Myogenic Process Research Articles

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on acetylcholinesterase during myogenic differentiation of contractile rat primary skeletal muscle cells

Emerging data indicate that prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could interfere with myogenic differentiation in vivo. Acetylcholinesterase (EC3.1.1.7; AChE), an enzyme critical for cholinergic neurotransmission, is abundantly expressed in neurons and mature myotubes, and we recently found that muscle AChE expression was suppressed in parallel with the inhibition of myogenic differentiation upon TCDD treatment in mouse C2C12 cells. This TCDD-induced suppression of muscle AChE was proposed to involve an aryl hydrocarbon receptor (AhR)-independent mechanism, but the precise underlying mechanism remains unclear. Considering the widely recognized role of muscular activity in AChE expression and its potential crosstalk with the AhR signaling pathway, we sought to investigate the effect of TCDD on muscle AChE expression in the presence of muscular activity. Therefore, we employed a highly contractile rat primary skeletal muscle culture system in which AChE activity and the expression of genes related to it (AChE T subunit and collagen Q (ColQ)) were increased during the myogenic differentiation process. Although TCDD treatment successfully induced the expression of genes regulated by AhR activation, the treatment exerted no notable effects on myogenic differentiation. Moreover, muscle AChE enzymatic activity and mRNA level remained unchanged following TCDD treatment, and only ColQ mRNA expression was slightly increased after 4-day treatment with TCDD (10−10 M). The compensatory role of muscle-contraction-related signaling pathways in this newly identified unresponsiveness of muscle AChE to TCDD warrants further investigation.

Integrated miRNA-mRNA transcriptomic analysis reveals epigenetic-mediated embryonic muscle growth differences between Wuzhishan and Landrace pigs1.

Pig is one of the major dietary protein sources for human consumption, from which muscle is the largest protein origin. However, molecular mechanisms concerning early porcine embryonic muscle development distinctions between pig breeds are still unclear. In this study, an integrated analysis of transcriptome and miRNAome was conducted using longissimus dorsi muscle of 4 early embryonic stages around the primary myofiber formation time (18-, 21-, 28-, and 35-d post coitus) from 2 pig breeds (Landrace [LR] and Wuzhishan [WZS]) differing in meat mass. The global miRNA/mRNA expression profile showed that WZS prepared for myogenic developmental processes earlier than LR. After identifying and analyzing the interaction network of top 100 up-/down-regulated miRNA and their target genes, we were able to find 3 gene clusters: chromatin modification-related (Chd2, H3f3a, Chd6, and Mll1), myogenesis-related (Pax3, Pbx1, Mef2a, and Znf423), and myosin component-related (Mylk, Myo5a, Mylk4, Myh9, and Mylk2) gene clusters. These genes may involve in miRNA-gene myogenic regulatory network that plays vital role in regulating distinct early porcine embryonic myogenic processes between LR and WZS. In summary, our study reveals an epigenetic-mediated myogenic regulatory axial that will help us to decipher molecular mechanisms concerning early porcine embryonic muscle development distinctions between pig breeds.

Dermatopontin in Skeletal Muscle Extracellular Matrix Regulates Myogenesis.

Dermatopontin (DPT) is an extensively distributed non-collagenous component of the extracellular matrix predominantly found in the dermis of the skin, and consequently expressed in several tissues. In this study, we explored the role of DPT in myogenesis and perceived that it enhances the cell adhesion, reduces the cell proliferation and promotes the myoblast differentiation in C2C12 cells. Our results reveal an inhibitory effect with fibronectin (FN) in myoblast differentiation. We also observed that DPT and fibromodulin (FMOD) regulate positively to each other and promote myogenic differentiation. We further predicted the 3D structure of DPT, which is as yet unknown, and validated it using state-of-the-art in silico tools. Furthermore, we explored the in-silico protein-protein interaction between DPT-FMOD, DPT-FN, and FMOD-FN, and perceived that the interaction between FMOD-FN is more robust than DPT-FMOD and DPT-FN. Taken together, our findings have determined the role of DPT at different stages of the myogenic process.

The Essentiality of Serine and Glycine for Skeletal Muscle Regeneration

Non‐essential amino acids (NEAA), serine (ser) and glycine (gly), are recognized as essential to support proliferation of some cell types, including skeletal muscle (SkM) progenitor cells (MPCs). MPCs and a well‐orchestrated myogenic program are essential for SkM regeneration following injury; impaired regeneration underlies pathological tissue remodeling and functional decline. It is appreciated that the metabolic microenvironment and nutrient availability influence the myogenic process. The objective of this research was to investigate the metabolic and cellular requirements of ser and gly for the regenerative process (MPC expansion, differentiation, and myotube formation) in vitro. Human primary MPCs (hMPCs) were cultured with and without ser/gly and markers of proliferation (live and dead staining, BrdU incorporation, Propidium Iodide cell‐cycle analysis) were assessed; markers of differentiation, and myotube formation (gene and protein expression of select myogenic regulatory factors [MRFs], myosin staining) are being investigated. Additionally, the regulation of the de novo ser synthesis pathway (SSP), ser/gly interconversion (SHMT1, SHMT2), and changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined with/without ser/gly using RNAseq, qPCR, immunoblotting, and a Seahorse bioanalyzer. Ser/gly depletion caused hMPCs to reside in a non‐proliferative state without inducing apoptosis and decreased OCR (p<0.05) but not ECAR (p=0.07), supporting a quiescent metabolic state. BrdU incorporation and cell‐cycle sorting with flow cytometry suggested that depletion of ser/gly inhibits hMPCs from leaving G1 phase. Intriguingly, proliferating hMPCs expressed all SSP enzymes (PHGDH, PSAT1, PSPH). The expression of SSP enzymes, SHMT2, ser transporters, and ATF4 (transcription factor involved in nutrient sensing) increased during ser/gly depletion suggesting ser/gly depletion is sensed by hMPCs and mechanisms to increase ser and gly availability are upregulated. Ser and gly are substrates for multiple metabolic pathways; building blocks for lipids, proteins, and nucleotides; one‐carbon donors; and help maintain cellular (epi)genetic status and redox state. Providing formate (a one‐carbon source), nucleotides, or additional glucose did not rescue proliferation. RNAseq analysis revealed an upregulation of genes involved in the glutathione metabolism pathway. In support of these findings a cell permeable version of glutathione provided a modest rescue to hMPC cell number and intracellular levels of total glutathione and the ratio of reduced to oxidized glutathione were diminished after the ser/gly depletion; glutathione may be a necessary product of ser/gly metabolism. Ongoing RNAseq analysis will allow us to identify potential mechanisms underlying the quiescent‐like state. Ongoing culture experiments will determine the effects of ser/gly depletion on differentiation and myotube formation. This research builds upon recent findings that clearly identify an exogenous requirement of ser and gly for hMPC expansion and challenges the notion that ser and gly are strictly NEAA, particularly in states of tissue regeneration.Support or Funding InformationPresident's Council of Cornell Women and Cornell Division of Nutritional SciencesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gene expression of Hanwoo satellite cell differentiation in longissimus dorsi and semimembranosus

BackgroundKorean Hanwoo cattle are known for their high meat quality, especially their high intramuscular fat compared to most other cattle breeds. Different muscles have very different meat quality traits and a study of the myogenic process in satellite cells can help us better understand the genes and pathways that regulate this process and how muscles differentiate.ResultsCell cultures of Longissimus dorsi muscle differentiated from myoblast into multinucleated myotubes faster than semimembranosus. Time-series RNA-seq identified a total of 13 differentially expressed genes between the two muscles during their development. These genes seem to be involved in determining muscle lineage development and appear to modulate the expression of myogenic regulatory factors (mainly MYOD and MYF5) during differentiation of satellite cells into multinucleate myotubes. Gene ontology enriched terms were consistent with the morphological changes observed in the histology. Most of the over-represented terms and genes expressed during myoblast differentiation were similar regardless of muscle type which indicates a highly conserved myogenic process albeit the rates of differentiation being different. There were more differences in the enriched GO terms during the end of proliferation compared to myoblast differentiation.ConclusionsThe use of satellite cells from newborn Hanwoo calves appears to be a good model to study embryonic myogenesis in muscle. Our findings provide evidence that the differential expression of HOXB2, HOXB4, HOXB9, HOXC8, FOXD1, IGFN1, ZIC2, ZIC4, HOXA11, HOXC11, PITX1, SIM2 and TBX4 genes could be involved in the differentiation of Longissimus dorsi and Semimembranosus muscles. These genes seem to modulate the muscle fate of the satellite cells during myogenesis through a differential expression profile that also controls the expression of some myogenic regulatory factors (MYOD and MYF5). The number of differentially expressed genes across time was unsurprisingly large. In relation to the baseline day 0, there were 631, 155, 175, 519 and 586 DE genes in LD, while in SM we found 204, 0, 615, 761 and 1154 DE genes at days 1, 2, 4, 7 and 14 respectively.

In vitro perfusion culture of adipose tissue and its myogenic induction

Objective To investigate the myogenic possibility and mechanism of adipose tissue block induced by in vitro perfusion. Methods Myogenic inducing liquid was perfused through the vascular pedicle to cultrue the rat inguinal fat fascia flap. After 3 weeks, the tissue viability was detected by the fluorescein diacetate and propidium iodide (FAD/PI) staining. The changes of histomorphology was observed by histological staining and immunofluorescence staining was used to observe the expression of Desmin and myogenic differentiation factor 1(MyoD1). The expression levels of Desmin, myogenic factor 5(Myf-5)and MyoD1 were detected by Western blotting. Results FAD/PI staining showed that the adipose tissue cultured in the induction fluid was still viable at 3 weeks. Histological observation showed that the adipose tissues induced by the inducing medium had formed some muscle-like tissues locally. Immunofluorescence staining showed positive expression of Desmin and MyoD1. Protein quantitative results showed that compared with non-induced group (0.028 2±0.027 1), the expression of muscle specific protein Desmin in induced group (0.114 5±0.153 1) increased slightly, yet the difference had no statistical significance (t=0.622, P>0.05). The expression level of myogenic differentiation factor MyoD1 was higher in induced group (0.255 2±0.103 2) than non-induced group (0.023 9±0.024 6) and the difference was statistically significant (t=4.877, P<0.01). The reduction of Myf-5 in non-induced group was also inhibit in the induced group (the expression level of Myf-5 were 1 time in induced group and 0.13 times in non-induced group after the induction compared to the control fat tissue group), the difference had statistical significance (t=5.215, P<0.01). Conclusion The adipose tissues cultured in the perfusion bioreactor can survive at least 3 weeks in vitro, and they can be partially transformed into muscle-like tissues. Myogenic factor Myf-5 and MyoD1 are both involved in the myogenic process of adipose tissue in vitro. Key words: Adipose; Myogenic induction; Tissue culture; In vitro perfusion

MiR-10b-5p Regulates C2C12 Myoblasts Proliferation and Differentiation.

The development of skeletal muscle is a complex process including myoblasts proliferation and differentiation. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at post-transcriptional level. Increasing evidences indicate that miRNAs are important regulators in myogenic processes. Here, we reported that the expression of miR-10b-5p steadily decreased during myoblasts proliferation, but significantly increased during myoblasts differentiation. The over-expression of miR-10b-5p promoted myoblasts proliferation and blunted myofiber formation in C2C12 cells, while miR-10b-5p down-regulation showed an opposite result. At the same time, we observed that the down-regulation of nuclear factor of activated T-cells 5 (NFAT5) repressed the differentiation of C2C12 cells, and interestingly, miR-10b-5p could suppress NFAT5 expression. Luciferase activity assays confirmed that miR-10b-5p directly target the 3'-untranslated region (3'-UTR) of NFAT5. Overall, we proposed here a novel insight that miR-10b-5p regulates the proliferation and differentiation of C2C12 myoblasts, and the impact on myogenic differentiation is partly through targeting NFAT5. Abbreviations: NFAT5: nuclear factor of activated T-cells 5; Cyclin B: cycle protein B; Cyclin D1: cycle protein D1; Cyclin E: cycle protein E; CDK4: cyclin-dependent kinase 4; MyoD: myogenic differentiation antigen; MyoG: myogenin; Myf5: myogenic factor 5; MRF4: myogenic regulatory factor 4; MyHC: myosin heavy chain; AQP5: aquaporin-5; CACNA1C: calcium voltage-gated channel subunit alpha1 C; SRF: serum response factor; Pax7: paired box 7; KLF4: Kruppel-like factor 4; 3'-UTR: 3'-untranslated region; GM: growth medium; DM: differentiation medium.

Morphology-Based Analysis of Myoblasts for Prediction of Myotube Formation

The development of new drugs depends on the efficiency of drug screening. Phenotype-based screening has attracted interest due to its considerable potency for the discovery of first-in-class drugs. In general, fluorescently labeled imagery is the leading technique for phenotype-based screening; however, there are growing requirements to understand total culture profiles, which are unclear after end-point assays. In this study, we demonstrate that morphology-based cellular evaluation of unlabeled cells is an efficient approach to evaluate myotube formation assays. One of our aims was to study the myogenic differentiation process in C2C12 cells to discern the differences between cellular responses to different medium conditions (serum concentrations and insulin dosages). Our results show that predictive morphological profiles that strongly correlate with myogenic differentiation can be generated from myotube images, even in the confluent stage. The differentiation rate after 14 days can be quantitatively predicted with the highest accuracy by means of images taken on days 0–11.5. In addition, for the application of our morphology-based cellular evaluation, the effect of cyclic guanosine monophosphate (cGMP) on myogenic differentiation was analyzed. Our results show that the quantitated morphological profile from these images can be an effective descriptor for analysis of the myotube-recovering effect of cGMP.

The isolation, culture and identification of skeletal muscle satellite cells from bactrian camel

In this experiment, the skeletal muscle tissue of Bactrian camel was used as sample material. The cells of primary generation were cultured by two-step enzymatic digestion and tissue block culture. The primary skeletal muscle satellite cells were purified by differential adherence methods, and then were determined by growth curve analysis. The advantages of mode of proliferation of cells were that the cells could maintain high activity, comparatively not tend to be contaminated, and gain a long culture period, which is more suitable for the in vitro culture of Bactrian camel satellite cells. When using the method called LASER Confocal imaging, with immunofluorescence labeling, to identify the specific gene in skeletal muscle satellite cells, the PAX7 expression was positive in cell lines. In addition, PCR amplification bands showed that PAX7, MYF5, and Desmin genes were all clearly expressed. After induced culture, satellite cells started the myogenic differentiation process. Through the above methods, comparatively pure Bactrian camel skeletal muscle satellite cells were obtained successfully and passage proces were carried out stably. In conclusion, the in vitro culture system of Bactrian camel skeletal muscle satellite cells was successfully established.

The overlap between regeneration and fibrosis in injured skeletal muscle is regulated by phosphatidylinositol 3-kinase/Akt signaling pathway - A bioinformatic analysis based on lncRNA microarray

Injured skeletal muscle would go through a sequence of the pathological phases of degeneration, myogenesis and fibrosis. Growing evidence indicated that fibrotic and myogenic phases might overlap within the injured skeletal muscle in the early time after injury. However, the mechanism underlying this overlapping remains unclear. Here, we performed an lncRNA microarray to identify the activated pathways in mice muscle seven days after contusion. KEGG analysis indicated that phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling cascade was predicted to be activated by lncRNAs. The top genes targeted by lncRNAs in PI3K/Akt signaling were subunits of laminin, collagen 5, and collagen 6, which participated in either myogenic or fibrotic process. Reverse transcriptase–polymerase chain reaction analysis and immunohistochemical stain further confirmed the prediction in silico. These results suggested that the overlap might be related to an activated PI3K/Akt pathway by lncRNA regulation.

MiR-204-5p regulates C2C12 myoblast differentiation by targeting MEF2C and ERRγ

Myogenic differentiation, which occurs in the process of muscle development, is a highly ordered process. Increasing evidence indicates that microRNAs (miRNAs) are important regulators in myogenic processes. In this study, we found that miR-204-5p expression gradually decreased when myoblasts were induced to differentiate. Our results suggested that miR-204-5p blunted myoblast differentiation, which was accompanied with a decreased proportion of myosin heavy chain (MyHC)-positive cells in myoblasts with augmented expression of miR-204-5p. Furthermore, overexpression of miR-204-5p significantly decreased the MyHC composition of slow-twitch fibers in myoblasts. Luciferase activity assays confirmed that miR-204-5p directly targeted the 3′-untranslated region (3′-UTR) of myocyte enhancer factor 2C (MEF2C) and estrogen-related receptor gamma (ERRγ). Small interfering RNA (siRNA) technology successfully inhibited the expression of MEF2C and ERRγ. Interference with MEF2C or ERRγ inhibited myoblast differentiation and the formation of slow-twitch fibers. Meanwhile, co-transfection of either si-MEF2C or si-ERRγ with miR-204-5p mimics resulted in a more severe attenuation of myogenic differentiation. In summary, this study demonstrates that miR-204-5p inhibits myoblast differentiation by targeting MEF2C and ERRγ. Our findings suggest that miR-204-5p is a potential regulator that could influence myogenesis.

Separation of functionally divergent muscle precursor cell populations from porcine juvenile muscles by discontinuous Percoll density gradient centrifugation

BackgroundSatellite cells (SC) and their descendants, muscle precursor cells (MPC), play a key role in postnatal muscle development, regeneration, and plasticity. Several studies have provided evidence that SC and MPC represent a heterogeneous population differing in their biochemical and functional properties. The identification and characterization of functionally divergent SC subpopulations should help to reveal the precise involvement of SC/MPC in these myogenic processes. The aim of the present work was therefore to separate SC subpopulations by using Percoll gradients and to characterize their myogenic marker profiles and their functional properties (adhesion, proliferation, and differentiation).ResultsSC/MPC from muscles of 4-day-old piglets were isolated by trypsin digestion and enriched by Percoll density gradient centrifugation. A mixed myogenic cell population was obtained from the 40/70% interface (termed: mixed P40/70) of a 25/40/70% Percoll gradient. Thereafter, by using a more stepped 25/40/50/70% Percoll gradient, mixed P40/70 was divided into subpopulation 40/50 (SP40/50) collected from the 40/50% interface and subpopulation 50/70 (SP50/70) collected from the 50/70% interface.All three isolated populations proliferated and showed a myogenic phenotype characterized by the ability to express myogenic markers (Pax7, MyoD1, Desmin, and MyoG) and to differentiate into myotubes. However, compared with mixed P40/70, SP40/50 and SP50/70 exhibited distinct functional behavior. Growth kinetic curves over 90 h obtained by the xCELLigence system and proliferation assays revealed that SP40/50 and mixed P40/70 constituted a fast adhering and fast proliferating phenotype. In contrast, SP50/70 showed considerably slower adhesion and proliferation. The fast-proliferating SP40/50 showed the highest myogenic differentiation potential with higher fusion rates and the formation of more middle-sized and large myotubes.ConclusionsThe described Percoll density gradient centrifugation represents a useful tool for subdividing pig SC/MPC populations with divergent myogenic functions. The physiological role of SC subpopulations during myogenesis and the interaction of these populations can now be analyzed to a greater extent, shedding light on postnatal growth variations in pigs and probably in other animals.

Co-delivery of micronized urinary bladder matrix damps regenerative capacity of minced muscle grafts in the treatment of volumetric muscle loss injuries.

Minced muscle grafts (MG) promote de novo muscle fiber regeneration and neuromuscular strength recovery in small and large animal models of volumetric muscle loss. The most noteworthy limitation of this approach is its reliance on a finite supply of donor tissue. To address this shortcoming, this study sought to evaluate micronized acellular urinary bladder matrix (UBM) as a scaffolding to promote in vivo expansion of this MG therapy in a rat model. Rats received volumetric muscle loss injuries to the tibialis anterior muscle of their left hind limb which were either left untreated or repaired with minced muscle graft at dosages of 50% and 100% of the defect mass, urinary bladder matrix in isolation, or a with an expansion product consisting of a combination of the two putative therapies in which the minced graft is delivered at a dosage of 50% of the defect mass. Rats survived to 2 and 8 weeks post injury before functional (in vivo neuromuscular strength), histological, morphological, and biochemical analyses were performed. Rats treated with the expansion product exhibited improved neuromuscular function relative to untreated VML after an 8 week time period following injury. This improvement in functional capacity, however, was accompanied with a concomitant reduction in graft mediated regeneration, as evidenced cell lineage tracing enable by a transgenic GFP expressing donor, and a mixed histological outcome indicating coincident fibrous matrix deposition with interspersed islands of nascent muscle fibers. Furthermore, quantitative immunofluorescence and transcriptional analysis following the 2 week time point suggests an exacerbated immune response to the UBM as a possible nidus for the observed suboptimal regenerative outcome. Moving forward, efforts related to the development of a MG expansion product should carefully consider the effects of the host immune response to candidate biomaterials in order to avoid undesirable dysregulation of pro-regenerative cross talk between the immune system and myogenic processes.

Creatine Supplementation and Skeletal Muscle Metabolism for Building Muscle Mass- Review of the Potential Mechanisms of Action.

Creatine, a very popular supplement among athletic populations, is of growing interest for clinical applications. Since over 90% of creatine is stored in skeletal muscle, the effect of creatine supplementation on muscle metabolism is a widely studied area. While numerous studies over the past few decades have shown that creatine supplementation has many favorable effects on skeletal muscle physiology and metabolism, including enhancing muscle mass (growth/hypertrophy); the underlying mechanisms are poorly understood. This report reviews studies addressing the mechanisms of action of creatine supplementation on skeletal muscle growth/hypertrophy. Early research proposed that the osmotic effect of creatine supplementation serves as a cellular stressor (osmosensing) that acts as an anabolic stimulus for protein synthesis signal pathways. Other reports indicated that creatine directly affects muscle protein synthesis via modulations of components in the mammalian target of rapamycin (mTOR) pathway. Creatine may also directly affect the myogenic process (formation of muscle tissue), by altering secretions of myokines, such as myostatin and insulin-like growth factor-1, and expressions of myogenic regulatory factors, resulting in enhanced satellite cells mitotic activities and differentiation into myofiber. Overall, there is still no clear understanding of the mechanisms of action regarding how creatine affects muscle mass/growth, but current evidence suggests it may exert its effects through multiple approaches, with converging impacts on protein synthesis and myogenesis.

Skeletal muscle regeneration involves macrophage-myoblast bonding

ABSTRACTRegeneration in adult skeletal muscle relies on the activation, proliferation, and fusion of myogenic precursor cells (MPC), mostly resident satellite cells (SC). However, the regulatory mechanism during this process is still under evaluation, with the final aim to manipulate regeneration when the intrinsic mechanism is corrupted. Furthermore, intercellular connections during skeletal muscle regeneration have not been previously thoroughly documented. Our hypothesis was that a direct and close cellular interaction between SC/MPC and invading myeloid cells is a key step to control regeneration. We tested this hypothesis during different steps of skeletal muscle regeneration: (a) the recruitment of activated SC; (b) the differentiation of MPC; (c) myotubes growth, in a mouse model of crush injury. Samples harvested (3 and 5 days) post-injury were screened by light and confocal microscopy. Ultrastructural analysis was performed by conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) followed by 3D modeling of electron tomography (ET) data. This revealed a new type of interaction between macrophages and myogenic cells by direct heterocellular surface apposition over large areas and long linear distances. In the analyzed volume, regions spaced below 20 nm, within molecular range, represented 31% of the macrophage membrane surface and more than 27% of the myotube membrane. The constant interaction throughout all stages of myogenesis suggests a potential new type of regulatory mechanism for the myogenic process. Thus, deciphering structural and molecular mechanisms of SC-macrophage interaction following injury might open promising perspectives for improving muscle healing.

MiR-23-TrxR1 as a novel molecular axis in skeletal muscle differentiation

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing protein involved in cellular redox homeostasis which is downregulated in skeletal muscle differentiation. Here we show that TrxR1 decrease occurring during myogenesis is functionally involved in the coordination of this cellular process. Indeed, TrxR1 depletion reduces myoblasts growth by inducing an early myogenesis -related gene expression pattern which includes myogenin and Myf5 up-regulation and Cyclin D1 decrease. On the contrary, the overexpression of TrxR1 during differentiation delays myogenic process, by negatively affecting the expression of Myogenin and MyHC. Moreover, we found that miR-23a and miR-23b - whose expression was increased in the early stage of C2C12 differentiation - are involved in the regulation of TrxR1 expression through their direct binding to the 3′ UTR of TrxR1 mRNA. Interestingly, the forced inhibition of miR-23a and miR-23b during C2C12 differentiation partially rescues TrxR1 levels and delays the expression of myogenic markers, suggesting the involvement of miR-23 in myogenesis via TrxR1 repression. Taken together, our results depict for the first time a novel molecular axis, which functionally acts in skeletal muscle differentiation through the modulation of TrxR1 by miR-23.

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Recombinant bovine growth hormone (rBGH) enhances somatic growth by regulating the GH-IGF axis in fingerlings of gilthead sea bream (Sparus aurata)

The growth hormone (GH)/insulin-like growth factors (IGFs) endocrine axis is the main growth-regulator system in vertebrates. Some authors have demonstrated the positive effects on growth of a sustained-release formulation of a recombinant bovine GH (rBGH) in different fish species. The aim of this work was to characterize the effects of a single injection of rBGH in fingerlings of gilthead sea bream on growth, GH-IGF axis, and both myogenic and osteogenic processes. Thus, body weight and specific growth rate were significantly increased in rBGH-treated fish respect to control fish at 6weeks post-injection, whereas the hepatosomatic index was decreased and the condition factor and mesenteric fat index were unchanged, altogether indicating enhanced somatic growth. Moreover, rBGH injection increased the plasma IGF-I levels in parallel with a rise of hepatic mRNA from total IGF-I, IGF-Ic and IGF-II, the binding proteins IGFBP-1a and IGFBP-2b, and also the receptors IGF-IRb, GHR-I and GHR-II. In skeletal muscle, the expression of IGF-Ib and GHR-I was significantly increased but that of IGF-IRb was reduced; the mRNA levels of myogenic regulatory factors, proliferation and differentiation markers (PCNA and MHC, respectively), or that of different molecules of the signaling pathway (TOR/AKT) were unaltered. Besides, the growth inhibitor myostatin (MSTN1 and MSTN2) and the hypertrophic marker (MLC2B) expression resulted significantly enhanced, suggesting altogether that the muscle is in a non-proliferative stage of development. Contrarily in bone, although the expression of most molecules of the GH/IGF axis was decreased, the mRNA levels of several osteogenic genes were increased. The histology analysis showed a GH induced lipolytic effect with a clear decrease in the subcutaneous fat layer. Overall, these results reveal that a better growth potential can be achieved on this species and supports the possibility to improve growth and quality through the optimization of its culture conditions.

Flavanol-Rich Lychee Fruit Extract Prevents Diabetes-induced Muscle Loss And Palmitate-induced Myotube Loss

Diabetes-induced muscle loss is associated with stimulated the muscle RING-finger protein 1 (MuRF1), a muscle specific E3 ubiquitin ligase. Elevated lipid metabolites impair myogenesis. Flavanol-rich lychee fruit extract (FRLFE) exhibited anti-diabetic and -obesity properties which would be an alternative approach for maintaining muscle homeostasis. PURPOSE: The underlying mechanisms of FRLFE on maintaining muscle homeostasis was studied in vivo and in vitro. METHODS: Dietary (10 wk) FRLFE supplementation (200 mg/kg diet) on the skeletal muscle size and transcription factors such as NF-κB and Foxo3a involved in regulation of MuRF1 were investigated in diabetic db/db mice (n=10/per group). The roles of FRLFE on cell cycle and senescent phenotype were investigated in palmitate-induced insulin resistance in C2C12 muscle cells. The statistical significance of the differences among the groups (P < 0.05) was determined by one-way ANOVA and following post hoc assessment by Student-Newman-Keuls Method. RESULTS: The average cross-sectional area was significantly increased by 1.6-fold after administration of FRLFE compared with non-treated db/db mice (420μm2). Prevention of muscle loss by FRLFE was associated with down regulation of MuRF1 mRNA expression (410% vs. 100%, p<0.05). Decreased NF-κB expression in the nuclear fraction was observed in FRLFE treated db/db mice compared with untreated mice (260% vs. 60%, p<0.05). At transcriptional level, FRLFE abated ceramide-induced MuRF1 promoter activity (200% vs. 100%, p<0.05). Restoration of SIRT1 expression (30% vs. 150%, p<0.05) prevented Foxo3a nuclear localization in FRLFE treated db/db mice. A strong β-galactosidase staining positive signal was observed in the palmitate-treated myoblasts. For cell cycle analysis, significantly increased proportion of cells in G2-phase by palmitate was observed compared with group without palmitate treatment (28.9% vs. 18.3%). Weak β-galactosidase staining positive signals in accordance with decreased proportion of cells in G2-phase (21.7%) were observed in FRLFE-treated myoblasts. CONCLUSION: The efficacy of FRLFE on preserving skeletal muscle mass and myogenic process indicates that FRLFE acts as an effective supplement for diabetes and/or obesity-induced muscle atrophy.

A Non‐Canonical Function of Leucyl tRNA Synthetase Negatively Regulates Skeletal Myogenesis

Aside from its house‐keeping function in protein synthesis, leucyl tRNA synthetase (LeuRS) has a non‐canonical function in regulating cell growth via the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway by sensing amino acid availability. mTOR also governs skeletal myogenesis, but its signaling network is distinct from that in cell growth regulation. We set out to investigate a potential role of LeuRS in skeletal myogenesis. Knockdown of LeuRS enhanced myoblast differentiation in vitro, whereas overexpression of LeuRS inhibited myogenesis. In a mouse model of injury‐induced adult muscle regeneration, local knockdown of LeuRS enhanced regeneration. Hence, LeuRS acts as a negative regulator of myogenesis both in vitro and in vivo. However, inhibition of the translational function of LeuRS by leucinol severely impaired myogenesis, consistent with the notion that protein synthesis is required for the myogenic process. These observations suggest a non‐translation role for LeuRS in suppressing myogenesis. Indeed, we found leucine‐binding but not tRNA charging activity of LeuRS to be necessary for its overexpression effect on myogenesis, suggesting that LeuRS may act as a leucine sensor in its function to ensure homeostasis of myogenesis. To probe the mechanism underlying this non‐canonical function of LeuRS, we examined the involvement of Rag GTPases, mediators of LeuRS activation of mTORC1 in cell growth. Constitutively active Rag reversed the effect of LeuRS knockdown on myogenesis and, conversely, Rag knockdown overcame the inhibitory effect of LeuRS overexpression. Rag knockdown also enhanced muscle regeneration in vivo, mirroring the effect of LeuRS knockdown. Taken together, our findings reveal for the first time a function for LeuRS in modulating the homeostasis of myogenesis. This new LeuRS function is mediated by a Rag‐mTORC1 pathway that negatively regulates the myogenic IRS1‐PI3K‐Akt pathway.Support or Funding InformationKeck Foundation, NIH/NIAMS R01AR048914