Search

Muscle satellite cells (SCs) are a quiescent (non-proliferative) stem cell population in uninjured skeletal muscle. Although SCs have been investigated for nearly 60 years, the molecular drivers that transform quiescent SCs into the rapidly dividing (activated) stem/progenitor cells that mediate muscle repair after injury remain largely unknown. Here we identify a prominent FBJ osteosarcoma oncogene (Fos) mRNA and protein signature in recently activated SCs that is rapidly, heterogeneously, and transiently induced by muscle damage. We further reveal a requirement for FOS to efficiently initiate key stem cell functions, including cell cycle entry, proliferative expansion, and muscle regeneration, via induction of "pro-regenerative" target genes that stimulate cell migration, division, and differentiation. Disruption of one of these Fos/AP-1 targets, NAD(+)-consuming mono-ADP-ribosyl-transferase 1 (Art1), in SCs delays cell cycle entry and impedes progenitor cell expansion and muscle regeneration. This work uncovers an early-activated FOS/ART1/mono-ADP-ribosylation (MARylation) pathway that is essential for stem cell-regenerative responses., Competing Interests: Declaration of interests A.J.W. is a scientific advisor for Frequency Therapeutics, and A.J.W. and L.L.R. are co-founders and scientific advisory board members and hold private equity in Elevian, Inc., a company that aims to develop medicines to restore regenerative capacity. Elevian also provides sponsored research to the Wagers lab., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In developing organisms, internal cellular processes generate mechanical stresses at the tissue scale. The resulting deformations depend on the material properties of the tissue, which can exhibit long-ranged orientational order and topological defects. It remains a challenge to determine these properties on the time scales relevant for developmental processes. Here, we build on the physics of liquid crystals to determine material parameters of cell monolayers. Specifically, we use a hydrodynamic description to characterize the stationary states of compressible active polar fluids around defects. We illustrate our approach by analyzing monolayers of C2C12 cells in small circular confinements, where they form a single topological defect with integer charge. We find that such monolayers exert compressive stresses at the defect centers, where localized cell differentiation and formation of three-dimensional shapes is observed.

Cultured meat is an emerging technology with the potential to solve huge challenges related to the environmental, ethical, and health implications of conventional meat production. Establishing the basic science of cultured meat has been the primary focus of the last decade but it is now feasible that cultured meat products will enter the market within the next 3 to 4 years. This proximity to market introduction demands an evaluation of aspects of the cultured meat production process that have not yet been outlined or discussed in significant detail. For example, one technological approach for the production of cultured meat uses adult muscle stem cells, the limited proliferative capacity of which necessitates repeated collection of tissue samples via biopsies of living donor animals. The selection of donor animals and the details of biopsy processes must be optimized, as this is a key bottleneck in the cultured meat production process. The number of stem cells harvested from a biopsy, together with their proliferative capacity, determines a 'multiplicity factor' achieved by a cultured meat production process, thus dictating the reduction in number of animals required to produce a given quantity of meat. This article considers potential scenarios for these critical upstream steps, focusing on the production of cultured beef as an example. Considerations related to donor selection and details of the biopsy process are discussed in detail. The practicalities of various scenarios for cultured beef production, the health of donor animals, and regulatory issues associated with the safety of cultured meat for consumers are also considered. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Beef is considered to be a good quality meat product because it contains linoleic acid and specific proteins, which can bring significant benefits to health. Circular RNAs (circRNAs) have been reported to regulate skeletal myogenesis. RNA-seq was used to investigate the circRNA molecular regulatory mechanisms with respect to differences in muscle quality between buffalo and cattle. A total of 10,449 circRNA candidates were detected, and 1128 of these were found to be differentially expressed between cattle and buffalo muscle tissue libraries. Differentially expressed 23 circRNAs were verified by qPCR. CircEch1, one of the most up-regulated circRNAs during muscle development, was subsequently characterized. CCK-8 (65.05 ± 2.33%, P < 0.0001), EdU (72.99 ± 0.04%, P < 0.001), and Western blotting assays showed that overexpression of circEch1 inhibited the proliferation of bovine myoblasts but promoted differentiation. In vivo studies suggested that circEch1 stimulates skeletal muscle regeneration. These results demonstrate that the novel regulator circEch1 induces myoblast differentiation and skeletal muscle regeneration. They also provide new insights into the mechanisms of circRNA regulation of beef quality.

Preoperative fasting aims to prevent pulmonary aspiration and improve bowel preparation, but it may induce profound systemic catabolic responses that lead to protein breakdown and insulin-resistant hyperglycemia after operation. However, the molecular mechanisms of catabolic reaction induced by prolonged preoperative fasting and surgical stress are undetermined. In this study, anesthetized rats were randomly assigned to receive a sham operation or laparotomy cecectomy. Fasting groups were restricted from food and water for 12 h before operation, while the feeding group had free access to food throughout the study period. Twenty-four hours after operation, the animals were sacrificed to collect blood samples and soleus muscles for analysis. Postoperative blood glucose level was significantly increased in the fasting group with elevated serum insulin and C-peptide. Continuous feeding reduced serum myoglobin and lactate dehydrogenase concentrations. Preoperative fasting activated inositol-requiring transmembrane kinase/endoribonuclease (IRE)-1α and c-Jun N-terminal kinase (JNK) mediated endoplasmic reticulum (ER)-stress, and reduced glucose transporter type 4 (Glut4) expression in the soleus muscle. Phospholamban phosphorylation was reduced and intracellular calcium levels were increased in the isolated skeletal muscle cells. Similar results were found in ER stress-induced C1C12 myoblasts. The expression of Glut4 was suppressed in the stressed C1C12, but was potentiated following inhibition of ER stress and chelation of intracellular free calcium. This study provides evidence demonstrating that prolonged preoperative fasting induces ER stress and generates insulin resistance in the skeletal muscle through suppression of Glut4 and inactivation of Ca 2+ -ATPase, leading to intracellular calcium homeostasis disruption and peripheral insulin resistance., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Volumetric muscle loss injuries overwhelm the endogenous regenerative capacity of skeletal muscle, and the associated oxidative damage can delay regeneration and prolong recovery. This study aimed to investigate the effect of silicon-ions on C2C12 skeletal muscle cells under normal and excessive oxidative stress conditions to gain insights into its role on myogenesis during the early stages of muscle regeneration. In vitro studies indicated that 0.1 mM Si-ions into cell culture media significantly increased cell viability, proliferation, migration, and myotube formation compared to control. Additionally, MyoG, MyoD, Neurturin, and GABA expression were significantly increased with addition of 0.1, 0.5, and 1.0 mM of Si-ion for 1 and 5 days of C2C12 myoblast differentiation. Furthermore, 0.1-2.0 mM Si-ions attenuated the toxic effects of H 2 O 2 within 24 h resulting in increased cell viability and differentiation. Addition of 1.0 mM of Si-ions significantly aid cell recovery and protected from the toxic effect of 0.4 mM H 2 O 2 on cell migration. These results suggest that ionic silicon may have a potential effect in unfavorable situations where reactive oxygen species is predominant affecting cell viability, proliferation, migration, and differentiation. Furthermore, this study provides a guide for designing Si-containing biomaterials with desirable Si-ion release for skeletal muscle regeneration.

Physical exercise is an effective strategy for improving human health. Various organs, including the heart, lung and kidney, can benefit from exercise. However, the underlying molecular mechanisms by which exercise protects organs remain unknown. Irisin, a myokine secreted from muscle in response to exercise, has attracted increased attention from researchers. The role of irisin in multiorgan protection has been gradually revealed, and this muscle-derived circulating factor is regarded as an essential bridge linking exercise and organ health. The mechanisms by which irisin protects diverse organs are different. Here, we review the research progress on the multiorgan protective effects of irisin and discuss the underlying molecular mechanisms.

In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .

By analyzing the effects of P53 inhibitors and ladder climbing exercise on P53 mRNA transcription in skeletal muscle of mice, the application of P53 mRNA in signal transduction mechanism of skeletal muscle cells was studied. Several clean ICR mice were fed for experiment. The experimental mice were divided into groups to analyze the effect of P53 inhibitor on P53 mRNA transcription in gastrocnemius muscle of mice. The mice were randomly divided into The application of P53 mRNA in signal transduction mechanism of skeletal muscle cells was studied, and the corresponding endurance exercise program and ladder climbing training program were designed. According to the research, exercise is to some extent a stimulating factor affecting P53 inhibitor. Endurance training and injection of P53 inhibitor affect P53 mRNA content. Exercise has a benign effect on ICR mice injected with P53 inhibitor. The expression of P53 mRNA in skeletal muscle was significantly affected by climbing training in youth, and decreased by climbing training in old age. However, there was no difference between long-term climbing training and short-term climbing training in the expression of P53 mRNA in skeletal muscle.

During passive heat stress, shifting of blood flow from the hepato-splanchnic to peripheral regions produces less favorable physiological conditions in the liver than in the skeletal muscle. We were wondering if the two organs differ in susceptibility to heat injury and thus examined the effects of heat shock exposure on apoptotic and heat stress-related markers in the gastrocnemius muscle and liver of mice. During heat exposure, mice had a peak core body temperature of 41.1 ± 0.7 °C. Heat-exposed mice showed higher levels of reactive oxygen species (ROS), cleaved caspases, fragmented DNA, and Drp1 protein expression in the gastrocnemius muscles than control mice. These changes were not observed in the livers of heat-exposed mice. Furthermore, the levels of glucocorticoid receptor, HSP70, and HSF1 proteins were significantly elevated in the gastrocnemius muscles of heat-exposed mice compared with that of control mice. The livers of heat-exposed mice also revealed increased expression of HSP70 but no changes in the other proteins. These results demonstrate that heat exposure induces significantly lower levels of the stress response and apoptosis in the liver than in the skeletal muscle of mice. The liver tissue resistance against heat stress is associated with low levels of heat-induced ROS production and mitochondrial fission protein expression.

Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte markers and are originally isolated from the embryonic dorsal aorta. From postnatal small vessels of skeletal muscle and heart, it is possible to isolate cells with similar characteristics to embryonic MABs. Adult MABs have the capacity to self-renew and to differentiate into cell types of mesodermal lineages upon proper culture conditions. To date, the origin of MABs and the relationship with other muscle stem cells are still debated. Recently, in a phase I-II clinical trial, intra-arterial HLA-matched MABs were proved to be relatively safe. Novel information on MAB pure populations is desirable, and implementation of their therapeutic potential is mandatory to approach efficacy in MAB-based treatments. This chapter provides an overview of the current techniques for isolation and characterization of rodent, canine, human, and equine adult MABs.

Mitochondrial reactive oxygen species (mtROS) and redox regulation play an important role in stem cell maintenance and cell fate decisions. Although changes in mtROS and redox homeostasis represent a physiological mechanism to drive stem cell commitment and differentiation, dysregulation of this system can lead to defects in stem cell maintenance and regenerative capacity. This chapter explains the methods used to assess mitochondrial superoxide levels and redox regulation in stem cell populations.

Background: Sarcopenia progresses in chronic kidney disease (CKD) and is positively correlated with mortality in end-stage kidney disease patients. Circulating irisin, an exercise-induced myokine, gradually decreases during CKD stage progression. Irisin inhibits the progression of kidney fibrosis, which is the final common outcome of CKD. Our preliminary study with C2C12 cells showed that Dojuksan, a herbal decoction, increases the expression of PGC1α (a regulator of irisin) and FNDC5 (a precursor of irisin)., Hypothesis: Dojuksan may increase circulating irisin and prevent the progression of kidney fibrosis., Study Design and Methods: Unilateral ureteral obstruction (UUO) was performed on seven-week-old male C57BL/6 mice to induce kidney tubulointerstitial fibrosis. Dojuksan (50, 100, or 200 mg/kg/day) or losartan (1.5 mg/kg/day), a standard clinical treatment for CKD, was administered orally one day prior to surgery and continued for seven days thereafter. To determine the role of irisin released from muscles, TGFβ-stimulated murine proximal tubular epithelial cells (mProx24 cells) were treated with conditioned media (CM) from Dojuksan-treated C2C12 muscle cells transfected with FNDC5 siRNA., Results: UUO mice exhibited muscle wasting along with progressive kidney injury. Similar to losartan, Dojuksan ameliorated kidney inflammation and fibrosis in UUO mice. Dojuksan, but not losartan, increased plasma irisin concentration in UUO mice. Dojuksan significantly increased basal FNDC5 expression and inhibited TNFα-induced and indoxyl sulfate-induced FNDC5 down-regulation in C2C12 cells. The TGFβ-induced collagen I (COL1) up-regulation in mProx24 cells was effectively inhibited by CM from C2C12 cells after Dojuksan treatment. Moreover, irisin inhibited TGFβ-induced COL1 in mProx24 cells, which was not affected by CM from C2C12 cells transfected with FNDC5 siRNA., Conclusion: Dojuksan ameliorates kidney fibrosis through irisin-mediated muscle-kidney crosstalk, suggesting that Dojuksan may be used as an alternative therapeutic agent against CKD., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The myoblast is a precursor cell that rebuilds muscle tissue after trauma in human and animal skeletal muscle tissue. Proliferation of myoblasts is important for skeletal muscle damage repair and is controlled by numerous transcription factors and signals. The regulation of these signaling pathways and their complex interactions are not fully understood. This study aims to determine the physiological functions of Activin A, Notch and Sonic Hedgehog (Shh) signaling in the proliferation of mouse C2C12 myoblasts and to explore their interactions. Activin A facilitated proliferation of C2C12 cells and promoted the conversion of G1 into S phase in cell cycle, whereas addition of the receptor inhibitor SB431542 attenuated the proliferation activity of rActA on C2C12 cells. Activin A also activated Notch and Shh signaling, while blockage of these pathways attenuated the function of Activin A in cell cycle. Inhibition of the Notch signaling by Notch response inhibitor DAPT significantly down-regulated the expression of Shh signaling molecules, whereas exogenous rShh reversed the inhibition of C2C12 cells proliferative activity induced by DAPT, indicating Notch signaling act upstream of the Shh pathway. Furthermore, inhibition of Notch signaling weakened the activation of Activin A-mediated Shh signaling. Taken together, our results provide a novel role of Activin A in regulating the proliferation of C2C12 skeletal muscle cells, which impacts ActivinA-Notch1-Shh signaling pathways., (Copyright © 2020 Elsevier B.V. All rights reserved.)

There has been an escalation in reports over the last decade examining the efficacy of bone marrow derived mesenchymal stem/stromal cells (BMSC) in bone tissue engineering and regenerative medicine-based applications. The multipotent differentiation potential, myelosupportive capacity, anti-inflammatory and immune-modulatory properties of BMSC underpins their versatile nature as therapeutic agents. This review addresses the current limitations and challenges of exogenous autologous and allogeneic BMSC based regenerative skeletal therapies in combination with bioactive molecules, cellular derivatives, genetic manipulation, biocompatible hydrogels, solid and composite scaffolds. The review highlights the current approaches and recent developments in utilizing endogenous BMSC activation or exogenous BMSC for the repair of long bone and vertebrae fractures due to osteoporosis or trauma. Current advances employing BMSC based therapies for bone regeneration of craniofacial defects is also discussed. Moreover, this review discusses the latest developments utilizing BMSC therapies in the preclinical and clinical settings, including the treatment of bone related diseases such as Osteogenesis Imperfecta.

Transdifferentiation of human non-muscle cells directly into myogenic cells by forced expression of MyoD represents one route to obtain highly desirable human myogenic cells. However, functional properties of the tissue constructs derived from these transdifferentiated cells have been rarely studied. Here, we report that three-dimensional (3D) tissue constructs engineered with iMyoD-hTERT-NHDFs, normal human dermal fibroblasts transduced with genes encoding human telomerase reverse transcriptase and doxycycline-inducible MyoD, generate detectable contractile forces in response to electrical stimuli upon MyoD expression. Withdrawal of doxycycline in the middle of 3D culture results in 3.05 and 2.28 times increases in twitch and tetanic forces, respectively, suggesting that temporally-controlled MyoD expression benefits functional myogenic differentiation of transdifferentiated myoblast-like cells. Treatment with CHIR99021, a Wnt activator, and DAPT, a Notch inhibitor, leads to further enhanced contractile forces. The ability of these abundant and potentially patient-specific and disease-specific cells to develop into functional skeletal muscle constructs makes them highly valuable for many applications, such as disease modeling.

Syncytial skeletal muscle cells contain hundreds of nuclei in a shared cytoplasm. We investigated nuclear heterogeneity and transcriptional dynamics in the uninjured and regenerating muscle using single-nucleus RNA-sequencing (snRNAseq) of isolated nuclei from muscle fibers. This revealed distinct nuclear subtypes unrelated to fiber type diversity, previously unknown subtypes as well as the expected ones at the neuromuscular and myotendinous junctions. In fibers of the Mdx dystrophy mouse model, distinct subtypes emerged, among them nuclei expressing a repair signature that were also abundant in the muscle of dystrophy patients, and a nuclear population associated with necrotic fibers. Finally, modifications of our approach revealed the compartmentalization in the rare and specialized muscle spindle. Our data identifies nuclear compartments of the myofiber and defines a molecular roadmap for their functional analyses; the data can be freely explored on the MyoExplorer server ( https://shiny.mdc-berlin.de/MyoExplorer/ ).

Mammalian cells exhibit remarkable diversity in cell size, but the factors that regulate establishment and maintenance of these sizes remain poorly understood. This is especially true for skeletal muscle, comprised of syncytial myofibers that each accrue hundreds of nuclei during development. Here, we directly explore the assumed causal relationship between multinucleation and establishment of normal size through titration of myonuclear numbers during mouse neonatal development. Three independent mouse models, where myonuclear numbers were reduced by 75, 55, or 25%, led to the discovery that myonuclei possess a reserve capacity to support larger functional cytoplasmic volumes in developing myofibers. Surprisingly, the results revealed an inverse relationship between nuclei numbers and reserve capacity. We propose that as myonuclear numbers increase, the range of transcriptional return on a per nuclear basis in myofibers diminishes, which accounts for both the absolute reliance developing myofibers have on nuclear accrual to establish size, and the limits of adaptability in adult skeletal muscle.

Muscle fibers are the largest cells in the body, and one of its few syncytia. Individual cell sizes are variable and adaptable, but what governs cell size has been unclear. We find that muscle fibers are DNA scarce compared to other cells, and that the nuclear number (N) adheres to the relationship N = aV b where V is the cytoplasmic volume. N invariably scales sublinearly to V (b < 1), making larger cells even more DNA scarce. N scales linearly to cell surface in adult humans, in adult and developing mice, and in mice with genetically reduced N, but in the latter the relationship eventually fails when they reach adulthood with extremely large myonuclear domains. Another exception is denervation-atrophy where nuclei are not eliminated. In conclusion, scaling exponents are remarkably similar across species, developmental stages and experimental conditions, suggesting an underlying scaling law where DNA-content functions as a limiter of muscle cell size.

Directional cell migration is a critical process underlying morphogenesis and post-natal tissue regeneration. During embryonic myogenesis, migration of skeletal myogenic progenitors is essential to generate the anlagen of limbs, diaphragm and tongue, whereas in post-natal skeletal muscles, migration of muscle satellite (stem) cells towards regions of injury is necessary for repair and regeneration of muscle fibres. Additionally, safe and efficient migration of transplanted cells is critical in cell therapies, both allogeneic and autologous. Although various myogenic cell types have been administered intramuscularly or intravascularly, functional restoration has not been achieved yet in patients with degenerative diseases affecting multiple large muscles. One of the key reasons for this negative outcome is the limited migration of donor cells, which hinders the overall cell engraftment potential. Here, we review mechanisms of myogenic stem/progenitor cell migration during skeletal muscle development and post-natal regeneration. Furthermore, strategies utilised to improve migratory capacity of myogenic cells are examined in order to identify potential treatments that may be applied to future transplantation protocols., (© 2020 The Authors Published under the terms of the CC BY 4.0 license.)

Myopathy is a muscle disease in which muscle fibers do not function properly, and eventually cause severe diseases, such as muscular dystrophy. The properly regeneration of skeletal muscle plays a pivotal role to maintain the muscle function after muscle injury. The aim of this study is to determine whether andrographolide plays an effect role on regulating skeletal muscle regeneration. Mouse satellite cells, C2C12 cells and Cardiotoxin (CTX) intramuscular injection induced acute skeletal muscle injury model were used to evaluate whether andrographolide is essential for skeletal muscle regeneration. The underling mechanism detected using immunohistochemistry stain, western blot, real time PCR. Andrographolide promotes mouse skeletal muscle regeneration. In cardiotoxin induced skeletal muscle injury model, andrographolide treatment enhanced myotube generation and promoted myotube fusion. Andrographolide treatment dramatically increased expression of myotube differentiation related genes, including Desmin, MyoD, MyoG, Myomaker, Tnni2, Dmd, Myoz1 and Myoz3. For the mechanism studies, we observed that andrographolide treatment significantly promoted histone modification, such as H3K4Me2, H3K4Me3 and H3K36Me2, both in vivo and in vitro. Treatment with DZNep, a Lysine methyltransferase EZH2 inhibitor, significantly attenuated andrographolide-induced expression of Myf5, Myomaker, Skeletal muscle α-actin, MyoD and MyoG. Taken together, our data in this study demonstrate andrographolide epigenetically drives differentiation and fusion of myotube, eventually promotes skeletal muscle regeneration. This should be a therapeutic treatment for skeletal muscle regeneration after muscle damage., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study aimed to verify if human myogenic cells could participate in muscle regeneration in macaques. This experimental setting would grant researchers a model that could better evaluate the effects of cell therapies in myopathies with a better translation to human patients. Human muscle precursor cells (MPCs) were cultured in vitro and transduced with ß-galactosidase. The cells were subsequently injected into 1-cm3 muscle regions of 6 macaques immunosuppressed with tacrolimus and dexamethasone. Allogeneic ß-galactosidase+ MPCs were injected in other regions as positive controls. Some cell-grafted regions were electroporated to induce extensive muscle regeneration. MPC-grafted regions were sampled 1 month later and analyzed by histology. There were ß-galactosidase+ myofibers in both the regions grafted with human and macaque MPCs. Electroporation increased the engraftment of human MPCs in the same way as in macaque allografts. The histological analysis (hematoxylin and eosin, CD8, and CD4 immunodetection) demonstrated an absence of cellular rejection in most MPC-grafted regions, as well as minimal lymphocytic infiltration in the regions transplanted with human MPCs in the individual with the lowest tacrolimus levels. Circulating de novo anti-donor antibodies were not detected. In conclusion, we report the successful engraftment of human myogenic cells in macaques, which was possible using tacrolimus-based immunosuppression., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterize at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. We identify key promoter-enhancer interaction motifs, namely, cliques and networks, and interactions that are dependent on Pax7 binding. Remarkably, Pax7 binds to a majority of super-enhancers, and together with a cadre of interacting transcription factors, assembles feed-forward regulatory loops. During differentiation, epigenetic memory and persistent looping are maintained at a subset of Pax7 enhancers in the absence of Pax7. We also identify and functionally validate a previously uncharacterized Pax7-bound enhancer hub that regulates the essential myosin heavy chain cluster during skeletal muscle cell differentiation. Our studies lay the groundwork for understanding the role of Pax7 in orchestrating changes in the three-dimensional chromatin conformation in muscle progenitors.

The myostatin (MSTN) gene is of interest in the livestock industry because mutations in this gene are closely related to growth performance and muscle differentiation. Thus, in this study, we established MSTN knockout (KO) quail myoblasts (QM7) and investigated the regulatory pathway of the myogenic differentiation process. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate MSTN KO QM7 cells and subsequently isolated a single cell-derived MSTN KO QM7 subline with 10- and 16-nucleotide deletions that induced translational frameshift mutations. The differentiation capacity and proliferation rate of MSTN KO QM7 cells were enhanced. We conducted next-generation-sequencing (NGS) analysis to compare the global gene expression profiles of wild-type (WT) QM7 and MSTN KO QM7 cells. Intriguingly, NGS expression profiles showed different expression patterns of p21 and p53 in MSTN KO QM7 cells. Moreover, we identified downregulated expression patterns of leukemia inhibitory factor and DNA Damage Inducible Transcript 4, which are genes in the p53 signaling pathway. Using quantitative RT-PCR (qRT-PCR) analysis and western blotting, we concluded that p53-related genes promote the cell cycle by upregulating p21 and enhancing muscle differentiation in MSTN KO QM7 cells. These results could be applied to improve economic traits in commercial poultry by regulating MSTN-related networks.

The objective of this study was to investigate whether the n-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) can directly regulate glucose and fat metabolism in skeletal muscle besides exerting anti-inflammatory effects. To accomplish this, L6 skeletal muscle cells were treated with 50 µM of either DHA or EPA for 1, 3, and 5 days. Here, we report that basal and insulin-stimulated rates of glucose uptake, glycogen synthesis, protein kinase B (AKT), and glycogen synthase kinase 3 (GSK3) phosphorylation were not affected by DHA or EPA. However, glucose and palmitate oxidation were consistently elevated by DHA treatment, whereas EPA only increased this variable transiently. Similarly, only DHA caused significant and sustained increases in AMP-activated protein kinase (AMPK) phosphorylation and protein levels of carnitine-palmitoyl transferase-1b (CPT1b) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in skeletal muscle cells. DHA also caused a larger anti-inflammatory effect than EPA in these cells. In conclusion, besides exerting anti-inflammatory effects, DHA and EPA directly regulated glucose and fat metabolism in skeletal muscle cells, although DHA was more effective in doing so than EPA. Thus, by directly enhancing glucose and fat oxidation, DHA may increase glucose disposal and reduce intramyocellular lipid accumulation.

A better understanding of the differential mechanisms regulating the deposition and release of fat between intramuscular and external adipose tissues is very important to the quality of beef. Resveratrol is a natural activator of sirtuin type 1 (SIRT1), a NAD-dependent deacetylase involved in regulating the cell cycle, energy homeostasis and apoptosis in adipose tissue. To compare the molecular mechanisms underlying differential apoptosis in bovine intramuscular and subcutaneous adipocytes, we evaluated the effect of resveratrol on differentiated adipocytes. We found that resveratrol-induced apoptosis in bovine adipocytes by regulating SIRT1 activity. In addition, we report that bovine intramuscular and subcutaneous adipocytes exhibited differential responses to resveratrol. In particular, gene and protein expression of Bcl-2 was higher, whereas that of SIRT1, AMPKα, FOXO1, Bax and caspase-3 were lower in bovine subcutaneous adipocytes than in intramuscular adipocytes. After resveratrol-treatment, the extent of up- or down-regulation was higher in subcutaneous adipocytes than in intramuscular adipocytes. These data indicate that bovine subcutaneous adipocytes are more sensitive to apoptosis than intramuscular adipocytes following treatment with resveratrol by regulating SIRT1 activity.

In cases of lower rectum-located tumor or severe disease, surgical resection is currently the effective management; however, it also carries increased risks of function loss. The interdisciplinary field of regenerative medicine offers strategies that can potentially restore severely diseased and injured tissues and organs. Adipose-derived stromal cells (ASCs) are an abundant and accessible source of adult stem cells and hold great promise as therapeutic agents for tissue regeneration. In this work, we transplanted cells isolated from human stromal tissues, including a 6%-7% ASC population, into heat-damaged femoral muscles of non-obese diabetic immunodeficient mice. The movement of the limbs was observed to determine the functional recovery 3 months after transplantation. Among the mice that did not receive cell transplantation, 20% were able to walk with the injured limb touching the ground, while all of the mice in the ASC-treatment group were able to walk. Furthermore, all ASC-treated mice were able to stand on both back paws, in contrast to the control group mice. The human stromal cell population containing ASCs was able to differentiate and engraft the injured muscle tissues successfully. Our results indicate that stromal material/ASC-based therapies are a promising strategy for the regeneration of tissues and function restoration after severe injury due to surgery.

The muscle-intrinsic clock machinery is required for the maintenance of muscle growth, remodeling and function. Our previous studies demonstrated that the essential transcription activator of the molecular clock feed-back loop, Brain and Muscle Arnt-Like 1(Bmal1), plays a critical role in myogenic progenitor behavior to promote and regenerative myogenesis. Using genetic approaches targeting Bmal1 in the DMD mdx (mdx) dystrophic mouse model, here we report that the loss of Bmal1 function significantly accelerated dystrophic disease progression. In contrast to the mild dystrophic changes in mdx mice, the genetic loss-of-function of Bmal1 aggravated muscle damage in this dystrophic disease background, as indicated by persistently elevated creatine kinase levels, increased injury area and reduced muscle grip strength. Mechanistic studies revealed that markedly impaired myogenic progenitor proliferation and myogenic response underlie the defective new myofiber formation in the chronic dystrophic milieu. Taken together, our study identified the function of pro-myogenic clock gene Bmal1 in protecting against dystrophic damage, suggesting the potential for augmenting Bmal1 function to ameliorate dystrophic or degenerative muscle diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Heparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear., Methods: We examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system., Results: CRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl 2 injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells., Significance: These results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.

Skeletal muscles produce secretory factors termed as myokines, which alter physiological functions of target tissues. We recently identified C-X-C chemokine ligand 10 (CXCL10) as a novel myokine, which is downregulated in response to exercise. In the present study, we investigated whether the nutritional changes affect CXCL10 expression in mouse skeletal muscle. Expression of CXCL10 was evaluated in mice fed a normal diet or a high fat diet for 10 weeks. In animals fed on HFD, Cxcl10 expression was significantly induced in fast-twitched muscles, and was accompanied by increased blood glucose and free fatty acid levels. In vitro experiments using C2C12 myotubes suggested that the increased levels of glucose and palmitic acids directly enhanced CXCL10 expression. Interestingly, the effect of palmitic acids was attenuated by palmitoleic acids. Considering its potent angiostatic activity, induction of CXCL10 by nutritional changes may contribute to the impairment of microvascular networks in skeletal muscles.

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca 2+ However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca 2+ channels (CaChs) and excitation-contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac , which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca 2+ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides., Competing Interests: The authors declare no competing interest.

Reptiles are the only amniotes that maintain the capacity to regenerate appendages. This study presents the first anatomical and histological evidence of tail repair with regrowth in an archosaur, the American alligator. The regrown alligator tails constituted approximately 6-18% of the total body length and were morphologically distinct from original tail segments. Gross dissection, radiographs, and magnetic resonance imaging revealed that caudal vertebrae were replaced by a ventrally-positioned, unsegmented endoskeleton. This contrasts with lepidosaurs, where the regenerated tail is radially organized around a central endoskeleton. Furthermore, the regrown alligator tail lacked skeletal muscle and instead consisted of fibrous connective tissue composed of type I and type III collagen fibers. The overproduction of connective tissue shares features with mammalian wound healing or fibrosis. The lack of skeletal muscle contrasts with lizards, but shares similarities with regenerated tails in the tuatara and regenerated limbs in Xenopus adult frogs, which have a cartilaginous endoskeleton surrounded by connective tissue, but lack skeletal muscle. Overall, this study of wild-caught, juvenile American alligator tails identifies a distinct pattern of wound repair in mammals while exhibiting features in common with regeneration in lepidosaurs and amphibia.

Chromatin remodeling complexes have functions in transcriptional regulation and chromosome maintenance, but it is mostly unknown how the function of these normally ubiquitous complexes is specified in the cellular context. Here, we describe that the evolutionary conserved long non-coding RNA linc-MYH regulates the composition of the INO80 chromatin remodeler complex in muscle stem cells and prevents interaction with WDR5 and the transcription factor YY1. Linc-MYH acts as a selective molecular switch in trans that governs the pro-proliferative function of the ubiquitous INO80 complex but does not affect its role in maintaining genomic stability. The molecular switch is essential for restricting generation of quiescent MuSCs and proliferation of myoblasts in homeostasis and regeneration. Since linc-MYH is expressed in proliferating myoblasts but not in quiescent MuSCs, we reason that the extent of myoblast proliferation has decisive effects on the size of the quiescent MuSC pool., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Background: Manual analysis of cross-sectional area, fiber-type distribution, and total and centralized nuclei in skeletal muscle cross sections is tedious and time consuming, necessitating an accurate, automated method of analysis. While several excellent programs are available, our analyses of skeletal muscle disease models suggest the need for additional features and flexibility to adequately describe disease pathology. We introduce a new semi-automated analysis program, MyoSight, which is designed to facilitate image analysis of skeletal muscle cross sections and provide additional flexibility in the analyses., Results: We describe staining and imaging methods that generate high-quality images of immunofluorescent-labelled cross sections from mouse skeletal muscle. Using these methods, we can analyze up to 5 different fluorophores in a single image, allowing simultaneous analyses of perinuclei, central nuclei, fiber size, and fiber-type distribution. MyoSight displays high reproducibility among users, and the data generated are in close agreement with data obtained from manual analyses of cross-sectional area (CSA), fiber number, fiber-type distribution, and number and localization of myonuclei. Furthermore, MyoSight clearly delineates changes in these parameters in muscle sections from a mouse model of Duchenne muscular dystrophy (mdx)., Conclusions: MyoSight is a new program based on an algorithm that can be optimized by the user to obtain highly accurate fiber size, fiber-type identification, and perinuclei and central nuclei per fiber measurements. MyoSight combines features available separately in other programs, is user friendly, and provides visual outputs that allow the user to confirm the accuracy of the analyses and correct any inaccuracies. We present MyoSight as a new program to facilitate the analyses of fiber type and CSA changes arising from injury, disease, exercise, and therapeutic interventions.

Branched-chain α-keto acids (BCKAs) are catabolites of branched-chain amino acids (BCAAs). Intracellular BCKAs are cleared by branched-chain ketoacid dehydrogenase (BCKDH), which is sensitive to inhibitory phosphorylation by BCKD kinase (BCKDK). Accumulation of BCKAs is an indicator of defective BCAA catabolism and has been correlated with glucose intolerance and cardiac dysfunction. However, it is unclear whether BCKAs directly alter insulin signaling and function in the skeletal and cardiac muscle cell. Furthermore, the role of excess fatty acids (FAs) in perturbing BCAA catabolism and BCKA availability merits investigation. By using immunoblotting and ultra-performance liquid chromatography MS/MS to analyze the hearts of fasted mice, we observed decreased BCAA-catabolizing enzyme expression and increased circulating BCKAs, but not BCAAs. In mice subjected to diet-induced obesity (DIO), we observed similar increases in circulating BCKAs with concomitant changes in BCAA-catabolizing enzyme expression only in the skeletal muscle. Effects of DIO were recapitulated by simulating lipotoxicity in skeletal muscle cells treated with saturated FA, palmitate. Exposure of muscle cells to high concentrations of BCKAs resulted in inhibition of insulin-induced AKT phosphorylation, decreased glucose uptake, and mitochondrial oxygen consumption. Altering intracellular clearance of BCKAs by genetic modulation of BCKDK and BCKDHA expression showed similar effects on AKT phosphorylation. BCKAs increased protein translation and mTORC1 activation. Pretreating cells with mTORC1 inhibitor rapamycin restored BCKA's effect on insulin-induced AKT phosphorylation. This study provides evidence for FA-mediated regulation of BCAA-catabolizing enzymes and BCKA content and highlights the biological role of BCKAs in regulating muscle insulin signaling and function., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Biswas et al.)

The protective effects of chronic moderate exercise-mediated autophagy include the prevention and treatment of several diseases and the extension of lifespan. In addition, physical exercise may impair cellular structures, requiring the action of the autophagy mechanism for clearance and renovation of damaged cellular components. For the first time, we investigated the adaptations on basal autophagy flux in vivo in mice's liver, heart, and skeletal muscle tissues submitted to four different chronic exercise models: endurance, resistance, concurrent, and overtraining. Measuring the autophagy flux in vivo is crucial to access the functionality of the autophagy pathway since changes in this pathway can occur in more than five steps. Moreover, the responses of metabolic, performance, and functional parameters, as well as genes and proteins related to the autophagy pathway, were addressed. In summary, the regular exercise models exhibited normal/enhanced adaptations with reduced autophagy-related proteins in all tissues. On the other hand, the overtrained group presented higher expression of Sqstm1 and Bnip3 with negative morphological and physical performance adaptations for the liver and heart, respectively. The groups showed different adaptions in autophagy flux in skeletal muscle, suggesting the activation or inhibition of basal autophagy may not always be related to improvement or impairment of performance.

Differentiation of mesenchymal stem cells (MSCs) derived from two different sources of fetal tissues such as umbilical cord blood (UCB) and tissue (UCT) into skeletal muscle have remained underexplored. Here, we present a comparative analysis of UCB and UCT MSCs, in terms of surface markers, proliferation and senescence marker expression. We find that CD45 - CD34 - MSCs obtained from UCT and UCB of term births display differences in the combinatorial expression of key MSC markers CD105 and CD90. Importantly, UCT MSCs display greater yield, higher purity, shorter culture time, and lower rates of senescence in culture compared to UCB MSCs. Using a robust myogenic differentiation protocol, we show that UCT MSCs differentiate more robustly into muscle than UCB MSCs by transcriptomic sequencing and specific myogenic markers. Functional assays reveal that CD90, and not CD105 expression promotes myogenic differentiation in MSCs and could explain the enhanced myogenic potential of UCT MSCs. These results suggest that in comparison to large volumes of UCB that are routinely used to obtain MSCs and with limited success, UCT is a more reliable, robust, and convenient source of MSCs to derive cells of the myogenic lineage for both therapeutic purposes and increasing our understanding of developmental processes.

Zinc is an essential metal ion involved in many biological processes. Studies have shown that zinc can activate several molecules in the insulin signalling pathway and the concomitant uptake of glucose in skeletal muscle cells. However, there is limited information on other potential pathways that zinc can activate in skeletal muscle. Accordingly, this study aimed to identify other zinc-activating pathways in skeletal muscle cells to further delineate the role of this metal ion in cellular processes. Mouse C2C12 skeletal muscle cells were treated with insulin (10 nM), zinc (20 µM), and the zinc chelator TPEN (various concentrations) over 60 min. Western blots were performed for the zinc-activation of pAkt, pErk, and pCreb. A Cignal 45-Reporter Array that targets 45 signalling pathways was utilised to test the ability of zinc to activate pathways that have not yet been described. Zinc and insulin activated pAkt over 60 min as expected. Moreover, the treatment of C2C12 skeletal muscle cells with TPEN reduced the ability of zinc to activate pAkt and pErk. Zinc also activated several associated novel transcription factor pathways including Nrf1/Nrf2, ATF6, CREB, EGR1, STAT1, AP-1, PPAR, and TCF/LEF, and pCREB protein over 120 min of zinc treatment. These studies have shown that zinc's activity extends beyond that of insulin signalling and plays a role in modulating novel transcription factor activated pathways. Further studies to determine the exact role of zinc in the activation of transcription factor pathways will provide novel insights into this metal ion actions.

Introduction: Skeletal muscle-derived stem cells (Sk-MDSCs) are considered promising sources of adult stem cell therapy. Skeletal muscle comprises approximately 40-50% of the total body mass with marked potential for postnatal adaptive response, such as muscle hypertrophy, hyperplasia, atrophy, and regenerative capacity. This strongly suggests that skeletal muscle contains various stem/progenitor cells related to muscle-nerve-vascular tissues, which would support the above postnatal events even in adulthood., Area Covered: The focus of this review is the therapeutic potential of the Sk-MDSCs as an adult stem cell autograft. For this purpose, the validity of cell isolation and purification, tissue reconstitution capacity in vivo after transplantation, comparison of the results of basic mouse and preclinical human studies, potential problematic and beneficial aspects, and effective usage have been discussed following the history of clinical applications., Expert Opinion: Although the clinical application of Sk-MDSCs began as a therapy for the systemic disease of Duchenne muscular dystrophy, here, through the unique local injection method, therapy for severely damaged peripheral nerves, particularly the long-gap nerve transection, has been introduced. The beneficial aspects of the use of Sk-MDSCs as the source of local tissue transplantation therapy have also been discussed.

Despite remarkable advancements in modern medicine, muscular atrophy remains as an unsolved problem. It is well known that pathological characteristics of different atrophy types could vary according to the pathophysiological causes. In fact, the lesion of atrophy is not always homogenously distributed but often predominantly evident in either fast or slow myofibers. As the focalization of the atrophic lesions, the existence and the functional impairment of each fast and slow progenitor/satellite cell (SC) are suspected though there are still controversies about this hypothesis. In this study, we isolated Pax7 positive (Pax7 +ve ) SCs from the tibia anterior (fast) and soleus (slow) muscles respectively and successfully demonstrated, for the first time, the difference between optimal exposure durations of photobiomodulation (PBM) which was known as low level laser irradiation (LLLI) in promoting proliferation of Pax7 +ve SC which were acquired from fast and slow muscles respectively. Moreover, a hypertrophy-accompanied bidirectional change in myofiber composition with neuromuscular junction alteration, either from slow to fast or fast to slow, were achieved by applying different PBM durations. Simultaneously, PBM exhibited a synergistic effect with muscle exercise on the increase in myofiber size. Our data suggested the existence of at least two different populations of Pax7 +ve SC which possess distinct sensitivities towards PBM. As our data revealed the capability of PBM in bidirectional changes of skeletal muscle composition and neuromuscular junction constitution thereby strengthen its contractility through altering the irradiation condition, we believe PBM showed the potential to be as a promising clinical treatment for muscular atrophy., (Copyright © 2020 Elsevier B.V. All rights reserved.)

To address the frequency of complex V defects, we systematically sequenced MT-ATP6/8 genes in 512 consecutive patients. We performed functional analysis in muscle or fibroblasts for 12 out of 27 putative homoplasmic mutations and in cybrids for four. Fibroblasts, muscle and cybrids with known deleterious mutations underwent parallel analysis. It included oxidative phosphorylation spectrophotometric assays, western blots, structural analysis, ATP production, glycolysis and cell proliferation evaluation. We demonstrated the deleterious nature of three original mutations. Striking gradation in severity of the mutations consequences and differences between muscle, fibroblasts and cybrids implied a likely under-diagnosis of human complex V defects., (Copyright © 2020 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Background: Myopathy affects nearly half of individuals with alcohol use disorder (AUD), and impaired skeletal muscle regenerative potential is a probable contributing factor. Previous findings from our laboratory indicate that chronic in vivo and in vitro ethanol (EtOH) treatment decreases myogenic potential of skeletal muscle myoblasts. Myogenesis, a highly coordinated process, requires shifts in cellular metabolic state allowing for myoblasts to proliferate and differentiate into mature myotubes. The objective of this study was to determine whether alcohol interferes with myoblast mitochondrial and glycolytic metabolism and impairs myogenic differentiation., Methods: Myoblasts were isolated from vastus lateralis muscle excised from alcohol-naïve adult male (n = 5) and female (n = 5) rhesus macaques. Myoblasts were proliferated for 3 days (day 0 differentiation; D0) and differentiated for 5 days (D5) with or without 50 mM EtOH. Metabolism was assessed using a mitochondrial stress test to measure oxygen consumption (OCR) and extracellular acidification (ECAR) rates at D0. Differentiation was examined at D5. Expression of mitochondrial and glycolytic genes and mitochondrial DNA (mtDNA) was measured at D0 and D5., Results: Ethanol significantly (p < 0.05) increased myoblast maximal OCR and decreased ECAR at D0, and decreased fusion index, myotubes per field, and total nuclei at D5. The EtOH-induced decrease in ECAR was associated with the EtOH-mediated decreases in fusion index and myotubes per field. EtOH did not alter the decrease in glycolytic gene expression and increase in mtDNA from D0 to D5., Conclusion: During myoblast proliferation, EtOH decreased glycolytic metabolism and increased maximal OCR, suggesting that myoblast metabolic phenotype was dysregulated with EtOH. The EtOH-induced decrease in ECAR was associated with decreased differentiation. These findings suggest that EtOH-mediated shifts in metabolic phenotype may underlie impaired differentiation, which has important clinical implications for myogenesis in those affected by alcoholic myopathy., (© 2020 by the Research Society on Alcoholism.)

Satellite cells (SCs) are myogenic progenitors responsible for skeletal muscle regeneration and maintenance. Upon activation, SCs enter a phase of robust proliferation followed by terminal differentiation. Underlying this myogenic progression, the sequential expression of muscle regulatory transcription factors (MRFs) and the downregulation of transcription factor paired box gene 7 (Pax7) are key steps regulating SC fate. In addition to transcriptional regulation, post-translational control of Pax7 and the MRFs provides another layer of spatiotemporal control to the myogenic process. In this context, previous work showed that Pax7 is ubiquitinated by the E3 ligase neural precursor cell-expressed developmentally downregulated protein 4 and interacts with several proteins related to the ubiquitin-proteasome system, including the deubiquitinase ubiquitin-specific protease 7 (USP7). Although USP7 functions in diverse cellular contexts, its role(s) during myogenesis remains poorly explored. Here, we show that USP7 is transiently expressed in adult muscle progenitors, correlating with the onset of myogenin expression, while it is downregulated in newly formed myotubes/myofibers. Acute inhibition of USP7 activity upon muscle injury results in persistent expression of early regeneration markers and a significant reduction in the diameter of regenerating myofibers. At the molecular level, USP7 downregulation or pharmacological inhibition impairs muscle differentiation by affecting myogenin stability. Co-immunoprecipitation and in vitro activity assays indicate that myogenin is a novel USP7 target for deubiquitination. These results suggest that USP7 regulates SC myogenic progression by enhancing myogenin stability., (© 2020 Federation of European Biochemical Societies.)

Thyroid hormones are important for homeostatic control of energy metabolism and body temperature. Although skeletal muscle is considered a key site for thyroid action, the contribution of thyroid hormone receptor signaling in muscle to whole-body energy metabolism and body temperature has not been resolved. Here, we show that T3-induced increase in energy expenditure requires thyroid hormone receptor alpha 1 (TRα 1 ) in skeletal muscle, but that T3-mediated elevation in body temperature is achieved in the absence of muscle-TRα 1 . In slow-twitch soleus muscle, loss-of-function of TRα 1 (TRα HSACre ) alters the fiber-type composition toward a more oxidative phenotype. The change in fiber-type composition, however, does not influence the running capacity or motivation to run. RNA-sequencing of soleus muscle from WT mice and TRα HSACre mice revealed differentiated transcriptional regulation of genes associated with muscle thermogenesis, such as sarcolipin and UCP3, providing molecular clues pertaining to the mechanistic underpinnings of TRα 1 -linked control of whole-body metabolic rate. Together, this work establishes a fundamental role for skeletal muscle in T3-stimulated increase in whole-body energy expenditure., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Small leucine rich proteoglycans (SLRPs), including Biglycan, have key roles in many organ and tissue systems. The goal of this article is to review the function of Biglycan and other related SLRPs in mineralizing tissues of the skeleton. The review is divided into sections that include Biglycan's role in structural biology, signaling, craniofacial and long bone homeostasis, remodeled skeletal tissues, and in human genetics. While many cell types in the skeleton are now known to be affected by Biglycan, there are still unanswered questions about its mechanism of action(s).

Introduction: Several studies investigated the changes in diffusion of water molecules in skeletal muscle cells of lifestyle-related-disease patients who performed a hybrid training (HYBT) for six months. They reported that the apparent diffusion coefficient (ADC) and all diffusion eigenvalues (λ 1 , λ 2 , and λ 3 ) increased after the HYBT, owing to the enlargement of the intramyocellular diffusion space (intracellular space) caused by the muscular hypertrophy. We assumed that the HYBT promoted metabolism of the whole skeletal muscle including lipids, which reduced the amount of intramyocellular lipid (IMCL), and led to a secondary enlargement of the diffusion space in the skeletal muscle cells. However, the IMCL has to be a diffusion limiting factor in order to verify this hypothesis. Until now, there is no report on whether IMCL is a diffusion limiting factor for water molecules. The objective of this study was to examine whether the IMCL is a diffusion limiting factor in skeletal muscle cells., Materials and Methods: We performed a three-dimensional quantification of the IMCL in triceps surae muscles of lifestyle-related-disease patients and healthy volunteers. In addition, we measured the ADC in the volume of interest (VOI), diffusion anisotropy (FA), and diffusion eigenvalues (λ 1 , λ 2 , and λ 3 ), and evaluated the correlations between these diffusion parameters and IMCL., Results: The results showed that the amount of IMCL was positively and negatively correlated with the FA and λ 3 , respectively, in lifestyle-related-disease patients. In addition, there was a weak negative correlation between IMCL and ADC, λ 1 , and λ 2 . There was no correlation between the amount of IMCL and diffusion parameters of healthy volunteers., Discussion: Above a certain amount, the IMCL correlates with the diffusion parameters. A higher amount of IMCL leads to smaller diffusion eigenvalues. This result suggested that IMCL possibility of influencing diffusion of water molecules in skeletal muscle cells. However, in order for the influence of IMCL to be reflected in the diffusion eigenvalues, it was needed large amount of IMCL existed, and we thought that the influence was smaller than the influence by the already reported cell membrane., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In addition to multi-nucleated muscle fibres, numerous resident and infiltrating mononuclear cells populate the muscle compartment. As most epigenetic assays in skeletal muscle are conducted on whole tissue homogenates, essentially nothing is known about regulatory processes exclusively within muscle fibres in vivo . Utilizing a novel genetically modified mouse model developed by our laboratory, we (1) outline a simple and rapid workflow for isolating pure myonuclei from small tissue samples via fluorescent activated cell sorting and extracting high-quality large-fragment DNA for downstream analyses, and (2) provide information on myonuclear and interstitial cell nuclear CpG DNA methylation via reduced representation bisulphite sequencing (RRBS) using mice that were subjected to an acute mechanical overload of the plantaris muscle. In 3-month-old mice, myonuclei are ~50% of total nuclei in sham and ~30% in 3-d overloaded muscle, the difference being attributable to mononuclear cell infiltration and proliferation with overload. In purified myonuclei, pathway analysis of hypomethylated promoter regions following overload was distinct from interstitial nuclei and revealed marked regulation of factors that converge on the master regulator of muscle growth mTOR, and on autophagy. Specifically, acute hypomethylation of Rheb, Rictor, Hdac1, and Hdac2 , in addition to a major driver of ribosome biogenesis Myc , reveals the epigenetic regulation of hypertrophic signalling within muscle fibres that may underpin the long-term growth response to loading. This study provides foundational information on global myonuclear epigenetics in vivo using RRBS, and demonstrates the importance of isolating specific nuclear populations to study the epigenetic regulation of skeletal muscle fibre adaptation.

Skeletal muscle, a significant contributor to resting energy expenditure and reactive oxygen species, may play a critical role in body-weight regulation and aging processes. Methionine restriction (MR) is a dietary intervention which extends lifespan, lowers body-weight and enhances energy expenditure in rodents, all of which have been linked to mitochondrial function in various tissues including liver, kidney, heart and brown adipose tissue; however, mitochondrial responses to MR in skeletal muscle is largely unknown. Given the importance of skeletal muscle on energy metabolism and aging-related processes, we investigated if there are changes in skeletal muscle mitochondrial energetics in response to MR. Although MR lowers body-weight in rats, neither respiration, proton leak nor hydrogen peroxide metabolism were altered in isolated skeletal muscle mitochondria. This suggests that mitochondrial function in skeletal muscle remains conserved while MR alters metabolism in other tissues., (Copyright © 2020. Published by Elsevier B.V.)

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells 1-4 . Macrophages and satellite cells communicate in different ways 1-5 , but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.