Differentiation Of Primary Myoblasts Research Articles

Nnos Expression Patterns In The Differentiation Of Primary Myoblasts.

Nnos Expression Patterns In The Differentiation Of Primary Myoblasts.

The Maintenance of AMPK Activity Eliminates Abnormally Accelerated Differentiation of Primary Myoblasts Isolated from Atrophied Rat Soleus Muscle

Mechanical unloading of skeletal muscles leads to the development of atrophic processes and a decrease in the total number of satellite cells (SCs) that are involved in muscle regeneration. In vitro studies revealed an increased differentiation of myoblasts derived from rat soleus muscle after an unloading-induced decrease in AMP-activated protein kinase (AMPK). AMPK is necessary for the activation of SCs and also participates in the regulation of myoblast proliferation and differentiation. It can be assumed that a decrease in the activity of AMPK after mechanical unloading can contribute to the acceleration of myoblast differentiation. The main purpose of this study was to elucidate a possible role of AMPK in the regulation of differentiation of myoblasts isolated from rat soleus muscle after mechanical unloading. To test this hypothesis, a specific AMPK activator, AICAR, was used to prevent a decrease in AMPK activity during differentiation of myoblasts isolated from rat soleus muscle after 7-day unloading. Immunocytochemistry, PCR-RT and Western blotting were used to assess changes during myoblast differentiation. In differentiating myoblasts derived from the unloaded soleus muscle there was a significant decrease in AMPK (Thr172) and ACC (Ser 79) phosphorylation levels, an increase in myotube differentiation index, myoblast fusion factors and the expression of myogenic regulatory factors (MRF). Furthermore, there was a decrease in the expression of slow myosin heavy chains (MyHC) and an increase in the expression of fast MyHC isoforms. AICAR treatment of differentiating myoblasts obtained from the unloaded soleus muscle prevented a decrease in AMPK and ACC phosphorylation, returned the expression levels of MRF and fast isoforms of MyHC to the control levels as well as maintained the expression of slow MyHC. Thus, abnormally accelerated differentiation of myoblasts isolated from atrophied rat soleus muscle can be compensated by maintaining the control levels of AMPK activity using AICAR.

AMPK Phosphorylation Impacts Apoptosis in Differentiating Myoblasts Isolated from Atrophied Rat Soleus Muscle

Regrowth of atrophied myofibers depends on muscle satellite cells (SCs) that exist outside the plasma membrane. Muscle atrophy appears to result in reduced number of SCs due to apoptosis. Given reduced AMP-activated protein kinase (AMPK) activity during differentiation of primary myoblasts derived from atrophic muscle, we hypothesized that there may be a potential link between AMPK and susceptibility of differentiating myoblasts to apoptosis. The aim of this study was to estimate the effect of AMPK activation (via AICAR treatment) on apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle. Thirty rats were randomly assigned to the following two groups: control (C, n = 10) and 7-day hindlimb suspension (HS, n = 20). Myoblasts derived from the soleus muscles of HS rats were divided into two parts: AICAR-treated cells and non-treated cells. Apoptotic processes were evaluated by using TUNEL assay, RT-PCR and WB. In differentiating myoblasts derived from the atrophied soleus, there was a significant decrease (p < 0.05) in AMPK and ACC phosphorylation in parallel with increased number of apoptotic nuclei and a significant upregulation of pro-apoptotic markers (caspase-3, -9, BAX, p53) compared to the cells derived from control muscles. AICAR treatment of atrophic muscle-derived myoblasts during differentiation prevented reductions in AMPK and ACC phosphorylation as well as maintained the number of apoptotic nuclei and the expression of pro-apoptotic markers at the control levels. Thus, the maintenance of AMPK activity can suppress enhanced apoptosis in differentiating myoblasts derived from atrophied rat soleus muscle.

The Maintenance of AMPK Activity Eliminates Abnormally Accelerated Differentiation of Primary Myoblasts Isolated from Atrophied Rat Soleus Muscle

The Maintenance of AMPK Activity Eliminates Abnormally Accelerated Differentiation of Primary Myoblasts Isolated from Atrophied Rat Soleus Muscle

PGC-1α senses the CBC of pre-mRNA to dictate the fate of promoter-proximally paused RNAPII

PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway.

ДИФФЕРЕНЦИРОВКА МИОБЛАСТОВ, ВЫДЕЛЕННЫХ ИЗ M. SOLEUS КРЫСЫ ПОСЛЕ АНТИОРТОСТАТИЧЕСКОГО ВЫВЕШИВАНИЯ, СОПРОВОЖДАЕТСЯ ДЕФОСФОРИЛИРОВАНИЕМ P27KIP1 И УСИЛЕНИЕМ АПОПТОЗА

Apoptosis and its control during differentiation of primary myoblasts isolated from rat soleus muscle after 7-d tail suspension were investigated. Primary myoblasts were first subjected to myogenic differentiation. TUNEL labeling of double-strand DNA breaks was applied to detect apoptotic cells during myoblast differentiation. Western blot was used to determine the apoptosis markers and a number of signaling molecules, i.e. protein BAX, activated caspase-3, phospho-AMPK (Thr172), phospho-AKT (Ser 437), phospho-p27Kip1 (Thr198), phospho-p27Kip1 (Thr157). PCR analysis was used to investigate the expression of pro-apoptotic markers in myoblasts. The investigation showed enhancement of apoptosis as well as АМРК and p27Kip1 dephosphorylation in the process of differentiation of myoblasts taken from m. soleus of tail-suspended rats.

The mitochondrial Cu+ transporter PiC2 (SLC25A3) is a target of MTF1 and contributes to the development of skeletal muscle in vitro.

The loading of copper (Cu) into cytochrome c oxidase (COX) in mitochondria is essential for energy production in cells. Extensive studies have been performed to characterize mitochondrial cuproenzymes that contribute to the metallation of COX, such as Sco1, Sco2, and Cox17. However, limited information is available on the upstream mechanism of Cu transport and delivery to mitochondria, especially through Cu-impermeable membranes, in mammalian cells. The mitochondrial phosphate transporter SLC25A3, also known as PiC2, binds Cu+ and transports the ion through these membranes in eukaryotic cells, ultimately aiding in the metallation of COX. We used the well-established differentiation model of primary myoblasts derived from mouse satellite cells, wherein Cu availability is necessary for growth and maturation, and showed that PiC2 is a target of MTF1, and its expression is both induced during myogenesis and favored by Cu supplementation. PiC2 deletion using CRISPR/Cas9 showed that the transporter is required for proliferation and differentiation of primary myoblasts, as both processes are delayed upon PiC2 knock-out. The effects of PiC2 deletion were rescued by the addition of Cu to the growth medium, implying the deleterious effects of PiC2 knockout in myoblasts may be in part due to a failure to deliver sufficient Cu to the mitochondria, which can be compensated by other mitochondrial cuproproteins. Co-localization and co-immunoprecipitation of PiC2 and COX also suggest that PiC2 may participate upstream in the copper delivery chain into COX, as verified by in vitro Cu+-transfer experiments. These data indicate an important role for PiC2 in both the delivery of Cu to the mitochondria and COX, favoring the differentiation of primary myoblasts.

Nnos Dna Methylation Level And Nnosμ Gene Expression In The Differentiation Of Primary Myoblasts.

Nnos Dna Methylation Level And Nnosμ Gene Expression In The Differentiation Of Primary Myoblasts.

Modulating Myogenesis: An Optimized In Vitro Assay to Pharmacologically Influence Primary Myoblast Differentiation.

The intentional pharmacological manipulation of myogenesis is an important technique for understanding the underlying mechanisms of muscle differentiation and disease etiology. Using the pharmacological agent metformin as an example molecule, we present a systematic approach to examine the impact of pharmacological agents on the myogenic program. This consists of optimizing the in vitro differentiation of primary myoblast cells followed by the generation of a dose-response curve for a respective pharmaceutical. To assess myogenic differentiation, we utilized three approaches (incorporating both transcriptional and protein techniques) to assess the effects of biologically active agents on the in vitro differentiation of primary myogenic progenitors. First, the immunofluorescent visualization of myosin heavy chain (MYHC), which is expressed in differentiated myofibers, is used to obtain the fusion index, a quantitative read-out of differentiation efficiency. Second, quantitative reverse transcription PCR (RT-qPCR) reveals the expression of myogenic factors (Pax7, Myf5, Myod, Myog, Myh2) at the transcript level. Third, western blotting is used to assess the protein expression levels of the myogenic markers (PAX7, MYF5, MYOD, MYOG, and MYHC). By monitoring the expression of these various myogenic factors during the differentiation process, the relative cellular state and differentiation status between samples can be determined. Combined, these approaches enable the successful assessment of the impact of pharmacological agents on myogenic differentiation. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Immunofluorescence assay for qualitative and quantitative assessment of pharmacological agents on in vitro myogenic differentiation Support Protocol 1: Evaluating myogenic gene expression by RT-qPCR Support Protocol 2: Evaluating myogenic protein expression by western blot.

Epigenetic Regulation Mechanisms of the Cofilin-1 Gene in the Development and Differentiation of Bovine Primary Myoblasts.

As the quality of beef products has received increasing attention, it is essential to explore the underlying transcriptional and epigenetic mechanisms of meat traits. Our project uses Qinchuan cattle as the research subject. First, we examined the spatiotemporal expression pattern of the CFL1 gene in a panel of fetal bovine, calf, and adult cattle samples. Then, we performed DNA methylation experiments of CFL1 on myogenesis and muscle maturation using the BSP amplification and COBRA sequencing techniques and found that high DNA methylation levels showed low expression levels. Next, we performed an assay between bta-miR-182 and the CFL1 gene and demonstrated that miR-182 could promote bovine primary myoblast differentiation by negatively regulated the expression of CFL1. Finally, we constructed an adenovirus overexpression and interference vector and found that CFL1 could suppress the differentiation of bovine primary myoblasts. In summary, our experiment comprehensively analyzes the epigenetic regulation mechanisms of the CFL1 gene in the development and differentiation of bovine primary myoblasts. This has far-reaching significance for improving the meat production and meat quality of Qinchuan cattle. This can provide reliable data support and a theoretical research basis for the rapid and efficient breeding selection of local yellow cattle and the genetic improvement of meat quality.

Immunofluorescence analysis of myogenic differentiation.

Skeletal muscle is a highly regenerative tissue that can efficiently recover from various damages caused by injuries and excessive exercises. In adult muscle, stem cells termed satellite cells are mitotically quiescent but activated upon muscle damages to enter the cell cycle as myogenic precursor cells or myoblasts. After several rounds of cell cycles, they exist the cycle and fuse to each other to form multinucleated myotubes, and eventually mature to become contractile myofibers. Satellite cells can be readily isolated from mouse skeletal muscle with enzymatic digestion and magnetic separation with antibodies against specific surface markers. C2C12 cells are an immortalized mouse myoblast cell line that is commercially available and more readily expandable than primary myoblasts. Both primary myoblasts and C2C12 cells have been extensively used as useful in vitro models for myogenic differentiation. Proper examination of this process requires monitoring specific protein expression in subcellular compartments, which can be accomplished through immunofluorescence staining. This chapter describes the workflow for the isolation of satellite cells from mouse skeletal muscle and subsequent immunofluorescence staining to assess the proliferation and differentiation of primary myoblasts and C2C12 cells.

Integrated Analysis of mRNA and MicroRNA Co-expressed Network for the Differentiation of Bovine Skeletal Muscle Cells After Polyphenol Resveratrol Treatment.

Resveratrol (RSV) has been confirmed to benefit human health. Resveratrol supplemented in the feeds of animals improved pork, chicken, and duck meat qualities. In this study, we identified differentially expressed (DE) messenger RNAs (mRNAs) (n = 3,856) and microRNAs (miRNAs) (n = 93) for the weighted gene co-expression network analysis (WGCNA) to investigate the co-expressed DE mRNAs and DE miRNAs in the primary bovine myoblasts after RSV treatment. The mRNA results indicated that RSV treatments had high correlations with turquoise module (0.91, P-value = 0.01) and blue module (0.93, P-value < 0.01), while only the turquoise module (0.96, P-value < 0.01) was highly correlated with the treatment status using miRNA data. After biological enrichment analysis, the 2,579 DE genes in the turquoise module were significantly enriched in the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The top two GO terms were actin filament-based process (GO:0030029) and actin cytoskeleton organization (GO:0030036). The top two KEGG pathways were regulation of actin cytoskeleton (bta04810) and tight junction (bta04530). Then, we constructed the DE mRNA co-expression and DE miRNA co-expression networks in the turquoise module and the mRNA–miRNA targeting networks based on their co-expressions in the key module. In summary, the RSV-induced miRNAs participated in the co-expression networks that could affect mRNA expressions to regulate the primary myoblast differentiation. Our study provided a better understanding of the roles of RSV in inducing miRNA and of the characteristics of DE miRNAs in the key co-expressed module in regulation of mRNAs and revealed new candidate regulatory miRNAs and genes for the beef quality traits.

Isolation, Culture, and Differentiation of Primary Myoblasts Derived from Muscle Satellite Cells

The skeletal muscle is key for body mobility and motor performance, but aging and diseases often lead to progressive loss of muscle mass due to wasting or degeneration of muscle cells. Muscle satellite cells (MuSCs) represent a population of tissue stem cells residing in the skeletal muscles and are responsible for homeostatic maintenance and regeneration of skeletal muscles. Growth, injury, and degenerative signals activate MuSCs, which then proliferate (proliferating MuSCs are called myoblasts), differentiate and fuse with existing multinuclear muscle cells (myofibers) to mediate muscle growth and repair. Here, we describe a protocol for isolating MuSCs from skeletal muscles of mice for in vitro analysis. In addition, we provide a detailed protocol on how to culture and differentiate primary myoblasts into myotubes and an immunofluorescent staining procedure to characterize the cells. These methods are essential for modeling regenerative myogenesis in vitro to understand the dynamics, function and molecular regulation of MuSCs.

NADPH Oxidase 4 Contributes to Myoblast Fusion and Skeletal Muscle Regeneration.

Myoblast fusion is an essential step in skeletal muscle development and regeneration. NADPH oxidase 4 (Nox4) regulates cellular processes such as proliferation, differentiation, and survival by producing reactive oxygen species (ROS). Insulin-like growth factor 1 induces muscle hypertrophy via Nox4, but its function in myoblast fusion remains elusive. Here, we report a ROS-dependent role of Nox4 in myoblast differentiation. Regenerating muscle fibers after injury by cardiotoxin had a lower cross-sectional area in Nox4-knockout (KO) mice than myofibers in wild-type (WT) mice. Diameters and fusion index values of myotubes differentiated from Nox4-KO primary myoblasts were significantly lower than those of myotubes derived from WT myoblasts. However, no difference was observed in the differentiation index and expression of MyoD, myogenin, and myosin heavy chain 3 (MHC) between KO and WT myotubes. The decreased fusion index was also observed during differentiation of primary myoblasts and C2C12 cells with suppressed Nox4 expression. In contrast, in C2C12 cells overexpressing Nox4, the fusion index was increased, whereas the differentiation index and MHC and myogenin protein expression were not affected compared to control. Interestingly, the expression of myomaker (Tmem8c), a fusogenic protein that controls myoblast fusion, was reduced in Nox4-knockdown C2C12 cells. The myomaker expression level was proportional to the cellular ROS level, which was regulated by of Nox4 expression level. These results suggests that Nox4 contributes to myoblast fusion, possibly through the regulation of myomaker expression via ROS production, and that Nox4-dependent ROS may promote skeletal muscle regeneration and growth.

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Role of Mutant TBP in Regulation of Myogenesis on Muscle Satellite Cells.

In polyglutamine (PolyQ) diseases, mutant proteins cause not only neurological problems but also peripheral tissue abnormalities. Among all systemic damages, skeletal muscle dystrophy is the severest. Previously by studying knock-in (KI) mouse models of spinal cerebellar ataxia 17 (SCA17), it was found that mutant TATA box binding protein (TBP) decreases its interaction with myogenic differentiation antigen, thus reducing the expression of skeletal muscle structural proteins and resulting in muscle degeneration. In this paper, the role of mutant TBP in myogenesis was investigated. Single myofibers were isolated from tibialis anterior muscles of wild type (WT) and SCA17KI mice. The 1TBP18 staining confirmed the expression of mutant TBP in muscle satellite cells in SCA17KI mice. In the BaCl2-induced TA muscle injury, H&E cross-section staining showed no significant change in myofibril size before and after BaCl2 treatment, and there was no significant difference in centralized nuclei between WT and SCA17KI mice, suggesting that mutant TBP had no significant effect on muscle regeneration. In the cultured primary myoblasts from WT and SCA17KI mice in vitro, representative BrdU immunostaining showed no significant difference in proliferation of muscle satellite cells. The primary myoblasts were then induced to differentiate and immunostained for eMyHC, and the staining showed there was no significant difference in differentiation of primary myoblasts between WT and SCA1KI mice. Our findings confirmed that mutant TBP had no significant effect on myogenesis.

Roles of Zip8 and Zip14 Transporters in Myogenesis

Transition metals like manganese (Mn) are necessary catalytic and structural cofactors in enzymes and as second messengers in cell signaling. Studies on the molecular mechanisms by which Mn contributes to development are focused on its role as co‐factor of the mitochondrial enzyme superoxide dismutase 2 (SOD2). However, Mn has been proposed to participate in protein glycosylation during bone and neural development. Our laboratory is investigating the biological relevance of Mn in the differentiation of skeletal muscle cells. During myogenesis, total mitochondrial content increases and cells increase expression of SOD2, which protects against oxidative stress. Our lab has shown that the gene and protein levels of ZIP8 and ZIP14, two membrane Zn/Mn transporters, increase during differentiation of the C2C12 immortalized mouse myoblasts. Preliminary data shows a significant increase in the cellular Mn quota during the differentiation of primary myoblasts derived from mouse satellite cells, which are the stem cell pool that sustains and regenerates myofibers in adult skeletal muscle. We hypothesized that ZIP8 and ZIP14 might be associated with the Mn influx during myogenesis. To determine the role of ZIP8, ZIP14, and Mn during myogenesis we used shRNA to delete Zip8 and Zip14 transporters in primary myoblasts derived from primary myoblasts. Partial deletion of these transporters resulted in changes in cellular Mn levels. Zip8 knockdown myoblasts are unable to differentiate compared to control and Zip14 knockdown myoblasts, as shown by the expression of muscle‐specific differentiation markers and alterations on the morphology of differentiating cells. Sod2 activity is reduced also in Zip8 knockdown myoblasts. This work will contribute considerably to the very limited literature related to Mn biology in myogenesis and would provide a foundation for future efforts to understand the contribution of Mn in other differentiation and disease models.Support or Funding InformationThis project was funded by the Faculty Diversity Scholars Award from the University of Massachusetts Medical School Awarded to TP‐B. S. Gordon is supported by the NIH Grant R25 HL092610‐11This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LncRNA-MEG3 promotes bovine myoblast differentiation by sponging miR-135.

Long noncoding RNA maternally expressed gene 3 (lncRNA-MEG3) is an important regulator in multiple biological functions. However, lncRNA-MEG3's function in cattle growth and regulatory mechanism on bovine skeletal muscle development has not yet been well studied. In this project, we first investigated lncRNA-MEG3's expression profile and detected that it was highly expressed in bovine skeletal muscle tissue and its RNA level was kept increasingly during the early phase of bovine primary myoblast differentiation. Using luciferase reporter assays, we identified the lncRNA-MEG3 core promoter containing putative transcription factor binding site for myocyte enhancer factor 2C (MEF2C). Interestingly, we found that LncRNA-MEG3 could significantly upregulate and downregulate myosin heavy chain ( MHC), myogenin ( MyoG), and MEF2C through overexpression and RNAi strategies, respectively. Using luciferase reporter assays, we also verified lncRNA-MEG3 as a miR-135 sponge. Overexpression of miR-135 markedly inhibited the wild type of lncRNA-MEG3, but not the mutant lncRNA-MEG3 reporter. The luciferase activity of miR-135 sensor could be rescued by lncRNA-MEG3 via competing for miRNA-135. In addition, the luciferase activity of MEF2C was significantly upregulated by the wild type of lncRNA-MEG3. This study, for the first time, revealed that lncRNA-MEG3 could promote bovine skeletal muscle differentiation via interacting with miRNA-135 and MEF2C. The results were valuable for further studies and applications of lncRNA related roles in beef molecular breeding.

β-Catenin is essential for differentiation of primary myoblasts via cooperation with MyoD and α-catenin.

Canonical Wnts promote myoblast differentiation; however, the role of β-catenin in adult myogenesis has been contentious, and its mechanism(s) unclear. Using CRISPR-generated β-catenin-null primary adult mouse myoblasts, we found that β-catenin was essential for morphological differentiation and timely deployment of the myogenic gene program. Alignment, elongation and fusion were grossly impaired in null cells, and myogenic gene expression was not coordinated with cytoskeletal and membrane remodeling events. Rescue studies and genome-wide analyses extended previous findings that a β-catenin-TCF/LEF interaction is not required for differentiation, and that β-catenin enhances MyoD binding to myogenic loci. We mapped cellular pathways controlled by β-catenin and defined novel targets in myoblasts, including the fusogenic genes myomaker and myomixer. We also showed that interaction of β-catenin with α-catenin was important for efficient differentiation. Overall the study suggests dual roles for β-catenin: a TCF/LEF-independent nuclear function that coordinates an extensive network of myogenic genes in cooperation with MyoD; and an α-catenin-dependent membrane function that helps control cell-cell interactions. β-Catenin-TCF/LEF complexes may function primarily in feedback regulation to control levels of β-catenin and thus prevent precocious/excessive myoblast fusion.