Search

Skeletal muscle growth is an economically important trait in the cattle industry. Secreted muscle-derived proteins, referred to as myokines, have important roles in regulating the growth, metabolism, and health of skeletal muscle in human and biomedical research models. Accumulating evidence supports the importance of myokines in skeletal muscle and whole-body health, though little is known about the potential presence and functional significance of these proteins in cattle. This study evaluates and confirms that secreted proteins acidic and rich in cysteine (SPARC), fibroblast growth factor 21 (FGF-21), myostatin (MSTN), and decorin (DCN) are expressed and SPARC, FGF-21, and DCN are secreted by primary bovine satellite cells from 3- (BSC3; n = 3) and 11- (BSC11; n = 3) month -old commercial angus steers. Cells were cultured and collected at zero, 12, 24, and 48 hours to characterize temporal expression and secretion from undifferentiated and differentiated cells. The expression of SPARC was higher in the undifferentiated (p = 0.04) and differentiated (p = 0.07) BSC11 than BSC3. The same was observed with protein secretion from undifferentiated (p <0.0001) BSC11 compared to BSC3. Protein secretion of FGF-21 was higher in undifferentiated BSC11 (p < 0.0001) vs. BSC3. DCN expression was higher in differentiated BSC11 (p = 0.006) vs. BSC3. Comparing undifferentiated vs. differentiated BSC, MSTN expression was higher in differentiated BSC3 (p ≤ 0.001) for 0, 12, and 24 hours and in BSC11 (p ≤ 0.03) for 0, 12, 24, and 48 hours. There is also a change over time for SPARC expression (p ≤ 0.03) in undifferentiated and differentiated BSC and protein secretion (p < 0.0001) in undifferentiated BSC, as well as FGF-21 expression (p = 0.007) in differentiated BSC. This study confirms SPARC, FGF-21, and DCN are secreted, and SPARC, FGF-21, MSTN, and DCN are expressed in primary bovine muscle cells with age and temporal differences., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glutathione peroxidase 2 (GPX2) is a selenium-dependent enzyme and protects cells against oxidative damage. Recently, GPX2 has been identified as a candidate gene for backfat and feed efficiency in pigs. However, it is unclear whether GPX2 regulates the development of porcine preadipocytes and skeletal muscle cells. In this study, adenoviral gene transfer was used to overexpress GPX2. Our findings suggest that overexpression of GPX2 gene inhibited proliferation of porcine preadipocytes. And the process is accompanied by the reduction of the p-p38. GPX2 inhibited adipogenic differentiation and promoted lipid degradation, while ERK1/2 was reduced and p-p38 was increased. Proliferation of porcine skeletal muscle cells was induced after GPX2 overexpression, was accompanied by activation in JNK, ERK1/2, and p-p38. Overexpression methods confirmed that GPX2 has a promoting function in myoblastic differentiation. ERK1/2 pathway was activated and p38 was suppressed during the process. This study lays a foundation for the functional study of GPX2 and provides theoretical support for promoting subcutaneous fat reduction and muscle growth., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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.

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.

Denervation reduces the abundance of Na+,K+-ATPase (NKA) in skeletal muscle, while reinnervation increases it. Primary human skeletal muscle cells, the most widely used model to study human skeletal muscle in vitro, are usually cultured as myoblasts or myotubes without neurons and typically do not contract spontaneously, which might affect their ability to express and regulate NKA. We determined how differentiation, de novo innervation, and electrical pulse stimulation affect expression of NKA (α and β) subunits and NKA regulators FXYD1 (phospholemman) and FXYD5 (dysadherin). Differentiation of myoblasts into myotubes under low serum conditions increased expression of myogenic markers CD56 (NCAM1), desmin, myosin heavy chains, dihydropyridine receptor subunit α1S, and SERCA2 as well as NKAα2 and FXYD1, while it decreased expression of FXYD5 mRNA. Myotubes, which were innervated de novo by motor neurons in co-culture with the embryonic rat spinal cord explants, started to contract spontaneously within 7-10 days. A short-term co-culture (10-11 days) promoted mRNA expression of myokines, such as IL-6, IL-7, IL-8, and IL-15, but did not affect mRNA expression of NKA, FXYDs, or myokines, such as musclin, cathepsin B, meteorin-like protein, or SPARC. A long-term co-culture (21 days) increased the protein abundance of NKAα1, NKAα2, FXYD1, and phospho-FXYD1Ser68 without attendant changes in mRNA levels. Suppression of neuromuscular transmission with α-bungarotoxin or tubocurarine for 24 h did not alter NKA or FXYD mRNA expression. Electrical pulse stimulation (48 h) of non-innervated myotubes promoted mRNA expression of NKAβ2, NKAβ3, FXYD1, and FXYD5. In conclusion, low serum concentration promotes NKAα2 and FXYD1 expression, while de novo innervation is not essential for upregulation of NKAα2 and FXYD1 mRNA in cultured myotubes. Finally, although innervation and EPS both stimulate contractions of myotubes, they exert distinct effects on the expression of NKA and FXYDs., Competing Interests: The authors have declared that no competing interests exist.

Short chain fatty acids (SCFAs) produced endogenously in the gut by bacterial fermentation of dietary fiber have been studied as nutrients that act as signaling molecules to activate G-protein coupled receptors (GPCRs) such as GPR41 and GPR43. GPR43 functioning involves the suppression of lipid accumulation and maintaining body energy homeostasis, and is activated by acetic acid or propionic acid. Previously, we reported that the orally administered acetic acid improves lipid metabolism in liver and skeletal muscles and suppresses obesity, thus improving glucose tolerance. Acetic acid stimulates AMP-activated protein kinase (AMPK) through its metabolic pathway in skeletal muscle cells. We hypothesized that acetic acid would stimulate GPR43 in skeletal muscle cells and has function in modulating gene expression related to muscle characteristics through its signal pathway. The objective of the current study was to clarify this effect of acetic acid. The GPR43 expression, observed in the differentiated myotube cells, was increased upon acetic acid treatment. Acetic acid induced the intracellular calcium influx in the cells and this induction was significantly inhibited by the GPR43-specific siRNA treatment. The calcineurin molecule is activated by calcium/calmodulin and is associated with proliferation of slow-twitch fibers. Calcineurin was activated by acetic acid treatment and inhibited by the concomitant treatment with GPR43-siRNA. Acetic acid induced nuclear localization of myocyte enhancer factor 2A (MEF2A), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and nuclear factor of activated t cells c1 (NFATc1). However, these localizations were abolished by the treatment with GPR43-siRNA. It was concluded that acetic acid plays a role in the activation of GPR43 and involves the proliferation of slow-twitch fibers in L6 skeletal muscles through the calcium-signaling pathway caused by induction of intracellular calcium influx., Competing Interests: Authors have declared that there is no conflict of interest.

It has been reported that Bach1-deficient mice show reduced tissue injuries in diverse disease models due to increased expression of heme oxygenase-1 (HO-1)that possesses an antioxidant function. In contrast, we found that Bach1 deficiency in mice exacerbated skeletal muscle injury induced by cardiotoxin. Inhibition of Bach1 expression in C2C12 myoblast cells using RNA interference resulted in reduced proliferation, myotube formation, and myogenin expression compared with control cells. While the expression of HO-1 was increased by Bach1 silencing in C2C12 cells, the reduced myotube formation was not rescued by HO-1 inhibition. Up-regulations of Smad2, Smad3 and FoxO1, known inhibitors of muscle cell differentiation, were observed in Bach1-deficient mice and Bach1-silenced C2C12 cells. Therefore, Bach1 may promote regeneration of muscle by increasing proliferation and differentiation of myoblasts., Competing Interests: The authors have declared that no competing interests exist.

Intramuscular fat content (IMF) is a complex trait influencing the technological and sensorial features of meat products and determining pork quality. Thus, we aimed at analyzing through RNA-sequencing the Semimembranosus muscle transcriptome of Italian Large White pigs to study the gene networks associated with IMF deposition. Two groups of samples were used; each one was composed of six unrelated pigs with extreme and divergent IMF content (0.67 ± 0.09% in low IMF vs. 6.81 ± 1.17% in high IMF groups) that were chosen from 950 purebred individuals. Paired-end RNA sequences were aligned to Sus scrofa genome assembly 11.1 and gene counts were analyzed using WGCNA and DeSeq2 packages in R environment. Interestingly, among the 58 differentially expressed genes (DEGs), several were related to primary cilia organelles (such as Lebercilin 5 gene), in addition to the genes involved in the regulation of cell differentiation, in the control of RNA-processing, and G-protein and ERK signaling pathways. Together with cilia-related genes, we also found in high IMF pigs an over-expression of the Fibroblast Growth Factor 2 (FGF2) gene, which in other animal species was found to be a regulator of ciliogenesis. Four WGCNA gene modules resulted significantly associated with IMF deposition: grey60 (P = 0.003), darkturquoise (P = 0.022), skyblue1 (P = 0.022), and lavenderblush3 (P = 0.030). The genes in the significant modules confirmed the results obtained for the DEGs, and the analysis with "cytoHubba" indicated genes controlling RNA splicing and cell differentiation as hub genes. Among the complex molecular processes affecting muscle fat depots, genes involved in primary cilia may have an important role, and the transcriptional reprogramming observed in high IMF pigs may be related to an FGF-related molecular cascade and to ciliogenesis, which in the literature have been associated with fibro-adipogenic precursor differentiation., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Roberta Davoli is part of the PLoS ONE Editorial Board. The other authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The reproduction of reliable in vitro models of human skeletal muscle is made harder by the intrinsic 3D structural complexity of this tissue. Here we coupled engineered hydrogel with 3D structural cues and specific mechanical properties to derive human 3D muscle constructs ("myobundles") at the scale of single fibers, by using primary myoblasts or myoblasts derived from embryonic stem cells. To this aim, cell culture was performed in confined, laminin-coated micrometric channels obtained inside a 3D hydrogel characterized by the optimal stiffness for skeletal muscle myogenesis. Primary myoblasts cultured in our 3D culture system were able to undergo myotube differentiation and maturation, as demonstrated by the proper expression and localization of key components of the sarcomere and sarcolemma. Such approach allowed the generation of human myobundles of ~10 mm in length and ~120 μm in diameter, showing spontaneous contraction 7 days after cell seeding. Transcriptome analyses showed higher similarity between 3D myobundles and skeletal signature, compared to that found between 2D myotubes and skeletal muscle, mainly resulting from expression in 3D myobundles of categories of genes involved in skeletal muscle maturation, including extracellular matrix organization. Moreover, imaging analyses confirmed that structured 3D culture system was conducive to differentiation/maturation also when using myoblasts derived from embryonic stem cells. In conclusion, our structured 3D model is a promising tool for modelling human skeletal muscle in healthy and diseases conditions., Competing Interests: The authors have declared that no competing interests exist.

Charcot-Marie-Tooth (CMT) disease is an inherited peripheral motor and sensory neuropathy. The disease is divided into demyelinating (CMT1) and axonal (CMT2) neuropathies, and although we have gained molecular information into the details of CMT1 pathology, much less is known about CMT2. Due to its clinical and genetic heterogeneity, coupled with a lack of animal models, common underlying mechanisms remain elusive. In order to gain an understanding of the normal function of genes associated with CMT2, and to draw direct comparisons between them, we have studied the behavioural, cellular and molecular consequences of mutating nine different genes in the nematode Caenorhabditis elegans (lin-41/TRIM2, dyn-1/DNM2, unc-116/KIF5A, fzo-1/MFN2, osm-9/TRPV4, cua-1/ATP7A, hsp-25/HSPB1, hint-1/HINT1, nep-2/MME). We show that C. elegans defective for these genes display debilitated movement in crawling and swimming assays. Severe morphological defects in cholinergic motors neurons are also evident in two of the mutants (dyn-1 and unc-116). Furthermore, we establish methods for quantifying muscle morphology and use these to demonstrate that loss of muscle structure occurs in the majority of mutants studied. Finally, using electrophysiological recordings of neuromuscular junction (NMJ) activity, we uncover reductions in spontaneous postsynaptic current frequency in lin-41, dyn-1, unc-116 and fzo-1 mutants. By comparing the consequences of mutating numerous CMT2-related genes, this study reveals common deficits in muscle structure and function, as well as NMJ signalling when these genes are disrupted., Competing Interests: The authors have declared that no competing interests exist.

Methods: Muscle sections were stained for cell boundary (laminin) and myofiber type (myosin heavy chain isoforms). Myosoft, running in the open access software platform FIJI (ImageJ), was used to analyze myofiber size and type in transverse sections of entire gastrocnemius/soleus muscles., Results: Myosoft provides an accurate analysis of hundreds to thousands of muscle fibers within 25 minutes, which is >10-times faster than manual analysis. We demonstrate that Myosoft is capable of handling high-content images even when image or staining quality is suboptimal, which is a marked improvement over currently available and comparable programs., Conclusions: Myosoft is a reliable, accurate, high-throughput, and convenient tool to analyze high-content muscle histology. Myosoft is freely available to download from Github at https://github.com/Hyojung-Choo/Myosoft/tree/Myosoft-hub., Competing Interests: The authors have declared that no competing interests exist.

The mitochondrial theory of aging attributes much of the aging process to mitochondrial DNA damage. The polymerase gamma (PolG) mutant mouse was designed to evaluate this theory and thus carries a mutated proofreading region of polymerase gamma (D257A) that exclusively transcribes the mitochondrial genome. As a result, PolGD257A mice accumulate mitochondrial DNA (mtDNA) mutations that lead to premature aging, as evidenced by hair loss, weight loss, kyphosis, increased rates of apoptosis, organ damage, and an early death, occurring around 12 months of age. Research has shown that exercise decreases skeletal muscle mtDNA mutations and normalizes protein levels in PolG mice. However, brain mtDNA changes with exercise in PolG mice have not been studied. We found no effects of exercise on mtDNA mutations or copy number in either the brain or liver of PolG mice, despite changes to body mass. Our results suggest that mitochondrial mutations play little role in exercise-brain interactions in the PolG model of accelerated aging. In addition to evaluating the effect of exercise on mtDNA outcomes, we also implemented novel methods for both extracting mtDNA and measuring mtDNA mutations, with aims for improving the efficiency and accuracy of these methods., Competing Interests: The authors have declared that no competing interests exist.

Human embryonic stem cell (hESC)-derived skeletal muscle progenitors (SMP)-defined as PAX7-expressing cells with myogenic potential-can provide an abundant source of donor material for muscle stem cell therapy. As in vitro myogenesis is decoupled from in vivo timing and 3D-embryo structure, it is important to characterize what stage or type of muscle is modeled in culture. Here, gene expression profiling is analyzed in hESCs over a 50 day skeletal myogenesis protocol and compared to datasets of other hESC-derived skeletal muscle and adult murine satellite cells. Furthermore, day 2 cultures differentiated with high or lower concentrations of CHIR99021, a GSK3A/GSK3B inhibitor, were contrasted. Expression profiling of the 50 day time course identified successively expressed gene subsets involved in mesoderm/paraxial mesoderm induction, somitogenesis, and skeletal muscle commitment/formation which could be regulated by a putative cascade of transcription factors. Initiating differentiation with higher CHIR99021 concentrations significantly increased expression of MSGN1 and TGFB-superfamily genes, notably NODAL, resulting in enhanced paraxial mesoderm and reduced ectoderm/neuronal gene expression. Comparison to adult satellite cells revealed that genes expressed in 50-day cultures correlated better with those expressed by quiescent or early activated satellite cells, which have the greatest therapeutic potential. Day 50 cultures were similar to other hESC-derived skeletal muscle and both expressed known and novel SMP surface proteins. Overall, a putative cascade of transcription factors has been identified which regulates four stages of myogenesis. Subsets of these factors were upregulated by high CHIR99021 or their binding sites were significantly over-represented during SMP activation, ranging from quiescent to late-activated stages. This analysis serves as a resource to further study the progression of in vitro skeletal myogenesis and could be mined to identify novel markers of pluripotent-derived SMPs or regulatory transcription/growth factors. Finally, 50-day hESC-derived SMPs appear similar to quiescent/early activated satellite cells, suggesting they possess therapeutic potential., Competing Interests: The authors have declared that no competing interests exist.

Although it is now recognized that women suffer from myofascial pain to a greater extent than men, and that the muscular fasciae can respond to hormonal stimuli, thanks to the expression of sex hormone receptors, how the fasciae can modify their structure under hormonal stimulation is not clear. In this work, an immunocytochemical analysis of collagen-I, collagen-III and fibrillin were carried out on fibroblasts isolated from human fascia lata after in vitro treatment with various levels of sex hormones β-estradiol and/or relaxin-1, according to the phases of a woman's period (follicular, periovulatory, luteal, post-menopausal phases and pregnancy). This study demonstrates for the first time that fascial cells can modulate the production of some components of the extracellular matrix according to hormone levels, when treated with β-estradiol: collagen-I falls from 6% of positivity in the follicular phase to 1.9 in the periovulatory phase. However, after the addition of relaxin-1 to the cell culture, the production of extracellular matrix decreased and remained at the same level (1.7% of collagen-I, at both follicular and periovulatory levels of hormones). These results confirm the antifibrotic function of relaxin-1, thanks to its ability to reduce matrix synthesis. They are also a first step in our understanding of how some hormonal dysfunctions in women can cause a dysregulation of extracellular matrix production in fasciae., Competing Interests: The authors have declared that no competing interests exist.

Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair., Competing Interests: The authors have declared that no competing interests exist.

Background: Green light penetrates the skull and has directly affected on the secretion of melatonin in plasma, which regulates the endocrine activities to influence the muscle growth, satellite cell mitotic activity and quality properties of meat from the embryonic period to posthatch in chick. Pituitary adenylate cyclase-activating polypeptide 6-38 (PACAP6-38) could inhibit the synthesis and secretion of pineal melatonin. Finding a new way for exploring the mechanism of light-regulated muscle growth in ovo is essential for promoting the productive performance in poultry., Methods: Chick embryos were exposed to darkness (D-group) and green light (G-group) throughout the embryonic period, and injected with PACAP6-38 or saline at embryonic day 8. Plasma hormone, skeletal muscle fiber areas, satellite cell proliferation activity, paired domain homeobox transcription factor 7 and myogenic regulatory factors were observed., Results: By saline treatment, the percentage of proliferating cell nuclear antigen immunoreactive cells and mitotic activity of satellite cells in skeletal muscle were higher in G-group than those of in D-group at post-hatching day 0. With the increase of plasma melatonin, green light promoted the secretion of growth hormone (GH) and insulin like factor 1 (IGF-1) in plasma, the satellite cell proliferation, the size of muscle fiber, as well as the mRNA expressions of Pax7, myogenic regulatory factors and IGF-1R. After PACAP6-38 treatment to inhibit the secretion of melatonin in ovo, aforementioned parameters were remarkably decreased and the difference of these parameters was disappeared between D-group and G-group., Conclusion: These data indicated that stimulation with monochromatic green light during incubation enhanced the secretion of melatonin and up-regulation of GH-IGF-1 axis to activate the satellite cells proliferation and myofiber formation, involving the expression of Pax7 and myogenic regulatory factors., Competing Interests: The authors have declared that no competing interests exist.

Phosphatidylcholine (PPC) formula has been therapeutically used to reduce areas of localized fat. However, no single research has been carried out on its effect on a variety of cells in adipose and muscle tissues. Herein, the current study aimed to explore the activity of PPC on different cells in adipose and muscle tissues and to investigate the molecular mechanisms contributing to the effects of PPC on lipolysis and apoptosis. mRNA expression levels of various genes were measured by quantitative real-time PCR. Protein expression levels were observed through Western blotting and cell viability was measured by MTT assay. Lipolysis and caspase 3 activity assay were performed using commercial kits. PPC induces lipolysis and apoptosis in adipocytes (3T3-L1), but not in the other tested cells, including skeletal muscle cells (C2C12 myocytes), endothelial cells (HUVEC), and fibroblasts (BJ). The possible role of TNFα and IL-1β-mediated pathways on the effects of PPC was also revealed. We confirmed that treatment with PPC caused lipolysis and apoptosis in a dose-dependent manner (only in 3T3-L1 adipocytes). The effect of PPC observed in 3T3-L1 adipocytes was not evident in C2C12 myocytes, HUVEC, and fibroblasts. PPC also increased TNFα and IL-1β expression and release in 3T3-L1 adipocytes in a dose-dependent fashion, but not in C2C12 myocytes, HUVEC, and BJ. Suppression of TNFα or IL-1β reversed PPC-induced lipolysis and apoptosis in 3T3-L1 adipocytes, suggesting that PPC could promote adipocyte-specific lipolysis and apoptosis through TNFα and IL-1β-mediated signaling. We conclude that the specific activity of PPC on adipocyte in adipose without other tissue damages can be an effective approach for melting lipid., Competing Interests: The authors have declared that no competing interests exist.

Background: Skeletal muscles include fast and slow muscle fibers. The tibialis anterior muscle (TA) is mainly composed of fast muscle fibers, whereas the soleus muscle (SOL) is mainly composed of slow muscle fibers. However, a noninvasive approach for appropriately investigating the characteristics of muscles is not available. Monitoring of skeletal muscle characteristics can help in the evaluation of the effects of strength training and diseases on skeletal muscles., Purpose: The present study aimed to determine whether q-space imaging can distinguish between TA and SOL in in vivo mice., Methods: In vivo magnetic resonance imaging of the right calves of mice (n = 8) was performed using a 7-Tesla magnetic resonance imaging system with a cryogenic probe. TA and SOL were assessed. q-space imaging was performed with a field of view of 10 mm × 10 mm, matrix of 48 × 48, and section thickness of 1000 μm. There were ten b-values ranging from 0 to 4244 s/mm2, and each b-value had diffusion encoding in three directions. Magnetic resonance imaging findings were compared with immunohistological findings., Results: Full width at half maximum and Kurtosis maps of q-space imaging showed signal intensities consistent with immunohistological findings for both fast (myosin heavy chain II) and slow (myosin heavy chain I) muscle fibers. With regard to quantification, both full width at half maximum and Kurtosis could represent the immunohistological findings that the cell diameter of TA was larger than that of SOL (P < 0.01)., Conclusion: q-space imaging could clearly differentiate TA from SOL using differences in cell diameters. This technique is a promising method to noninvasively estimate the fiber type ratio in skeletal muscles, and it can be further developed as an indicator of muscle characteristics., Competing Interests: Daisuke Nakashima and Yasushi Sera are directors of Grace imaging Inc. There are no patents, products in development or marketed products associated with this research to declare. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

We clarified in our previous study that hypoxic training promotes angiogenesis in skeletal muscle, but the mechanism of angiogenesis in skeletal muscle remains unknown. In this study, we investigated the influence of differences in hypoxia exposure on angiogenesis in skeletal muscles at differing ages and metabolic characteristics at which the production of reactive oxygen species and nitric oxide may differ. Ten-week-old (young) and 20-month-old (old) mice were separated into control (N), continuous hypoxia (H), and intermittent hypoxia (IH) groups. The H group was exposed to 16% O2 hypoxia for 5 days and the IH group was exposed to 16% O2 hypoxia at one-hour intervals during the light period for 5 days. After completion of hypoxia exposure, the soleus and gastrocnemius muscles were immediately excised, and mRNA expression of angiogenesis- and satellite cell-related genes was investigated using real-time RT-PCR. In addition, muscle fiber type composition, muscle fiber area, number of satellite cells, and capillary density were measured immunohistochemically. In the young soleus muscle, the muscle fiber area was decreased in the H group, and mRNA expression of satellite cell activation-related MyoD, MHCe, and BDNF was significantly increased. On the other hand, in the old soleus muscle, nNOS and VEGF-A mRNA expression, and the capillary density were significantly increased in the H group. In the superficial portion of the gastrocnemius, mRNA expression of FGF2, an angiogenic factor secreted by satellite cells, was significantly increased in the young IH group. In addition, a positive correlation between VEGF-A mRNA expression and nNOS mRNA expression in the soleus muscle and eNOS mRNA expression in the superficial portion of the gastrocnemius was noted. These data demonstrated that age, hypoxia exposure method and muscle metabolic characteristics are related, which results in significant differences in angiogenesis., Competing Interests: The authors have declared that no competing interests exist.

Skeletal muscle is considered a secretory organ that produces bioactive proteins known as myokines, which are released in response to various stimuli. However, no experimental evidence exists regarding the mechanism by which acute muscle contraction regulates myokine secretion. Here, we present evidence that acute contractions induced myokine secretion from C2C12 myotubes. Changes in the cell culture medium unexpectedly triggered the release of large amounts of proteins from the myotubes, and these proteins obscured the contraction-induced myokine secretion. Once protein release was abolished, the secretion of interleukin-6 (IL-6), the best-known regulatory myokine, increased in response to a 1-hour contraction evoked by electrical stimulation. Using this experimental condition, intracellular calcium flux, rather than the contraction itself, triggered contraction-induced IL-6 secretion. This is the first report to show an evidence for acute contraction-induced myokine secretion by skeletal muscle cells., Competing Interests: The authors have declared that no competing interests exist.

Ankyrin repeat and kinase domain containing 1 (ANKK1) gene has been widely related to neuropsychiatry disorders. The localization of ANKK1 in neural progenitors and its correlation with the cell cycle has suggested its participation in development. However, ANKK1 functions still need to be identified. Here, we have further characterized the ANKK1 localization in vivo and in vitro, by using immunolabeling, quantitative real-time PCR and Western blot in the myogenic lineage. Histologic investigations in mice and humans revealed that ANKK1 is expressed in precursors of embryonic and adult muscles. In mice embryos, ANKK1 was found in migrating myotubes where it shows a polarized cytoplasmic distribution, while proliferative myoblasts and satellite cells show different isoforms in their nuclei and cytoplasm. In vitro studies of ANKK1 protein isoforms along the myogenic progression showed the decline of nuclear ANKK1-kinase until its total exclusion in myotubes. In adult mice, ANKK1 was expressed exclusively in the Fast-Twitch muscles fibers subtype. The induction of glycolytic metabolism in C2C12 cells with high glucose concentration or treatment with berberine caused a significant increase in the ANKK1 mRNA. Similarly, C2C12 cells under hypoxic conditions caused the increase of nuclear ANKK1. These results altogether show a relationship between ANKK1 gene regulation and the metabolism of muscles during development and in adulthood. Finally, we found ANKK1 expression in regenerative fibers of muscles from dystrophic patients. Future studies in ANKK1 biology and the pathological response of muscles will reveal whether this protein is a novel muscle disease biomarker.

A large body of evidence suggests that persistent dietary fat overload causes mitochondrial dysfunction and systemic metabolic gridlock. Mitochondrial and lipid metabolism in skeletal muscle (SkM) are severely affected upon persistent high fat diet (HFD) leading to premature tissue aging. Here, we designed weekly cycles of fasting (called as time-controlled fasting, TCF) and showed that they were effective in limiting mitochondrial damage and metabolic disturbances induced by HFD. Specifically, TCF was able to prevent the decline of adipose triglyceride lipase (Atgl), maintain efficient mitochondrial respiration in SkM as well as improve blood glucose and lipid profile. Atgl was found to be the mediator of such preventive effects as its downregulation or up-regulation in C2C12 myotubes triggers mitochondrial alteration or protects against the deleterious effects of high fat levels respectively. In conclusion, TCF could represent an effective strategy to limit mitochondrial impairment and metabolic inflexibility that are typically induced by modern western diets or during aging.

From whole organisms to individual cells, responses to environmental conditions are influenced by genetic makeup, where the effect of genetic variation on a trait depends on the environmental context. RNA-sequencing quantifies gene expression as a molecular trait, and is capable of capturing both genetic and environmental effects. In this study, we explore opportunities of using allele-specific expression (ASE) to discover cis-acting genotype-environment interactions (GxE)-genetic effects on gene expression that depend on an environmental condition. Treating 17 common, clinical traits as approximations of the cellular environment of 267 skeletal muscle biopsies, we identify 10 candidate environmental response expression quantitative trait loci (reQTLs) across 6 traits (12 unique gene-environment trait pairs; 10% FDR per trait) including sex, systolic blood pressure, and low-density lipoprotein cholesterol. Although using ASE is in principle a promising approach to detect GxE effects, replication of such signals can be challenging as validation requires harmonization of environmental traits across cohorts and a sufficient sampling of heterozygotes for a transcribed SNP. Comprehensive discovery and replication will require large human transcriptome datasets, or the integration of multiple transcribed SNPs, coupled with standardized clinical phenotyping.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterized by progressive weakness of the skeletal and cardiac muscles. This X-linked disorder is caused by open reading frame disrupting mutations in the DMD gene, resulting in strong reduction or complete absence of dystrophin protein. In order to use dystrophin as a supportive or even surrogate biomarker in clinical studies on investigational drugs aiming at correcting the primary cause of the disease, the ability to reliably quantify dystrophin expression in muscle biopsies of DMD patients pre- and post-treatment is essential. Here we demonstrate the application of the ProteinSimple capillary immunoassay (Wes) method, a gel- and blot-free method requiring less sample, antibody and time to run than conventional Western blot assay. We optimized dystrophin quantification by Wes using 2 different antibodies and found it to be highly sensitive, reproducible and quantitative over a large dynamic range. Using a healthy control muscle sample as a reference and α-actinin as a protein loading/muscle content control, a panel of skeletal muscle samples consisting of 31 healthy controls, 25 Becker Muscle dystrophy (BMD) and 17 DMD samples was subjected to Wes analysis. In healthy controls dystrophin levels varied 3 to 5-fold between the highest and lowest muscle samples, with the reference sample representing the average of all 31 samples. In BMD muscle samples dystrophin levels ranged from 10% to 90%, with an average of 33% of the healthy muscle average, while for the DMD samples the average dystrophin level was 1.3%, ranging from 0.7% to 7% of the healthy muscle average. In conclusion, Wes is a suitable, efficient and reliable method for quantification of dystrophin expression as a biomarker in DMD clinical drug development.

Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening.

Secreted protein, acidic and rich in cysteine (SPARC) is differentially associated with cell proliferation and extracellular matrix (ECM) assembly. We show here the effect of exogenous SPARC inhibition/induction on ECM and mitochondrial proteins expression and on the differentiation of C2C12 cells. The cells were cultured in growth medium (GM) supplemented with different experimental conditions. The differentiation of myoblasts was studied for 5 days, the expressions of ECM and mitochondrial proteins were measured and the formation of the myotubes was quantified after exogenous induction/inhibition of SPARC. The results indicate that the addition of recombinant SPARC protein (rSPARC) in cell culture medium increased the differentiation of C2C12 myoblasts and myogenin expression during the myotube formation. However, the treatment with antibody specific for SPARC (anti-SPARC) prevented the differentiation and decreased myogenin expression. The induction of SPARC in the proliferating and differentiating C2C12 cells increased collagen 1a1 protein expression, whereas the inhibition decreased it. The effects on fibronectin protein expression were opposite. Furthermore, the addition of rSPARC in C2C12 myoblast increased the expression of mitochondrial proteins, ubiquinol-cytochrome c reductase core protein II (UQCRC2) and succinate dehydrogenase iron-sulfur subunit (SDHB), whereas the anti-SPARC decreased them. During the differentiation, only the anti-SPARC had the effects on mitochondrial proteins, NADH dehydrogenase ubiquinone 1 beta subcomplex subunit 8 (NADHB8), SDHB and cytochrome c oxidase 1 (MTCO1). Thus, SPARC plays a crucial role in the proliferation and differentiation of C2C12 and may be involved in the link between the ECM remodeling and mitochondrial function.

The effects of actin dependent molecular mechanisms in coordinating cellular proliferation, migration and differentiation during embryogenesis are not well-understood. We have previously shown that actin-binding Filamin A (FlnA) and actin-nucleating Formin 2 (Fmn2) influence the development of the brain causing microcephaly in mice. In this study, we broaden this phenotype to explore the effects of these two proteins in the development of extra-CNS organ systems, including the gut, muscle, and skeleton. We observed defects in rib and sternum midline closure leading to thoracoabdominal schisis in FlnA+Fmn2 knockout mice, reminiscent of the pentalogy of Cantrell syndrome. These mice exhibit shortened guts, as well as thinned thoracic muscle mass. Immunostaining showed these changes are partially caused by a decrease in the number of presumptive mesenchymal proliferating cells with loss of either FlnA or FlnA+Fmn2. This proliferation defect appears to be in part due to delayed differentiation in these regions. While both FlnA and FlnA+Fmn2 mice show reduced cell death relative to WT control, increased caspase staining was seen in the double null relative to FlnA null suggesting that this could also contribute to the FlnA+Fmn2 phenotype. Therefore FlnA and Fmn2 are likely essential to cell proliferation, differentiation and cell death in a variety of tissues and organs, further reiterating the importance of vesicle trafficking in regulation of development.

Objective: Caveolin-3 (CAV3) protein is known to be expressed specifically in various myocytes, but its physiological function remains unclear. CAV3, located at the cell membrane, may promote the sensitivity of the Akt signaling pathway, which is closely related to glucose metabolism and to cell growth and proliferation., Methods: The CAV3 gene was stably transfected into C2C12 muscle cells, and the effects were evaluated by biochemical assays, WB and confocal microscopy for the observation of cellular glucose metabolism, growth and proliferation, and the effect of CAV3 on the Akt signaling pathway with no insulin stimulation., Results: After C2C12 cells were transfected with the mouse CAV3 gene, which increased CAV3 expression, the abundance of the CAV3 and GLUT4 proteins on the cell membrane increased, but the total GLUT4 protein content of the cell was unchanged. Glucose uptake was increased, and this did not affect the glycogen synthesis, but the cell surface area and cell proliferation increased. While there were significant increases in p-Akt and p-p70s6K, which is a downstream component of Akt signaling, the level of GSK3β protein, another component of Akt signaling did not change., Conclusions: The muscle, CAV3 protein can activate Akt signaling, increase GLUT4 protein localization in the cell membrane, increase glucose uptake, and promote myocyte growth and proliferation. CAV3 protein has a physiological role in glycometabolism, growth and proliferation, independent of insulin stimulation.

Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, "mesenchymal stem cells") in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146+ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells.

Oxidative and glycolytic myofibers have different structures and metabolic characteristics and their ratios are important in determining poultry meat quality. However, the molecular mechanisms underlying their differences are unclear. In this study, global gene expression profiling was conducted in oxidative skeletal muscle (obtained from the soleus, or SOL) and glycolytic skeletal muscle (obtained from the extensor digitorum longus, or EDL) of Chinese Qingyuan partridge chickens, using the Agilent Chicken Gene Expression Chip. A total of 1224 genes with at least 2-fold differences were identified (P < 0.05), of which 654 were upregulated and 570 were downregulated in SOL. GO, KEGG pathway, and co-expressed gene network analyses suggested that PRKAG3, ATP2A2, and PPARGC1A might play important roles in myofiber composition. The function of PPARGC1A gene was further validated. PPARGC1A mRNA expression levels were higher in SOL than in EDL muscles throughout the early postnatal development stages. In myoblast cells, shRNA knockdown of PPARGC1A significantly inhibited some muscle development and transition-related genes, including PPP3CA, MEF2C, and SM (P < 0.01 or P < 0.05), and significantly upregulated the expression of FWM (P < 0.05). Our study demonstrates strong transcriptome differences between oxidative and glycolytic myofibers, and the results suggest that PPARGC1A is a key gene involved in chicken myofiber composition and transition.

The experiment was conducted to study the effect of the glutamate (Glu) on muscle protein loss through toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain proteins (NODs), Akt/Forkhead Box O (Akt/FOXO) and mammalian target of rapamycin (mTOR) signaling pathways in LPS-challenged piglets. Twenty-four weaned piglets were assigned into four treatments: (1) Control; (2) LPS+0% Glu; (3) LPS + 1.0% Glu; (4) LPS + 2.0% Glu. The experiment was lasted for 28 days. On d 28, the piglets in the LPS challenged groups were injected with LPS on 100 μg/kg body weight (BW), and the piglets in the control group were injected with the same volume of 0.9% NaCl solution. After 4 h LPS or saline injection, the piglets were slaughtered and the muscle samples were collected. Glu supplementation increased the protein/DNA ratio in gastrocnemius muscle, and the protein content in longissimus dorsi (LD) muscle after LPS challenge (P<0.05). In addition, Glu supplementation decreased TLR4, IL-1 receptor-associated kinase (IRAK) 1, receptor-interacting serine/threonine-protein kinase (RIPK) 2, and nuclear factor-κB (NF-κB) mRNA expression in gastrocnemius muscle (P<0.05), MyD88 mRNA expression in LD muscle, and FOXO1 mRNA expression in LD muscle (P<0.05). Moreover, Glu supplementation increased p-Akt/t-Akt ratio (P<0.05) in gastrocnemius muscle, and p-4EBP1/t-4EBP1 ratio in both gastrocnemius and LD muscles (P<0.05). Glu supplementation in the piglets' diets might be an effective strategy to alleviate LPS-induced muscle protein loss, which might be due to suppressing the mRNA expression of TLR4 and NODs signaling-related genes, and modulating Akt/FOXO and mTOR signaling pathways.

Endurance exercise improves walking performance in patients with peripheral artery disease (PAD), which is characterized by skeletal muscle dysfunction caused by lower extremity ischemia. Although transcriptional analyses of exercise-induced changes in normal animals and healthy volunteers have been reported, no detailed study has explored exercise-induced alterations in gene expression in PAD animal models. Here, we determined the acute and chronic effects of exercise on mRNA expression in the skeletal muscles of two mouse models of PAD. Three particular gene categories were investigated: known exercise-responsive genes (Pgc1a, Il6, Nr4a1, Nr4a2, and Nr4a3); myogenic and muscle regeneration-related genes (Myf5, Myogenin, Myomaker, and Myh3); and Gpr56 and its ligand Col3a1. PAD was induced by bilateral femoral artery ligation in normal C57BL/6 and diabetic KK-Ay mice. From 1 week after surgery, repetitive twice-weekly 30-min treadmill endurance exercise sessions were applied. Altered mRNA expression in the soleus muscles was measured in both the acute and chronic phases. In the acute phase, transcript levels of exercise-inducible genes showed significant increases in both C57BL/6 and diabetic KK-Ay PAD mice; levels of regeneration-related genes showed little alteration, and those of Gpr56 increased immediately and significantly after exercise in both models. In the chronic phase, transcript levels of Pgc1a, Myf5, Myogenin, Myomaker, Myh3, Gpr56, and Col3a1 were upregulated significantly in sedentary C57BL/6 PAD mice compared with that in sham-operated mice. Exercise training inhibited the upregulation of Col3a1, Myf5, and Myogenin significantly. In KK-Ay PAD mice, only Gpr56 mRNA levels increased significantly compared with those in sham-operated mice. RNA sequence analysis revealed 33 and 166 differentially upregulated, and 363 and 99 downregulated, genes after exercise training in C57BL/6 PAD and KK-Ay PAD mice, respectively. In summary, we detected significant alterations of skeletal muscle genes after exercise in PAD mouse models and characterized their expression patterns.

Igfn1 is a complex locus that codes for multiple splicing variants of Immunoglobulin- and Fibronectin-like domain containing proteins predominantly expressed in skeletal muscle. To reveal possible roles for Igfn1, we applied non-selective knock-down by shRNAs as well as specific targeting of Igfn1 exon 13 by CRISPR/Cas9 mutagenesis in C2C12 cells. Decreased expression of Igfn1 variants via shRNAs against the common 3'-UTR region caused a total blunting of myoblast fusion, but did not prevent expression of differentiation markers. Targeting of N-terminal domains by elimination of exon 13 via CRISPR/Cas9 mediated homologous recombination, also resulted in fusion defects as well as large multinucleated cells. Expression of IGFN1&#95;v1 partially rescued fusion and myotube morphology in the Igfn1 exon 13 knock-out cell line, indicating a role for this variant in myoblast fusion and differentiation. However, in vivo overexpression of IGFN1&#95;v1 or the Igfn1 Exon 13 CRISPR/Cas9 targeting vector did not result in significant size changes in transfected fibres.

A decline in the regenerative capacity of adult stem cells with aging is well documented. As a result of this decline, the efficacy of autologous stem cell therapies is likely to decline with increasing donor age. In these cases, strategies to restore the function of aged stem cells would have clinical utility. Globally, the transcription factor NF-κB is up-regulated in aged tissues. Given the negative role that NF-κB plays in myogenesis, we investigated whether the age-related decline in the function of muscle-derived stem/progenitor cells (MDSPCs) could be improved by inhibition of NF-κB. Herein, we demonstrate that pharmacologic or genetic inhibition of NF-κB activation increases myogenic differentiation and improves resistance to oxidative stress. Our results suggest that MDSPC "aging" may be reversible, and that pharmacologic targeting of pathways such as NF-κB may enhance the efficacy of cell-based therapies.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is responsible for a devastating immunosuppressive disease affecting juvenile domestic chickens. IBDV particles are naked icosahedrons enclosing a bipartite double-stranded RNA genome harboring three open reading frames (ORF). One of these ORFs codes for VP5, a non-structural polypeptide dispensable for virus replication in tissue culture but essential for IBDV pathogenesis. Using two previously described recombinant viruses, whose genomes differ in a single nucleotide, expressing or not the VP5 polypeptide, we have analyzed the role of this polypeptide during the IBDV replication process. Here, we show that VP5 is not involved in house-keeping steps of the virus replication cycle; i.e. genome transcription/replication, protein translation and virus assembly. Although infection with the VP5 expressing and non-expressing viruses rendered similar intracellular infective progeny yields, striking differences were detected on the ability of their progenies to exiting infected cells. Experimental data shows that the bulk of the VP5-expressing virus progeny efficiently egresses infected cells during the early phase of the infection, when viral metabolism is peaking and virus-induced cell death rates are as yet minimal, as determined by qPCR, radioactive protein labeling and quantitative real-time cell death analyses. In contrast, the release of the VP5-deficient virus progeny is significantly abridged and associated to cell death. Taken together, data presented in this report show that IBDV uses a previously undescribed VP5-dependent non-lytic egress mechanism significantly enhancing the virus dissemination speed. Ultrastructural analyses revealed that newly assembled IBDV virions associate to a vesicular network apparently facilitating their trafficking from virus assembly factories to the extracellular milieu, and that this association requires the expression of the VP5 polypeptide., Competing Interests: The authors have declared that no competing interests exist.

The effects of sciatic nerve crush (SNC) and treatment with Riluzole on muscle activity during unrestrained locomotion were identified in an animal model by analysis of the EMG activity recorded from soleus (Sol) and extensor digitorum longus (EDL) muscles of both hindlimbs; in intact rats (IN) and in groups of rats treated for 14 days with saline (S) or Riluzole (R) after right limb nerve crush at the 1st (1S and 1R) or 2nd (2S and 2R) day after birth. Changes in the locomotor pattern of EMG activity were correlated with the numbers of survived motor units (MUs) identified in investigated muscles. S rats with 2-8 and 10-28 MUs that survived in Sol and EDL muscles respectively showed increases in the duration and duty factor of muscle EMG activity and a loss of correlation between the duty factors of muscle activity, and abnormal flexor-extensor co-activation 3 months after SNC. R rats with 5, 6 (Sol) and 15-29 MUs (EDL) developed almost normal EMG activity of both Sol and control EDL muscles, whereas EDL muscles with SNC showed a lack of recovery. R rats with 8 (Sol) and 23-33 (EDL) MUs developed almost normal EMG activities of all four muscles. A subgroup of S rats with a lack of recovery and R rats with almost complete recovery that had similar number of MUs (8 and 24-28 vs 8 and 23-26), showed that the number of MUs was not the only determinant of treatment effectiveness. The results demonstrated that rats with SNC failed to develop normal muscle activity due to malfunction of neuronal circuits attenuating EDL muscle activity during the stance phase, whereas treatment with Riluzole enabled almost normal EMG activity of Sol and EDL muscles during locomotor movement., Competing Interests: The authors have declared that no competing interests exist.

Aryl Hydrocarbon Receptor Nuclear Translocator/ hypoxia-inducible factor 1 beta (ARNT/ HIF1β), a member of bHLH-PAS family of transcriptional factors, plays a critical role in metabolic homeostasis, insulin resistance and glucose intolerance. The contributions of ARNT in pancreas, liver and adipose tissue to energy balance through gene regulation have been described. Surprisingly, the impact of ARNT signaling in the skeletal muscles, one of the major organs involved in glucose disposal, has not been investigated, especially in type II diabetes. Here we report that ARNT is expressed in the skeletal muscles, particularly in the energy-efficient oxidative slow-twitch myofibers, which are characterized by increased oxidative capacity, mitochondrial content, vascular supply and insulin sensitivity. However, muscle-specific deletion of ARNT did not change myofiber type distribution, oxidative capacity, mitochondrial content, capillarity, or the expression of genes associated with these features. Consequently, the lack of ARNT in the skeletal muscle did not affect weight gain, lean/fat mass, insulin sensitivity and glucose tolerance in lean mice, nor did it impact insulin resistance and glucose intolerance in high fat diet-induced obesity. Therefore, skeletal muscle ARNT is dispensable for controlling muscle fiber type and metabolic regulation, as well as diet-induced weight control, insulin sensitivity and glucose tolerance., Competing Interests: The authors have declared that no competing interests exist.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

This study appraised the effects of dietary blend of 80% canola oil and 20% palm oil and postmortem ageing on oxidative stability, fatty acids and quality attributes of gluteus medius (GM) muscle in goats. Twenty-four Boer bucks were randomly allotted to diet supplemented with 0, 4 and 8% oil blend, fed for 100 days and slaughtered, and the GM muscle was subjected to a 7 d chill storage (4±1°C). Diet had no effect (P> 0.05) on the colour, drip loss, thiobarbituric acid-reactive substances (TBARS) value, free thiol, carbonyl, myoglobin and metmyoglobin contents, metmyoglobin reducing activity (MRA), antioxidant enzyme activities and abundance of myosin heavy chain (MHC) and actin in the GM muscle in goats. The meat from goats fed 4 and 8% oil blend had higher (P< 0.05) concentration of α and γ-tocopherol and abundance of troponin T compared with that from the control goats. The GM muscle from the oil-supplemented goats had lower (P< 0.05) concentration of C16:0 and greater (P< 0.05) concentration of C18:1n-9, C18:3n-3 and C20:5n-3 compared with that from the control goats. Nonetheless, diet did not affect (P< 0.05) the total fatty acid in the GM muscle in goats. Regardless of the diet, the free thiol and myoglobin contents, concentration of tocopherol and total carotenoids, MHC and MRA in the GM muscle decreased (P< 0.05) while carbonyl content, TBARS, drip loss and metmyoglobin content increased over storage. Dietary blend of 80% canola oil and 20% palm oil beneficially altered tissue lipids without hampering the oxidative stability of chevon.

Gonadal maturation is an extremely energy consuming process for batch spawners and it is associated with a significant decrease in growth and seasonal deterioration in flesh quality. Our knowledge about the molecular mechanisms linking sexual maturation and muscle growth is still limited. In the present study, we performed RNA-Seq using 454 GS-FLX pyrosequencing in fast skeletal muscle sampled from two-year-old Atlantic cod (Gadus morhua) at representative time points throughout the reproductive cycle (August, March and May). In total, 126,937 good quality reads were obtained, with 546 nucleotide length and 52% GC content on average. RNA-Seq analysis using the CLC Genomics Workbench with the Atlantic cod reference UniGene cDNA data revealed 59,581 (46.9%) uniquely annotated reads. Pairwise comparison for expression levels identified 153 differentially expressed UniGenes between time points. Notably, we found a significant suppression of myh13 and myofibrillar gene isoforms in fast skeletal muscle during the spawning season. This study uncovered a large number of differentially expressed genes that may be influenced by gonadal maturation, thus representing a significant contribution to our limited understanding of the molecular mechanisms regulating muscle wasting and regeneration in batch spawners during their reproductive cycle.

Regular physical exercise is central to a healthy lifestyle. However, exercise-related muscle contraction can induce reactive oxygen species and reactive nitrogen species (ROS/RNS) production in skeletal muscle. The nuclear factor-E2-related factor-2 (Nrf2) transcription factor is a cellular sensor for oxidative stress. Regulation of nuclear Nrf2 signaling regulates antioxidant responses and protects organ structure and function. However, the role of Nrf2 in exercise- or contraction-induced ROS/RNS production in skeletal muscle is not clear. In this study, using differentiated C2C12 cells and electrical pulse stimulation (EPS) of muscle contraction, we explored whether Nrf2 plays a role in the skeletal muscle response to muscle contraction-induced ROS/RNS. We found that EPS (40 V, 1 Hz, 2 ms) stimulated ROS/RNS accumulation and Nrf2 activation. We also showed that expression of NQO1, HO-1 and GCLM increased after EPS-induced muscle contraction and was remarkably suppressed in cells with Nrf2 knockdown. We also found that the antioxidant N-acetylcysteine (NAC) significantly attenuated Nrf2 activation after EPS, whereas the nitric oxide synthetase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) did not. Furthermore, Nrf2 knockdown after EPS markedly decreased ROS/RNS redox potential and cell viability and increased expression of the apoptosis marker Annexin V in C2C12 myotubes. These results indicate that Nrf2 activation and expression of Nrf2 regulated-genes protected muscle against the increased ROS caused by EPS-induced muscle contraction. Thus, our findings suggest that Nrf2 may be a key factor for preservation of muscle function during muscle contraction.

Head and neck cancer is often diagnosed at advanced stages, and surgical resection with wide margins is generally indicated, despite this treatment being associated with poor postoperative quality of life (QOL). We have previously reported on the therapeutic effects of skeletal muscle-derived multipotent stem cells (Sk-MSCs), which exert reconstitution capacity for muscle-nerve-blood vessel units. Recently, we further developed a 3D patch-transplantation system using Sk-MSC sheet-pellets. The aim of this study is the application of the 3D Sk-MSC transplantation system to the reconstitution of facial complex nerve-vascular networks after severe damage. Mouse experiments were performed for histological analysis and rats were used for functional examinations. The Sk-MSC sheet-pellets were prepared from GFP-Tg mice and SD rats, and were transplanted into the facial resection model (ST). Culture medium was transplanted as a control (NT). In the mouse experiment, facial-nerve-palsy (FNP) scoring was performed weekly during the recovery period, and immunohistochemistry was used for the evaluation of histological recovery after 8 weeks. In rats, contractility of facial muscles was measured via electrical stimulation of facial nerves root, as the marker of total functional recovery at 8 weeks after transplantation. The ST-group showed significantly higher FNP (about three fold) scores when compared to the NT-group after 2-8 weeks. Similarly, significant functional recovery of whisker movement muscles was confirmed in the ST-group at 8 weeks after transplantation. In addition, engrafted GFP+ cells formed complex branches of nerve-vascular networks, with differentiation into Schwann cells and perineurial/endoneurial cells, as well as vascular endothelial and smooth muscle cells. Thus, Sk-MSC sheet-pellet transplantation is potentially useful for functional reconstitution therapy of large defects in facial nerve-vascular networks.

Background: Skeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs). Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM), which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential., Results: We showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM) indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers., Conclusions: These findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by a selective loss of motor neurons together with a progressive muscle weakness. Albeit the pathophysiological mechanisms of the disease remain unknown, growing evidence suggests that skeletal muscle can be a target of ALS toxicity. In particular, the two main intracellular degradation mechanisms, autophagy and the ubiquitin-proteasome degradative system (UPS) have been poorly studied in this tissue. In this study we investigated the activation of autophagy and the UPS as well as apoptosis in the skeletal muscle from SOD1G93A mice along disease progression. Our results showed a significant upregulation of proteasome activity at early symptomatic stage, while the autophagy activation was found at presymptomatic and terminal stages. The mRNA upregulated levels of LC3, p62, Beclin1, Atg5 and E2f1 were only observed at symptomatic and terminal stages, which reinforced the time-point activation of autophagy. Furthermore, no apoptosis activation was observed along disease progression. The combined data provided clear evidence for the first time that there is a time-point dependent activation of autophagy and UPS in the skeletal muscle from SOD1G93A mice.

Dystrophin (Dmd) is a structural protein that links the extracellular matrix to actin filaments in muscle fibers and is required for the maintenance of muscles integrity. Mutations in Dmd lead to muscular dystrophies in humans and other vertebrates. Here, we report the characterization of a zebrafish gene trap line that fluorescently labels the endogenous Dmd protein (Dmd-citrine, Gt(dmd-citrine) ct90a). We show that the Dmd-citrine line recapitulates endogenous dmd transcript expression and Dmd protein localization. Using this Dmd-citrine line, we follow Dmd localization to the myosepta in real-time using time-lapse microscopy, and find that the accumulation of Dmd protein at the transverse myosepta coincides with the onset of myotome formation, a critical stage in muscle maturation. We observed that Dmd protein localizes specifically to the myosepta prior to dmd mRNA localization. Additionally, we demonstrate that the Dmd-citrine line can be used to assess muscular dystrophy following both genetic and physical disruptions of the muscle.

The present study evaluated low-level laser therapy (LLLT) effects on some physiological pathways that may lead to muscle damage or regeneration capacity in dystrophin-deficient muscle cells of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). Primary cultures of mdx skeletal muscle cells were irradiated only one time with laser and analyzed after 24 and 48 hours. The LLLT parameter used was 830 nm wavelengths at 5 J/cm² fluence. The following groups were set up: Ctrl (untreated C57BL/10 primary muscle cells), mdx (untreated mdx primary muscle cells), mdx LA 24 (mdx primary muscle cells - LLLT irradiated and analyzed after 24 h), and mdx LA 48 (mdx primary muscle cells - LLLT irradiated and analyzed after 48 h). The mdx LA 24 and mdx LA 48 groups showed significant increase in cell proliferation, higher diameter in muscle cells and decreased MyoD levels compared to the mdx group. The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups. The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca2+]i. The mdx group showed significant increase in H2O2 production and 4-HNE levels compared to the Ctrl group and LLLT treatment reduced this increase. GSH levels and GPx, GR and SOD activities increased in the mdx group. Laser treatment reduced the GSH levels and GR and SOD activities in dystrophic muscle cells. The mdx group showed significant increase in the TNF-α and NF-κB levels, which in turn was reduced by the LLLT treatment. Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.

Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

Introduction: Hypertonic media causes cells to shrink due to water loss through aquaporin channels. After acute shrinkage, cells either regulate their volume or, alternatively, undergo a number of metabolic changes which ultimately lead to cell death. In many cell types, hypertonic shrinkage is followed by apoptosis. Due to the complex 3D morphology of skeletal muscle and the difficulty in obtaining isolated human tissue, we have begun skeletal muscle volume regulation studies using the human skeletal muscle cell line TE671RD. In this study we investigated whether hypertonic challenge of the human skeletal muscle cell line TE671RD triggered cell death or evoked a cell volume recovery response., Methods: The cellular volume of TE671RD cells was calculated from the 2D surface area. Cell death was assessed by both the trypan blue live/dead assay and the TUNEL assay., Results: Medium osmolality was increased by addition of up to 200 mM sucrose. Addition of 200 mM sucrose resulted in mean cell shrinkage of 44±1% after 30 mins. At later time points (2 and 4 hrs) two separate cell subpopulations with differing mean cell volume became apparent. The first subpopulation (15±2% of the total cell number) continued to shrink whereas the second subpopulation had an increased cell volume. Cell death was observed in a small proportion of cells (approximately 6-8%)., Conclusion: We have established that a substantial proportion of TE671RD cells respond to hypertonic challenge with RVI, but that these cells are resistant to hypertonicity triggered cell death.

In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine effects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly characterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and alpha-sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs account for 1-5% of the total and that 60-65% of these EVs were also positive for the exosomal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and -181a-5p were significantly up-regulated after acute exercise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis.

The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.