Myoblast Cells Research Articles

Fibrillar Hydrogel Inducing Cell Mechanotransduction for Tissue Engineering.

One of the key strategies for tissue engineering is to design multifunctional bioinks that balance printability with cytocompatibility. Here, we describe fibrillar hydrogels produced by Schiff base formation between B-type gelatin and oxidized sodium alginate, followed by the incorporation of type I collagen, yielding a new gel (MyoColl). The resulting hydrogel exhibits a temperature- and mass-ratio-dependent sol-gel transition, showing variability of hydrogel properties depending on the component ratio. MyoColl composition provides a convenient platform for biofabrication in terms of shear thinning, yielding, Young's modulus, and shape accuracy. Metabolic activity tests and fluorescent microscopy of 2D hydrogel-based mouse C2C12 myoblast cell culture show significant cytocompatibility of the developed carriers. In addition, primary signs of cell mechanotransduction and myofilament formation of 3D printed MyoColl-based cell cultures were detected and described. Due to these promising results, the described hydrogel composition has shown itself as a convenient platform for muscle tissue engineering.

Predictive model of spatial nematic order in confined cell populations

Two-dimensional tissues made of spindle-shaped cells have many applications in the fields of tissue engineering and biotechnology. The uniformity of the tissues is critically affected by topological defects, which are singular points of cell alignment. For systematic control and analysis of defect distributions, this paper proposes a numerical method to predict and quantify spatial distributions of defects in two-dimensional domains. In the proposed method, spindle-shaped cells are modeled as nematic liquid crystals, whose alignment and Frank elastic energy are explicitly expressed. The equilibrium distributions of the defects can then be calculated using a Markov chain Monte Carlo method. The proposed method was experimentally verified by culturing mouse myoblast (C2C12) cells on microwells. The order of the defect scattering was almost the same as for the proposed estimation method, indicating that the proposed method can be used for the systematic design of topographical guides for controlling defect distributions.

Myotoxicity of Crotoxin on C2C12 Myoblasts and its Inhibition by Crotalus Neutralizing Factor versus Enhanced Resistance in Myotubes: Exploring Toxicity and Membrane Potential.

Crotalus Neutralizing Factor (CNF) is a γ-type Phospholipase A2 (PLA2) inhibitor present in the blood of Crotalus durissus terrificus snake. Particularly, CNF inhibits the toxic action of Crotoxin (CTX), which is a major neurotoxin found in C. d. terrificus venom. CTX induces also myotoxic action and demonstrates high selectivity for skeletal muscle fibers. Consequently, CTX can diffuse beyond the site of infection, which can potentially evoke rhabdomyolysis. The present study has evaluated the effects of CTX on myoblasts and myotubes of muscle cells C2C12 in vitro and the effect of CNF on CTX-induced damage. Cytotoxicity assays were performed by measuring the mitochondrial enzyme dehydrogenase levels. Furthermore, creatine kinase and lactate dehydrogenase levels were used as indicators of muscle damage. Crotoxin has been found to have cytotoxic effects on C2C12 myoblast cells, while CNF has not shown toxic effects on these cells. Furthermore, the findings have shown CNF (50 μg/mL) to abolish CTX toxicity in myoblasts. The myotubes, differentiated cells, showed no change in mitochondrial respiration when exposed to CNF or CTX, showing greater resistance to the toxic actions of crotoxin. The data have confirmed the potential of CNF as an anti-myotoxic agent to prevent CTX-damaged myoblasts and increase resistance to the toxic effects of crotoxin on differentiated cells.

Extracellular matrix-mimicking cryogels composed of methacrylated fucoidan enhance vascularized skeletal muscle regeneration following volumetric muscle loss

Volumetric muscle loss (VML) significantly impairs the inherent regenerative ability of skeletal muscle and results in chronic functional impairment. Polysaccharides in the muscle extracellular matrix are crucial for regulating cell proliferation and differentiation. Recent studies indicate that fucoidan has beneficial effects on musculoskeletal conditions. However, the impact of fucoidan on skeletal muscle regeneration remains poorly understood. In this study, methacrylated fucoidan (FuMA) was synthesized through chemical grafting of the methacryloyl group onto fucoidan. In vitro experiments demonstrated that treatment with FuMA significantly up-regulated the expression of myogenic markers and promoted the formation of myotubes in C2C12 myoblast cells. Importantly, FuMA treatment led to a significant enhancement in mitochondrial energy metabolism of myoblasts via activation of the NRF2 antioxidant signaling pathway. To further investigate the regenerative properties in repairing skeletal muscle defects, we fabricated a dual crosslinked cryogel consisting of FuMA and methacrylated gelatin (GelMA) with a porous and interconnected structure. In a rat tibialis anterior muscle VML model, implantation of the FuMA/GelMA cryogel effectively promoted the regeneration of muscle fibers, reduced collagen deposition, and facilitated the formation of new blood vessels. Hence, polysaccharide-based cryogels represent a promising implantable biomimetic scaffold for facilitating skeletal muscle regeneration following severe injuries.

Fiber-based Biomaterial Scaffolds for Cell Support towards the Production of Cultivated Meat

The in vitro production of animal-derived foods via cellular agriculture is emerging as a key solution to global food security challenges. Here, the potential for fiber-based scaffolds, including silk and cotton, in the cultivation of muscle cells for tissue formation was pursued. Mechanical properties and cytocompatibility with the mouse myoblast cell line C2C12 and immortalized bovine muscle satellite cells (iBSCs) were assessed, as well as pre-digestion options for the materials due to their resilience within the human digestive track. The fibers supported cell adhesion, proliferation, and guided muscle cell orientation, facilitating myotube formation per differentiation. A progressive increase in biomass was also documented. Interestingly, iBSC proliferation was enhanced with coatings of recombinant proteins while C2C12 cells showed minimal response. Thus, both cotton and silk yarns were suitable as fiber-based scaffolds towards cell supportive goals, suggesting an alternative path toward structured protein-rich foods via this initial stage of textile engineering for food. Biomass prediction models were generated, enabling forecasts of cell growth and maturation across various scaffold conditions and cell types. This capability enhances the precision of the cultivation process towards an engineering approach, building on the inherent benefits of hierarchical muscle tissue structure, but here via textile engineering with these initial muscle-coated edible fibers. Further, the approach offers to reduce costs by optimizing cultivation time and media needs. These approaches are part of a foundation for future scalable and sustainable cultivated meat production. Statement of SignificanceThis research investigates the use of one-dimensional fiber-based scaffolds for cultivated meat production, contributing to advancements in cellular agriculture. It introduces a method to measure changes in biomass and scaffold degradation throughout the cultivation process. Additionally, our development of biomass prediction models improves the precision and predictability of cultivated meat production. This research not only aids in scaling up cultivated meats but also enhances the use of textile engineering techniques in tissue engineering, paving the way for producing complex, three-dimensional meat structures more sustainably.

Aquo-ethanolic extract of Lilii Bulbus attenuates dexamethasone-induced muscle loss and enhances muscle strength in experimental mice

Traditionally, Lilium lancifolium bulb is known for its ability to nourish yin, nourish the lungs, clear the heart, soothe coughs, reduce irritability, and calm the mind. In Oriental Medicine, it is categorized as a tonic remedy for alleviating symptoms of fatigue and enhancing the strength of bones and muscles.In this study, we aimed to validate the effectiveness of the aquo-ethanolic extract of Lilli Bulbus (LBE) in a dexamethasone (DEX)-induced muscle atrophy model, both in vitro and in vivo, and elucidate its mechanism of action through muscle transcriptome analysis. The effects of LBE on the viability and myotube density of C2C12 myoblasts and differentiated C2C12 myotubes with and without DEX treatment were investigated. LBE pretreatment protected C2C12 myoblast cells and increased the muscle density of C2C12 myotubes in response to DEX. LBE showed potent free radical scavenging activities in cell-free biochemical assays as well as antioxidant activity in C2C12 myoblasts. LBE also exhibited protective effects in an experimental animal model of DEX-induced muscle atrophy, showing muscular function and motor coordination recovery. Transcriptomic analysis of three different muscle tissues from mice with DEX-induced muscle atrophy showed that the regulation of the extracellular matrix was perturbed by glucocorticoid treatment, and this perturbation was reversed by LBE treatment.Collectively, LBE alleviated skeletal muscle loss and maintained muscle function from the chronic toxicity of DEX by protecting muscle cells from various stressful conditions, as well as DEX itself, inhibiting muscle protein degradation, and preserving the muscle tissue microenvironment.

Experimental evaluation and simulation of myoblast cell alignment on the UV-irradiated liquid crystalline elastomers as a versatile tissue-engineered Scaffolding material

Efforts to develop versatile tissue-engineered scaffolds mimicking the functions of extracellular matrices (ECMs) have led to the emergence of liquid crystalline elastomers (LCEs) as promising materials. Here, thiol-acrylate-based LCEs forming a nematic phase upon UV irradiation were synthesized, and their biological compatibility was evaluated using C2C12 myoblast cells. Prior to UV exposure, some characteristics, e.g., phase transition temperature (TNI), order parameter, and reaction time, of the as-synthesized LCEs are characterized for property optimization. Fourier-transform infrared spectroscopy revealed that thiol-acrylate Michael addition polymerization was successful, judged by the disappearance of the characteristic peaks of thiol and acrylate groups. Differential scanning calorimetry thermograms and dynamic mechanical thermal analysis curves showed TNI values at ca. 49 °C, at which point the nematic phase is the dominant morphology. Moreover, the cross-linking density, the fixing, and the recovery ratios for the sample with optimal properties (i.e., LCE3) were determined to be 10.3 mol/cm3, 97.2 % and 91 %, respectively. Assessment of mesomorphism and roughness demonstrated the suitability of LCE3 for cell culture. Subsequent studies on adhesion, viability, differentiation, and proliferation of cultured myoblast cells, coupled with particle image velocimetry simulation, highlighted the potential of UV-irradiated LCE3 as a promising scaffold material for inducing surface morphology-induced cellular alignment and interactions.

Developing endoscopic cell sheet delivery device using a beating human heart simulator and a pig model for minimally invasive cardiac regenerative therapy

Abstract Introduction Recently, clinical trials have commenced for cardiac regenerative therapy utilizing stem cells in patients with heart failure. Among the various transplantation approaches, there is a growing demand for less invasive methods to transplant sheet/patch-shaped cell products onto the heart surface, rather than resorting to conventional open surgery. This is particularly important considering the need for immunosuppressants due to allogeneic cell transplantation. Objective This study aims to develop a device for endoscopic transplantation of cell sheets onto the beating heart surface. Methods Prototype devices, initially developed in our prior study using a static heart model (Regenerative Therapy, 2020), were refined to better suit the cell sheet transplantation procedure onto a beating heart. We constructed a three-dimensional (3D) model of a beating heart located inside a thoracic cavity model, based on human computed tomography data and a 3D printer dedicated to simulating endoscopic surgical procedures. Human mesenchymal stromal cell (MSC)-derived cell sheets were prepared using temperature-responsive culture dishes and transferred onto a beating heart model pulsating at 80 beats per minute. We assessed the technical feasibility (N=8). Furthermore, skeletal muscle tissue was harvested from the thigh of the Clawn miniature pigs, and skeletal myoblasts were cultured from enzymatically digested tissue to create a cell sheet for autologous transplantation (8.0×10⁶ cells/sheet). Subsequently, the cell sheets labeled with Hoechst 33342 were endoscopically transplanted onto the pig heart surface (N=2). The pigs were sacrificed one week later, and the engraftment of the cell sheet was histologically evaluated. Results Successful deployment of MSC sheets onto the beating heart model was achieved in all procedures with one trial (100% success rate). The procedure duration from device insertion to the completion of cell sheet transplantation averaged 142±19 seconds. In experiments on autologous transplantation of myoblast cell sheets, two cases were successfully accomplished, with the transplanted cell sheet positive for Hoechst 33342 observed on the heart surface. Conclusion We have developed an endoscopic cell sheet delivery device using a pulsating heart model and a pig model, which holds promise for minimally invasive cardiac regenerative therapy in the future. Further investigations into the safety of the procedure are warranted for the clinical application of this system.

Morphodynamics of interface between dissimilar cell aggregations

Tissue interfaces are essential for development and their disruption often leads to diseases such as tumor invasion. Here, we combine experiments, theoretical modeling, and numerical simulations to quantify the morphodynamics of interface in a biphasic system composed of Madin Darby canine kidney (MDCK) and mouse myoblast (C2C12) cells. We show that cellular activity regulates the interface morphodynamics and drives wave propagation along the interface. Based on the dispersion relationship, we identify that the wave dynamics results from the activity-mediated instability of the interface and coherent flow. It is found that the topological defects accumulate around and destabilize the interface and +1/2 topological defects are more likely to aggregate in MDCK cell clusters. A biphasic active nematic theory is employed to reproduce our experimental observations and decipher the underlying mechanisms. These findings provide physical insights into the interfacial evolution that could be implicated in tissue morphogenesis and tumor invasion.

Epigenetic regulation of myogenesis by vitamin C.

The micronutrient vitamin C is essential for the maintenance of skeletal muscle health and homeostasis. The pro-myogenic effects of vitamin C have long been attributed to its role as a general antioxidant agent, as well as its role in collagen matrix synthesis and carnitine biosynthesis. Here, we show that vitamin C also functions as an epigenetic compound, facilitating chromatin landscape transitions during myogenesis through its activity as an enzymatic cofactor for histone H3 and DNA demethylation. Utilizing C2C12 myoblast cells to investigate the epigenetic effects of vitamin C on myogenesis, we observe that treatment of cells with vitamin C decreases global H3K9 methylation and increases 5-hmC levels. Furthermore, vitamin C treatment enhances myoblast marker gene expression and myotube formation during differentiation. We identify KDM7A as a histone lysine demethylase markedly upregulated during myogenesis. Accordingly, knockdown of Kdm7a prevents the pro-myogenic effects of vitamin C. Chromatin immunoprecipitation analysis showed that KDM7A occupies the promoter region of the myogenic transcription factor MyoD1 where it facilitates histone demethylation. We also confirm that the methylcytosine dioxygenases TET1 and TET2 are required for myogenic differentiation and that their loss blunts stimulation of myogenesis by vitamin C. In conclusion, our data suggest that an epigenetic mode of action plays a major role in the myogenic effects of vitamin C.

Small Molecule Screening Identifies HSP90 as a Modifier of RNA Foci in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG triplet repeat expansion within the 3' untranslated region of the DMPK gene. Expression of the expanded allele generates RNA containing long tracts of CUG repeats (CUGexp RNA) that form hairpin structures and accumulate in nuclear RNA foci; however, the factors that control DMPK expression and the formation of CUGexp RNA foci remain largely unknown. We performed an unbiased small molecule screen in an immortalized human DM1 skeletal muscle myoblast cell line and identified HSP90 as a modifier of endogenous RNA foci. Small molecule inhibition of HSP90 leads to enhancement of RNA foci and upregulation of DMPK mRNA levels. Knockdown and overexpression of HSP90 in undifferentiated DM1 myoblasts validated the impact of HSP90 with upregulation and downregulation of DMPK mRNA, respectively. Furthermore, we identified p-STAT3 as a downstream mediator of HSP90 impacting levels of DMPK mRNA and RNA foci. Interestingly, differentiated cells exhibited an opposite effect of HSP90 inhibition displaying downregulation of DMPK mRNA through a mechanism independent of p-STAT3 involvement. This study has revealed a novel mediator for DMPK mRNA and foci regulation in DM1 cells with the potential to identify targets for future therapeutic intervention.

Inhibition of CILP2 Improves Glucose Metabolism and Mitochondrial Dysfunction in Sarcopenia via the Wnt Signalling Pathway.

Skeletal muscle is the primary organ involved in insulin-mediated glucose metabolism. Elevated levels of CILP2 are a significant indicator of impaired glucose tolerance and are predominantly expressed in skeletal muscle. It remains unclear whether CILP2 contributes to age-related muscle atrophy through regulating the glucose homeostasis and insulin sensitivity. Initially, the expression levels of CILP2 were assessed in elderly mice and patients with sarcopenia. Lentiviral vectors were used to induce either silencing or overexpression of CILP2 in C2C12 myoblast cells. The effects of CILP2 on proliferation, myogenic differentiation, insulin sensitivity and glucose uptake were evaluated using immunofluorescence, western blotting, real-time quantitative polymerase chain reaction, RNA sequencing, glucose uptake experiments, dual-luciferase reporter assays and co-immunoprecipitation (CO-IP). An adeno-associated virus-9 containing a muscle-specific promoter was injected into SAMP8 senile mice to observe the efficacy of CILP2 knockout. We found that there was more CLIP2 expressed in the skeletal muscle of ageing mice (+1.1-fold, p < 0.01) and in patients with sarcopenia (+2.5-fold, p < 0.01) compared to the control group. Following the overexpression of CILP2, Ki67 (-65%, p < 0.01), PCNA (-32%, p < 0.05), MyoD1 (-89%, p < 0.001), MyoG (-31%, p < 0.05) and MyHC (-85%, p < 0.001), which indicate proliferation and differentiation potential, were significantly reduced. In contrast, MuRF-1 (+59%, p < 0.05), atrogin-1 (+43%, p < 0.05) and myostatin (+31%, p < 0.05), the markers of muscular atrophy, were significantly increased. Overexpression of CILP2 decreased insulin sensitivity, glucose uptake (-18%, p < 0.001), GLUT4 translocation to the membrane and the maximum respiratory capacity of mitochondria. Canonical Wnt signalling was identified through RNA sequencing as a potential pathway for CILP2 regulation in C2C12, and Wnt3a was confirmed as an interacting protein of CILP2 in the CO-IP assay. The addition of recombinant Wnt3a protein reversed the inhibitory effects on myogenesis and glucose metabolism caused by CILP2 overexpression. Conversely, CILP2 knockdown promoted myogenesis and glucose metabolism. CILP2 knockdown improved muscle atrophy in mice, characterized by significant increases in time to exhaustion (+42%, p < 0.001), grip strength (+19%, p < 0.01), muscle mass (+15%, p < 0.001) and mean muscle cross-sectional area (+37%, p < 0.01). CILP2 knockdown enhanced glycogen synthesis (+83%, p < 0.001) and the regeneration of oxidative and glycolytic muscle fibres in SAMP8 ageing mice via the Wnt/β-catenin signalling pathway. Our results indicate that CILP2 interacts with Wnt3a to suppress the Wnt/β-catenin signalling pathway and its downstream cascade, leading to impaired insulin sensitivity and glucose metabolism in skeletal muscle. Targeting CILP2 inhibition could offer potential therapeutic benefits for sarcopenia.

Streamlining RNA Aptamer Selection via Unique Molecular Identifiers and High-Throughput Sequencing.

Aptamers are valuable tools for applications such as cell imaging, drug delivery, and therapeutics. RNA aptamers, in particular, exhibit complex structural diversity and flexibility, affording higher affinity and specificity, broader target recognition, and easier chemical modification compared with DNA aptamers. However, traditional selection methods for RNA aptamers are time-consuming and involve numerous rounds of screening, thus limiting their widespread application. To overcome this challenge, we propose an efficient truncated selection approach termed ID-SELEX. This method incorporates a molecular identification marker whereby each template is labeled with a unique molecular identifier, or UMI. Such incorporation helps mitigate biases introduced by multiple polymerase chain reaction (PCR) amplification during high-throughput sequencing, ensuring accurate identification of aptamer candidates. Utilizing ID-SELEX, we successfully identified a panel of high-quality aptamers targeting the human colon cancer cell line HCT-8 in just 2 rounds of selection. Furthermore, we demonstrated the versatility of this strategy by selecting 6 RNA aptamers targeting mouse myoblast cell line C2C12 with only one round of selection. In summary, RNA aptamer selection based on ID-SELEX utilizes high-throughput sequencing and UMI labeling to enable the rapid screening of RNA aptamers across human and murine cell lines. As such, ID-SELEX has the potential to facilitate RNA aptamer discovery, providing a novel molecular tool for biomedical research, clinical applications, and precision medicine.

9291 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

8670 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

Development of a novel Japanese eel myoblast cell line for application in cultured meat production

The present study investigates the isolation, analysis, and characterization of primary cultured cells derived from the muscle tissue of Japanese eel (Anguilla japonica), culminating in establishing a spontaneously immortalized myoblast cell line, JEM1129. We isolated satellite cells from eel muscle tissue to establish a foundation for cultured eel meat production. While initial cell cultures contained myoblasts, continued passaging led to a decline in myoblast characteristics and an increase in fibroblast-like cells. RNA-Seq and RT-qPCR analyses showed significant downregulation of well-established markers for satellite cells and myoblasts, such as pax7a and myoD, over successive passages, highlighting a loss of myoblastic traits. Single-cell cloning was employed to overcome this challenge and maintain myoblast purity, leading to the successful creation of the JEM1129 cell line. These JEM1129 cells demonstrated enhanced expression of myoblast marker genes, exceeding the initial primary culture cell population. The cells showed strong myotube formation, particularly when cultured in a differentiation medium, indicating their robust potential for muscle development. The JEM1129 cell line represents a significant advancement in the cultivation of eel muscle cells, offering a promising avenue for cultured meat production. The findings contribute to a deeper understanding of muscle cell biology and provide valuable insights into using fish-derived myoblasts for cultured meat production.

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.

Non-cytotoxic, biocompatible pyrochlore Cerium Zirconium Oxide nanoparticles for asymmetric supercapacitor applications

A novel biocompatible and non-cytotoxic Ce2Zr2O7 (CZO) nano-sized particles were developed by a straightforward hydrothermal method. Cubic structure with Fd-3m space group (227) of prepared nanoparticles has been confirmed from X-ray diffraction (XRD) patterns. Nano-sphere like particle morphology was conspicuously noticed from the FE-SEM and TEM investigations thoroughly. Particle size, inter-planar distance, SAED pattern and elemental presence were affirmatively evaluated. The elemental presence in the sample and their ionic states were systematically investigated through X-ray photoelectron spectroscopy analysis. We have investigated the cytotoxicity of the Ce2Zr2O7 nanoparticles on C2C12 (mouse myoblast cell line) cells and observed to be non-cytotoxicity on C2C12 cells which supports the eco-friendly and biocompatible nanoparticles. We have investigated the electrochemical performance of Ce2Zr2O7 nanoparticles using various electrolytes such as Na2SO4, Li2SO4 and KOH. We have obtained the predominant electrochemical performance of 250 F g−1 at 0.05 A g−1 along with appreciable cycling durability from KOH electrolyte interestingly. In addition, we fabricated asymmetric supercapacitor device and evaluated its energy and power densities systematically. We have evaluated maximum energy and power densities are in the order of 13 Wh kg−1 and 3692 W kg−1, respectively. Therefore, eco-friendly and biocompatible Ce2Zr2O7 nanoparticles could be suggested as a contender for the electrode substance for supercapacitor applications. This investigation has also been an usher hope of further technological advancement in the field of nano-biomedical along with energy storage research fields.

Myoblast-Derived Galectin 3 Impairs the Early Phases of Osteogenesis Affecting Notch and Akt Activity.

Galectin-3 (Gal-3) is a pleiotropic lectin produced by most cell types, which regulates multiple cellular processes in various tissues. In bone, depending on its cellular localization, Gal-3 has a dual and opposite role. If, on the one hand, intracellular Gal-3 promotes bone formation, on the other, its circulating form affects bone remodeling, antagonizing osteoblast differentiation and increasing osteoclast activity. From an analysis of the secretome of cultured differentiating myoblasts, we interestingly found the presence of Gal-3. After that, we confirmed that Gal-3 was expressed and released in the extracellular environment from myoblast cells during their differentiation into myotubes, as well as after mechanical strain. An in vivo analysis revealed that Gal-3 was triggered by trained exercise and was specifically produced by fast muscle fibers. Speculating a role for this peptide in the muscle-to-bone cross talk, a direct co-culture in vitro system, simultaneously combining media that were obtained from differentiated myoblasts and osteoblast cells, confirmed that Gal-3 is a mediator of osteoblast differentiation. Molecular and proteomic analyses revealed that the secreted Gal-3 modulated the biochemical processes occurring in the early phases of bone formation, in particular impairing the activity of the STAT3 and PDK1/Akt signaling pathways and, at the same time, triggering that one of Notch. Circulating Gal-3 also affected the expression of the most common factors involved in osteogenetic processes, including BMP-2, -6, and -7. Intriguingly, Gal-3 was able to interfere with the ability of differentiating osteoblasts to interact with the components of the extracellular bone matrix, a crucial condition required for a proper osteoblast differentiation. All in all, our evidence lays the foundation for further studies to present this lectin as a novel myokine involved in muscle-to-bone crosstalk.

Melatonin can mitigate H2O2-induced atrophy and promote muscle fiber hypertrophy in morphological level in mouse myoblast cell line

Objective: It is known that oxidative stress is the main factor in the formation of disuse muscle atrophy which is the most common type of muscle atrophy. The utilization of antioxidants as supplements, particularly the category known as mitochondrial targeting antioxidants (MTA), such as melatonin, have demonstrated significant potential as an advanced therapeutic approach. In this study, we aimed to investigate the effect of melatonin application on the cellular morphology of the C2C12 cell line. Materials and Methods: In our experiment, we induced oxidative stress using hydrogen peroxide (H2O2) to create a model of skeletal muscle atrophy. We established four distinct groups of C2C12 cells, all exposed to the same conditions. These groups included Control (C), Melatonin (M), H2O2 (H), and Melatonin + H2O2 (MH). The aim was to examine morphological features, specifically myotube diameters, to assess atrophy. Results: The analysis revealed significant differences in diameters among the groups (p &lt; 0.05). The melatonin treatment group not only showed a mitigation of diameter change due to atrophy but also exhibited a significant increase in diameter. Conclusion: The results suggest that H2O2 induces muscle atrophy, and melatonin plays a dual role in maintaining muscle health, protecting against atrophy and promoting hypertrophy, particularly at the morphological level. However, additional research is needed to figure out the details of underlying mechanisms.