Expression Of Myogenic Marker Genes Research Articles

Metabolomic changes in culture media with varying passage numbers of pig muscle stem cell culture for cultured meat production

Selecting the primary cells in an optimal state for cultured meat production is a crucial challenge in commercializing cultured meat. We investigated the metabolomic changes in culture media according to passage numbers for indirectly assessing the state of primary cells. Pig skeletal muscle stem cells (PSCs) harvested from the biceps femoris muscles of 7-d-old crossbred pigs (Landrace × Yorkshire × Duroc, LYD) were used for cell characterization. Fresh media (FM) and spent media (SM) of PSCs during passages 1 to 3 in vitro culture were prepared for metabolomics analysis. SM was collected on the third day of proliferation for each passage of PSCs. Cell characterization analysis revealed that the proliferation rate was highest at passage 2; however, a significant loss of expression of myogenic marker genes was observed at passage 3. Based on metabolomic profiles of culture media, FM and SM groups (SM1, SM2, and SM3) were clearly separated by partial least squares-discriminant analysis. A total of seven differentially abundant metabolites (DAMs) were identified from FM and SM for each passage, based on the following criteria: P < 0.05, fold change > 1.5 or < 0.66, and a variable importance in projection score > 1.5. All seven DAMs and their interconnected metabolites might be primarily used as substrates for energy production and most of them were relatively abundant in SM3. Among the seven DAMs, the three potential biomarkers (γ-glutamyl-L-leucine, cytosine, and ketoleucine), which showed significant changes exclusively in SM3, each had an area under the curve value of 1. Therefore, monitoring the levels of these key metabolites in culture media could serve as a quality control measure for cultured meat production by enabling the indirect detection of suboptimal PSCs based on their proliferation ability.

Isolation, identification, and induced differentiation of satellite cells from skeletal muscle of adult tree shrews.

A method for the in vitro isolation, purification, identification, and induced differentiation of satellite cells from adult tree shrew skeletal muscle was established. The mixed enzyme digestion method and differential adhesion method were used to obtain skeletal muscle satellite cells, which were identified and induced to differentiate to verify their pluripotency. The use of a mixture of collagenase II, hyaluronidase IV, and DNase I is an efficient method for isolating adult tree shrew skeletal muscle satellite cells. The P3 generation of cells had good morphology, rapid proliferation, high viability, and an "S"-shaped growth curve. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence staining indicated that marker genes or proteins were expressed in skeletal muscle satellite cells. After myogenic differentiation was induced, multiple-nucleated myotubes were observed, and the MyHC protein was expressed. The expression of myogenic marker genes changed with the differentiation process. After the induction of adipogenic differentiation, orange-red lipid droplets were observed, and the expression of adipogenic marker genes increased gradually with the differentiation process. In summary, satellite cells from adult tree shrew skeletal muscle were successfully isolated using a mixed enzyme digestion method, and their potential for differentiation into myogenic and adipogenic cells was confirmed, laying a foundation for further in vitro study of tree shrew muscle damage.

Optimization of Culture Conditions for Maintaining Pig Muscle Stem Cells In Vitro.

Muscle stem cells isolated from domestic animals, including cows and pigs, were recently spotlighted as candidates for the production of alternative protein resources, so-called cultured meat or lab-grown meat. In the present study, we aimed to optimize the in vitro culture conditions for the long-term expansion of pig muscle stem cells via the screening of various signaling molecules. Pig muscle stem cells were collected from the biceps femoris muscles of 3-d-old crossbred pigs (Landrace×Yorkshire×Duroc, LYD) and cultured in minimum essential medium-based growth media. However, the pig muscle stem cells gradually lost their proliferation ability and featured morphologies during the long-term culture over two weeks. To find suitable in vitro culture conditions for an extended period, skeletal muscle growth medium-2, including epidermal growth factor (EGF), dexamethasone, and a p38 inhibitor (SB203580), was used to support the stemness of the pig muscle stem cells. Interestingly, pig muscle stem cells were stably maintained in a long-term culture without loss of the expression of myogenic marker genes as determined by PCR analysis. Immunostaining analysis showed that the stem cells were capable of myogenic differentiation after multiple passaging. Therefore, we found that basal culture conditions containing EGF, dexamethasone, and a p38 inhibitor were suitable for maintaining pig muscle stem cells during expanded culture in vitro. This culture method may be applied for the production of cultured meat and further basic research on muscle development in the pig.

Neonatal vitamin A injection promotes cattle muscle growth and increases oxidative muscle fibers

BackgroundVitamin A and its metabolite, retinoic acid (RA), are important regulators of cell differentiation and organ morphogenesis. Its impact on beef cattle muscle growth remains undefined.MethodAngus steer calves were administrated with 0 (control) or 150,000 IU vitamin A (retinyl palmitate in glycerol, i.m.) per calf at birth and 1 month of age. At 2 months of age, a biopsy of the Biceps femoris muscle was obtained to analyze the immediate effects of vitamin A injection on myogenic capacity of muscle cells. The resulting steers were harvested at 14 months of age.ResultsVitamin A administration increased cattle growth at 2 months. At 2 months of age, Vitamin A increased PAX7 positive satellite cells and the expression of myogenic marker genes including PAX7, MYF5, MYOD and MYOG. Muscle derived mononuclear cells were further isolated and induced myogenesis in vitro. More myotubes and a higher degree of myogenesis was observed in vitamin A groups. Consistently, vitamin A increased Latissimus dorsi (LD) muscle fiber size at harvest. In addition, vitamin A increased the ratio of oxidative type I and type IIA fibers and reduced the glycolic type IIX fibers. Furthermore, we found that RA, a key bioactive metabolite of vitamin A, activated PPARGC1A promoter, which explains the upregulated expression of PPARGC1A in skeletal muscle.ConclusionVitamin A administration to neonatal calves enhanced postnatal muscle growth by promoting myogenesis and increasing satellite cell density, accompanied with a shift to oxidative muscle fibers.

Effect of glutamine on heat-shock protein beta 1 (HSPB1) expression during myogenic differentiation in bovine embryonic fibroblast cells.

The objective of this study was to examine the effects of glutamine on heat-shock protein beta 1 (HSPB1) expression in bovine embryonic fibroblast cells during myogenesis. First, to elucidate the role of glutamine on HSPB1 expression during myogenesis, we treated with glutamine in myogenic lineage determinant (MyoD) over-expressed bovine embryonic fibroblast cells (BEFS-MyoD cells). Second, knockdown of HSPB1 using small interference RNA was performed to evaluate whether muscle development by glutamine is dependent on HSPB1 in BEFS-MyoD cells. As a result, glutamine promoted the mRNA level of HSPB1, Myogenin, Desmin, and mTOR as well as myotube formation, and protein synthesis (p<0.05). The inhibition of HSPB1 expression during myogenesis has shown to repress the expression of myogenic marker genes (MyoD, Myogenin, Desmin) (p<0.01), formation of myotubes and protein synthesis (p<0.05). According to the results, it is concluded that glutamine regulates HSPB1 expression during myogenesis.

Evodiamine promotes differentiation and inhibits proliferation of C2C12 muscle cells.

Evodiamine is a botanical alkaloid compound extracted from Tetradium plants. Previous studies have reported that evodiamine (Evo) treatment can reduce food uptake and improve insulin resistance in animals . The skeletal muscle comprises about 40% of the body mass of adults and has a vital role in regulating whole body glucose metabolism and energy metabolism. However, the effect of Evo on skeletal muscle is unclear. The main aim of the present study was to investigate the effect of Evo on the differentiation and proliferation of the mouse C2C12 muscle cell line. The results demonstrated that Evo promoted the expression of myogenic marker genes (Myogenin and muscle myosin heavy chain) and increased myoblast differentiation, potentially via activation of the Wnt/β‑catenin pathway. Furthermore, Evo increased mRNA expression of p21, reduced mRNA expression of CyclinB, CyclinD and CyclinE and reduced the percentage of proliferating cells. Also, phosphorylation of ERK1/2 was decreased by Evo treatment during cell proliferation. In conclusion, these findings indicated that Evo has marked effects on skeletal muscle development.

Squalene epoxidase plays a critical role in determining pig meat quality by regulating adipogenesis, myogenesis, and ROS scavengers

In mammals, Squalene epoxidase (SQLE) is an enzyme that converts squalene to 2,3-oxidosqualene, in the early stage of cholesterol generation. Here, we identified single nucleotide polymorphisms (SNPs) in the SQLE gene (c.2565 G > T) by RNA Sequencing from the liver tissue of Berkshire pigs. Furthermore, we found that homozygous GG pigs expressed more SQLE mRNA than GT heterozygous and TT homozygous pigs in longissimus dorsi tissue. Next, we showed that the SNP in the SQLE gene was associated with several meat quality traits including backfat thickness, carcass weight, meat colour (yellowness), fat composition, and water-holding capacity. Rates of myogenesis and adipogenesis induced in C2C12 cells and 3T3-L1 cells, respectively, were decreased by Sqle knockdown. Additionally, the expression of myogenic marker genes (Myog, Myod, and Myh4) and adipogenic marker genes (Pparg, Cebpa, and Adipoq) was substantially downregulated in cells transfected with Sqle siRNA. Moreover, mRNA expression levels of ROS scavengers, which affect meat quality by altering protein oxidation processes, were significantly downregulated by Sqle knockdown. Taken together, our results suggest the molecular mechanism by which SNPs in the SQLE gene can affect meat quality.

(-)-Epigallocatechin-3-gallate stimulates myogenic differentiation through TAZ activation

Muscle loss is a typical process of aging. Green tea consumption is known to slow down the progress of aging. Their underlying mechanisms, however, remain largely unknown. In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG), a polyphenolic compound of green tea, on myogenic differentiation and found that EGCG significantly increases myogenic differentiation. After EGCG treatment, the expression of myogenic marker genes, such as myosin heavy chain, are increased through activation of TAZ, a transcriptional coactivator with a PDZ-binding motif. TAZ-knockdown does not stimulate EGCG-induced myogenic differentiation. EGCG facilitates the interaction between TAZ and MyoD, which stimulates MyoD-mediated gene transcription. EGCG induces nuclear localization of TAZ through the dephosphorylation of TAZ at its Ser89 residue, which relieves 14-3-3 binding in the cytosol. Interestingly, inactivation of Lats kinase is observed after EGCG treatment, which is responsible for the production of dephosphorylated TAZ. Together, these results suggest that EGCG induces myogenic differentiation through TAZ, suggesting that TAZ plays an important role in EGCG induced muscle regeneration.

Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

Differentiation of muscle satellite cells (MSCs) involves interaction of the proteins present in the extracellular matrix (ECM) with MSCs to regulate their activity, and therefore phenotype. Herein, we report fibromodulin (FMOD), a member of the proteoglycan family participating in the assembly of ECM, as a novel regulator of myostatin (MSTN) during myoblast differentiation. In addition to having a pronounced effect on the expression of myogenic marker genes [myogenin (MYOG) and myosin light chain 2 (MYL2)], FMOD was found to maintain the transcriptional activity of MSTN Moreover, coimmunoprecipitation and in silico studies performed to investigate the interaction of FMOD helped confirm that it antagonizes MSTN function by distorting its folding and preventing its binding to activin receptor type IIB. Furthermore, in vivo studies revealed that FMOD plays an active role in healing by increasing satellite cell recruitment to sites of injury. Together, these findings disclose a hitherto unrecognized regulatory role for FMOD in MSCs and highlight new mechanisms whereby FMOD circumvents the inhibitory effects of MSTN and triggers myoblast differentiation. These findings offer a basis for the design of novel MSTN inhibitors that promote muscle regeneration after injury or for the development of pharmaceutical agents for the treatment of different muscle atrophies.-Lee, E. J., Jan, A. T., Baig, M. H., Ashraf, J. M., Nahm, S.-S., Kim, Y.-W., Park, S.-Y., Choi, I. Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

A novel adipokine C1q/TNF-related protein 3 is expressed in developing skeletal muscle and controls myoblast proliferation and differentiation.

Several hormones and growth factors, including adipokines, play important roles during muscle development and regeneration. CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor-related protein (CTRP) superfamily. CTRP3 is a novel adipokine previously reported to reduce glucose output in hepatocytes and lower glucose levels in mice models. In the present study, we provide the first evidence for a physiological role of the CTRP3 in myogenesis using C2C12 myoblasts. CTRP3 was expressed in developing skeletal muscle tissues, and the expression level of CTRP3 was increased during myogenic differentiation of C2C12 cells. Recombinant CTRP3 (rCTRP3) promoted the proliferation of undifferentiated C2C12 myoblasts and this response required activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. In contrary, rCTRP3 inhibited myogenic differentiation and fusion of C2C12 cells by suppressing the expression of myogenic marker genes (myogenin and myosin heavy chain). CTRP3 mRNA expression was increased in C2C12 myoblasts treated with transforming growth factor-β3 (TGF-β3), suggesting that TGF-β3 is one of the extracellular factors regulating CTRP3 expression during myogenesis. These results indicate a novel physiological role for CTRP3 during skeletal myogenesis.

MYOD mediates skeletal myogenic differentiation of human amniotic fluid stem cells and regeneration of muscle injury

IntroductionHuman amniotic fluid stem (hAFS) cells have been shown to differentiate into multiple lineages, including myoblasts. However, molecular mechanisms underlying the myogenic differentiation of hAFS cells and their regenerative potential for muscle injury remain to be elucidated.MethodsIn order to induce myogenic differentiation of hAFS cells, lentiviruses for MYOD were constructed and transduced into hAFS cells. Formation of myotube-like cells was analyzed by immunocytochemistry, and expression of molecular markers for myoblasts was analyzed by reverse transcription polymerase chain reaction and Western blotting. For in vivo muscle regeneration, MYOD transduced hAFS cells were injected into left tibialis anterior (TA) muscles injured with cardiotoxin, and muscle regeneration was analyzed using hematoxylin and eosin, immunocytochemistry and formation of neuro-muscular junction.ResultsMYOD expression in hAFS cells successfully induced differentiation into multinucleated myotube-like cells. Consistently, significant expression of myogenic marker genes, such as MYOG, DES, DMD and MYH, was induced by MYOD. Analysis of pre-myogenic factors showed that expression of PAX3, MEOX1 and EYA2 was significantly increased by MYOD. MYOD was phosphorylated and localized in the nucleus. These results suggest that in hAFS cells, MYOD is phosphorylated and localized in the nucleus, thus inducing expression of myogenic factors, resulting in myogenic differentiation of hAFS cells. To test regenerative potential of MYOD-transduced hAFS cells, we transplanted them into injured muscles of immunodeficient BALB/cSlc-nu mice. The results showed a substantial increase in the volume of TA muscle injected with MYOD-hAFS cells. In addition, TA muscle tissue injected with MYOD-hAFS cells has more numbers of neuro-muscular junctions compared to controls, indicating functional restoration of muscle injury by MYOD-hAFS cells.ConclusionsCollectively, our data suggest that transduction of hAFS cells with MYOD lentiviruses induces skeletal myogenic differentiation in vitro and morphological and functional regeneration of injured muscle in vivo.

Porcine peroxisome proliferator-activated receptor γ induces transdifferentiation of myocytes into adipocytes1

Peroxisome proliferator-activated receptor gamma2 (PPARgamma) is a nuclear transcription factor that regulates adipocyte differentiation and lipogenic genes during adipogenesis. The activity of rodent PPARgamma is regulated by phosphorylation of serine 112. The current experiment was designed to study the ability of porcine PPARgamma to stimulate transdifferentiation of myoblasts to adipocytes by overexpressing wild-type PPARgamma or mutated PPARgamma (serine 112 was mutated to alanine) in mouse myoblast cells. The expression of adipogenic marker genes (adipocyte fatty acid binding protein, lipoprotein lipase, and glycerol-3 phosphate dehydrogenase) in cells stably expressing mutated porcine PPARgamma was greater than in cells with wild-type PPARgamma, indicating that the mutated PPARgamma has greater adipogenic capability than the wild-type PPARgamma. Under treatment with a ligand, both wild-type and mutant porcine PPARgamma-expressing C2C12 myoblasts differentiated into adipocytes in 10 d. The expression of myogenic marker genes (myogenin, myogenic regulatory factor-4) was suppressed in cells transfected with the mutated PPARgamma or wild-type PPARgamma. Moreover, wild-type and mutant PPARgamma were able to inhibit myogenesis without addition of a ligand. Our results suggest that porcine wild-type PPARgamma and mutated PPARgamma can both convert myoblast cells into adipocytes, and also that the ability to transdifferentiate was greater in cells containing the mutated PPARgamma than in cells containing the wild-type PPARgamma. Therefore, the existence of serine 112 in PPARgamma may have a role in regulating adipocyte differentiation.