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Skeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Studies in differentiating skeletal muscle cells in vitro have revealed that the microtubule-organizing center shifts from the centrosome to the perinuclear sites. As the Golgi apparatus surrounds the nucleus in a myotube, it is unclear whether microtubules are nucleated at the nuclear envelope or at the surrounding Golgi apparatus. In this study, we investigated the positional relationship between the microtubule nucleating sites and the Golgi apparatus in C2C12 myotubes and in primary cultured mouse skeletal myotubes. We focused on gaps in the perinuclear Golgi apparatus where the nuclear envelope was not covered with the Golgi apparatus. In microtubule regrowth assay, microtubule regrowth after cold-nocodazole depolymerization of preexisting microtubules was not found at the gap of the perinuclear Golgi apparatus. Most of the microtubule regrowth was detected at the CDK5RAP2 (CDK5 regulatory subunit-associated protein 2)-rich spots on the perinuclear Golgi apparatus. Disruption of the perinuclear Golgi apparatus with brefeldin A treatment eliminated the perinuclear microtubule regrowth. The Golgi apparatus of undifferentiated myoblasts and those at the cytoplasm of myotubes were also the microtubule nucleating sites. From these observations, we concluded that most of the perinuclear microtubule nucleation occurs on the Golgi apparatus surrounding the nucleus., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Resistance training (RT) dynamically alters the skeletal muscle nuclear DNA methylome. However, no study has examined if RT affects the mitochondrial DNA (mtDNA) methylome. Herein, ten older, Caucasian untrained males (65 ± 7 y.o.) performed six weeks of full-body RT (twice weekly). Body composition and knee extensor torque were assessed prior to and 72 h following the last RT session. Vastus lateralis (VL) biopsies were also obtained. VL DNA was subjected to reduced representation bisulfite sequencing providing excellent coverage across the ~16-kilobase mtDNA methylome (254 CpG sites). Biochemical assays were also performed, and older male data were compared to younger trained males (22 ± 2 y.o., n = 7, n = 6 Caucasian & n = 1 African American). RT increased whole-body lean tissue mass (p = .017), VL thickness (p = .012), and knee extensor torque (p = .029) in older males. RT also affected the mtDNA methylome, as 63% (159/254) of the CpG sites demonstrated reduced methylation (p < .05). Several mtDNA sites presented a more "youthful" signature in older males after RT in comparison to younger males. The 1.12 kilobase mtDNA D-loop/control region, which regulates replication and transcription, possessed enriched hypomethylation in older males following RT. Enhanced expression of mitochondrial H- and L-strand genes and complex III/IV protein levels were also observed (p < .05). While limited to a shorter-term intervention, this is the first evidence showing that RT alters the mtDNA methylome in skeletal muscle. Observed methylome alterations may enhance mitochondrial transcription, and RT evokes mitochondrial methylome profiles to mimic younger men. The significance of these findings relative to broader RT-induced epigenetic changes needs to be elucidated., (© 2021 Federation of American Societies for Experimental Biology.)

The FOXP3 + CD4 + regulatory T (T reg ) cells located in non-lymphoid tissues differ in phenotype and function from their lymphoid organ counterparts. Tissue T reg cells have distinct transcriptomes, T cell receptor repertoires and growth and survival factor dependencies that arm them to survive and operate in their home tissue. Their functions extend beyond immune surveillance to tissue homeostasis, including regulation of local and systemic metabolism, promotion of tissue repair and regeneration, and control of the proliferation, differentiation and fate of non-lymphoid cell progenitors. T reg cells in diverse tissues share a common FOXP3 + CD4 + precursor located within lymphoid organs. This precursor undergoes definitive specialization once in the home tissue, following a multilayered array of common and tissue-distinct transcriptional programmes. Our deepening knowledge of tissue T reg cell biology will inform ongoing attempts to harness T reg cells for precision immunotherapeutics., (© 2021. Springer Nature Limited.)

Skeletal muscle anatomy and physiology are sexually dimorphic but molecular underpinnings and muscle-specificity are not well-established. Variances in metabolic health, fitness level, sedentary behavior, genetics, and age make it difficult to discern inherent sex effects in humans. Therefore, mice under well-controlled conditions were used to determine female and male (n = 19/sex) skeletal muscle fiber type/size and capillarity in superficial and deep gastrocnemius (GA-s, GA-d), soleus (SOL), extensor digitorum longus (EDL), and plantaris (PLT), and transcriptome patterns were also determined (GA, SOL). Summed muscle weight strongly correlated with lean body mass (r 2  = 0.67, p < 0.0001, both sexes). Other phenotypes were muscle-specific: e.g., capillarity (higher density, male GA-s), myofiber size (higher, male EDL), and fiber type (higher, lower type I and type II prevalences, respectively, in female SOL). There were broad differences in transcriptomics, with >6000 (GA) and >4000 (SOL) mRNAs differentially-expressed by sex; only a minority of these were shared across GA and SOL. Pathway analyses revealed differences in ribosome biology, transcription, and RNA processing. Curation of sexually dimorphic muscle transcripts shared in GA and SOL, and literature datasets from mice and humans, identified 11 genes that we propose are canonical to innate sex differences in muscle: Xist, Kdm6a, Grb10, Oas2, Rps4x (higher, females) and Ddx3y, Kdm5d, Irx3, Wwp1, Aldh1a1, Cd24a (higher, males). These genes and those with the highest "sex-biased" expression in our study do not contain estrogen-response elements (exception, Greb1), but a subset are proposed to be regulated through androgen response elements. We hypothesize that innate muscle sexual dimorphism in mice and humans is triggered and then maintained by classic X inactivation (Xist, females) and Y activation (Ddx3y, males), with coincident engagement of X encoded (Kdm6a) and Y encoded (Kdm5d) demethylase epigenetic regulators that are complemented by modulation at some regions of the genome that respond to androgen., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Objective: MicroRNAs (miRNA) are known to regulate the expression of genes involved in several physiological processes including metabolism, mitochondrial biogenesis, proliferation, differentiation, and cell death., Methods: Using "in silico" analyses, we identified 219 unique miRNAs that potentially bind to the 3'UTR region of a critical mitochondrial regulator, the peroxisome proliferator-activated receptor gamma coactivator (PGC) 1 alpha (Pgc1α). Of the 219 candidate miRNAs, miR-696 had one of the highest interactions at the 3'UTR of Pgc1α, suggesting that miR-696 may be involved in the regulation of Pgc1α., Results: Consistent with this hypothesis, we found that miR-696 was highly expressed in the skeletal muscle of STZ-induced diabetic mice and chronic high-fat-fed mice. C2C12 muscle cells exposed to palmitic acid also exhibited a higher expression of miR-696. This increased expression corresponded with a reduced expression of oxidative metabolism genes and reduced mitochondrial respiration. Importantly, reducing miR-696 reversed decreases in mitochondrial activity in response to palmitic acid. Using C2C12 cells treated with the AMP-activated protein kinase (AMPK) activator AICAR and skeletal muscle from AMPKα2 dominant-negative (DN) mice, we found that the signaling mechanism regulating miR-696 did not involve AMPK. In contrast, overexpression of SNF1-AMPK-related kinase (SNARK) in C2C12 cells increased miR-696 transcription while knockdown of SNARK significantly decreased miR-696. Moreover, muscle-specific transgenic mice overexpressing SNARK exhibited a lower expression of Pgc1α, elevated levels of miR-696, and reduced amounts of spontaneous activity., Conclusions: Our findings demonstrate that metabolic stress increases miR-696 expression in skeletal muscle cells, which in turn inhibits Pgc1α, reducing mitochondrial function. SNARK plays a role in this process as a metabolic stress signaling molecule inducing the expression of miR-696., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Loss of muscle mass and strength after disuse followed by impaired muscle recovery commonly occurs with aging. Metformin (MET) and leucine (LEU) individually have shown positive effects in skeletal muscle during atrophy conditions but have not been evaluated in combination nor tested as a remedy to enhance muscle recovery following disuse atrophy in aging. The purpose of this study was to determine if a dual treatment of metformin and leucine (MET + LEU) would prevent disuse-induced atrophy and/or promote muscle recovery in aged mice and if these muscle responses correspond to changes in satellite cells and collagen remodeling. Aged mice (22-24 months) underwent 14 days of hindlimb unloading (HU) followed by 7 or 14 days of reloading (7 or 14 days RL). MET, LEU, or MET + LEU was administered via drinking water and were compared to Vehicle (standard drinking water) and ambulatory baseline. We observed that during HU, MET + LEU resolved whole body grip strength and soleus muscle specific force decrements caused by HU. Gastrocnemius satellite cell abundance was increased with MET + LEU treatment but did not alter muscle size during disuse or recovery conditions. Moreover, MET + LEU treatment alleviated gastrocnemius collagen accumulation caused by HU and increased collagen turnover during 7 and 14 days RL driven by a decrease in collagen IV content. Transcriptional pathway analysis revealed that MET + LEU altered muscle hallmark pathways related to inflammation and myogenesis during HU. Together, the dual treatment of MET and LEU was able to increase muscle function, satellite cell content, and reduce collagen accumulation, thus improving muscle quality during disuse and recovery in aging., (© 2021 Federation of American Societies for Experimental Biology.)

Dynamic regulation of gene expression is often governed by progression through transient cell states. Bulk RNA-seq analysis can only detect average change in expression levels and is unable to identify this dynamics. Single cell RNA-seq presents an unprecedented opportunity that helps in placing the cells on a hypothetical time trajectory that reflects gradual transition of their transcriptomes. This continuum trajectory or 'pseudotime', may reveal the developmental pathway and provide us with information on dynamic transcriptomic changes and other biological processes. Existing approaches to build pseudotime heavily depend on reducing huge dimension to extremely low dimensional subspaces and may lead to loss of information. We propose PseudoGA, a genetic algorithm based approach to order cells assuming that gene expressions vary according to a smooth curve along the pseudotime trajectory. We observe superior accuracy of our method in simulated as well as benchmarking real datasets. Generality of the assumption behind PseudoGA and no dependence on dimensionality reduction technique make it a robust choice for pseudotime estimation from single cell transcriptome data. PseudoGA is also time efficient when applied to a large single cell RNA-seq data and adaptable to parallel computing. R code for PseudoGA is freely available at https://github.com/indranillab/pseudoga., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Skeletal muscle regeneration is mediated by myoblasts that undergo epigenomic changes to establish the gene expression program of differentiated myofibers. mSWI/SNF chromatin remodeling enzymes coordinate with lineage-determining transcription factors to establish the epigenome of differentiated myofibers. Bromodomains bind to acetylated lysines on histone N-terminal tails and other proteins. The mutually exclusive ATPases of mSWI/SNF complexes, BRG1 and BRM, contain bromodomains with undefined functional importance in skeletal muscle differentiation. Pharmacological inhibition of mSWI/SNF bromodomain function using the small molecule PFI-3 reduced differentiation in cell culture and in vivo through decreased myogenic gene expression, while increasing cell cycle-related gene expression and the number of cells remaining in the cell cycle. Comparative gene expression analysis with data from myoblasts depleted of BRG1 or BRM showed that bromodomain function was required for a subset of BRG1- and BRM-dependent gene expression. Reduced binding of BRG1 and BRM after PFI-3 treatment showed that the bromodomain is required for stable chromatin binding at target gene promoters to alter gene expression. Our findings demonstrate that mSWI/SNF ATPase bromodomains permit stable binding of the mSWI/SNF ATPases to promoters required for cell cycle exit and establishment of muscle-specific gene expression., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Muscle stem cells (MuSCs) are a rare stem cell population that provides myofibers with a remarkable capacity to regenerate after tissue injury. Here, we have adapted the Cleavage Under Target and Tagmentation technology to the mapping of the chromatin landscape and transcription factor binding in 50,000 activated MuSCs isolated from injured mouse hindlimb muscles. We have applied this same approach to human CD34 + hematopoietic stem and progenitor cells. This protocol could be adapted to any rare stem cell population. For complete details on the use and execution of this protocol, please refer to Robinson et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The growth of fishes and their metabolism is highly variable in fish species and is an indicator for fish fitness. Therefore, somatic growth, as a main biological process, is ecologically and economically significant. The growth differences of two closely related salmonids, rainbow trout (Oncorhynchus mykiss) and maraena whitefsh (Coregonus maraena), have not been adequately studied as a comparative study and are therefore insufficiently understood. For this reason, our aim was to examine muscle growth in more detail and provide a first complex insight into the growth and muscle metabolism of these two fish species at slaughter size. In addition to skeletal muscle composition (including nuclear counting and staining of stem and progenitor cells), biochemical characteristics, and enzyme activity (creatine kinase, lactate dehydrogenase, isocitrate dehydrogenase) of rainbow trout and maraena whitefish were determined. Our results indicate that red muscle contains cells with a smaller diameter compared to white muscle and those fibres had more stem and progenitor cells as a proportion of total nuclei. Interestingly, numerous interspecies differences were identified; in rainbow trout muscle RNA content, intermediate fibres and fibre diameter and in whitefish red muscle cross-sectional area, creatine kinase activity were higher compared to the other species at slaughter weight. The proportional reduction in red muscle area, accompanied by an increase in DNA content and a lower activity of creatine kinase, exhibited a higher degree of hypertrophic growth in rainbow trout compared to maraena whitefish, which makes this species particularly successful as an aquaculture species., Competing Interests: The authors have declared that no competing interests exist.

Depending on metabolic requirements, skeletal muscle mitochondria integrate O 2 consumption and ATP production with lipid, glucose, or amino acid metabolism. Free fatty acids (FFAs) are the main source of energy during rest and mild-intensity exercise. We present a detailed protocol for measuring FFA-β-oxidation coupled with O 2 respiration by a Clark-type electrode in isolated mitochondria from mouse soleus oxidative muscle. We optimized the procedure, including buffer composition, protease treatment, and quantifiable parameters (P/O, Phosphate/Oxygen Ratio; OCR, Oxygen Consumption Rate; RCR,Respiration Control Rate; OSR, Oligomycin Sensitive Respiration). For complete details on the use and execution of this protocol, please refer to Sanchez-Gonzalez et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Repeated mechanical stress causes injuries in the adult skeletal muscle that need to be repaired. Although muscle regeneration is a highly efficient process, it fails in some pathological conditions, compromising tissue functionality. This may be caused by aberrant cell-cell communication, resulting in the deposition of fibrotic and adipose infiltrates. Here, we investigate in vivo changes in the profile of skeletal muscle secretome during the regeneration process to suggest new targetable regulatory circuits whose failure may lead to tissue degeneration in pathological conditions. We describe the kinetic variation of expression levels of 76 secreted proteins during the regeneration process. In addition, we profile the gene expression of immune cells, endothelial cells, satellite cells, and fibro-adipogenic progenitors. This analysis allowed us to annotate each cell-type with the cytokines and receptors they have the potential to synthetize, thus making it possible to draw a cell-cell interaction map. We next selected 12 cytokines whose receptors are expressed in FAPs and tested their ability to modulate FAP adipogenesis and proliferation. We observed that IL1α and IL1β potently inhibit FAP adipogenesis, while EGF and BTC notably promote FAP proliferation. In addition, we characterized the cross-talk mediated by extracellular vesicles (EVs). We first monitored the modulation of muscle EV cargo during tissue regeneration. Using a single-vesicle flow cytometry approach, we observed that EVs differentially affect the uptake of RNA and proteins into their lumen. We also investigated the EV capability to interact with SCs and FAPs and to modulate their proliferation and differentiation. We conclude that both cytokines and EVs secreted during muscle regeneration have the potential to modulate adipogenic differentiation of FAPs. The results of our approach provide a system-wide picture of mechanisms that control cell fate during the regeneration process in the muscle niche.

Purpose: Recent work has highlighted the therapeutic potential of targeting autophagy to modulate cell survival in a variety of diseases including cancer. Recently, we found that the RNA-binding protein Staufen1 (STAU1) is highly expressed in alveolar rhabdomyosarcoma (ARMS) and that this abnormal expression promotes tumorigenesis. Here, we asked whether STAU1 is involved in the regulation of autophagy in ARMS cells., Methods: We assessed the impact of STAU1 expression modulation in ARMS cell lines (RH30 and RH41), non-transformed skeletal muscle cells (C2C12) and STAU1-transgenic mice using complementary techniques., Results: We found that STAU1 silencing reduces autophagy in the ARMS cell lines RH30 and RH41, while increasing their apoptosis. Mechanistically, this inhibitory effect was found to be caused by a direct negative impact of STAU1 depletion on the stability of Beclin-1 (BECN1) and ATG16L1 mRNAs, as well as by an indirect inhibition of JNK signaling via increased expression of Dual specificity phosphatase 8 (DUSP8). Pharmacological activation of JNK or expression silencing of DUSP8 was sufficient to restore autophagy in STAU1-depleted cells. By contrast, we found that STAU1 downregulation in non-transformed skeletal muscle cells activates autophagy in a mTOR-dependent manner, without promoting apoptosis. A similar effect was observed in skeletal muscles obtained from STAU1-overexpressing transgenic mice., Conclusions: Together, our data indicate an effect of STAU1 on autophagy regulation in ARMS cells and its differential role in non-transformed skeletal muscle cells. Our findings suggest a cancer-specific potential of targeting STAU1 for the treatment of ARMS., (© 2021. Springer Nature Switzerland AG.)

A large number of studies have shown that polyphenols can regulate skeletal muscle fiber type transformation through AMPK signal. However, the effects and mechanism of naringin (a natural polyphenol) on muscle fiber type transformation still remains unclear. Thus, we hypothesized that naringin would induce the transformation of skeletal muscle fibers from type II to type I by AMPK signaling. C2C12 myotubes and BALB/c mice models were used to test this hypothesis. We found that naringin significantly increased the protein expression of slow myosin heavy chain (MyHC), myoglobin and troponin I type I slow skeletal (Troponin I-SS) and the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), and significantly decreased fast MyHC protein expression and lactate dehydrogenase (LDH) activity, accompanied by the activation of AMPK and the activity of peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) in mice and C2C12 myotubes. Further inhibition of AMPK activity by compound C showed that the above effects were significantly inhibited in C2C12 myotubes. In conclusion, naringin promotes the transformation of skeletal muscle fibers from type II to type I through AMPK/PGC-1α signaling pathway, which not only enriches the nutritional and physiological functions of naringin, but also provides a theoretical basis for the regulation of muscle fiber type transformation by nutritional approaches., Competing Interests: Author Declarations The authors declare that there are no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chronic diseases are associated with considerable morbidity and mortality. Therefore, new therapeutic strategies are warranted. Here, we provide a brief review outlining the rationale and feasibility for the generation of intraspecies and interspecies chimeras, which one day may serve as a platform for organ transplantation. These strategies are further associated with consideration of scientific and ethical issues.

Animal models clearly illustrate that the maintenance of skeletal muscle mass depends on the function and interaction of a heterogeneous population of resident and infiltrating mononuclear cells. Several lines of evidence suggest that mononuclear cells also play a role in muscle wasting in humans, and targeting these cells may open new treatment options for intervention or prevention in sarcopenia. Methodological and ethical constraints have perturbed exploration of the cellular characteristics and function of mononuclear cells in human skeletal muscle. Thus, investigations of cellular phenotypes often depend on immunohistochemical analysis of small tissue samples obtained by needle biopsies, which do not match the deep phenotyping of mononuclear cells obtained from animal models. Here, we have developed a protocol for fluorescence-activated cell sorting (FACS), based on single-cell RNA-sequencing data, for quantifying and characterizing mononuclear cell populations in human skeletal muscle. Muscle stem cells, fibro-adipogenic progenitors, and two subsets of macrophages (CD11c +/- ) are present in needle biopsies in comparable quantities per milligram tissue to open surgical biopsies. We find that direct cell isolation is preferable due to a substantial shift in transcriptome when using preculture before the FACS procedure. Finally, in vitro validation of the cellular phenotype of muscle stem cells, fibro-adipogenic progenitors, and macrophages confirms population-specific traits. This study demonstrates that mononuclear cell populations can be quantified and subsequently analyzed from needle biopsy material and opens the perspective for future clinical studies of cellular mechanisms in muscle wasting.

Skeletal muscle (SM) weakness occurs in hypothyroidism and resistance to thyroid hormone α (RTHα) syndrome. However, the cell signaling and molecular mechanism(s) underlying muscle weakness under these conditions is not well understood. We thus examined the role of thyroid hormone receptor α (TRα), the predominant TR isoform in SM, on autophagy, mitochondrial biogenesis, and metabolism to demonstrate the molecular mechanism(s) underlying muscle weakness in these two conditions. Two genetic mouse models were used in this study: TRα1PV/+ mice, which express the mutant Thra1PV gene ubiquitously, and SM-TRα1L400R/+ mice, which express TRα1L400R in a muscle-specific manner. Gastrocnemius muscle from TRα1PV/+, SM-TRα1L400R/+, and their control mice was harvested for analyses. We demonstrated that loss of TRα1 signaling in gastrocnemius muscle from both the genetic mouse models led to decreased autophagy as evidenced by accumulation of p62 and decreased expression of lysosomal markers (lysosomal-associated membrane protein [LAMP]-1 and LAMP-2) and lysosomal proteases (cathepsin B and cathepsin D). The expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), mitochondrial transcription factor A (TFAM), and estrogen-related receptor α (ERRα), key factors contributing to mitochondrial biogenesis as well as mitochondrial proteins, were decreased, suggesting that there was reduced mitochondrial biogenesis due to the expression of mutant TRα1. Transcriptomic and metabolomic analyses of SM suggested that lipid catabolism was impaired and was associated with decreased acylcarnitines and tricarboxylic acid cycle intermediates in the SM from the mouse line expressing SM-specific mutant TRα1. Our results provide new insight into TRα1-mediated cell signaling, molecular, and metabolic changes that occur in SM when TR action is impaired., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Megaconial congenital muscular dystrophy (CMD)(OMIM #602541), related to CHKB mutation, is a rare autosomal recessive disorder. To date, only 35 confirmed patients are recorded. We present a detailed description of the clinical, histopathological, imaging, and genetic findings of five children from four Indian families. The children had moderate-to-severe autistic behavior, hand stereotypies, and global developmental delay mimicking atypical Rett syndrome. In addition, generalized hypotonia was a common initial finding. The progression of muscle weakness was variable, with two patients having a milder phenotype and three having a severe form. Interestingly, the majority did not attain sphincter control. Only patient 1 had classical ichthyotic skin changes. Muscle biopsy in two patients showed a myopathic pattern with characteristic peripherally placed enlarged mitochondria on modified Gomori trichrome stain and electron microscopy. Genetic analysis in these patients identified three novel null mutations in CHKB [c.1027dupA (p.Ser343LysfsTer86);c.224 + 1G > T (5' splice site); c.1123C > T (p.Gln375Ter)] and one reported missense mutation, c.581G > A (p.Arg194Gln), all in the homozygous state. Megaconial CMD, although rare, forms an important group with a complex phenotypic presentation and accounted for 5.5% of our genetically confirmed CMD patients. Atypical Rett syndrome-like presentation may be a clue towards CHKB-related disorder., (© 2021. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Thyroid hormones (THs) are key regulators of different biological processes. Their action involves genomic and non-genomic mechanisms, which together mediate the final effects of TH in target tissues. However, the proportion of the two processes and their contribution to the TH-mediated effects are still poorly understood. Skeletal muscle is a classical target tissue for TH, which regulates muscle strength and contraction, as well as energetic metabolism of myofibers. Here we address the different contribution of genomic and non-genomic action of TH in skeletal muscle cells by specifically silencing the deiodinase Dio2 or the β3-Integrin expression via CRISPR/Cas9 technology. We found that myoblast proliferation is inversely regulated by integrin signal and the D2-dependent TH activation. Similarly, inhibition of the nuclear receptor action reduced myoblast proliferation, confirming that genomic action of TH attenuates proliferative rates. Contrarily, genomic and non-genomic signals promote muscle differentiation and the regulation of the redox state. Taken together, our data reveal that integration of genomic and non-genomic signal pathways finely regulates skeletal muscle physiology. These findings not only contribute to the understanding of the mechanisms involved in TH modulation of muscle physiology but also add insight into the interplay between different mechanisms of action of TH in muscle cells.

High mechanical load of muscles may induce muscular apoptosis on the one hand and adaptation to exercise on the other. This study aimed to explore whether changes of circulatory levels of inflammation, apoptosis and heat shock proteins (HSPs) messenger RNA (mRNA) following single bout of high-intensity interval exercise (HIIE) differs between physically active (PA) and inactive (PI) men. Nine PA and 9 PI (peak oxygen consumption: 2.6 ± 0.4 vs. 2.0 ± 0.2 L·min -1 ) healthy men (age: 28.7 ± 6.3 vs. 30.2 ± 4.5 years and body mass index: 2.6 ± 2.1 vs. 23.3 ± 2.8 kg·m -2 ) performed HIIE, comprising 4 repeats of a Wingate test (load: 0.050 kg·kg -1 body weight). Blood samples were collected before exercise, 5 min after HIIE, and 24 h after HIIE for measuring mRNA of inflammation markers interleukin-6 ( IL-6 ) and tumor necrosis factor alpha ( TNFα ), apoptosis markers including Bcl-2, Bax , and HSP27 , HSP60 , HSP70 , HSP90 using quantitative real-time PCR analysis. Post-HIIE IL-6 , TNFα and HSP60 were higher in the PI than the PA group 5 min after exercise ( p = 0.003, effect size (ES) = 1.59; p = 0.007, ES = 1.59 and p = 0.027, ES = 1.10 respectively). HSP70 acutely increased only in the PA group ( p = 0.024, ES = 1.20). The increase in Bcl-2 ( p = 0.047, ES = 1.08) and Bax ( p = 0.024, ES = 1.20) levels were higher in the PI group 5 min after HIIE. The present study indicated that the response of inflammatory, apoptosis and HSP gene expressions to HIIE in blood of healthy male volunteers strongly depends on their level of regular physical activity. Novelty: Blood IL-6 and HSP60 mRNA levels following high intensity exercise may indicate metabolic stress. Increased blood HSP70 mRNA in physically active men may show an alternative apoptosis suppression pathway.

Weight loss and exercise reduce colorectal cancer (CRC) risk in persons with obesity. Whether weight loss and exercise effect myofiber characteristics and muscle stem/progenitor cell populations in mice with preneoplastic colorectal lesions, a model of CRC risk, is unknown. To address this gap, male C57Bl/6J mice were fed a high-fat diet (HFD) to induce obesity or a control (CON) diet prior to azoxymethane injection to induce preneoplastic colorectal lesions. The HFD group was then randomized to weight loss conditions that included (1) switching to the CON diet only (HFD-SED) or switching to the CON diet with treadmill exercise training (HFD-EX). Average myofiber cross-sectional area was not different between groups. There were more smaller-sized fibres in HFD-EX ( p < 0.05 vs. CON), and more fibrosis in HFD-SED ( p < 0.05 vs. HFD-EX and CON). There was a trend for more committed (Pax7 + MyoD + ) myoblasts ( p = 0.059) and more fibro-adipogenic progenitors in HFD-EX ( p < 0.05 vs. CON). Additionally, the canonical pro-inflammatory marker p-NF-κB was markedly reduced in the interstitium of HFD-EX ( p < 0.05 vs. CON and HFD-SED). Our findings suggest that in mice with preneoplastic colorectal lesions, HFD followed by weight loss with exercise reduces muscle fibrosis and results in a higher content of muscle stem/progenitor cells. Novelty: Exercise improves muscle architecture in mice with preneoplastic colorectal lesion Exercise increases fibro/adipogenic progenitors and reduces inflammatory signaling in mice with preneoplastic colorectal lesions.

We report on the first regulatory approved clinical trial of a prospective open-label physician-initiated study assessing the safety and efficacy of intradetrusor injected Autologous Muscle Derived Cells (AMDC) treatment for underactive bladder (UAB). 20 non-neurogenic UAB patients were treated. Approximately 50-250 mg of quadriceps femoris muscle was collected using a spirotome 8-gauge needle. The muscle biopsy samples were sent to Cook MyoSite (Pittsburgh, PA) for processing, isolation, and propagation of cells. Research patients received approximately 30 intradetrusor injections of 0.5 mL delivered to the bladder, for a total of 15 mL and 125 million AMDC, performed utilizing a flexible cystoscope under direct vision using topical local anesthesia. Follow-up assessments included adverse events and efficacy via voiding diary and urodynamic testing at 1, 3, 6 and 12 months post-injection. An optional second injection was offered at the end of the 6 months visit. 20 patients received the first injection and all 20 patients requested and received a second injection. Median patient age was 65 years old (range 41-82 years). There were 16 male (80%) and 4 female (20%) patients. Etiology included 7 men (35%) with persistent urinary retention after transurethral resection of the prostate for benign prostatic hyperplasia and 13 patients (65%) with idiopathic chronic urinary retention. At the primary outcome time point of 12 months, 11/19 patients (58%) reported a global response assessment (GRA) ≥ 5, showing slight to marked improvement in their UAB symptoms, compared to 6/20 (30%) patients at 3 months post-injection. No serious procedure or treatment-related adverse events occurred. Noted improvements included: decreased post void residual urine volume, increased voiding efficiency, and decreased catheter use. Intradetrusor-injected AMDC as a treatment for UAB was successfully completed in a 20-patient trial without serious adverse event and with signal of efficacy. Cellular therapy may be a promising novel treatment for catheter-dependent chronic urinary retention. A multicenter controlled trial is needed to further assess the promise of regenerative medicine in the treatment of lower urinary tract dysfunction.

To understand muscular steatosis observed in beef carcasses, physicochemical and histochemical characteristics were compared between abnormal (massive fatty replaced) and normal regions of beef striploin. Fat content in the abnormal region (48.02%) was approximately twice than that in the normal region (22.01%). However, fatty acids did not show significant (P > 0.05) differences in their compositions between the two regions. Tenderness was significantly (P < 0.05) higher in the abnormal region. However, other meat quality traits were not significantly (P > 0.05) different between the two regions. Massive accumulation of adipocytes was accompanied by muscle fiber atrophy regardless of muscle fiber types. Without a change in total muscle fiber density, oxidative fiber composition was significantly increased, whereas glycolytic fiber composition was decreased (P < 0.05). These findings suggest that adipogenic transdifferentiation and muscle fiber type switching can occur within the muscle due to muscular steatosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Understanding the signaling pathways that regulate the final differentiation of human myoblasts is essential for successful cell transplantation and drug screening for the treatment of muscular dystrophy. In an effort to improve myotube formation from hiPSC-derived myoblasts, we validated a collection of 13 small molecules in a newly established in vitro screening platform for the assessment of myotube formation. The analysis of myotube formation as measured by the fusion index showed that the combinational inhibition of the TGFβ signaling with NOTCH signaling enhances the ability of multi-nucleated myotube production. Combinational treatment of inhibitors for TGFβ and NOTCH signaling pathways improved myotube formation in a dose-dependent manner. This effect was achieved by inhibiting the combinatorial mechanism of signaling. The combination treatment of small molecules effective in inducing multinucleated myotubes was validated in healthy human primary myoblasts. In addition, it was also applied to DMD patient iPSC-derived myoblasts to enhance the generation of multinucleated myotubes.

Materials and Methods: Rat L6 skeletal muscle cells were cultured in 25 cm 2 flasks. These differentiated cells were treated, and then, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (probe-based) was used to measure the relative mRNA expression level for metabolic, inflammatory, and nuclear receptor genes including peroxisome proliferator-activated receptor gamma (PGC-1 α ), carnitine palmitoyl transferase 1 beta (CPT1B), long-chain acyl-CoA de hydrogenase (LCAD), acetyl-CoA carboxylase beta (ACC β ), pyruvate dehydrogenase kinase 4 (PDK4), hexokinase II (HKII), phosphofructokinase (PFK), interleukin-6 (IL-6), and nuclear receptor subfamily 4, group A (NR4A) at different treatment conditions., Results: Adenosine-5'-N-ethyluronamide (NECA), a stable adenosine analogue, significantly stimulate inflammatory mediator (IL-6) ( p < 0.001) and nuclear receptors (NR4A) ( p < 0.05) and significantly modulate metabolic (PFK, LCAD, PGC-1 α , and CPT1B) gene expressions in skeletal muscle cells ( p < 0.05, p < 0.05, p < 0.001, and p < 0.01, respectively). This present study shows that there is a noteworthy crosstalk between NECA and insulin at various metabolic levels including glycolysis (HKII), fatty acid oxidation (ACC β ), and insulin sensitivity (PDK4)., Conclusions: A novel crosstalk between adenosine analogue and insulin has been demonstrated for the first time; evidence has been gathered in vitro for the effects of NECA and insulin treatment on intracellular signaling pathways, in particular glycolysis and insulin sensitivity in skeletal muscle cells., Competing Interests: The author declares that he has no conflicts of interest., (Copyright © 2021 Mansour Haddad.)

Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at successive developmental stages identifies a population displaying a dual muscle and connective tissue signature. BMP signalling is active in this dual population and at the tendon/muscle interface. In vivo and in vitro gain- and loss-of-function experiments show that BMP signalling regulates a fibroblast-to-myoblast conversion. These results suggest a scenario in which BMP signalling converts a subset of lateral plate mesoderm-derived cells to a myogenic fate in order to create a boundary of fibroblast-derived myonuclei at the myotendinous junction that controls limb muscle patterning.

Our recent in vivo human studies showed that colonic administration of sodium acetate (SA) resulted in increased circulating acetate levels, which was accompanied by increments in whole-body fat oxidation in overweight-obese men. Since skeletal muscle has a major role in whole-body fat oxidation, we aimed to investigate effects of SA on fat oxidation and underlying mechanisms in human primary skeletal muscle cells (HSkMC). We investigated the dose (0-5 mmol/L) and time (1, 4, 20, and 24 h) effect of SA on complete and incomplete endogenous and exogenous oxidation of 14 C-labeled palmitate in HSkMC derived from a lean insulin sensitive male donor. Both physiological (0.1 and 0.25 mmol/L) and supraphysiological (0.5, 1 and 5 mmol/L) concentrations of SA neither increased endogenous nor exogenous fat oxidation over time in HSkMC. In addition, no effect of SA was observed on Thr 172 -AMPKα phosphorylation. In conclusion, our previously observed in vivo effects of SA on whole-body fat oxidation in men may not be explained via direct effects on HSkMC fat oxidation. Nevertheless, SA-mediated effects on whole-body fat oxidation may be triggered by other mechanisms including gut-derived hormones or may occur in other metabolically active tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 González Hernández, Blaak, Hoebers, Essers, Canfora and Jocken.)

Sarcopenia, the age-related loss of skeletal muscle mass and function, contributes to high morbidity and mortality in the older population. Regular exercise is necessary to avoid the initiation and progression of sarcopenia, in which the underlying molecular mechanism is still not clear. Our data revealed that the outcomes induced by sarcopenia, including muscle mass and strength loss, decreased cross-sectional area of gastrocnemius fiber, chronic inflammation, and increased dysfunctional mitochondria, were reversed by regulation exercise. Knockout or silencing of Sestrin2 (Sesn2) resulted in imbalanced mitochondrial fusion and fission, mitochondrial biogenesis, and mitophagy damage in vivo and in vitro, which was attenuated by aerobic exercise or overexpression of Sesn2. Moreover, we found that the effects of Sesn2 on mitochondrial function are dependent on AMP-activated protein kinase α2 (AMPKα2). This study indicates that aerobic exercise alleviates the negative effects resulting from sarcopenia via the Sesn2/AMPKα2 pathway and provides new insights into the molecular mechanism by which the Sesn2/AMPKα2 signaling axis mediates the beneficial impact of exercise on sarcopenia., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Skeletal muscle has the remarkable ability to regenerate. However, with age and disease muscle strength and function decline. Myofiber size, which is affected by injury and disease, is a critical measurement to assess muscle health. Here, we test and apply Cellpose, a recently developed deep learning algorithm, to automatically segment myofibers within murine skeletal muscle. We first show that tissue fixation is necessary to preserve cellular structures such as primary cilia, small cellular antennae, and adipocyte lipid droplets. However, fixation generates heterogeneous myofiber labeling, which impedes intensity-based segmentation. We demonstrate that Cellpose efficiently delineates thousands of individual myofibers outlined by a variety of markers, even within fixed tissue with highly uneven myofiber staining. We created a novel ImageJ plugin (LabelsToRois) that allows processing of multiple Cellpose segmentation images in batch. The plugin also contains a semi-automatic erosion function to correct for the area bias introduced by the different stainings, thereby identifying myofibers as accurately as human experts. We successfully applied our segmentation pipeline to uncover myofiber regeneration differences between two different muscle injury models, cardiotoxin and glycerol. Thus, Cellpose combined with LabelsToRois allows for fast, unbiased, and reproducible myofiber quantification for a variety of staining and fixation conditions.

Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis through stabilization of the actin network. MYH9 accumulates in NEK9 LIR mutant cells and mice, and depletion of MYH9 restores ciliogenesis in NEK9 LIR mutant cells. These results suggest that NEK9 regulates ciliogenesis by acting as an autophagy adaptor for MYH9. Given that the LIR in NEK9 is conserved only in land vertebrates, the acquisition of the autophagic regulation of the NEK9-MYH9 axis in ciliogenesis may have possible adaptive implications for terrestrial life.

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in dystrophin gene, affecting 1/3000 males. Gene therapy has been proven to ameliorate dystrophic pathology. To investigate therapeutic benefits from long-term effect of human mini-dystrophin and functional outcomes, transgenic mdx mice (Tg-mdx) containing a single copy of human mini-dystrophin (∆hDys3849) gene, five rods (Rods1-2, Rods22-24), and two hinges (H1 and H4) driven by a truncated creatine-kinase promoter (dMCK) in a recombinant adeno-associated viral vector (rAAV) backbone, were generated and used to determine gene expression and improvement of muscle function. Human mini-dystrophin gene expression was found in a majority of the skeletal muscles, but no expression in cardiac muscle. Dystrophin-associated glycoproteins (DAGs) such as sarcoglycans and nNOS were restored at the sarcolemma and coincided with human mini-dystrophin gene expression at the ages of 6, 10, and 20 months; Morphology of dystrophic muscle expressing the human mini-dystrophin gene was improved and central nuclei were reduced. Myofiber membrane integrity was improved by Evans blue dye test. Improvement in treadmill running and grip force was observed in transgenic mice at 6 months. Tetanic force and specific force of tibialis anterior (TA) muscle were significantly increased at the ages of 6, 10, and 20 months. Pseudohypertrophy was not found in TA muscle at 10 and 20 months when compared with wild-type C57 (WT) group. This study demonstrated that the long-term effects of human mini-dystrophin effectively ameliorated pathology and improved the functions of the dystrophic muscles in the transgenic DMD mouse model., (© 2021 Federation of American Societies for Experimental Biology.)

Fibroblast growth factor 21 (FGF21) plays a critical role in the regulation of lipid metabolism; however, its function in goat intramuscular fat (IMF) deposition remains unknown. To explore the role of FGF21 for goat IMF deposition, we performed gain and loss function of FGF21 in intramuscular adipocyte. Our results showed that overexpression of FGF21 mediated by adenovirus inhibits lipid accumulation of goat intramuscular adipocyte, accompanied by down-regulating the mRNA levels of peroxisome proliferator activated receptor γ (PPARγ), adipocyte fatty acid-binding protein 2 (aP2) and sterol regulatory element-binding protein 1 (SREBP1), but up-regulating counterpart of preadipocyte factor1 (Pref1). Conversely, siRNAs knocking down FGF21 promotes the expression of PPARγ and CCAAT/enhancer binding protein-α (C/EBPα) but suppressed that of lipoprotein lipase (LPL) and Pref1. Meanwhile, we found that FGF21 regulates the expression of many KLFs transcription factors, such as KLF3, 7, 9, 11, 14, and 16. These findings demonstrate a key role of FGF21 as a negative factor in the regulation of adipogenic differentiation in goat intramuscular preadipocyte.

Microfluidic-based technologies enable the development of cell culture systems that provide tailored microenvironmental inputs to mammalian cells. Primary myoblasts can be induced to differentiate into multinucleated skeletal muscle cells, myotubes, which are a relevant model system for investigating skeletal muscle metabolism and physiology in vitro. However, it remains challenging to differentiate primary myoblasts into mature myotubes in microfluidics devices. Here we investigated the effects of integrating continuous (solid) and intermittent (dashed) walls in microfluidic channels as topological constraints in devices designed to promote the alignment and maturation of primary myoblast-derived myotubes. The topological constraints caused alignment of the differentiated myotubes, mimicking the native anisotropic organization of skeletal muscle cells. Interestingly, dashed walls facilitated the maturation of skeletal muscle cells, as measured by quantifying myotube cell area and the number of nuclei per myotube. Together, our results suggest that integrating dashed walls as topographic constraints in microfluidic devices supports the alignment and maturation of primary myoblast-derived myotubes., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscle tissue engineering is established, the role of FGF-2 in endothelial function to promote angiogenesis after transplantation is unclear. Here, a culture system comprising a five-layered sheet of human skeletal muscle cells co-incubated on green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) mimicking in vivo angiogenesis was used to investigate the role of FGF-2 in vascularization of engineered tissues. The basal level of FGF-2 in cultured media of skeletal muscle cell sheets was undetectable. Therefore, cell sheets co-incubated with GFP-HUVECs were exogenously treated with 10 ng/mL FGF-2, and endothelial network formation was evaluated. After prolonged culture, the endothelial network length and connectivity increased following treatment with FGF-2 as compared with control treatment. The numbers of medium and long endothelial networks significantly increased inside the sheet longer than 0.2 and 0.4 cm, respectively, after FGF-2 treatment. Time-lapse microscopy monitoring dynamic endothelial behavior revealed that FGF-2-mediated maintenance of endothelial connection and retardation of endothelial network disconnection after 72 h. The present study suggests the precise role of FGF-2 in maintaining endothelial connection and the extent of the endothelial network in skeletal muscle cell sheets. This understanding can be applied to design in vitro pre-vascularized tissue and graft integration prospects., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue.

The aim was to examine the efficiency of a scaffold made of poly (L-lactic acid)-co-poly(ϵ-caprolactone), collagen (COL), polyaniline (PANI), and enriched with adipose-derived stem cells (ASCs) as a nerve conduit in a rat model. P(LLA-CL)-COL-PANI scaffold was optimized and electrospun into a tubular-shaped structure. Adipose tissue from 10 Lewis rats was harvested for ASCs culture. A total of 28 inbred male Lewis rats underwent sciatic nerve transection and excision of a 10 mm nerve trunk fragment. In Group A, the nerve gap remained untouched; in Group B, an excised trunk was used as an autograft; in Group C, nerve stumps were secured with P(LLA-CL)-COL-PANI conduit; in Group D, P(LLA-CL)-COL-PANI conduit was enriched with ASCs. After 6 months of observation, rats were sacrificed. Gastrocnemius muscles and sciatic nerves were harvested for weight, histology analysis, and nerve fiber count analyses. Group A showed advanced atrophy of the muscle, and each intervention (B, C, D) prevented muscle mass decrease ( p < 0.0001); however, ASCs addition decreased efficiency vs. autograft ( p < 0.05). Nerve fiber count revealed a superior effect in the nerve fiber density observed in the groups with the use of conduit (D vs. B p < 0.0001, C vs. B p < 0.001). P(LLA-CL)-COL-PANI conduits with ASCs showed promising results in managing nerve gap by decreasing muscle atrophy.

Estrogenic molecules have been reported to regulate glucose homeostasis and may be beneficial for diabetes management. Here, we investigated the estrogenic effect of β- sitosterol-3-O-D-glucopyranoside (BSD), isolated from the fruits of Cupressus sempervirens and monitored its ability to regulate glucose utilization in skeletal muscle cells. BSD stimulated ERE-mediated luciferase activity in both ERα and ERβ-ERE luc expression system with greater response through ERβ in HEK-293T cells, and induced the expression of estrogen-regulated genes in estrogen responsive MCF-7 cells. In silico docking and molecular interaction studies revealed the affinity and interaction of BSD with ERβ through hydrophobic interaction and hydrogen bond pairing. Furthermore, prolonged exposure of L6-GLUT4 myc myotubes to BSD raised the glucose uptake under basal conditions without affecting the insulin-stimulated glucose uptake, the effect associated with enhanced translocation of GLUT4 to the cell periphery. The BSD-mediated biological response to increase GLUT4 translocation was obliterated by PI-3-K inhibitor wortmannin, and BSD significantly increased the phosphorylation of AKT (Ser-473). Moreover, BSD-induced GLUT4 translocation was prevented in the presence of fulvestrant. Our findings reveal the estrogenic activity of BSD to stimulate glucose utilization in skeletal muscle cells via PI-3K/AKT-dependent mechanism.

Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early-onset, severe, and rapidly progressive form of muscular dystrophy affecting both male and female patients. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and heart. We isolated human muscle stem cells from 2 donors, with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of 2 coregulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with > 90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base-edited patient cells regenerated muscle and contributed to the Pax7+ satellite cell compartment in vivo in mouse xenografts. Here, we provide the first evidence to our knowledge that autologous gene-repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.

During aging and menopausal transition in women, a progressive muscle degeneration ( i.e. decrease in quality and muscle function) occurs. This muscle dysfunction, caused by decreased proliferation of muscle satellite cells, increased levels of inflammatory markers, and altered levels of sex hormones, exposes women to a raised incidence of sarcopenia. In this regard, hormonal balance and, in particular, estradiol, seems to be essential in skeletal muscle function. The role of the estradiol on satellite cells and the release of inflammatory cytokines in menopausal women are reviewed. In particular, estradiol has a beneficial effect on the skeletal muscle by stimulating satellite cell proliferation. Skeletal muscle can respond to estrogenic hormonal control due to the presence of specific receptors for estradiol at the level of muscle fibers. Additionally, estradiol can limit inflammatory stress damage on skeletal muscle. In this review, we primarily focused on the role of estradiol in sarcopenia and on the possibility of using Estradiol Replacement Therapy, which combined with nutritional and physical activity programs, can counteract this condition representing a valid tool to treat sarcopenia in women., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer BS declared a shared affiliation with some of the authors, RC and EM, to the handling editor at time of review., (Copyright © 2021 Geraci, Calvani, Ferri, Marzetti, Arosio and Cesari.)

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.

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.

The prevalence of prediabetes is rapidly increasing, and this can lead to an increased risk for individuals to develop type 2 diabetes and associated diseases. Therefore, it is necessary to develop nutritional strategies to maintain healthy glucose levels and prevent glucose metabolism dysregulation in the general population. Functional ingredients offer great potential for the prevention of various health conditions, including blood glucose regulation, in a cost-effective manner. Using an artificial intelligence (AI) approach, a functional ingredient, NRT&#95;N0G5IJ, was predicted and produced from Pisum sativum (pea) protein by hydrolysis and then validated. Treatment of human skeletal muscle cells with NRT&#95;N0G5IJ significantly increased glucose uptake, indicating efficacy of this ingredient in vitro. When db/db diabetic mice were treated with NRT&#95;N0G5IJ, we observed a significant reduction in glycated haemoglobin (HbA1c) levels and a concomitant benefit on fasting glucose. A pilot double-blinded, placebo controlled human trial in a population of healthy individuals with elevated HbA1c (5.6% to 6.4%) showed that HbA1c percentage was significantly reduced when NRT&#95;N0G5IJ was supplemented in the diet over a 12-week period. Here, we provide evidence of an AI approach to discovery and demonstrate that a functional ingredient identified using this technology could be used as a supplement to maintain healthy glucose regulation.

METTL3 increasing the mature miRNA levels via N6-Methyladenosine (m6A) modification of primary miRNA (pri-miRNA) transcripts has emerged as an important post-transcriptional regulation of miRNA biogenesis. Our previous studies and others have showed that muscle specific miRNAs are essential for skeletal muscle differentiation. Whether these miRNAs are also regulated by METTL3 is still unclear. Here, we found that m6A motifs were present around most of these miRNAs, which were indeed m6A modified as confirmed by m6A-modified RNA immunoprecipitation (m6A RIP). However, we surprisingly found that these muscle specific miRNAs were repressed instead of increased by METTL3 in C2C12 in vitro differentiation and mouse skeletal muscle regeneration after injury in vivo model. To elucidate the underlined mechanism, we performed reporter assays in 293T cells and validated METTL3 increasing these miRNAs at post-transcriptional level as expected. Furthermore, in myogenic C2C12 cells, we found that METTL3 not only repressed the expression of myogenic transcription factors (TFs) which can enhance the muscle specific miRNAs, but also increased the expression of epigenetic regulators which can repress these miRNAs. Thus, METTL3 could repress the muscle specific miRNAs at transcriptional level indirectly. Taken together, our results demonstrated that skeletal muscle specific miRNAs were repressed by METTL3 and such repression is likely synthesized transcriptional and post-transcriptional regulations., 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 © 2021 Elsevier Inc. All rights reserved.)

The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships., Competing Interests: The authors have declared that no competing interests exist.

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.

Selenoprotein N (SEPN1) is a type II glycoprotein of the endoplasmic reticulum (ER) that senses calcium levels to tune the activity of the sarcoplasmic reticulum calcium pump (SERCA pump) through a redox-mediated mechanism, modulating ER calcium homeostasis. In SEPN1-depleted muscles, altered ER calcium homeostasis triggers ER stress, which induces CHOP-mediated malfunction, altering excitation-contraction coupling. SEPN1 is localized in a region of the ER where the latter is in close contact with mitochondria, i.e., the mitochondria-associated membranes (MAM), which are important for calcium mobilization from the ER to mitochondria. Accordingly, SEPN1-depleted models have impairment of both ER and mitochondria calcium regulation and ATP production. SEPN1-related myopathy (SEPN1-RM) is an inherited congenital muscle disease due to SEPN1 loss of function, whose main histopathological features are minicores, i.e., areas of mitochondria depletion and sarcomere disorganization in muscle fibers. SEPN1-RM presents with weakness involving predominantly axial and diaphragmatic muscles. Since there is currently no disease-modifying drug to treat this myopathy, analysis of SEPN1 function in parallel with that of the muscle phenotype in SEPN1 loss of function models should help in understanding the pathogenic basis of the disease and possibly point to novel drugs for therapy. The present essay recapitulates the novel biological findings on SEPN1 and how these reconcile with the muscle and bioenergetics phenotype of SEPN1-related myopathy.

In Pompe disease, the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) causes skeletal and cardiac muscle weakness, respiratory failure, and premature death. While enzyme replacement therapy using recombinant human GAA (rhGAA) can significantly improve patient outcomes, detailed disease mechanisms and incomplete therapeutic effects require further studies. Here we report a three-dimensional primary human skeletal muscle ("myobundle") model of infantile-onset Pompe disease (IOPD) that recapitulates hallmark pathological features including reduced GAA enzyme activity, elevated glycogen content and lysosome abundance, and increased sensitivity of muscle contractile function to metabolic stress. In vitro treatment of IOPD myobundles with rhGAA or adeno-associated virus (AAV)-mediated hGAA expression yields increased GAA activity and robust glycogen clearance, but no improvements in stress-induced functional deficits. We also apply RNA sequencing analysis to the quadriceps of untreated and AAV-treated GAA -/- mice and wild-type controls to establish a Pompe disease-specific transcriptional signature and reveal novel disease pathways. The mouse-derived signature is enriched in the transcriptomic profile of IOPD vs. healthy myobundles and partially reversed by in vitro rhGAA treatment, further confirming the utility of the human myobundle model for studies of Pompe disease and therapy.

Mesenchymal stem cells are increasingly studied for their use as drug-carrier in addition to their intrinsic potential for regenerative medicine. They could be used to transport molecules with a poor bioavailability such as curcumin in order to improve their clinical usage. This natural polyphenol, well-known for its antioxidant and anti-inflammatory properties, has a poor solubility that limits its clinical potential. For this purpose, the use of NDS27, a curcumin salt complexed with hydroxypropyl-beta-cyclodextrin (HPβCD), displaying an increased solubility in aqueous solution, is preferred. This study aims to evaluate the uptake of NDS27 into skeletal muscle-derived mesenchymal stem cells (mdMSCs) and the effects of such uptake onto their mesenchymal properties. It appeared that the uptake of NDS27 into mdMSCs is concentration-dependent and not time-dependent. The use of a concentration of 7 µmol/L which does not affect the viability and proliferation also allows preservation of their adhesion, invasion and T cell immunomodulatory abilities., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)