Differentiation Of Myoblast Cells Research Articles

C2C12 myoblasts differentiate into myofibroblasts via the TGF-β1 signaling pathway mediated by Fibulin2

Myoblast cells play a critical role in the regeneration of skeletal muscle following injury. It has been reported that local elevation of transforming growth factor-β1(TGF-β1) after skeletal muscle injury induces differentiation of myoblast cells into myofibroblasts.However, the mechanisms underlying this differentiation process remain incompletely understood. In this study, we found that Fibulin2 expression significantly increases in myoblast cells in response to TGF-β1 stimulation.Elevated Fibulin2 levels enhance the expression of fibrotic markers, mediated through phosphorylation of Smad2.Conversely, downregulation of Fibulin2 in myoblast cells inhibits the upregulation of fibrotic markers induced by TGF-β1 stimulation.Extracellular secretion of Fibulin2 activates the TGF-β1-Smad2 pathway, thereby promoting the upregulation of fibrotic markers.Hence, Fibulin2 and TGF-β1 form a positive feedback loop that facilitates differentiation of myoblast cells into myofibroblasts.

Ginkgolic acid regulates myogenic development by influencing the proliferation and differentiation of C2C12 myoblast cells

Ginkgolic acid regulates myogenic development by influencing the proliferation and differentiation of C2C12 myoblast cells

MULTILINEAGE DIFFERENTIATION OF C2C12 CELLS AND MONOCYTES IN VITRO WITHIN A 6-BROMOINDIRUBIN-3’-OXIME-INCORPORATED CHITOSAN-BASED SCAFFOLD FOR ENHANCED BONE REGENERATION

Critical size bone defects deriving from large bone loss are an unmet clinical challenge1. To account for disadvantages with clinical treatments, researchers focus on designing biological substitutes, which mimic endogenous healing through osteogenic differentiation promotion. Some studies have however suggested that this notion fails to consider the full complexity of native bone with respect to the interplay between osteoclast and osteoblasts, thus leading to the regeneration of less functional tissue2. The objective of this research is to assess the ability of our laboratory's previously developed 6-Bromoindirubin-3’-Oxime (BIO) incorporated guanosine diphosphate crosslinked chitosan scaffold in promoting multilineage differentiation of myoblastic C2C12 cells and monocytes into osteoblasts and osteoclasts1, 3, 4. BIO addition has been previously demonstrated to promote osteogenic differentiation in cell cultures5, but implementation of a co-culture model here is expected to encourage crosstalk thus further supporting differentiation, as well as the secretion of regulatory molecules and cytokines2.Biocompatibility testing of both cell types is performed using AlamarBlue for metabolic activity, and nucleic acid staining for distribution. Osteoblastic differentiation is assessed through quantification of ALP and osteopontin secretion, as well as osteocalcin and mineralization staining. Differentiation into osteoclasts is verified using SEM and TEM, qPCR, and TRAP staining.Cellular viability of C2C12 cells and monocytes was maintained when cultured separately in scaffolds with and without BIO for 21 days. Both scaffold variations showed a characteristic increase in ALP secretion from day 1 to 7, indicating early differentiation but BIO-incorporated sponges yielded higher values compared to controls. SEM and TEM imaging confirmed initial aggregation and fusion of monocytes on the scaffold's surface, but BIO addition appeared to result in smoother cell surfaces indicating a change in morphology. Late-stage differentiation assessment and co-culture work in the scaffold are ongoing, but initial results show promise in the material's ability to support multilineage differentiation.Acknowledgements: The authors would like to acknowledge the financial support of the Collaborative Health Research Program (CHRP) through CIHR and NSERC, as well as Canada Research Chair – Tier 1 in Regenerative Medicine and Nanomedicine, and the FRQ-S.

Dipyridamole activates adenosine A2B receptor and AMPK/cAMP signaling and promotes myogenic differentiation of myoblastic C2C12 cells.

Introduction: Sarcopenia is defined as a loss of muscle mass and strength. ATP homeostasis is crucial during myogenesis. We determined how the purinergic system modulates myogenesis using dipyridamole (blocks adenosine taken up by the cells) and tenofovir (inhibits ATP release) in a myoblast cell line. Methods: C2C12 cells were differentiated in the presence/absence of tenofovir/dipyridamole, with/without the A2B selective inhibitor PSB-603. Extra-/intracellular nucleotides were examined via HPLC. The expression of muscle differentiation proteins (Pax7, Mif5, MyoD, MyoG, and MHC), PKA/CREB, adenosine receptors (A1, A2A, A2B, and A3), ATP-channel pannexin-1 and the P2X7 receptor was analyzed via WB and RT-PCR. cAMP and AMPK activation was measured. Results: Tenofovir increased intracellular ATP and reduced extracellular adenosine, decreasing Pax7 expression and increasing MHC expression prematurely. Dipyridamole increased intracellular AMP and extracellular adenosine, counteracting the premature myogenesis promoted by tenofovir. All adenosine receptors were expressed during differentiation with dipyridamole, increasing A2B expression. Tenofovir maintained inactive AMPK and decreased cAMP levels, as well as PKAα and pCREB expression, which were recovered with dipyridamole. Discussion: Adenosine and ATP act as mediators in muscle myogenesis. The blockade of ATP release by tenofovir promotes premature myogenesis, with dipyridamole counteracting the premature differentiation promoted by tenofovir via the adenosine A2B receptor and cAMP/AMPK pathways. Therefore, dipyridamole might be of interest as a therapeutic approach in sarcopenia.

The ensured proliferative capacity of myoblast in serum-reduced conditions with Methyl-β-cyclodextrin.

To produce muscle fibers for cultured meat on a large scale, it is important to expand myoblasts in a serum-reduced or serum-free medium to avoid cost, ethical, and environmental issues. Myoblasts such as C2C12 cells differentiate quickly into myotubes and lose their ability to proliferate when the serum-rich medium is replaced with a serum-reduced medium. This study demonstrates that Methyl-β-cyclodextrin (MβCD), a starch-derived agent that depletes cholesterol, can inhibit further differentiation of myoblasts at the MyoD-positive stage by reducing plasma membrane cholesterol on C2C12 cells and primary cultured chick muscle cells. Furthermore, MβCD efficiently blocks cholesterol-dependent apoptotic cell death of myoblasts, which is one of the mechanisms by which it inhibits the differentiation of C2C12 myoblast cells, as dead cells of myoblast are necessary for the fusion of adjacent myoblasts during the differentiation process into myotubes. Importantly, MβCD maintains the proliferative capacity of myoblasts only under differentiation conditions with a serum-reduced medium, suggesting that its mitogenic effect is due to its inhibitory effect on myoblast differentiation into myotube. In conclusion, this study provides significant insights into ensuring the proliferative capacity of myoblasts in a future serum-free condition for cultured meat production.

IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

Myoblast fusion is essential for skeletal muscle development, growth, and regeneration. However, the molecular mechanisms underlying myoblast fusion and differentiation are not fully understood. Previously, we reported that interleukin-4 (IL-4) promotes myoblast fusion; therefore, we hypothesized that IL-4 signaling might regulate the expression of the molecules involved in myoblast fusion. In this study, we showed that in addition to fusion, IL-4 promoted the differentiation of C2C12 myoblast cells by inducing myoblast determination protein 1 (MyoD) and myogenin, both of which regulate the expression of myomerger and myomaker, the membrane proteins essential for myoblast fusion. Unexpectedly, IL-4 treatment increased the expression of myomerger, but not myomaker, in C2C12 cells. Knockdown of IL-4 receptor alpha (IL-4Rα) in C2C12 cells by small interfering RNA impaired myoblast fusion and differentiation. We also demonstrated a reduction in the expression of MyoD, myogenin, and myomerger by knockdown of IL-4Rα in C2C12 cells, while the expression level of myomaker remained unchanged. Finally, cell mixing assays and the restoration of myomerger expression partially rescued the impaired fusion in the IL-4Rα-knockdown C2C12 cells. Collectively, these results suggest that the IL-4/IL-4Rα axis promotes myoblast fusion and differentiation via the induction of myogenic regulatory factors, MyoD and myogenin, and myomerger.

CircRNA Profiling of Skeletal Muscle in Two Pig Breeds Reveals CircIGF1R Regulates Myoblast Differentiation via miR-16.

Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation.

The Dynamic and Crucial Role of the Arginine Methylproteome in Myoblast Cell Differentiation.

Protein arginine methylation is an extensive and functionally significant post-translational modification. However, little is known about its role in differentiation at the systems level. Using stable isotope labeling by amino acids in cell culture (SILAC) proteomics of whole proteome analysis in proliferating or five-day differentiated mouse C2C12 myoblasts, followed by high-resolution mass spectrometry, biochemical assays, and specific immunoprecipitation of mono- or dimethylated arginine peptides, we identified several protein families that were differentially methylated on arginine. Our study is the first to reveal global changes in the arginine mono- or dimethylation of proteins in proliferating myoblasts and differentiated myocytes and to identify enriched protein domains and novel short linear motifs (SLiMs). Our data may be crucial for dissecting the links between differentiation and cancer growth.

In Vitro Differentiation of Myoblast Cell Lines on Spider Silk Scaffolds in a Rotating Bioreactor for Vascular Tissue Engineering.

Functional construction of tissue-engineered vessels as an alternative to autologous vascular grafts has been shown to be feasible, however the proliferation of seeded smooth-muscle cells remains a limiting factor. We employed a rotating bioreactor system to improve myoblast cell differentiation on a spider silk scaffold for tissue-engineered vessel construction. C2C12 myofibroblast cells were seeded on the surface of spider silk scaffold constructs and cultivated in a rotating bioreactor system with a continuous rotation speed (1 rpm). Cell function, cell growth and morphological structure and expression of biomarkers were analyzed using scanning electron microscopy, the LIVE/DEAD® assay, Western blot and quantitative real-time PCR analyses. A dense myofibroblast cell sheet could be developed which resembled native blood vessel muscular tissue in morphological structure and in function. Bioreactor perfusion positively affected cell morphology, and increased cell viability and cell differentiation. The expression of desmin, MYF5 and MEF2D surged as an indication of myoblast differentiation. Cell-seeded scaffolds showed a tear-down at 18 N when strained at a set speed (20 mm min-1). Spider silk scaffolds appear to offer a reliable basis for engineered vascular constructs and rotating bioreactor cultivation may be considered an effective alternative to complex bioreactor setups to improve cell viability and biology.

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.

A Temporary Pause in the Replication Licensing Restriction Leads to Rereplication during Early Human Cell Differentiation.

Gene amplifications in amphibians and flies are known to occur during development and have been well characterized, unlike in mammalian cells, where they are predominantly investigated as an attribute of tumors. Recently, we first described gene amplifications in human and mouse neural stem cells, myoblasts, and mesenchymal stem cells during differentiation. The mechanism leading to gene amplifications in amphibians and flies depends on endocycles and multiple origin-firings. So far, there is no knowledge about a comparable mechanism in normal human cells. Here, we describe rereplication during the early myotube differentiation of human skeletal myoblast cells, using fiber combing and pulse-treatment with EdU (5′-Ethynyl-2′-deoxyuridine)/CldU (5-Chlor-2′-deoxyuridine) and IdU (5-Iodo-2′-deoxyuridine)/CldU. We found rereplication during a restricted time window between 2 h and 8 h after differentiation induction. Rereplication was detected in cells simultaneously with the amplification of the MDM2 gene. Our findings support rereplication as a mechanism enabling gene amplification in normal human cells.

Biocompatibility Computation of Muscle Cells on Polyhedral Oligomeric Silsesquioxane-Grafted Polyurethane Nanomatrix.

This study was performed to appraise the biocompatibility of polyhedral oligomeric silsesquioxane (POSS)-grafted polyurethane (PU) nanocomposites as potential materials for muscle tissue renewal. POSS nanoparticles demonstrate effectual nucleation and cause noteworthy enhancement in mechanical and thermal steadiness as well as biocompatibility of resultant composites. Electrospun, well-aligned, POSS-grafted PU nanofibers were prepared. Physicochemical investigation was conducted using several experimental techniques, including scanning electron microscopy, energy dispersive X-ray spectroscopy, electron probe microanalysis, Fourier transform infrared spectroscopy, and X-ray diffraction pattern. Adding POSS molecules to PU did not influence the processability and morphology of the nanocomposite; however, we observed an obvious mean reduction in fiber diameter, which amplified specific areas of the POSS-grafted PU. Prospective biomedical uses of nanocomposite were also appraised for myoblast cell differentiation in vitro. Little is known about C2C12 cellular responses to PU, and there is no information regarding their interaction with POSS-grafted PU. The antimicrobial potential, anchorage, proliferation, communication, and differentiation of C2C12 on PU and POSS-grafted PU were investigated in this study. In conclusion, preliminary nanocomposites depicted superior cell adhesion due to the elevated free energy of POSS molecules and anti-inflammatory potential. These nanofibers were non-hazardous, and, as such, biomimetic scaffolds show high potential for cellular studies and muscle regeneration.

Involvement of DPY19L3 in Myogenic Differentiation of C2C12 Myoblasts.

DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation.

Male-Biased gga-miR-2954 Regulates Myoblast Proliferation and Differentiation of Chicken Embryos by Targeting YY1.

Previous studies have shown that gga-miR-2954 was highly expressed in the gonads and other tissues of male chickens, including muscle tissue. Yin Yang1 (YY1), which has functions in mammalian skeletal muscle development, was predicted to be a target gene of gga-miR-2954. The purpose of this study was to investigate whether gga-miR-2954 plays a role in skeletal muscle development by targeting YY1, and evaluate its function in the sexual dimorphism development of chicken muscle. Here, all the temporal and spatial expression profiles in chicken embryonic muscles showed that gga-miR-2954 is highly expressed in males and mainly localized in cytoplasm. Gga-miR-2954 exhibited upregulated expression of in vitro myoblast differentiation stages. Next, through the overexpression and loss-of-function experiments performed in chicken primary myoblasts, we found that gga-miR-2954 inhibited myoblast proliferation but promoted differentiation. During myogenesis, gga-miR-2954 could suppress the expression of YY1, which promoted myoblast proliferation and inhibited the process of myoblast cell differentiation into multinucleated myotubes. Overall, these findings reveal a novel role of gga-miR-2954 in skeletal muscle development through its function of the myoblast proliferation and differentiation by suppressing the expression of YY1. Moreover, gga-miR-2954 may contribute to the sex difference in chicken muscle development.

A digital light processing 3D printed magnetic bioreactor system using silk magnetic bioink

Among various bioreactors used in the field of tissue engineering and regenerative medicine, a magnetic bioreactor is more capable of providing steady force to the cells while avoiding direct manipulation of the materials. However, most of them are complex and difficult to fabricate, with drawbacks in terms of consistency and biocompatibility. In this study, a magnetic bioreactor system and a magnetic hydrogel were manufactured by single-stage three-dimensional (3D) printing with digital light processing (DLP) technique for differentiation of myoblast cells. The hydrogel was composed of a magnetic part containing iron oxide and glycidyl-methacrylated silk fibroin, and a cellular part printed by adding mouse myoblast cell (C2C12) to gelatin glycidyl methacrylate, that was placed in the magnetic bioreactor system to stimulate the cells in the hydrogel. The composite hydrogel was steadily printed by a one-stage layering technique using a DLP printer. The magnetic bioreactor offered mechanical stretching of the cells in the hydrogel in 3D ways, so that the cellular differentiation could be executed in three dimensions just like the human environment. Cell viability, as well as gene expression using quantitative reverse transcription-polymerase chain reaction, were assessed after magneto-mechanical stimulation of the myoblast cell-embedded hydrogel in the magnetic bioreactor system. Comparison with the control group revealed that the magnetic bioreactor system accelerated differentiation of mouse myoblast cells in the hydrogel and increased myotube diameter and length in vitro. The DLP-printed magnetic bioreactor and the hydrogel were simply manufactured and easy-to-use, providing an efficient environment for applying noninvasive mechanical force via FDA-approved silk fibroin and iron oxide biocomposite hydrogel, to stimulate cells without any evidence of cytotoxicity, demonstrating the potential for application in muscle tissue engineering.

Potential Roles of Silicon/Silica‐Based Nanoparticles in 3D Printed Hydrogels for Skeletal Muscle Regeneration

Introduction : Skeletal muscle constitutes about 40% of the total body mass and plays a significant role in the movement of the human body. Although skeletal muscles have remarkable endogenous regenerative capacity, this capacity is overwhelmed following acute severe traumatic injuries. Furthermore, the associated oxidative damage can delay regeneration process and prolong recovery. These traumatic injuries result from combat- and/or trauma-induced muscle injuries and often lead to irreversible tissue damage and impaired vascularization. Volumetric muscle loss (VML) is a severe traumatic injury that results in a critical loss (≥ 20%) of the native muscle mass leading to permanent disability. Our hypothesis is that using 3D bioprinted hydrogel modified with silica-based nanoparticles (NPs) laden human skeletal muscle cells will provide the required architecture and enhance muscle regeneration in VML defects where high levels of reactive oxygen species (ROS) is predominant. Materials and Methods Sodium metasilicate powder was used to adjust and optimize the effective concentration of ionic silicon, while hydrogen peroxide was used as sources of ROS to simulate the oxidative damage conditions of VML injuries. Silica based nanoparticles were embedded into GelMA-based hydrogels for 3D printing of scaffolds that mimic the skeletal muscle architecture. Results Our preliminary data using ionic silicon indicated that Si-ions are not cytotoxic to myoblast cells under tested concentrations (0.1-2.0 mM) (Figure 1). Furthermore, Si-ions significantly enhanced myoblast cell viability, proliferation, and differentiation into myotubes as indicated by a higher fusion index compared to the control. In-vitro studies indicated that0.4 mM of H2O2 into the growth media significantly decreases the cell viability after 6 and 24 hr. compared to the control (**p < 0.01, n=4 per group). Addition of 0.5-1.0 mM of Si into the growth media significantly enhances the cell viability under conditions that mimic high ROS (i.e., H202 treated group). The 3D printed scaffolds of GelMA + NPs indicated a higher myoblast cell viability significantly reducing cell death compared to the bare GelMA scaffolds as shown at Figure 2. Conclusion Our preliminary findings conclude that 0.1 mM Si-ions enhance myoblast viability, proliferation, and differentiation of C2C12 myoblast cells. Using 0.4 mM of H2O2 can simulate the oxidative damage condition in myoblast cells in-vitro. Using 0.1-0.5 mM silicon ions can attenuate the oxidative damage (0.4 mM H2O2) on C2C12 myoblast cells. 3D printed hydrogels loaded with silica-based nanoparticles are promising materials for 3D printing of muscle constructs.

Mechanism of Heat-Induced Differentiation of Myoblast Cells

Mechanism of Heat-Induced Differentiation of Myoblast Cells

Msx1 cooperates with Runx1 for inhibiting myoblast differentiation

Myogenesis is an important and complicated biological process, especially during the process of embryonic development. The homeoprotein Msx1 is a crucial transcriptional repressor of myogenesis and maintains myogenic precursor cells in an undifferentiated, proliferative state. However, the molecular mechanism through which Msx1 coordinates myogenesis remains to be elucidated. Here, we determine the interacting partner proteins of Msx1 in myoblast cells by a proteomic screening method. Msx1 is found to interact with 55 proteins, among which our data demonstrate that the cooperation of Runt-related transcription factor 1 (Runx1) with Msx1 is required for myoblast cell differentiation. Our findings provide important insights into the mechanistic roles of Msx1 in myoblast cell differentiation, and lays foundation for the myogenic differentiation process.

Injectable muscle-adhesive antioxidant conductive photothermal bioactive nanomatrix for efficiently promoting full-thickness skeletal muscle regeneration

The completed skeletal muscle regeneration resulted from severe injury and muscle-related disease is still a challenge. Here, we developed an injectable muscle-adhesive antioxidant conductive bioactive photothermo-responsive nanomatrix for regulating the myogenic differentiation and promoting the skeletal muscle regeneration in vivo. The multifunctional nanomatrix was composed of polypyrrole@polydopamine (PPy@PDA, 342 ± 5.6 nm) nanoparticles-crosslinked Pluronic F-127 (F127)-polycitrate matrix (FPCP). The FPCP nanomatrix demonstrated inherent multifunctional properties including excellent photothermo-responsive and shear-thinning behavior, muscle-adhesive feature, injectable ability, electronic conductivity (0.48 ± 0.03 S/m) and antioxidant activity and photothermal function. The FPCP nanomatrix displayed better photothermal performance with near-infrared irradiation, which could provide the photo-controlled release of protein (91% ± 2.6% of BSA was released after irradiated 3 times). Additionally, FPCP nanomatrix could significantly enhance the cell proliferation and myogenic differentiation of mouse myoblast cells (C2C12) by promoting the expressions of myogenic genes (MyoD and MyoG) and myosin heavy chain (MHC) protein with negligible cytotoxicity. Based on the multifunctional properties, FPCP nanomatrix efficiently promoted the full-thickness skeletal muscle repair and regeneration in vivo, through stimulating the angiogenesis and myotube formation. This study firstly indicated the vital role of multifunctional PPy@PDA nanoparticles in regulating myogenic differentiation and skeletal muscle regeneration. This work also suggests that rational design of bioactive matrix with multifunctional feature would greatly enhance the development of regenerative medicine.

LRTM1 promotes the differentiation of myoblast cells by negatively regulating the FGFR1 signaling pathway

The proliferation and differentiation of myoblast cells are regulated by the fibroblast growth factor receptor (FGFR) signaling pathway. Although the regulation of FGFR signaling cascades has been widely investigated, the inhibitory mechanism that particularly function in skeletal muscle myogenesis remains obscure. In this study, we determined that LRTM1, an inhibitory regulator of the FGFR signaling pathway, negatively modulates the activation of ERK and promotes the differentiation of myoblast cells. LRTM1 is dynamically expressed during myoblast differentiation and skeletal muscle regeneration after injury. In mouse myoblast C2C12 cells, knockout (KO) of Lrtm1 significantly prevents the differentiation of myoblast cells; this effect is associated with the reduction of MyoD transcriptional activity and the overactivation of ERK kinase. Notably, further studies demonstrated that LRTM1 associates with p52Shc and inhibits the recruitment of p52Shc to FGFR1. Taken together, our findings identify a novel negative regulator of FGFR1, which plays an important role in regulating the differentiation of myoblast cells.