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American Journal of Physiology. Cell... Aug 2022The development of skeletal muscle (myogenesis) is a well-orchestrated process where myoblasts withdraw from the cell cycle and differentiate into myotubes. Signaling by...
The development of skeletal muscle (myogenesis) is a well-orchestrated process where myoblasts withdraw from the cell cycle and differentiate into myotubes. Signaling by fluxes in intracellular calcium (Ca) is known to contribute to myogenesis, and increased mitochondrial biogenesis is required to meet the metabolic demand of mature myotubes. However, gaps remain in the understanding of how intracellular Ca signals can govern myogenesis. Polycystin-2 (PC2 or TRPP1) is a nonselective cation channel permeable to Ca. It can interact with intracellular calcium channels to control Ca release and concurrently modulates mitochondrial function and remodeling. Due to these features, we hypothesized that PC2 is a central protein in mediating both the intracellular Ca responses and mitochondrial changes seen in myogenesis. To test this hypothesis, we created CRISPR/Cas9 knockout (KO) C2C12 murine myoblast cell lines. PC2 KO cells were unable to differentiate into myotubes, had impaired spontaneous Ca oscillations, and did not develop depolarization-evoked Ca transients. The autophagic-associated pathway beclin-1 was downregulated in PC2 KO cells, and direct activation of the autophagic pathway resulted in decreased mitochondrial remodeling. Re-expression of full-length PC2, but not a calcium channel dead pathologic mutant, restored the differentiation phenotype and increased the expression of mitochondrial proteins. Our results establish that PC2 is a novel regulator of in vitro myogenesis by integrating PC2-dependent Ca signals and metabolic pathways.
Topics: Animals; Calcium; Calcium Channels; Mice; Mice, Knockout; Muscle Development; Muscle, Skeletal; Proprotein Convertase 2; Signal Transduction; TRPP Cation Channels
PubMed: 35675637
DOI: 10.1152/ajpcell.00159.2021 -
The Journal of Physiology Apr 2022Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal...
Intrauterine growth restriction (IUGR) is a leading cause of neonatal morbidity and mortality in humans and domestic animals. Developmental adaptations of skeletal muscle in IUGR lead to increased risk of premature muscle loss and metabolic disease in later life. Here, we identified β-Klotho (KLB), a fibroblast growth factor 21 (FGF21) co-receptor, as a novel regulator of muscle development in IUGR. Using the pig as a naturally-occurring disease model, we performed transcriptome-wide profiling of fetal muscle (day 90 of pregnancy) from IUGR and normal-weight (NW) littermates. We found that, alongside large-scale transcriptional changes comprising multiple developmental, tissue injury and metabolic gene pathways, KLB was increased in IUGR muscle. Moreover, FGF21 concentrations were increased in plasma in IUGR fetuses. Using cultures of fetal muscle progenitor cells (MPCs), we showed reduced myogenic capacity of IUGR compared to NW muscle in vitro, as evidenced by differences in fusion indices and myogenic transcript levels, as well as mechanistic target of rapamycin (mTOR) activity. Moreover, transfection of MPCs with KLB small interfering RNA promoted myogenesis and mTOR activation, whereas treatment with FGF21 had opposite and dose-dependent effects in porcine and also in human fetal MPCs. In conclusion, our results identify KLB as a novel and potentially critical mediator of impaired muscle development in IUGR, through conserved mechanisms in pigs and humans. Our data shed new light onto the pathogenesis of IUGR, a significant cause of lifelong ill-health in humans and animals. KEY POINTS: Intrauterine growth restriction (IUGR) is associated with large-scale transcriptional changes in developmental, tissue injury and metabolic gene pathways in fetal skeletal muscle. Levels of the fibroblast growth factor 21 (FGF21) co-receptor, β-Klotho (KLB) are increased in IUGR fetal muscle, and FGF21 concentrations are increased in IUGR fetal plasma. KLB mediates a reduction in muscle development through inhibition of mechanistic target of rapamycin signalling. These effects of KLB on muscle cells are conserved in pig and human, suggesting a vital role of this protein in the regulation of muscle development and function in mammals.
Topics: Animals; Female; Fetal Growth Retardation; Mammals; Muscle Development; Muscle, Skeletal; Pregnancy; Signal Transduction; Swine
PubMed: 35081669
DOI: 10.1113/JP281647 -
Cells Feb 2021IgLON5 is a cell adhesion protein belonging to the immunoglobulin superfamily and has important cellular functions. The objective of this study was to determine the role...
IgLON5 is a cell adhesion protein belonging to the immunoglobulin superfamily and has important cellular functions. The objective of this study was to determine the role played by IgLON5 during myogenesis. We found IgLON5 expression progressively increased in C2C12 myoblasts during transition from the adhesion to differentiation stage. IgLON5 knockdown (IgLON5) cells exhibited reduced cell adhesion, myotube formation, and maturation and reduced expressions of different types of genes, including those coding for extracellular matrix (ECM) components (COL1a1, FMOD, DPT, THBS1), cell membrane proteins (ITM2a, CDH15), and cytoskeletal protein (WASP). Furthermore, decreased IgLON5 expression in FMOD, DPT, COL1a1, and ITM2a cells suggested that IgLON5 and these genes mutually control gene expression during myogenesis. IgLON5 immunoneutralization resulted in significant reduction in the protein level of myogenic markers (MYOD, MYOG, MYL2). IgLON5 expression was higher in the CTX-treated gastrocnemius mice muscles (day 7), which confirmed increase expression of IgLON5 during muscle. Collectively, these results suggest IgLON5 plays an important role in myogenesis, muscle regeneration, and that proteins in ECM and myoblast membranes form an interactive network that establishes an essential microenvironment that ensures muscle stem cell survival.
Topics: Animals; Cell Adhesion; Cell Differentiation; Membrane Proteins; Mice, Inbred C57BL; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; MyoD Protein; Myoblasts; Mice
PubMed: 33671182
DOI: 10.3390/cells10020417 -
Mediators of Inflammation 2019Anti-inflammatory cytokine interleukin-4 (IL-4) promotes glucose tolerance and insulin sensitivity while reduces lipid deposits. However, the effects of IL-4 on energy...
Anti-inflammatory cytokine interleukin-4 (IL-4) promotes glucose tolerance and insulin sensitivity while reduces lipid deposits. However, the effects of IL-4 on energy metabolism in muscle, the largest insulin-targeting organ, remain obscure. The study aimed at addressing the roles of IL-4 in myocyte differentiation (myogenesis) and energy metabolism of muscle cells. Effects of IL-4 on myogenesis, and interaction between IL-4 and insulin on glucose metabolism of C2C12 myoblasts and the terminal differentiated myocytes were analyzed. IL-4 improved GLUT4 translocation and tended to elevate glucose uptake by boosting insulin signaling. In diabetic mice, transient and long-term IL-4 showed differential effects on insulin signaling and efficacy. The study provides evidence to address the roles of IL-4 in mediating whole-body muscle reservoir and glucose metabolism, as well as the interaction between immune responses and energy homeostasis. IL-4 has dual potential to act as an adjuvant therapeutic target for sarcopenia to preserve muscle mass and insulin resistance to improve insulin sensitivity, which implicates the regulation of immune system to the muscle differentiation and exercise performance.
Topics: Animals; Biological Transport; Blotting, Western; Cell Differentiation; Cell Line; Glucose; Glucose Transporter Type 4; Interleukin-4; Male; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Muscle Cells; Muscle Development; Myoblasts; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 31814802
DOI: 10.1155/2019/4182015 -
Biochemical and Biophysical Research... Jul 2022Myogenesis is a complex process that is regulated by a variety of factors. We have previously shown that vitamin C and mild endoplasmic reticulum stress synergistically...
Myogenesis is a complex process that is regulated by a variety of factors. We have previously shown that vitamin C and mild endoplasmic reticulum stress synergistically enhance myogenesis. The present study evaluated the effects of vitamin C (ascorbic acid (AsA) and AsA 2-phosphate (AsAp)) and extracellular signal-regulated kinase (ERK) 1/2 pathway on myogenesis. Treatment with U0126, an inhibitor of MEK1/2 that phosphorylates and activates ERK1/2, during the differentiation, increased the mRNA levels of Myod and Myog with an increase in the protein level of myosin heavy chain (MYH)1/2. Treatment with AsA or AsAp alone had minimal effects on myogenesis in C2C12 cells. However, combination treatment with vitamin C and U0126 greatly enhanced myogenesis; the number of thick and long myotubes was increased, and the expression of MYH1/2 was also increased. PD98059, another MEK1/2 inhibitor, also enhanced myogenesis in combination with vitamin C. These results indicate that relief of endogenous ERK1/2 activity enhances vitamin C-mediated myogenesis, suggesting a functional interaction between endogenous ERK1/2 activity and vitamin C. In addition, inhibition of p38 mitogen-activated protein kinase repressed myogenesis in the presence of vitamin C. Thus, vitamin C is a conditional factor that modulates myogenesis.
Topics: Ascorbic Acid; Extracellular Signal-Regulated MAP Kinases; Muscle Development; Muscle Fibers, Skeletal; p38 Mitogen-Activated Protein Kinases
PubMed: 35504090
DOI: 10.1016/j.bbrc.2022.04.103 -
BMB Reports Feb 2022Skeletal myogenesis is essential to keep muscle mass and integrity, and impaired myogenesis is closely related to the etiology of muscle wasting. Recently, miR-141-3p...
Skeletal myogenesis is essential to keep muscle mass and integrity, and impaired myogenesis is closely related to the etiology of muscle wasting. Recently, miR-141-3p has been shown to be induced under various conditions associated with muscle wasting, such as aging, oxidative stress, and mitochondrial dysfunction. However, the functional significance and mechanism of miR-141-3p in myogenic differentiation have not been explored to date. In this study, we investigated the roles of miR-141-3p on CFL2 expression, proliferation, and myogenic differentiation in C2C12 myoblasts. MiR-141-3p appeared to target the 3'UTR of CFL2 directly and suppressed the expression of CFL2, an essential factor for actin filament (F-actin) dynamics. Transfection of miR-141-3p mimic in myoblasts increased F-actin formation and augmented nuclear Yes-associated protein (YAP), a key component of mechanotransduction. Furthermore, miR-141-3p mimic increased myoblast proliferation and promoted cell cycle progression throughout the S and G2/M phases. Consequently, miR-141-3p mimic led to significant suppressions of myogenic factors expression, such as MyoD, MyoG, and MyHC, and hindered the myogenic differentiation of myoblasts. Thus, this study reveals the crucial role of miR-141-3p in myogenic differentiation via CFL2-YAP-mediated mechanotransduction and provides implications of miRNA-mediated myogenic regulation in skeletal muscle homeostasis. [BMB Reports 2022;55(2): 104-109].
Topics: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Cofilin 2; Mechanotransduction, Cellular; Mice; MicroRNAs; Muscle Development; Myoblasts
PubMed: 35000671
DOI: 10.5483/BMBRep.2022.55.2.142 -
Biochemical and Biophysical Research... Sep 2021Myogenesis is a complex process regulated by several factors. This study evaluated the functional interaction between vitamin C and a high dose of capsaicin (a potential...
Myogenesis is a complex process regulated by several factors. This study evaluated the functional interaction between vitamin C and a high dose of capsaicin (a potential endoplasmic reticulum (ER) stress inducer) on myogenesis. After the induction of differentiation, treatment with ascorbic acid or ascorbic acid phosphate (AsAp) alone had minimal effects on myogenesis in C2C12 cells. However, treatment with capsaicin (300 μM) in undifferentiated C2C12 cells increased the expression levels of genes related to ER stress as well as oxidative stress. Myogenesis was effectively enhanced in C2C12 cells treated with a combination of capsaicin (300 μM) for one day before differentiation stimulation and AsAp for four days post-differentiation; subsequently, thick and long myotubes formed, and the expression levels of myosin heavy chain (MYH) 1/2 and Myh1, Myh4, and Myh7 increased. Considering that mild ER stress stimulates myogenesis, AsAp may elicit myogenesis through the alleviation of oxidative stress-induced negative effects in capsaicin-pretreated cells. The enhanced expression of Myh1 and Myh4 coincided with the expression of Col1a1, a type I collagen, suggesting that the fine-tuning of the myogenic cell microenvironment is responsible for efficient myogenesis. Our results indicate that vitamin C is a potential stimulator of myogenesis in cells, depending on the cell context.
Topics: Animals; Ascorbic Acid; Capsaicin; Cell Differentiation; Cell Line; Endoplasmic Reticulum Stress; Mice; Muscle Development; Muscle Fibers, Skeletal; Myoblasts
PubMed: 34198164
DOI: 10.1016/j.bbrc.2021.06.067 -
Biomolecules Sep 2023Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a superfamily of RNA-binding proteins consisting of more than 20 members. These proteins play a crucial role in... (Review)
Review
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a superfamily of RNA-binding proteins consisting of more than 20 members. These proteins play a crucial role in various biological processes by regulating RNA splicing, transcription, and translation through their binding to RNA. In the context of muscle development and regeneration, hnRNPs are involved in a wide range of regulatory mechanisms, including alternative splicing, transcription regulation, miRNA regulation, and mRNA stability regulation. Recent studies have also suggested a potential association between hnRNPs and muscle-related diseases. In this report, we provide an overview of our current understanding of how hnRNPs regulate RNA metabolism and emphasize the significance of the key members of the hnRNP family in muscle development. Furthermore, we explore the relationship between the hnRNP family and muscle-related diseases.
Topics: Heterogeneous-Nuclear Ribonucleoproteins; RNA-Binding Proteins; RNA Splicing; MicroRNAs; Muscle Development
PubMed: 37892116
DOI: 10.3390/biom13101434 -
Cell Proliferation Feb 2024Alternative splicing (AS) disruption has been linked to disorders of muscle development, as well as muscular atrophy. However, the precise changes in AS patterns that...
Alternative splicing (AS) disruption has been linked to disorders of muscle development, as well as muscular atrophy. However, the precise changes in AS patterns that occur during myogenesis are not well understood. Here, we employed isoform long-reads RNA-seq (Iso-seq) and single-cell RNA-seq (scRNA-seq) to investigate the AS landscape during myogenesis. Our Iso-seq data identified 61,146 full-length isoforms representing 11,682 expressed genes, of which over 52% were novel. We identified 38,022 AS events, with most of these events altering coding sequences and exhibiting stage-specific splicing patterns. We identified AS dynamics in different types of muscle cells through scRNA-seq analysis, revealing genes essential for the contractile muscle system and cytoskeleton that undergo differential splicing across cell types. Gene-splicing analysis demonstrated that AS acts as a regulator, independent of changes in overall gene expression. Two isoforms of splicing factor TRA2B play distinct roles in myogenic differentiation by triggering AS of TGFBR2 to regulate canonical TGF-β signalling cascades differently. Our study provides a valuable transcriptome resource for myogenesis and reveals the complexity of AS and its regulation during myogenesis.
Topics: Alternative Splicing; Protein Isoforms; RNA Splicing; Transcriptome; Muscle Development; Gene Expression Profiling
PubMed: 37705195
DOI: 10.1111/cpr.13545 -
The CRISPR Journal Aug 2022Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to...
Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin ()-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein. Myogenesis assay and immunofluorescence staining were performed to evaluate muscle differentiation, while CCK8 assay, cell cycle assay, and 5-ethynyl-2'-deoxyuridine staining were used to measure muscle proliferation. Reverse transcription-polymerase chain reaction and Western blotting were also performed to examine cellular signaling. Myogenic factors (including myosin heavy chain, MSTN, myocardin, and serum response factor) increased significantly after day 5 during myogenesis. MSTN was efficiently silenced after transfecting the dCas9 RNP complex, which significantly promoted more myotube formation and a higher fusion index for L6 cells. In cellular signaling, MSTN repression enhanced the expression of MyoG and MyoD, phosphorylation of Smad2, and the activity of Wnt1/GSK-3β/β-catenin pathway. Moreover, MSTN repression accelerated L6 cell growth with a higher cell proliferation index as well as a higher expression of cyclin D1 and cyclin E. Nonviral delivery of the dCas9 RNP complex significantly promoted myoblast differentiation and proliferation, providing a promising approach to improve muscle regeneration for SUI and PFD. Further characterization and validation of this approach are needed.
Topics: Animals; CRISPR-Cas Systems; Female; Gene Editing; Glycogen Synthase Kinase 3 beta; Humans; Muscle Development; Myostatin; Rats; Ribonucleoproteins; Urinary Incontinence, Stress
PubMed: 35758824
DOI: 10.1089/crispr.2022.0009