Saururus chinensis (Lour.) Baill. extract promotes skeletal muscle cell differentiation by positively regulating mitochondrial biogenesis and AKT/mTOR signaling in vitro.

Promotion of myoblast differentiation by activating mitochondrial biogenesis and protein synthesis signaling pathways provides a potential alternative strategy to balance energy and overcome muscle loss and muscle disorders. Saururus chinensis (Lour.) Baill. extract (SCE) has been used extensively as a traditional herbal medicine and has several physiological activities, including anti­asthmatic, anti­oxidant, anti­inflammatory, anti­atopic, anticancer and hepatoprotective properties. However, the effects and mechanisms of action of SCE on muscle differentiation have not yet been clarified. In the present study, it was investigated whether SCE affects skeletal muscle cell differentiation through the regulation of mitochondrial biogenesis and protein synthesis in murine C2C12 myoblasts. The XTT colorimetric assay was used to determine cell viability, and myosin heavy chain (MyHC) levels were determined using immunocytochemistry. SCE was applied to C2C12 myotube at different concentrations (1, 5, or 10 ng/ml) and times (1,3, or 5 days). Reverse transcription­quantitative PCR and western blotting were used to analyze the mRNA and protein expression change of factors related to differentiation, mitochondrial biogenesis and protein synthesis. Treatment of C2C12 cells with SCE at 1,5, and 10 ng/ml did not affect cell viability. SCE promoted C2C12 myotube formation and significantly increased MyHC expression in a concentration­ and time­dependent manner. SCE significantly increased the mRNA and protein expression of muscle differentiation­specific markers, such as MyHC, myogenic differentiation 1, myogenin, Myogenic Factor 5, and ß­catenin, mitochondrial biosynthesis­related factors, such as peroxisome proliferator­activated receptor­gamma coactivator­1α, nuclear respirator factor­1, AMP­activated protein kinase phosphorylation, and histone deacetylase 5 and AKT/mTOR signaling factors related to protein synthesis. SCE may prevent skeletal muscle dysfunction by enhancing myoblast differentiation through the promotion of mitochondrial biogenesis and protein synthesis.

Amber (Succinite) Extract Enhances Glucose Uptake through the Up-Regulation of ATP and Down-Regulation of ROS in Mouse C2C12 Cells.

Traditionally, amber (Succinite) has been used to alleviate all types of pain, skin allergies, and headaches. However, no studies have been conducted on its antidiabetic and antioxidant effects. In this study, differentiated skeletal muscle C2C12 cells were used to demonstrate the protective effects of amber (AMB) against H2O2-induced cell death. In addition, the effects of AMB on glucose uptake and ATP production were investigated. Our results showed that AMB at 10, 25, and 50 µg/mL suppressed the elevation of ROS production induced by H2O2 in a dose-dependent manner. Moreover, AMB enhanced glucose utilization in C2C12 cells through the improvement of ATP production and an increase in PGC-1α gene expression resulting in an amelioration of mitochondrial activity. On the other hand, AMB significantly increased the gene expression of glucose transporters GLUT4 and GLUT1. Our finding suggests that AMB can be used as a natural supplement for diabetes treatment and for the promotion of skeletal muscle function.

Muscle-specific PGC-1α modulates mitochondrial oxidative stress in aged sarcopenia through regulating Nrf2.

BACKGROUND: Aged sarcopenia is characterized by loss of skeletal muscle mass and strength, and mitochondrial dysregulation in skeletal myocyte is considered as a major factor. Here, we aimed to analyze the effects of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) on mitochondrial reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) in aged skeletal muscles. METHODS: C2C12 cells were stimulated by 50 µM 7ß-hydroxycholesterol (7ß-OHC) to observe the changes of cellular ROS, mitochondrial ROS, and expression of PGC-1α and Nrf2. Different PGC-1α expression in cells was established by transfection with small interfering RNA (siRNA) or plasmids overexpressing PGC-1α (pEX-3-PGC-1α). The effects of different PGC-1α expression on cellular ROS, mitochondrial ROS and Nrf2 expression were measured in cells. Wild type (WT) mice and PGC-1α conditional knockout (CKO) mice were used to analyze the effects of PGC-1α on aged sarcopenia and expression of Nrf2 and CD38 in gastrocnemius muscles. Diethylmaleate, a Nrf2 activator, was used to analyze the connection between PGC-1α and Nrf2 in cells and in mice. RESULTS: In C2C12 cells, the expressions of PGC-1α and Nrf2 were declined by the 7ß-OHC treatment or PGC-1α silence. Moreover, PGC-1α silence increased the harmful ROS and decreased the Nrf2 protein expression in the 7ß-OHC-treated cells. PGC-1α overexpression decreased the harmful ROS and increased the Nrf2 protein expression in the 7ß-OHC-treated cells. Diethylmaleate treatment decreased the harmful ROS in the 7ß-OHC-treated or PGC-1α siRNA-transfected cells. At the same age, muscle-specific PGC-1α deficiency aggravated aged sarcopenia, decreased Nrf2 expression and increased CD38 expression in gastrocnemius muscles compared with the WT mice. Diethylmaleate treatment improved the muscle function and decreased the CD38 expression in the old two genotypes. CONCLUSIONS: Our study demonstrated that PGC-1α modulated mitochondrial oxidative stress in aged sarcopenia through regulating Nrf2.

Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12.

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (kcat). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Vibration acceleration enhances proliferation, migration, and maturation of C2C12 cells and promotes regeneration of muscle injury in male rats.

Vibration acceleration (VA) using a whole-body vibration device is beneficial for skeletal muscles. However, its effect at the cellular level remains unclear. We aimed to investigate the effects of VA on muscles in vitro and in vivo using the C2C12 mouse myoblast cell line and cardiotoxin-induced injury in male rat soleus muscles. Cell proliferation was evaluated using the WST/CCK-8 assay and proportion of Ki-67 positive cells. Cell migration was assessed using wound-healing assay. Cell differentiation was examined by the maturation index in immunostained cultured myotubes and real-time polymerase chain reaction. Regeneration of soleus muscle in rats was assessed by recruitment of satellite cells, cross-sectional area of regenerated muscle fibers, number of centrally nucleated fibers, and conversion of regenerated muscle from fast- to slow-twitch. VA at 30 Hz with low amplitude for 10 min promoted C2C12 cell proliferation, migration, and myotube maturation, without promoting expression of genes related to differentiation. VA significantly increased Pax7-stained satellite cells and centrally nucleated fibers in injured soleus muscles on Day 7 and promoted conversion of fast- to slow-twitch muscle fibers with an increase in the mean cross-sectional area of regenerated muscle fibers on Day 14. VA enhanced the proliferation, migration, and maturation of C2C12 myoblasts and regeneration of injured rat muscles.

Effects of Horse Meat Hydrolysate on Oxidative Stress, Proinflammatory Cytokines, and the Ubiquitin-Proteasomal System of C2C12 Cells.

Sarcopenia, the age-related muscle atrophy, is a serious concern as it is associated with frailty, reduced physical functions, and increased mortality risk. Protein supplementation is essential for preserving muscle mass, and horse meat can be an excellent source of proteins. Since sarcopenia occurs under conditions of oxidative stress, this study aimed to investigate the potential anti-muscle atrophy effect of horse meat hydrolysate using C2C12 cells. A horse meat hydrolysate less than 3 kDa (A4<3kDa) significantly increased the viability of C2C12 myoblasts against H2O2-induced cytotoxicity. Exposure of C2C12 myoblasts to lipopolysaccharide led to an elevation of cellular reactive oxygen species levels and mRNA expression of proinflammatory cytokines, including tumor necrosis factor-α and interleukin 6, and these effects were attenuated by A4<3kDa treatment. Additionally, A4<3kDa activated protein synthesis-related proteins through the protein kinase B/mechanistic target of rapamycin pathway, while decreasing the expression of activity and degradation-related proteins, such as Forkhead box O3, muscle RING finger protein-1, and Atrogin-1 in dexamethasone-treated C2C12 myotubes. Therefore, the natural material A4<3kDa has the potential ofprotecting against muscle atrophy, while further in vivo study is needed.

Intracellular pyruvate-lactate-alanine cycling detected using real-time nuclear magnetic resonance spectroscopy of live cells and isolated mitochondria.

Pyruvate, an end product of glycolysis, is a master fuel for cellular energy. A portion of cytosolic pyruvate is transported into mitochondria, while the remaining portion is converted reversibly into lactate and alanine. It is suggested that cytosolic lactate and alanine are transported and metabolized inside mitochondria. However, such a mechanism continues to be a topic of intense debate and investigation. As a part of gaining insight into the metabolic fate of the cytosolic lactate and alanine; in this study, the metabolism of mouse skeletal myoblast cells (C2C12) and their isolated mitochondria was probed utilizing stable isotope-labeled forms of the three glycolysis products, viz. [3-13 C1 ]pyruvate, [3-13 C1 ]lactate, and [3-13 C1 ]alanine, as substrates. The uptake and metabolism of each substrate was monitored, separately, in real-time using 1 H-13 C 2D nuclear magnetic resonance (NMR) spectroscopy. The dynamic variation of the levels of the substrates and their metabolic products were quantitated as a function of time. The results demonstrate that all three substrates were transported into mitochondria, and each substrate was metabolized to form the other two metabolites, reversibly. These results provide direct evidence for intracellular pyruvate-lactate-alanine cycling, in which lactate and alanine produced by the cytosolic pyruvate are transported into mitochondria and converted back to pyruvate. Such a mechanism suggests a role for lactate and alanine to replenish mitochondrial pyruvate, the primary source for adenosine triphosphate (ATP) synthesis through oxidative phosphorylation and the electron transport chain. The results highlight the potential of real-time NMR spectroscopy for gaining new insights into cellular and subcellular functions.

Uncoupling Protein 3 Promotes the Myogenic Differentiation of Type IIb Myotubes in C2C12 Cells.

Uncoupling protein 3 (Ucp3) is an important transporter within mitochondria and is mainly expressed in skeletal muscle, brown adipose tissue and the myocardium. However, the effects of Ucp3 on myogenic differentiation are still unclear. This study evaluated the effects of Ucp3 on myogenic differentiation, myofiber type and energy metabolism in C2C12 cells. Gain- and loss-of-function studies revealed that Ucp3 could increase the number of myotubes and promote the myogenic differentiation of C2C12 cells. Furthermore, Ucp3 promoted the expression of the type IIb myofiber marker gene myosin heavy chain 4 (Myh4) and decreased the expression of the type I myofiber marker gene myosin heavy chain 7 (Myh7). In addition, energy metabolism related to the expression of PPARG coactivator 1 alpha (Pgc1-α), ATP synthase, H+ transportation, mitochondrial F1 complex, alpha subunit 1 (Atp5a1), lactate dehydrogenase A (Ldha) and lactate dehydrogenase B (Ldhb) increased with Ucp3 overexpression. Ucp3 could promote the myogenic differentiation of type IIb myotubes and accelerate energy metabolism in C2C12 cells. This study can provide the theoretical basis for understanding the role of Ucp3 in energy metabolism.

Prior Treatment with AICAR Causes the Selective Phosphorylation of mTOR Substrates in C2C12 Cells.

Metabolic stress in skeletal muscle cells causes sustained metabolic changes, but the mechanisms of the prolonged effects are not fully known. In this study, we tested C2C12 cells with the AMP-activated protein kinase (AMPK) stimulator AICAR and measured the changes in the metabolic pathways and signaling kinases. AICAR caused an acute increase in the phosphorylation of the AMPK target ULK1, the mTORC1 substrate S6K, and the mTORC2 target Akt. Intriguingly, prior exposure to AICAR only decreased glucose-6 phosphate dehydrogenase activity when it underwent three-hour recovery after exposure to AICAR in a bicarbonate buffer containing glucose (KHB) instead of Dulbecco's Minimum Essential Medium (DMEM). The phosphorylation of the mTORC1 target S6K was increased after recovery in DMEM but not KHB, although this appeared to be specific to S6K, as the phosphorylation of the mTORC1 target site on ULK1 was not altered when the cells recovered in DMEM. The phosphorylation of mTORC2 target sites was also heterogenous under these conditions, with Akt increasing at serine 473 while other targets (SGK1 and PKCα) were unaffected. The exposure of cells to rapamycin (an mTORC1 inhibitor) and PP242 (an inhibitor of both mTOR complexes) revealed the differential phosphorylation of mTORC2 substrates. Taken together, the data suggest that prior exposure to AICAR causes the selective phosphorylation of mTOR substrates, even after prolonged recovery in a nutrient-replete medium.

Vitisin A-13-O-ß-D-glucoside and Vitisin A from Iris lactea inhibit lipogenesis and promote lipolysis via the PKA/HSL pathway during adipogenic transdifferentiation of C2C12 cells.

Oligostilbenes are a group of natural products derived from the polymerization of stilbene monomers. Despite the demonstration of their activities in regulating lipid metabolism, the function of oligostilbenes in the adipogenic transdifferentiation of multipotent myoblast cells remains unknown. Hence, the five oligostilbenes from Iris lactea were tested for their regulatory effects on adipogenic transdifferentiation of C2C12 myoblast cells. As a result, it was shown that Vitisin A-13-O-ß-D-glucoside (VitAOG), Vitisin A (VitA) and Hopeaphenol (Hop) can greatly inhibit the adipogenic transdifferentiation of C2C12 cells by reducing lipid accumulation and downregulating the expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein beta (C/EBPß) and fatty acid binding proteins 4 (FABP4). In contrast, Vitisin D (VitD) and Isohopeaphenol (Isohop) promote adipogenic transdifferentiation of C2C12 cells by increasing lipid accumulation and upregulating the expression of adipogenesis and lipogenesis markers. Further research found that the lipolytic protein levels of adipocyte triglyceride lipase (ATGL) and phosphorylation of hormone-sensitive lipase (HSL) were elevated by VitAOG and VitA. Additionally, VitAOG and VitA maintain lipid homeostasis by improving mitochondrial function. Taken together, our study reveals an effect of oligostilbenes on lipid metabolism in C2C12 cells, and VitAOG and VitA can be regarded as potential candidates for the treatment of obesity and other disorders of lipid metabolism.

Rab44 negatively regulates myoblast differentiation by controlling fusogenic protein transport and mTORC1 signaling.

Skeletal muscle is composed of multinucleated myotubes formed by the fusion of mononucleated myoblasts. Skeletal muscle differentiation, termed as myogenesis, have been investigated using the mouse skeletal myoblast cell line C2C12. It has been reported that several "small" Rab proteins, major membrane-trafficking regulators, possibly regulate membrane protein transport in C2C12 cells; however, the role of Rab proteins in myogenesis remains unexplored. Rab44, a member of "large" Rab GTPases, has recently been identified as a negative regulator of osteoclast differentiation. In this study, using C2C12 cells, we found that Rab44 expression was upregulated during myoblast differentiation into myotubes. Knockdown of Rab44 enhanced myoblast differentiation and myotube formation. Consistent with these results, Rab44 knockdown in myoblasts increased expression levels of several myogenic marker genes. Rab44 knockdown increased the surface accumulation of myomaker and myomixer, two fusogenic proteins required for multinucleation, implying enhanced cell fusion. Conversely, Rab44 overexpression inhibited myoblast differentiation and tube formation, accompanied by decreased expression of some myogenic markers. Furthermore, Rab44 was found to be predominantly localized in lysosomes, and Rab44 overexpression altered the number and size of lysosomes. Considering the underlying molecular mechanism, Rab44 overexpression impaired the signaling pathway of the mechanistic target of rapamycin complex1 (mTORC1) in C2C12 cells. Namely, phosphorylation levels of mTORC1 and downstream mTORC1 substrates, such as S6 and P70-S6K, were notably lower in Rab44 overexpressing cells than those in control cells. These results indicate that Rab44 negatively regulates myoblast differentiation into myotubes by controlling fusogenic protein transport and mTORC1 signaling.

MiR-222-3p suppresses C2C12 myoblast proliferation and differentiation via the inhibition of IRS-1/PI3K/Akt pathway.

Numerous studies have revealed the profound impact of microRNAs on regulating skeletal muscle development and regeneration. However, the biological function and regulation mechanism of miR-222-3p in skeletal muscle remains largely unknown. In this study, miR-222-3p was found to be abundantly expressed in the impaired skeletal muscles, indicating that it might have function in the development and regeneration process of the skeletal muscle. MiR-222-3p overexpression impeded C2C12 myoblast proliferation and myogenic differentiation, whereas inhibition of miR-222-3p got the opposite results. The dual-luciferase reporter assay showed that insulin receptor substrate-1 (IRS-1) was the target gene of miR-222-3p. We next found that knockdown of IRS-1 could obviously suppress C2C12 myoblast proliferation and differentiation. Additionally, miR-222-3p-induced repression of myoblast proliferation and differentiation was verified to be associated with a decrease in phosphoinositide 3-kinase (PI3K)-Akt signaling. Overall, we demonstrated that miR-222-3p inhibited C2C12 cells myogenesis via IRS-1/PI3K/Akt pathway. Therefore, miR-222-3p may be used as a therapeutic target for alleviating muscle loss caused by inherited and nonhereditary diseases.

Physicochemical, technofunctional, in vitro antioxidant, and in situ muscle protein synthesis properties of a sprat (Sprattus sprattus) protein hydrolysate.

Introduction: Sprat (Sprattus sprattus) is an underutilized fish species that may act as an economic and sustainable alternative source of protein due to its good amino acid (AA) profile along with its potential to act as a source of multiple bioactive peptide sequences. Method and results: This study characterized the physicochemical, technofunctional, and in vitro antioxidant properties along with the AA profile and score of a sprat protein enzymatic hydrolysate (SPH). Furthermore, the impact of the SPH on the growth, proliferation, and muscle protein synthesis (MPS) in skeletal muscle (C2C12) myotubes was examined. The SPH displayed good solubility and emulsion stabilization properties containing all essential and non-essential AAs. Limited additional hydrolysis was observed following in vitro-simulated gastrointestinal digestion (SGID) of the SPH. The SGID-treated SPH (SPH-SGID) displayed in vitro oxygen radical antioxidant capacity (ORAC) activity (549.42 µmol TE/g sample) and the ability to reduce (68%) reactive oxygen species (ROS) production in C2C12 myotubes. Muscle growth and myotube thickness were analyzed using an xCELLigence™ platform in C2C12 myotubes treated with 1 mg protein equivalent.mL-1 of SPH-SGID for 4 h. Anabolic signaling (phosphorylation of mTOR, rpS6, and 4E-BP1) and MPS (measured by puromycin incorporation) were assessed using immunoblotting. SPH-SGID significantly increased myotube thickness (p < 0.0001) compared to the negative control (cells grown in AA and serum-free medium). MPS was also significantly higher after incubation with SPH-SGID compared with the negative control (p < 0.05). Conclusions: These preliminary in situ results indicate that SPH may have the ability to promote muscle enhancement. In vivo human studies are required to verify these findings.

Characteristics of microRNAs in Skeletal Muscle of Intrauterine Growth-Restricted Pigs.

microRNAs are a class of small RNAs that have been extensively studied, which are involved in many biological processes and disease occurrence. The incidence of intrauterine growth restriction is higher in mammals, especially multiparous mammals. In this study, we found that the weight of the longissimus dorsi of intrauterine growth-restricted pigs was significantly lower than that of normal pigs. Then, intrauterine growth-restricted pig longissimus dorsi were used to characterize miRNA expression profiles by RNA sequencing. A total of 333 miRNAs were identified, of which 26 were differentially expressed. Functional enrichment analysis showed that these differentially expressed miRNAs regulate the expression of their target genes (such as PIK3R1, CCND2, AKT3, and MAP3K7), and these target genes play an important role in the proliferation and differentiation of skeletal muscle through signaling pathways such as the PI3K-Akt, MAPK, and FoxO signaling pathways. Furthermore, miRNA-451 was significantly upregulated in IUGR pig skeletal muscle. Overexpression of miR-451 in C2C12 cells significantly promoted the expression of Mb, Myod, Myog, Myh1, and Myh7, suggesting that miR-451 may be involved in the regulation of the myoblastic differentiation of C2C12 cells. Our results reveal the role of miRNA-451 in regulating myogenic differentiation of skeletal muscle in pigs with intrauterine growth restriction.

Cordycepin inhibits myogenesis via activating the ERK1/2 MAPK signalling pathway in C2C12 cells.

Cordycepin (with a molecular formula of C10H13N5O3), a natural adenosine isolated from Cordyceps militaris, has an important regulatory effect on skeletal muscle remodelling and quality maintenance. The aim of this study was to investigate the effect of cordycepin on myoblast differentiation and explore the underlying molecular mechanisms of this effect. Our results showed that cordycepin inhibited myogenesis by downregulating myogenic differentiation (MyoD) and myogenin (MyoG), preserved undifferentiated reserve cell pools by upregulating myogenic factor 5 (Myf5) and retinoblastoma-like protein p130 (p130), and enhanced energy reserves by decreasing intracellular reactive oxygen species (ROS) and enhancing mitochondrial membrane potential, mitochondrial mass, and ATP content. The effect of cordycepin on myogenesis was associated with increased phosphorylation of extracellular signal-regulated kinase 1/2 (p-ERK1/2). PD98059 (a specific inhibitor of p-ERK1/2) attenuated the inhibitory effect of cordycepin on C2C12 differentiation. The present study reveals that cordycepin inhibits myogenesis through ERK1/2 MAPK signalling activation accompanied by an increase in skeletal muscle energy reserves and improving skeletal muscle oxidative stress, which may have implications for its further application for the prevention and treatment of degenerative muscle diseases caused by the depletion of depleted muscle stem cells.

Zeb1 and Tle3 are trans-factors that differentially regulate the expression of myosin heavy chain-embryonic and skeletal muscle differentiation.

Myosin heavy chain-embryonic encoded by the Myh3 gene is a skeletal muscle-specific contractile protein expressed during mammalian development and regeneration, essential for proper myogenic differentiation and function. It is likely that multiple trans-factors are involved in this precise temporal regulation of Myh3 expression. We identify a 4230 bp promoter-enhancer region that drives Myh3 transcription in vitro during C2C12 myogenic differentiation and in vivo during muscle regeneration, including sequences both upstream and downstream of the Myh3 TATA-box that are necessary for complete Myh3 promoter activity. Using C2C12 mouse myogenic cells, we find that Zinc-finger E-box binding homeobox 1 (Zeb1) and Transducin-like Enhancer of Split 3 (Tle3) proteins are crucial trans-factors that interact and differentially regulate Myh3 expression. Loss of Zeb1 function results in earlier expression of myogenic differentiation genes and accelerated differentiation, whereas Tle3 depletion leads to reduced expression of myogenic differentiation genes and impaired differentiation. Tle3 knockdown resulted in downregulation of Zeb1, which could be mediated by increased expression of miR-200c, a microRNA that binds to Zeb1 transcript and degrades it. Tle3 functions upstream of Zeb1 in regulating myogenic differentiation since double knockdown of Zeb1 and Tle3 resulted in effects seen upon Tle3 depletion. We identify a novel E-box in the Myh3 distal promoter-enhancer region, where Zeb1 binds to repress Myh3 expression. In addition to regulation of myogenic differentiation at the transcriptional level, we uncover post-transcriptional regulation by Tle3 to regulate MyoG expression, mediated by the mRNA stabilizing Human antigen R (HuR) protein. Thus, Tle3 and Zeb1 are essential trans-factors that differentially regulate Myh3 expression and C2C12 cell myogenic differentiation in vitro.

Serum-free alginate-C2C12 cells microcapsule as a model of alternative animal protein source.

Due to the climate change crisis, and environmental impacts of the traditional meat sector, the production of artificial animal protein based on in vitro cell culture technology is proposed as an alternative. Furthermore, since traditional animal serum-supplemented cultures pose scientific challenges such as batch variation and contamination risks, artificial animal protein cultures are currently in urgent need of not only serum-free cultures, but also microcarrier culture systems for scalability. However, serum-free microcarrier-based culture system for the differentiation of muscle cells is not available to date. Therefore, we established an edible alginate microcapsules culture system for the differentiation of C2C12 cells in serum-free conditions. Furthermore, metabolites related to central carbon metabolism were profiled based on targeted metabolomics using mass spectrometry. The C2C12 cells cultured in alginate microcapsules displayed high viability throughout 7 days and successfully differentiated within 4 days in serum and serum-free cultures except for AIM-V cultures, which was confirmed by CK activity and MHC immunostaining. Lastly, to the best of our knowledge, this is the first report to compare metabolite profiles between monolayer and alginate microcapsule culture systems. Alginate microcapsule culture showed higher levels of intracellular glycolysis and TCA cycle intermediates, lactate, and the contribution of essential amino acids compared to the monolayer culture. We believe our serum-free alginate microcapsule culture system is adaptable to different species of muscle cells and contributes to future food technology as a proof of concept for the scalability of alternative animal protein source production.

Manipulation of porous poly(l-lactide-co-ε-caprolactone) microcarriers via microfluidics for C2C12 expansion.

The growth and repair of skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. In order to acquire enough cells for neoskeletal muscle regeneration, it is urgent to develop microcarriers for skeletal myoblasts proliferation with a considerable efficiency. The current study was thus proposed to develop a microfluidic technology to manufacture porous poly(l-lactide-co-ε-caprolactone) (PLCL) microcarriers of high uniformity, and porosity was manipulated via camphene to suit the proliferation of C2C12 cells. A co-flow capillary microfluidic device was first designed to obtain PLCL microcarriers with different porosity. The attachment and proliferation of C2C12 cells on these microcarriers were evaluated and the differentiation potential of expanded cells were verified. The obtained porous microcarriers were all uniform in size with a high mono-dispersity (CV < 5 %). The content of camphene rendered effects on the size, porosity, and pore size of microcarriers, and porous structure addition produced a softening of their mechanical properties. The one of 10 % camphene (PM-10) exhibited the superior expansion for C2C12 cells with the number of cells after 5 days of culture reached 9.53 times of the adherent cells on the first day. The expanded cells from PM-10 still retained excellent myogenic differentiation performance as the expressions of MYOD, Desmin and MYH2 were intensively enhanced. Hence, the current developed porous PLCL microcarriers could offer as a promising type of substrates not only for in vitro muscular precursor cells expansion without compromising any multipotency but also have the potential as injectable constructs to mediate muscle regeneration.

Glyoxylic Acid, an α-Keto Acid Metabolite Derived from Glycine, Promotes Myogenesis in C2C12 Cells.

α-Keto acids may help prevent malnutrition in patients with chronic kidney disease (CKD), who consume protein-restricted diets, because they serve as amino acid sources without producing nitrogenous waste compounds. However, the physiological roles of α-keto acids, especially those derived from non-essential amino acids, remain unclear. In this study, we examined the effect of glyoxylic acid (GA), an α-keto acid metabolite derived from glycine, on myogenesis in C2C12 cells. Differentiation and mitochondrial biogenesis were used as myogenesis indicators. Treatment with GA for 6 d resulted in an increase in the expression of differentiation markers (myosin heavy chain II and myogenic regulatory factors), mitochondrial biogenesis, and intracellular amounts of amino acids (glycine, serine, and alanine) and their metabolites (citric acid and succinic acid). In addition, GA treatment suppressed the 2.5-µM dexamethasone (Dex)-induced increase in mRNA levels of ubiquitin ligases (Trim63 and Fbxo32), muscle atrophy markers. These results indicate that GA promotes myogenesis, suppresses Dex-induced muscle atrophy, and is metabolized to amino acids in muscle cells. Although further in vivo experiments are needed, GA may be a beneficial nutrient for ameliorating the loss of muscle mass, strength, and function in patients with CKD on a strict dietary protein restriction.