL relationship, but only if the precise contractile filament lengths are known, and if passive forces from parallel elastic components and activation-associated sarcomere shortening are considered properly. As SLs are not uniformly distributed across the whole muscle and changes in SLs with muscle length are location-dependent, our results may not be valid for the proximal or distal parts of the muscle. The approach described here, and our findings, may encourage future studies to determine the role of SL non-uniformity in influencing sarcomere FL properties in different muscles and for different locations within single muscles.
    Keywords:  Cross-bridge theory; Force-length relationship; Multi-photon microscopy; Muscle contraction; Sarcomere length instability; Sarcomere length non-uniformity; Skeletal muscle properties; Sliding filament theory; in situ
  32. Front Physiol. 2021 ;12 653060
      Objective: Disuse atrophy (DA) describes inactivity-induced skeletal muscle loss, through incompletely defined mechanisms. An intriguing observation is that individual muscles exhibit differing degrees of atrophy, despite exhibiting similar anatomical function/locations. We aimed to develop an innovative experimental paradigm to investigate Atrophy Resistant tibialis anterior (TA) and Atrophy Susceptible medial gastrocnemius (MG) muscles (aRaS) with a future view of uncovering central mechanisms.Method: Seven healthy young men (22 ± 1 year) underwent 15 days unilateral leg immobilisation (ULI). Participants had a single leg immobilised using a knee brace and air-boot to fix the leg (75° knee flexion) and ankle in place. Dual-energy X-ray absorptiometry (DXA), MRI and ultrasound scans of the lower leg were taken before and after the immobilisation period to determine changes in muscle mass. Techniques were developed for conchotome and microneedle TA/MG muscle biopsies following immobilisation (both limbs), and preliminary fibre typing analyses was conducted.
    Results: TA/MG muscles displayed comparable fibre type distribution of predominantly type I fibres (TA 67 ± 7%, MG 63 ± 5%). Following 15 days immobilisation, MG muscle volume (-2.8 ± 1.4%, p < 0.05) and muscle thickness decreased (-12.9 ± 1.6%, p < 0.01), with a positive correlation between changes in muscle volume and thickness (R2 = 0.31, p = 0.038). Importantly, both TA muscle volume and thickness remained unchanged.
    Conclusion: The use of this unique "aRaS" paradigm provides an effective and convenient means by which to study the mechanistic basis of divergent DA susceptibility in humans, which may facilitate new mechanistic insights, and by extension, mitigation of skeletal muscle atrophy during human DA.
    Keywords:  aRaS; disuse atrophy; medial gastrocnemius muscle; skeletal muscle atrophy; tibialis anterior muscle
  33. J Appl Physiol (1985). 2021 May 20.
      Diseases or conditions where diaphragm muscle (DIAm) function is impaired, including chronic obstructive pulmonary disease, cachexia, asthma and aging are associated withincreased risks of pulmonary symptoms, longer duration of hospitalizations and increasing requirements for mechanical ventilation. Vitamin-D deficiency is associated with proximal muscle weakness that resolves following therapy with vitamin D3. Skeletal muscle expresses the vitamin D receptor (VDR), which responds to the active form of vitamin D, 1,25-dihydroxyvitamin D3 by altering gene expression in target cells. In knock-out mice without skeletal muscle VDRs, there is marked atrophy of muscle fibers and a change in skeletal muscle biochemistry. We used a tamoxifen-inducible skeletal muscle Cre recombinase in Vdrfl/fl mice (Vdrfl/fl actin.iCre+) to assess the role of muscle-specific VDR signaling on DIAm specific force, fatigability and fiber type-dependent morphology. Vdrfl/fl actin.iCre+ mice treated with vehicle and Vdrfl/fl mice treated with tamoxifen served as controls. 7 days following the final treatment, mice were euthanized, the DIAm was removed and isometric force and fatigue were assessed in DIAm strips using direct muscle stimulation. The proportion and cross-sectional areas of DIAm fiber types were evaluated by immunolabeling with myosin heavy chain antibodies differentiating type I, IIa and IIx and/or IIb fibers. We show that in mice with skeletal muscle-specific VDR deletion, maximum specific force and residual force following fatigue are impaired, along with a selective atrophy of type IIx and/or IIb fibers. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.
    Keywords:  fatigue; muscle fiber type; muscle specific force; vitamin D; vitamin D receptor
  34. Connect Tissue Res. 2021 May 20. 1-10
      Purpose: We investigate the underlying biological effects and mechanisms of rESWT on myogenic contracture and muscle atrophy in a rabbit model of extending knee joint contracture.Materials and Methods: In group control, the knee joint was not fixed. In group I-4w, the knee joint was only fixed for 4 weeks. In groups SR-1 w, SR-2 w, and SR-4 w, the knee joint was fixed for 4 weeks before the rabbits underwent 1, 2, and 4 weeks of self-recovery, respectively. In groups rESWT-1 w, rESWT 2 w, and rESWT-4 w, the knee joint was fixed for 4 weeks before the rabbits underwent 1, 2, and 4 weeks of rESWT, respectively. The myogenic contracture was measured, the cross-sectional area and key protein levels for NF-κB/HIF-1α signaling pathway and myogenic regulatory factors were evaluated.Results: During the recovery period, biological findings showed that the levels of myogenic contracture and muscle atrophy were milder in group rESWT by compared with group SR after 2 weeks. Molecular biological analysis showed that MyoD protein levels in the group rESWT was significantly higher than those in the group SR, and importantly, phospho-NF-κB p65 and HIF-1α protein levels in the group rESWT were significantly lower than those in the group SR at the same time point.Conclusions: This is the first study demonstrated that rESWT has the potential to reduce myogenic contracture and muscle atrophy after long-term immobilization in animal model. It is a possible mechanism that changing the low oxygen environment in skeletal muscle through rESWT may inhibit activation of NF-κB/HIF-1α signaling pathway.
    Keywords:  NF-κB/HIF-1α signaling pathway; Radial extracorporeal shock wave; muscle atrophy; myod; myogenic contracture
  35. J Cell Sci. 2020 Jan 01. pii: jcs.240879. [Epub ahead of print]
      Sarcopenia, the loss of muscle mass and strength associated to age, has been linked to impairment of the cytosolic Ca2+ peak that triggers muscle contraction, but mechanistic details remain unknown. Here we explore the hypothesis that a reduction in sarcoplasmic reticulum Ca2+ concentration ([Ca2+]SR) is at the origin of this loss of Ca2+ homeostasis. We engineered Drosophila melanogaster to express the Ca2+ indicator GAP3 targeted to muscle SR, and we developed a new method to calibrate the signal into [Ca2+]SR in vivo. [Ca2+]SR dropped with age from ∼600 µM down to 50 µM in close correlation to muscle function, which declined monotonically when [Ca2+]SR was <400 µM. [Ca2+]SR results from the pump-leak steady-state at the SR membrane. However, changes in expression of the SERCA pump and of the ryanodine receptor leak, were too modest to explain the large changes seen in [Ca2+]SR. Instead, these changes are compatible with increased leakiness through the ryanodine receptor as the main determinant of the [Ca2+]SR decline in aging muscle. In contrast, there were no changes in endoplasmic reticulum [Ca2+] with age in brain neurons.
    Keywords:  Aging; Calcium homeostasis; Calcium imaging; Drosophila melanogaster; Endoplasmic reticulum; Fly; Ryanodine receptor; Sarco/endoplasmic reticulum calcium ATPase (SERCA); Sarcopenia; Sarcoplasmic reticulum
  36. Nat Commun. 2021 05 19. 12(1): 2942
      The association between reduced myofilament force-generating capacity (Fmax) and heart failure (HF) is clear, however the underlying molecular mechanisms are poorly understood. Here, we show impaired Fmax arises from reduced BAG3-mediated sarcomere turnover. Myofilament BAG3 expression decreases in human HF and positively correlates with Fmax. We confirm this relationship using BAG3 haploinsufficient mice, which display reduced Fmax and increased myofilament ubiquitination, suggesting impaired protein turnover. We show cardiac BAG3 operates via chaperone-assisted selective autophagy (CASA), conserved from skeletal muscle, and confirm sarcomeric CASA complex localization is BAG3/proteotoxic stress-dependent. Using mass spectrometry, we characterize the myofilament CASA interactome in the human heart and identify eight clients of BAG3-mediated turnover. To determine if increasing BAG3 expression in HF can restore sarcomere proteostasis/Fmax, HF mice were treated with rAAV9-BAG3. Gene therapy fully rescued Fmax and CASA protein turnover after four weeks. Our findings indicate BAG3-mediated sarcomere turnover is fundamental for myofilament functional maintenance.
  37. Exp Physiol. 2021 May 16.
      NEW FINDINGS: What is the central question of the study? Do interindividual differences in trainability exist for morphological and molecular skeletal muscle responses to aerobic exercise training? What is the main finding and its importance? Interindividual differences in trainability were present for some, but not all, morphological and molecular outcomes included in our study. Our findings suggest that is inappropriate, and perhaps erroneous, to assume that variability in observed responses reflects interindividual differences in trainability in skeletal muscle responses to aerobic exercise training.ABSTRACT: Studies have interpreted a wide range of morphological and molecular changes in human skeletal muscle as evidence of interindividual differences in trainability. However, these interpretations fail to account for the influence of random measurement error and within-subject variability. The purpose of the present study was to use the standard deviation of individual response (SDIR ) statistic to test the hypothesis that interindividual differences in trainability are present for some but not all skeletal muscle outcomes. Twenty-nine recreationally-active males (age: 21±2 years; BMI: 24±3; VO2 peak: 45±7 mL/kg/min) completed four weeks of continuous training (REL; n = 14) or control (CTRL; n = 15). Maximal enzyme activities (citrate synthase and β-HAD), capillary density, fibre type composition, fibre-specific SDH activity and substrate storage (IMTG and glycogen), and markers of mitophagy (BNIP3, NIX, PRKN, and PINK1) were measured in vastus lateralis samples collected before and after the intervention. We also calculated SDIR values for VO2 peak, peak work rate, and the onset of blood lactate accumulation for REL and a separate group that exercised at the negative talk test (TT) stage. Although positive SDIR values - indicating interindividual differences in trainability - were obtained for aerobic capacity outcomes, maximal enzyme activities, capillary density, all fibre-specific outcomes, and BNIP3 protein content, the remaining outcomes produced negative SDIR values indicating a large degree of random measurement error and/or within-subject variability. Our findings question the interpretation of heterogeneity in observed responses as evidence of interindividual differences in trainability and highlight the importance of including control groups when analyzing individual skeletal muscle response to exercise training. This article is protected by copyright. All rights reserved.
    Keywords:  SDIR; aerobic exercise training; individual responses; interindividual variability
  38. Physiol Res. 2021 04 30. 70(2): 119-151
      The purpose of this systematic review is twofold: 1) to identify, evaluate, and synthesize the heretofore disparate scientific literatures regarding the effects of direct exposure to microgravity on the musculoskeletal system, taking into account for the first time both bone and muscle systems of both humans and animals; and 2) to investigate the efficacy and limitations of exercise countermeasures on the musculoskeletal system under microgravity in humans.The Framework for Scoping Studies (Arksey and O'Malley 2005) and the Cochrane Handbook for Systematic Reviews of Interventions (Higgins JPT 2011) were used to guide this review. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist was utilized in obtaining the combined results (Moher, Liberati et al. 2009). Data sources, PubMed, Embase, Scopus, and Web of Science were searched for published articles through October 2019 using the Mesh terms of microgravity, musculoskeletal system, and exercise countermeasures. A total of 84 references were selected, including 40 animal studies and 44 studies with human participants. The heterogeneity in the study designs, methodologies, and outcomes deemed this review unsuitable for a meta-analysis. Thus, we present a narrative synthesis of the results for the key domains under five categories: 1) Skeletal muscle responses to microgravity in humans 2) Skeletal muscle responses to microgravity in animals 3) Adaptation of the skeletal system to microgravity in humans 4) Adaptation of the skeletal system to microgravity in animals 5) Effectiveness of exercise countermeasures on the human musculoskeletal system in microgravity. Existing studies have produced only limited data on the combined effects on bone and muscle of human spaceflight, despite the likelihood that the effects on these two systems are complicated due to the components of the musculoskeletal system being anatomically and functionally interconnected. Bone is directly affected by muscle atrophy as well as by changes in muscle strength, notably at muscle attachments. Given this interplay, the most effective exercise countermeasure is likely to be robust, individualized, resistive exercise, primarily targeting muscle mass and strength.
  39. J Therm Biol. 2021 May;pii: S0306-4565(21)00097-8. [Epub ahead of print]98 102930
      Proteins secreted from skeletal muscle serving a signalling role have been termed myokines. Many of the myokines are exercise factors, produced and released in response to muscle activity. Cold exposures affecting muscle may occur in recreational, occupational and therapeutic settings. Whether muscle temperature independently affects myokine profile, is still to be elucidated. We hypothesized that manipulating muscle temperature by means of external cooling would change myokine production and release. In the present study we have established new models for cold exposure of muscle in vivo and in vitro where rat hind limb or cultured human myotubes were cooled to 18 °C. After a recovery period, muscle tissue, cells and culture media were harvested for further analysis by qPCR and immunoassays. Expression of several myokine genes were significantly increased after cold exposure in both models: in rat muscle, mRNA levels of CCL2 (p = 0.04), VEGFA (p = 0.02), CXCL1 (p = 0.02) and RBM3 (p = 0.02) increased while mRNA levels of IL-6 (p = 0.03) were decreased; in human myotubes, mRNA levels of IL6 (p = 0.01), CXCL8 (p = 0.04), VEGFA (p = 0.03) and CXCL1 (p < 0.01) were significantly increased, as well as intracellular protein levels of IL-8 (CXCL8 gene product; p < 0.01). The corresponding effect on myokine secretion was not observed, on the contrary, IL-8 (p = 0.02) and VEGF (VEGFA gene product) p < 0.01) concentrations in culture media were reduced after cold exposure in vitro. In conclusion, cold exposure of muscle in vivo and in vitro had an effect on the production and release of several known exercise-related myokines. Myokine expression at the level of mRNA and protein was increased by cold exposure, whereas secretion tended to be decreased.
    Keywords:  Cold exposure; Exercise; In vitro; In vivo; Myokines; Skeletal muscle
  40. Biol Open. 2020 Jan 01. pii: bio.054262. [Epub ahead of print]
      The mitochondrial contact site and cristae organizing system (MICOS) is a multi-protein interaction hub that helps define mitochondrial ultrastructure. While the functional importance of MICOS is mostly characterized in yeast and mammalian cells in culture, the contributions of MICOS to tissue homeostasis in vivo remain further elucidation. In this study, we examined how knocking down expression of Drosophila MICOS genes affects mitochondrial function and muscle tissue homeostasis. We found that CG5903/MIC26-MIC27 colocalizes and functions with Mitofilin/MIC60 and QIL1/MIC13 as a Drosophila MICOS component; knocking down expression of any of these three genes predictably altered mitochondrial morphology, causing loss of cristae junctions, and disruption of cristae packing. Furthermore, the knockdown flies exhibited low mitochondrial membrane potential, fusion/fission imbalances, increased mitophagy, and limited cell death. Reductions in climbing ability indicated deficits in muscle function. Knocking down MICOS genes also caused reduced mtDNA content and fragmented mitochondrial nucleoid structure in Drosophila. Together, our data demonstrate an essential role of Drosophila MICOS in maintaining proper homeostasis of mitochondrial structure and function to promote the function of muscle tissue.
    Keywords:  Drosophila; MICOS; Mitochondria
  41. BMC Geriatr. 2021 May 17. 21(1): 314
      BACKGROUND: The rapidly aging societies worldwide and in Singapore present a unique challenge, requiring an integrated multidisciplinary approach to address high-value targets such as muscle health. We propose pragmatic evidence-based multidisciplinary consensus recommendations for the assessment and multi-modal management of muscle health in older adults (≥65 years) across the continuum of care.METHODS: The recommendations are derived from an in-depth review of published literature by a multidisciplinary working group with clinical experience in the care of the older population in both acute and community settings.
    RESULTS: The panel recommends screening for muscle impairment using the SARC-F questionnaire, followed by assessment for low muscle strength (handgrip strength or 5-times chair stand test ≥10 s as a surrogate for lower limb strength) to diagnose possible/probable sarcopenia. For uncomplicated cases, lifestyle modifications in exercise and diet can be initiated in the community setting without further assessment. Where indicated, individuals diagnosed with possible/probable sarcopenia should undergo further assessment. Diagnosis of sarcopenia should be based on low muscle strength and low muscle mass (bioimpedance analysis, dual-energy X-ray absorptiometry or calf circumference as a surrogate). The severity of sarcopenia should be determined by assessment of physical performance (gait speed or 5-times chair stand test ≥12 s as a surrogate for gait speed). To treat sarcopenia, we recommend a combination of progressive resistance-based exercise training and optimization of nutritional intake (energy, protein and functional ingredients). High quality protein in sufficient quantity, to overcome anabolic resistance in older adults, and distributed throughout the day to enable maximum muscle protein synthesis, is essential. The addition of resistance-based exercise training is synergistic in improving the sensitivity of muscle protein synthesis response to the provision of amino acids and reducing anabolic resistance. An expected dose-response relationship between the intensity of resistance-based training, lean mass and muscle strength is described.
    CONCLUSIONS: Reviewed and endorsed by the Society of Rehabilitation Medicine Singapore and the Singapore Nutrition and Dietetics Association, these multidisciplinary consensus recommendations can provide guidance in the formulation of comprehensive and pragmatic management plans to improve muscle health in older adults in Singapore and Asia.
    Keywords:  Consensus; Exercise; Multidisciplinary; Muscle; Nutrition; Older adult; Sarcopenia
  42. Int J Biol Sci. 2021 ;17(7): 1682-1692
      N6-methyladenosine (m6A) is one of the most widespread and highly conserved chemical modifications in cellular RNAs of eukaryotic genomes. Owing to the development of high-throughput m6A sequencing, the functions and mechanisms of m6A modification in development and diseases have been revealed. Recent studies have shown that RNA m6A methylation plays a critical role in skeletal muscle development, which regulates myoblast proliferation and differentiation, and muscle regeneration. Exploration of the functions of m6A modification and its regulators provides a deeper understanding of the regulatory mechanisms underlying skeletal muscle development. In the present review, we aim to summarize recent breakthroughs concerning the global landscape of m6A modification in mammals and examine the biological functions and mechanisms of enzymes regulating m6A RNA methylation. We describe the interplay between m6A and other epigenetic modifications and highlight the regulatory roles of m6A in development, especially that of skeletal muscle. m6A and its regulators are expected to be targets for the treatment of human muscle-related diseases and novel epigenetic markers for animal breeding in meat production.
    Keywords:  RNA N6-methyladenosine; development; myogenesis; skeletal muscle
  43. Dis Model Mech. 2020 Jan 01. pii: dmm.047886. [Epub ahead of print]
      Myosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/ß-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases with myosin storage myopathy. We have developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of R1845W mutation. This study shows that higher expression level of the mutated allele is concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was supressed by expression of Abba/Thin, an E3 ubiquitin ligase. This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities including extensive loss of thick filaments with selective A-band loss and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Further, the impaired muscle morphology associated with the mutant allele was supressed by expression of Abba/Thin, an E3 ubiquitin ligase. These findings suggest that modification of ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients.
    Keywords:  Drosophila model; E3 ubiquitin ligase; MYH7; Myosin storage myopathy; Potential therapeutic approach; Slow/ß-cardiac myosin heavy chain; Ubiquitin proteasome system
  44. G3 (Bethesda). 2021 May 16. pii: jkab172. [Epub ahead of print]
      The mechanisms that determine the final topology of skeletal muscles remain largely unknown. We have been developing Drosophila body wall musculature as a model to identify and characterize the pathways that control muscle size, shape, and orientation during embryogenesis (Johnson et al., 2013; Williams et al., 2015; Yang et al., 2020a; Yang et al., 2020b). Our working model argues muscle morphogenesis is regulated by (1) extracellular guidance cues that direct muscle cells toward muscle attachment sites, and (2) contact dependent interactions between muscles and tendon cells. While we have identified several pathways that regulate muscle morphogenesis, our understanding is far from complete. Here we report the results of a recent EMS-based forward genetic screen that identified a myriad of loci not previously associated with muscle morphogenesis. We recovered new alleles of known muscle morphogenesis genes, including back seat driver, kon-tiki, thisbe, and tumbleweed, arguing our screen had the depth and precision to uncover myogenic genes. We also identified new alleles of spalt-major, barren, and patched that presumably disrupt independent muscle morphogenesis pathways. Equally as important, our screen shows that at least 11 morphogenetic loci remain to be mapped and characterized. Our screen has developed exciting new tools to study muscle morphogenesis, which may provide future insights into the mechanisms that regulate skeletal muscle topology.
    Keywords:  Drosophila; bsd; myogenesis; myotube guidance; salm
  45. Physiol Rep. 2021 May;9(9): e14842
      Mechanistic target of rapamycin complex 1 (mTORC1) plays a central role in muscle protein synthesis and repeated bouts of resistance exercise (RE) blunt mTORC1 activation. However, the changes in the proteolytic signaling when recurrent RE bouts attenuate mTORC1 activation are unclear. Using a RE model of electrically stimulated rat skeletal muscle, this study aimed to clarify the effect of repeated RE bouts on acute proteolytic signaling, particularly the calpain, autophagy-lysosome, and ubiquitin-proteasome pathway. p70S6K and rpS6 phosphorylation, indicators of mTORC1 activity, were attenuated by repeated RE bouts. Calpain 3 protein was decreased at 6 h post-RE in all exercised groups regardless of the bout number. Microtubule-associated protein 1 light chain 3 beta-II, an indicator of autophagosome formation, was increased at 3 h and repeated RE bouts increased at 6 h, post-RE. Ubiquitinated proteins were increased following RE, but these increases were independent of the number of RE bouts. These results suggest that the magnitude of autophagosome formation was increased following RE when mTORC1 activity was attenuated with repeated bouts of RE.
    Keywords:  LC3; calpain; mTORC1; muscle protein breakdown; resistance exercise; ubiquitinated protein
  46. J Exp Biol. 2021 May 15. pii: jeb242496. [Epub ahead of print]224(10):
      Skeletal muscle has numerous nuclei within a cell. The nucleus is considered as the central organelle for muscle protein synthesis (MPS). However, it is unclear whether myonuclear number is associated with MPS capacity within the individual muscle fibres. Therefore, the purpose of the present study was to reveal the relationship between myonuclear number per unit muscle fibre length and MPS under basal and conditions of elevated MPS by high-intensity muscle contraction (HiMC) using an in vivo nascent protein labelling technique (SUnSET) in rodents. We found that myonuclear number was positively correlated with MPS in individual muscle fibres in the basal condition. Similarly, ribosomal protein S6 (rpS6) content, which is a rough estimate of ribosome content, was positively correlated with MPS. However, myonuclear number was not associated with rpS6 content. In contrast to the basal condition, when MPS was increased by acute HiMC, no correlation was observed between myonuclear number and MPS, but the association between rpS6 and MPS was maintained. Importantly, these observations indicate that the number of nuclei in individual myofibers is related only to MPS at rest. However, the ribosome content in individual fibres is related to MPS of individual myofibers both at rest and following HiMC.
    Keywords:  Muscle contraction; Myonuclei; Protein synthesis
  47. J Cell Sci. 2020 Jan 01. pii: jcs.244855. [Epub ahead of print]
      Imbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionary conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing, ER structures. We demonstrate that, the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process which does not depend on autophagosome-lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation.
    Keywords:  Apoptosis; ER stress; Glycogen; Glycogen synthase; Glycogenin; UPR
  48. Front Immunol. 2021 ;12 674242
      Recombinant adeno-associated virus (rAAV) platforms hold promise for in vivo gene therapy but are undermined by the undesirable transduction of antigen presenting cells (APCs), which in turn can trigger host immunity towards rAAV-expressed transgene products. In light of recent adverse events in patients receiving high systemic AAV vector doses that were speculated to be related to host immune responses, development of strategies to mute innate and adaptive immunity is imperative. The use of miRNA binding sites (miR-BSs) to confer endogenous miRNA-mediated regulation to detarget transgene expression from APCs has shown promise for reducing transgene immunity. Studies have shown that designing miR-142BSs into rAAV1 vectors were able to repress costimulatory signals in dendritic cells (DCs), blunt the cytotoxic T cell response, and attenuate clearance of transduced muscle cells in mice to allow sustained transgene expression in myofibers with negligible anti-transgene IgG production. In this study, we screened individual and combinatorial miR-BS designs against 26 miRNAs that are abundantly expressed in APCs, but not in skeletal muscle. The highly immunogenic ovalbumin (OVA) transgene was used as a proxy for foreign antigens. In vitro screening in myoblasts, mouse DCs, and macrophages revealed that the combination of miR-142BS and miR-652-5pBS strongly mutes transgene expression in APCs but maintains high myoblast and myocyte expression. Importantly, rAAV1 vectors carrying this novel miR-142/652-5pBS cassette achieve higher transgene levels following intramuscular injections in mice than previous detargeting designs. The cassette strongly inhibits cytotoxic CTL activation and suppresses the Th17 response in vivo. Our approach, thus, advances the efficiency of miRNA-mediated detargeting to achieve synergistic reduction of transgene-specific immune responses and the development of safe and efficient delivery vehicles for gene therapy.
    Keywords:  adeno-associated virus vectors; antigen presenting cells; gene therapy; miR-142; miR-223-3p; miR-652-5p; miR-BS; microRNA
  49. Development. 2020 Jan 01. pii: dev.183855. [Epub ahead of print]
      Murine muscle stem cells (MuSCs) experience a transition from quiescence to activation that is required for regeneration, but it remains unknown if the trajectory and dynamics of activation change with age. Here, we use timelapse imaging and single cell RNA-seq to measure activation trajectories and rates in young and aged MuSCs. We find that the activation trajectory is conserved in aged cells, and develop effective machine learning classifiers for cell age. Using cell behavior analysis and RNA velocity, we find that activation kinetics are delayed in aged MuSCs, suggesting that changes in stem cell dynamics may contribute to impaired stem cell function with age. Intriguingly, we also find that stem cell activation appears to be a random walk like process, with frequent reversals, rather than a continuous, linear progression. These results support a view of the aged stem cell phenotype as a combination of differences in the location of stable cell states and differences in transition rates between them.
    Keywords:  Aging; Muscle stem cell; Single cell RNA-seq; State transition; Stem cell activation; Timelapse imaging
  50. Development. 2020 Jan 01. pii: dev.183004. [Epub ahead of print]
      Despites several decades of studies on the neuromuscular system, the relationship between muscle stem cells and motor neurons remains elusive. Using the Drosophila model, we provide evidences that adult muscle precursors (AMPs), the Drosophila muscle stem cells, interact with the motor axons during embryogenesis. AMPs not only hold the capacity to attract the navigating intersegmental (ISN) and segmental a (SNa) nerve branches, but are also mandatory to the innervation of muscles in the lateral field. This so far ignored AMPs role involves their filopodia-based interactions with nerve growth cones. In parallel, we report the previously undetected expression of encoding guidance molecules sidestep and side IV in AMPs. Altogether, our data support the view that Drosophila muscle stem cells represent spatial landmarks for navigating motor neurons and reveal that their positioning is critical for the muscles innervation in the lateral region. Furthermore, AMPs and motor axons are interdependent as the genetic ablation of SNa leads to a specific loss of SNa-associated lateral AMPs.
    Keywords:  Adult Muscle Precursors (AMPs); Drosophila; Guidance; Motor axon; Side
  51. Biol Open. 2020 Jan 01. pii: bio.048975. [Epub ahead of print]
      Calpain-3 (CAPN3) is a muscle specific type of calpain whose protease activity is triggered by Ca2+. Here, we developed CAPN3 sensor probes (SPs) to detect activated-CAPN3 using a fluorescence/Förster resonance energy transfer (FRET) technique. In our SPs, partial amino acid sequence of calpastatin, endogenous CAPN inhibitor but CAPN3 substrate, is inserted between two different fluorescence proteins which cause FRET. Biochemical and spectral studies revealed that CAPN3 cleaved SPs and changed emission wavelengths of SPs. Importantly, SPs were scarcely cleaved by CAPN1 and CAPN2. Furthermore, our SP successfully captured the activation of endogenous CAPN3 in living myotubes treated with ouabain. Our SPs would become a promising tool to detect the dynamics of CAPN3 protease activity in living cells.
    Keywords:  Calpain; Calpain-3; Calpainopathy; Limb-girdle muscular dystrophy type 2A; Proteolysis; Skeletal muscle
  52. Acta Physiol (Oxf). 2021 May 22. e13690
      AIM: The skeletal muscle Cl- channels, the ClC-1 channels, stabilize the resting membrane potential and dampen muscle fibre excitability. This study explored whether ClC-1 inhibition can recover nerve-stimulated force in isolated muscle under conditions of compromised neuromuscular transmission akin to disorders of myasthenia gravis and Lambert-Eaton syndrome.METHODS: Nerve-muscle preparations were isolated from rats. Preparations were exposed to pre-or post-synaptic inhibitors (ω-agatoxin, elevated extracellular Mg2+ , α-bungarotoxin or tubocurarine). The potential of ClC-1 inhibition (9-AC or reduced extracellular Cl- ) to recover nerve-stimulated force under these conditions was assessed.
    RESULTS: ClC-1 inhibition recovered force in both slow-twitch soleus and fast-twitch EDL muscles exposed to 0.2 µM tubocurarine or 3.5 mM Mg2+ . Similarly, ClC-1 inhibition recovered force in soleus muscles exposed to α-bungarotoxin or ω-agatoxin. Moreover, the concentrations of tubocurarine and Mg2+ required for reducing force to 50 % rose from 0.14±0.02 µM and 4.2±0.2 mM in control muscles to 0.45±0.03 µM and 4.7±0.3 mM in muscles with 9-AC, respectively (P<0.05, paired T-test). Inhibition of acethylcholineesterase (neostigmine) and inhibition of voltage gated K+ channels (4-AP) relieve symptoms in myasthenia gravis and Lambert-Eaton syndrome, respectively. Neostigmine and 9-AC additively increased the tubocurarine concentration required to reduce nerve-stimulated force to 50 % (0.56±0.05 µM with 9-AC and neostigmine) and, similarly, 4-AP and 9-AC additively increased the Mg2+ concentration required to reduce nerve-stimulated force to 50 % (6.5±0.2 mM with 9-AC and 4-AP).
    CONCLUSION: This study shows that ClC-1 inhibition can improve neuromuscular function in pharmacological models of compromised neuromuscular transmission.
    Keywords:  ClC-1 Cl- channels; muscle contractions; neuromuscular transmission disorders
  53. J Cell Sci. 2020 Jan 01. pii: jcs.248336. [Epub ahead of print]
      Lysosomes are compartments for the degradation of both endocytic and autophagic cargoes. The shape of lysosomes changes with cellular degradative demands, however, there is limited knowledge about the mechanisms or significance that underlies distinct lysosomal morphologies. Here, we found an extensive tubular autolysosomal network in Drosophila abdominal muscle remodeling during metamorphosis. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling.
    Keywords:  Atrophy; Autolysosome; Drosophila; Metamorphosis; Muscle; Syntaxin17
  54. J Gerontol A Biol Sci Med Sci. 2021 May 16. pii: glab139. [Epub ahead of print]
      Barriers associated with direct muscle quantification have prevented a consistent implementation of therapeutic measures for sarcopenia. Recently, the relevance of circulating C-terminal agrin fragment (CAF) as an accessible screening method alternative for sarcopenia has gained credence. Accordingly, this study aimed to verify the pertinence of plasma CAF as a biomarker for sarcopenia. Three-hundred healthy adults aged between 50-83 years took part in this study. Sarcopenia was diagnosed according to the European Working Group on Sarcopenia in Older People criteria. Body composition was assessed using dual-energy x-ray absorptiometry, while muscle strength was examined using hand dynamometry. Plasma CAF concentrations were determined using a commercially available ELISA kit. CAF concentrations were significantly associated with appendicular lean mass (ALM), but not grip strength (p=0.028, p=0.575, respectively). Plasma CAF concentrations were significantly elevated in sarcopenic individuals compared to non-sarcopenic (p<0.001). Overall, individuals with low grip strength or low ALM displayed significantly higher CAF levels compared to healthy controls, after adjusting for age and body mass index (p=0.027, p=0.003, respectively). In males, those with low grip strength or low ALM had significantly elevated CAF levels (p=0.039, p=0.027, respectively), while in females, only those with low ALM had significantly raised CAF concentrations, compared to healthy controls (p=0.035). Our findings illuminate the potential relevance of CAF as an accessible biomarker for skeletal muscle health. CAF determination may enhance clinical practise by facilitating more widespread treatment strategies for sarcopenia. Nevertheless, future research is needed to confirm the diagnostic pertinence of CAF concentrations in screening for sarcopenia.
    Keywords:  Agrin; Muscle strength; Muscle wasting; Neuromuscular junction; Skeletal muscle mass
  55. Trends Endocrinol Metab. 2021 May 13. pii: S1043-2760(21)00116-8. [Epub ahead of print]
      Soluble mediators secreted by skeletal muscles (collectively referred to as myokines) exert systemic effects by signaling to distant organs. Rai et al. have now uncovered a muscle-to-central nervous system (CNS) signaling axis, which is engaged in response to proteostatic stress. These adaptations confer protection against pathological protein accumulation in the CNS, a hallmark of neurodegenerative diseases.