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Abstract Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available. In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC–MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided. In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.

ABSTRACTTargeting disorders of glucose metabolism and the occurrence of hyperglycemia provides a potential therapeutic strategy for balancing energy to reduce insulin resistance such as type 2 diabetes. The present study investigated whether theasaponin E1 regulate energy metabolism in skeletal muscle cells. C2C12 mouse myoblasts were differentiated into myotubes . An MTT colorimetric assay verified cell viability. Theasaponin E1 (30, 45 or 60 mg/ ml), or metformin (2.5 mM), insulin (150 nM) for reference were used to treat the C2C12 myotubes. The expressions of energy metabolism were measured by western blotting in C2C12 myotubes. 2-NBDG kit to detect the glucose uptake capacity. Theasaponin E1 Treatment increases glucose uptake,GSK 3 β, as well as increases to the glycogen synthesis through the PI3K/AKT/mTOR signaling pathway, while reducing AMPK and ACC activities, and reducing the adipose synthesis pathway. These results suggest that theasaponin E1 may be increases glucose uptake by improving muscle energy metabolism.

Objective To investigate the effect and mechanism of bromodomain containing 2(Brd2) on mouse C2C12 myoblast differentiation. Methods The expression of Brd2 in skeletal muscle tissues of muscular dystrophy patients was identified via Gene Expression Omnibus (GEO). Using differentiation medium that contained 2% horse serum to induce C2C12 myogenic differentiation， Brd2 protein level was detected by Western blot assay during the progression. Brd2 was knocked down or overexpressed in C2C12 cells by lentivirus-mediated transfection， cell morphological changes and protein levels of some differentiation markers were determined by immunofluorescence or Western blot assay during the differentiation process. Additionally， transcriptome sequence analysis and its related experiments were used to explore the possible regulatory signaling pathways of Brd2 in C2C12 myogenic differentiation. Results Brd2 is low expressed in skeletal muscle of muscular dystrophy patients. In the process of inducing myogenic differentiation， Brd2 expression increased on day 2 and decreased on day 8. Knockdown of Brd2 inhibited the differentiation of myoblasts， while overexpression of Brd2 promoted the process. Wnt/β -catenin signaling pathway was regulated by Brd2. Conclusion Brd2 promotes myogenic differentiation via Wnt/β -catenin signaling pathway， suggesting that Brd2 may be beneficial for skeletal muscle regeneration. [ABSTRACT FROM AUTHOR]

Albizia julibrissin saponin active fraction (AJSAF), is a prospective adjuvant with dual Th1/Th2 and Tc1/Tc2 potentiating activity. Its adjuvant activity has previously been proven to be strictly dependent on its spatial co-localization with antigens, highlighting the role of local innate immunity in its mechanisms. However, its potential targets and pathways remain unclear. Here, its intracellular molecular mechanisms of innate immune response were explored using mouse C2C12 myoblast by integrative analysis of the in vivo and in vitro transcriptome in combination with experimental validations. AJSAF elicited a temporary cytotoxicity and inflammation towards C2C12 cells. Gene set enrichment analysis demonstrated that AJSAF regulated similar cell death- and inflammatory response-related genes in vitro and in vivo through activating second messenger–MAPK–CREB pathways. AJSAF markedly enhanced the Ca2+, cAMP, and reactive oxygen species levels and accelerated MAPK and CREB phosphorylation in C2C12 cells. Furthermore, Ca2+ chelator, CREB inhibitor, and MAPK inhibitors dramatically blocked the up-regulation of IL-6, CXCL1, and COX2 in AJSAF-treated C2C12 cells. Collectively, these results demonstrated that AJSAF induced innate immunity via Ca2+–MAPK–CREB pathways. This study is beneficial for insights into the molecular mechanisms of saponin adjuvants. [ABSTRACT FROM AUTHOR]

Skeletal muscle maintenance depends largely on muscle stem cells (satellite cells) that supply myoblasts required for muscle regeneration and growth. The ubiquitin–proteasome system is the major intracellular protein degradation pathway. We previously reported that proteasome dysfunction in skeletal muscle significantly impairs muscle growth and development. Furthermore, the inhibition of aminopeptidase, a proteolytic enzyme that removes amino acids from the termini of peptides derived from proteasomal proteolysis, impairs the proliferation and differentiation ability of C2C12 myoblasts. However, no evidence has been reported on the role of aminopeptidases with different substrate specificities on myogenesis. In this study, therefore, we investigated whether the knockdown of aminopeptidases in differentiating C2C12 myoblasts affects myogenesis. The knockdown of the X‐prolyl aminopeptidase 1, aspartyl aminopeptidase, leucyl‐cystinyl aminopeptidase, methionyl aminopeptidase 1, methionyl aminopeptidase 2, puromycine‐sensitive aminopeptidase, and arginyl aminopeptidase like 1 gene in C2C12 myoblasts resulted in defective myogenic differentiation. Surprisingly, the knockdown of leucine aminopeptidase 3 (LAP3) in C2C12 myoblasts promoted myogenic differentiation. We also found that suppression of LAP3 expression in C2C12 myoblasts resulted in the inhibition of proteasomal proteolysis, decreased intracellular branched‐chain amino acid levels, and enhanced mTORC2‐mediated AKT phosphorylation (S473). Furthermore, phosphorylated AKT induced the translocation of TFE3 from the nucleus to the cytoplasm, promoting myogenic differentiation through increased expression of myogenin. Overall, our study highlights the association of aminopeptidases with myogenic differentiation. [ABSTRACT FROM AUTHOR]

The growth and development of skeletal muscle also determine the meat production of yak, ultimately affecting the economic benefits. Hence, improving growth performance is a top priority in the yak industry. Skeletal muscle development is a complex process involving the regulation of several genes, including microRNAs (miRNAs). However, the transcription of miRNAs in yak skeletal muscle during prenatal to postnatal stages is unknown. We used small RNA sequencing (small RNA-Seq) to determine the global miRNAs of longissimus dorsi muscle from yak (the samples were collected from three fetuses and three adults). Totally 264 differently expressed miRNAs (|log2(fold change)|>1 and P-value≤0.05) were detected between the two groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differently expressed miRNAs-targeted genes participated in pathways associated with muscle development, such as MAPK, PI3K-Akt, and Hippo signaling pathways, etc. MiR-652, which was up-regulated in the fetal group, was transfected into C2C12 myoblasts to examine its role. miR-652 promoted (P≤0.05) proliferation and differentiation, but inhibited (P≤0.001) apoptosis at early period. Furthermore, miR-652 reduced (P≤0.001) the proportion of C2C12 myoblasts in the G1 phase while increasing (P≤0.01) the proportion of cells in the S and G2 phases. Dual-luciferase reporter assays indicated that ISL1 served as a target of miR-652. In general, these findings expand our understanding of yak skeletal muscle miRNAs, and suggested that miR-652 probably regulated myogenesis by regulating ISL1.

Background: Sarcopenia is a new and emerging risk factor aggravating the quality of life of elderly population. Because Korean Red Ginseng (RG) is known to have a great effect on relieving fatigue and enhancing physical performance, it is invaluable to examine its potential as an anti-sarcopenic drug. Methods: Anti-sarcopenic effect of non-saponin fraction of Korean Red Ginseng (RGNS) was evaluated in C2C12 myoblasts treated with C2-ceramide to induce senescence phenotypes, and 22-month-old mice fed with chow diet containing 2% RGNS (w/w) for 4 further months. Results: The RGNS treatment significantly alleviated cellular senescence indicated by intracellular lipid accumulation, increased amount of lysosomal β-galactosidase, and reduced proliferative capacity in C2C12 myoblasts. This effect was not observed with saponin fraction. In an aged mouse, the 4-month-RGNS diet significantly improved aging-associated loss of muscle mass and strength, assessed by the weights of hindlimb skeletal muscles such as tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (GN) and soleus (SOL), and the cross-sectional area (CSA) of SOL muscle, and the behaviors in grip strength and hanging wire tests, respectively. During the same period, an aging-associated shift of fast-to slow-twitch muscle in SOL muscle was also retarded by the RGNS treatment. Conclusions: These findings suggested that the long-term diet of RGNS significantly prevented aging-associated muscle atrophy and reduced physical performance, and thus RGNS has a strong potential to be developed as a drug that prevents or improves sarcopenia.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial regulatory roles in many biological processes, including the growth and development of skeletal muscle. miRNA-100-5p is often associated with tumor cell proliferation and migration. This study aimed to uncover the regulatory mechanism of miRNA-100-5p in myogenesis. In our study, we found that the miRNA-100-5p expression level was significantly higher in muscle tissue than in other tissues in pigs. Functionally, this study shows that miR-100-5p overexpression significantly promotes the proliferation and inhibits the differentiation of C2C12 myoblasts, whereas miR-100-5p inhibition results in the opposite effects. Bioinformatic analysis predicted that Trib2 has potential binding sites for miR-100-5p at the 3′UTR region. A dual-luciferase assay, qRT-qPCR, and Western blot confirmed that Trib2 is a target gene of miR-100-5p. We further explored the function of Trib2 in myogenesis and found that Trib2 knockdown markedly facilitated proliferation but suppressed the differentiation of C2C12 myoblasts, which is contrary to the effects of miR-100-5p. In addition, co-transfection experiments demonstrated that Trib2 knockdown could attenuate the effects of miR-100-5p inhibition on C2C12 myoblasts differentiation. In terms of the molecular mechanism, miR-100-5p suppressed C2C12 myoblasts differentiation by inactivating the mTOR/S6K signaling pathway. Taken together, our study results indicate that miR-100-5p regulates skeletal muscle myogenesis through the Trib2/mTOR/S6K signaling pathway. [ABSTRACT FROM AUTHOR]

Excessive mechanical traction damages the levator ani muscle (LAM), increasing the incidence of pelvic floor dysfunction (PFD). In this study, we explored the effects of oxidized nicotinamide adenine dinucleotide (NAD+) on the damage to both muscle cells and LAM tissue induced by mechanical stress (MS) at the cellular and animal levels. The cell damage model was established using a four-point bending system. The LAM damage model was established using vaginal distention and traction. Exogenous addition of PJ34, an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1), and the nicotinamide mononucleotide (NMN) precursor of NAD+ increased NAD+ levels. ATP content and mitochondrial membrane potential were measured to assess mitochondrial function. NAD+ levels, cell viability, and PARP-1 activity were detected using commercial kits. DNA damage in cells was detected with immunofluorescence staining, and LAM damage was detected with tissue TUNEL staining. PARP-1 activity and DNA damage of LAM were detected by immunohistochemistry. A small amount of DNA damage and PARP-1 activation did not affect NAD+ levels, while excessive DNA damage and PARP-1 activation led to an imbalance of NAD+ homeostasis. Furthermore, increasing NAD+ levels in vivo and in vitro could rescue mitochondrial dysfunction and damage to both muscle cells and LAM tissue induced by MS. In conclusion, MS can induce damage to both C2C12 cells and LAM tissue. Restoring NAD+ homeostasis can rescue this damage by improving mitochondrial function.

Targeting disorders of glucose metabolism and the occurrence of hyperglycemia provides a potential therapeutic strategy for balancing energy to reduce insulin resistance such as type 2 diabetes. The present study investigated whether theasaponin E1 regulate energy metabolism in skeletal muscle cells. C2C12 mouse myoblasts were differentiated into myotubes. An MTT colorimetric assay verified cell viability. Theasaponin E1 (30, 45 or 60 mg/ ml), or metformin (2.5 mM), insulin (150 nM) for reference were used to treat the C2C12 myotubes. The expressions of energy metabolism were measured by western blotting in C2C12 myotubes. 2-NBDG kit to detect the glucose uptake capacity. Theasaponin E1 Treatment increases glucose uptake,GSK 3 β, as well as increases to the glycogen synthesis through the PI3K/AKT/mTOR signaling pathway, while reducing AMPK and ACC activities, and reducing the adipose synthesis pathway. These results suggest that theasaponin E1 may be increases glucose uptake by improving muscle energy metabolism. [ABSTRACT FROM AUTHOR]

Albizia julibrissin saponin active fraction (AJSAF), is a prospective adjuvant with dual Th1/Th2 and Tc1/Tc2 potentiating activity. Its adjuvant activity has previously been proven to be strictly dependent on its spatial co-localization with antigens, highlighting the role of local innate immunity in its mechanisms. However, its potential targets and pathways remain unclear. Here, its intracellular molecular mechanisms of innate immune response were explored using mouse C2C12 myoblast by integrative analysis of the in vivo and in vitro transcriptome in combination with experimental validations. AJSAF elicited a temporary cytotoxicity and inflammation towards C2C12 cells. Gene set enrichment analysis demonstrated that AJSAF regulated similar cell death- and inflammatory response-related genes in vitro and in vivo through activating second messenger–MAPK–CREB pathways. AJSAF markedly enhanced the Ca2+, cAMP, and reactive oxygen species levels and accelerated MAPK and CREB phosphorylation in C2C12 cells. Furthermore, Ca2+ chelator, CREB inhibitor, and MAPK inhibitors dramatically blocked the up-regulation of IL-6, CXCL1, and COX2 in AJSAF-treated C2C12 cells. Collectively, these results demonstrated that AJSAF induced innate immunity via Ca2+–MAPK–CREB pathways. This study is beneficial for insights into the molecular mechanisms of saponin adjuvants.

Age-associated loss of skeletal muscle mass and function is one of the main causes of the loss of independence and physical incapacitation in the geriatric population. This study used the D-galactose-induced C2C12 myoblast aging model to explore whether nobiletin (Nob) could delay skeletal muscle aging and determine the associated mechanism. The results showed that Nob intervention improved mitochondrial function, increased ATP production, reduced reactive oxygen species (ROS) production, inhibited inflammation, and prevented apoptosis as well as aging. In addition, Nob improved autophagy function, removed misfolded proteins and damaged organelles, cleared ROS, reduced mitochondrial damage, and improved skeletal muscle atrophy. Moreover, our results illustrated that Nob can not only enhance mitochondrial function, but can also enhance autophagy function and the protein synthesis pathway to inhibit skeletal muscle atrophy. Therefore, Nob may be a potential candidate for the prevention and treatment of age-related muscle decline. [ABSTRACT FROM AUTHOR]

Excessive mechanical traction damages the levator ani muscle (LAM), increasing the incidence of pelvic floor dysfunction (PFD). In this study, we explored the effects of oxidized nicotinamide adenine dinucleotide (NAD+) on the damage to both muscle cells and LAM tissue induced by mechanical stress (MS) at the cellular and animal levels. The cell damage model was established using a four-point bending system. The LAM damage model was established using vaginal distention and traction. Exogenous addition of PJ34, an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1), and the nicotinamide mononucleotide (NMN) precursor of NAD+ increased NAD+ levels. ATP content and mitochondrial membrane potential were measured to assess mitochondrial function. NAD+ levels, cell viability, and PARP-1 activity were detected using commercial kits. DNA damage in cells was detected with immunofluorescence staining, and LAM damage was detected with tissue TUNEL staining. PARP-1 activity and DNA damage of LAM were detected by immunohistochemistry. A small amount of DNA damage and PARP-1 activation did not affect NAD+ levels, while excessive DNA damage and PARP-1 activation led to an imbalance of NAD+ homeostasis. Furthermore, increasing NAD+ levels in vivo and in vitro could rescue mitochondrial dysfunction and damage to both muscle cells and LAM tissue induced by MS. In conclusion, MS can induce damage to both C2C12 cells and LAM tissue. Restoring NAD+ homeostasis can rescue this damage by improving mitochondrial function. [ABSTRACT FROM AUTHOR]

It is already well known that castration improves marbling quality but exact timing of castration is still highly debated in beef cattle production industry. After castration, blood hormonal changes occur in steer and objective of this study was to investigate the effects of growth hormone (GH) levels on adipocyte differentiation in stromal vascular cells (SVCs) and transdifferentiation into adipocytes in C2C12 myoblasts. Total GH concentrations were measured via enzyme-linked immunosorbent assay (ELISA) in 24 male calves and 4 female calves. Cell proliferation, cellular triglyceride (TG) accumulation, and the cell’s lipolytic capability were measured in C2C12 myoblasts and SVCs. Myogenic, adipogenic, and brown adipocyte-specific gene expression was measured via real-time polymerase chain reaction (PCR) using SYBR green. Serum GH levels were the highest in late-castrated calves. Treatment with 5 ng/mL GH resulted in greater TG accumulation as well as increased CCAAT-enhancer-binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ expression compared to that after treatment with 15 ng/mL GH. Treatment with 5 ng/mL GH also resulted in lower myogenin (myo)G and myoD expression compared to that after treatment with 15 ng/mL GH. The expression of bone morphogenetic protein (BMP) 7 after treatment with 5 ng/mL GH was higher than that after treatment with 15 ng/mL GH. But carcass characteristics data showed no significant difference between early and late castrated steers. Therefore, our results indicate that castration timing does not seem to be inevitable determinate of carcass qualities, particularly carcass weight and marbling score in Hanwoo beef cattle.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial regulatory roles in many biological processes, including the growth and development of skeletal muscle. miRNA-100-5p is often associated with tumor cell proliferation and migration. This study aimed to uncover the regulatory mechanism of miRNA-100-5p in myogenesis. In our study, we found that the miRNA-100-5p expression level was significantly higher in muscle tissue than in other tissues in pigs. Functionally, this study shows that miR-100-5p overexpression significantly promotes the proliferation and inhibits the differentiation of C2C12 myoblasts, whereas miR-100-5p inhibition results in the opposite effects. Bioinformatic analysis predicted that Trib2 has potential binding sites for miR-100-5p at the 3′UTR region. A dual-luciferase assay, qRT-qPCR, and Western blot confirmed that Trib2 is a target gene of miR-100-5p. We further explored the function of Trib2 in myogenesis and found that Trib2 knockdown markedly facilitated proliferation but suppressed the differentiation of C2C12 myoblasts, which is contrary to the effects of miR-100-5p. In addition, co-transfection experiments demonstrated that Trib2 knockdown could attenuate the effects of miR-100-5p inhibition on C2C12 myoblasts differentiation. In terms of the molecular mechanism, miR-100-5p suppressed C2C12 myoblasts differentiation by inactivating the mTOR/S6K signaling pathway. Taken together, our study results indicate that miR-100-5p regulates skeletal muscle myogenesis through the Trib2/mTOR/S6K signaling pathway.

Skeletal muscle is an important metabolic organ of the body, and impaired skeletal muscle differentiation can result in a wide range of metabolic diseases. It has been shown that microRNAs (miRNAs) play an important role in skeletal muscle differentiation. The aim of this study was to investigate the role of mmu-miR-324-5p in the differentiation of C2C12 myoblasts and lipid droplet deposition in myotubes for future targeted therapies. We found that mmu-miR-324-5p was highly expressed in mouse skeletal muscle. Overexpression of miR-324-5p significantly inhibited C2C12 myoblast differentiation while promoting oleate-induced lipid accumulation and β-oxidation in C2C12 myoblasts. Conversely, inhibition of mmu-miR-324-5p promoted C2C12 myoblast differentiation and inhibited lipid deposition in myotubes. Mechanistically, mmu-miR-324-5p negatively regulated the expression of long non-coding Dum (lncDum) and peptidase M20 domain containing 1 (Pm20d1) in C2C12 myoblasts. Reduced lncDum expression was associated with a significant decrease in the expression of myogenesis-related genes. Knockdown of mmu-miR-324-5p increased the levels of lncDum and myogenesis-related gene expression. Following oleate-induced lipid deposition in C2C12 myoblasts, overexpression of mmu-miR-324-5p decreased the expression of Pm20d1 while increasing the expression of mitochondrial β-oxidation and long-chain fatty acid synthesis-related genes. In conclusion, we provide evidence that miR-324-5p inhibits C2C12 myoblast differentiation and promotes intramuscular lipid deposition by targeting lncDum and Pm20d1, respectively.

Skeletal muscle constitutes the largest percentage of tissue in the animal body and plays a pivotal role in the development of normal life activities in the organism. However, the regulation mechanism of skeletal muscle growth and development remains largely unclear. This study investigated the effects of Ankrd1 on the proliferation and differentiation of C2C12 myoblasts. Here, we identified Ankrd1 as a potential regulator of muscle cell development, and found that Ankrd1 knockdown resulted in the proliferation ability decrease but the differentiation level increase of C2C12 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyzes as well as RNA-seq results showed that Ankrd1 knockdown activated focal adhesion kinase (FAK)/F-actin signal pathway with most genes significantly enriched in this pathway upregulated. The integrin subunit Itga6 promoter activity is increased when Ankrd1 knockdown, as demonstrated by a dual-luciferase reporter assay. This study revealed the molecular mechanism by which Ankrd1 knockdown enhanced FAK phosphorylation activity through the alteration of integrin subunit levels, thus activating FAK/Rho-GTPase/F-actin signal pathway, eventually promoting myoblast differentiation. Our data suggested that Ankrd1 might serve as a potential regulator of muscle cell development. Our findings provide new insights into skeletal muscle growth and development and valuable references for further study of human muscle-related diseases., (© 2024 Wiley Periodicals LLC.)

Scope: Kaempferol (KMP), a bioactive flavonoid compound found in fruits and vegetables, contributes to human health in many ways but little is known about its relationship with muscle mass. The effect of KMP on C2C12 myoblast differentiation and the mechanisms that might underlie that effect are studied., Methods and Results: This study finds that KMP (1, 10 µM) increases the migration and differentiation of C2C12 myoblasts in vitro. Studying the possible mechanism underlying its effect on migration, the study finds that KMP activates Integrin Subunit Beta 1 (ITGB1) in C2C12 myoblasts, increasing p-FAK (Tyr398) and its downstream cell division cycle 42 (CDC42), a protein previously associated with cell migration. Regarding differentiation, KMP upregulates the expression of myosin heavy chain (MHC) and activates IGF1/AKT/mTOR/P70S6K. Interestingly, pretreatment with an AKT inhibitor (LY294002) and siRNA knockdown of IGF1R leads to a decrease in cell differentiation, suggesting that IGF1/AKT activation is required for KMP to induce C2C12 myoblast differentiation., Conclusion: Together, the findings suggest that KMP enhances the migration and differentiation of C2C12 myoblasts through the ITG1B/FAK/paxillin and IGF1R/AKT/mTOR pathways. Thus, KMP supplementation might potentially be used to prevent or delay age-related loss of muscle mass and help maintain muscle health., (© 2024 Wiley‐VCH GmbH.)

Age-associated loss of skeletal muscle mass and function is one of the main causes of the loss of independence and physical incapacitation in the geriatric population. This study used the D-galactose-induced C2C12 myoblast aging model to explore whether nobiletin (Nob) could delay skeletal muscle aging and determine the associated mechanism. The results showed that Nob intervention improved mitochondrial function, increased ATP production, reduced reactive oxygen species (ROS) production, inhibited inflammation, and prevented apoptosis as well as aging. In addition, Nob improved autophagy function, removed misfolded proteins and damaged organelles, cleared ROS, reduced mitochondrial damage, and improved skeletal muscle atrophy. Moreover, our results illustrated that Nob can not only enhance mitochondrial function, but can also enhance autophagy function and the protein synthesis pathway to inhibit skeletal muscle atrophy. Therefore, Nob may be a potential candidate for the prevention and treatment of age-related muscle decline.

Lactate serves not merely as an energy substrate for skeletal muscle but also regulates myogenic differentiation, leading to an elevation of reactive oxygen species (ROS) levels. The present study was focused on exploring the effects of lactate and ROS/p38 MAPK in promoting C2C12 myoblasts differentiation. Our results demonstrated that lactate increased C2C12 myoblasts differentiation at a range of physiological concentrations, accompanied by enhanced ROS contents. We used n-acetylcysteine (NAC, a ROS scavenger) pretreatment and found that it delayed lactate-induced C2C12 myoblast differentiation by upregulating Myf5 expression on days 5 and 7 and lowering MyoD and MyoG expression. The finding implies that lactate accompanies ROS-dependent manner to promote C2C12 myoblast differentiation. Additionally, lactate significantly increased p38 MAPK phosphorylation to promote C2C12 cell differentiation, but pretreatment with SB203580 (p38 MAPK inhibitor) reduced lactate-induced C2C12 myoblasts differentiation. whereas lactate pretreatment with NAC inhibited p38 MAPK phosphorylation in C2C12 cells, demonstrating that lactate mediated ROS and regulated the p38 MAPK signalling pathway to promote C2C12 cell differentiation. In conclusion, our results suggest that the promotion of C2C12 myoblasts differentiation by lactate is dependent on ROS and the p38 MAPK signalling pathway. These observations reveal a beneficial role for lactate in increasing myogenesis through ROS-sensitive mechanisms as well as providing new ideas regarding the positive impact of ROS in improving the function of skeletal muscle. [Display omitted] • Lactate promotes the differentiation of C2C12 cells. • Lactate promotes C2C12 cell differentiation in a ROS-dependent manner. • Lactate regulates C2C12 differentiation through the p38 MAPK signalling pathway. • ROS mediates the regulation of lactate on C2C12 cell differentiation through the p38 MAPK signalling pathway. [ABSTRACT FROM AUTHOR]

The catabolism of branched chain amino acids (BCAAs) is mainly carried out in skeletal muscle myofibers. It is mediated by branched chain aminotransferase 2 and branched chain alpha ketoacid dehydrogenase (BCKDH) in mitochondria for energy supply, especially during exercise. BCKDH kinase (BCKDK) is a negative regulator of BCAAs catabolism by its inhibitory phosphorylation of the BCKDH E1a subunit. The data presented in this article are related to the research article that we previously have reported entitled “Energy metabolism profile of the effects of amino acid treatment on skeletal muscle cells: Leucine inhibits glycolysis of myotubes” (Suzuki et al., 2020)[1]. In this report, we have demonstrated that 1hour treatment of BT2, an inhibitor of BCKDK, decreased the glycolysis of C2C12 differentiated myotubes compared to the control. Although BCAAs metabolism is basically assumed to be carried out in differentiated myofibers, BCKDK is expressed in both undifferentiated myoblasts and differentiated myotubes, and the biological and physiological significance of BCAAs metabolism in myoblasts is still unclear. Present data demonstrate an in vitro assessment of BT2 on C2C12 myoblasts proliferation and differentiation. The data suggest that activation of BCAAs catabolism by the BCKDK inhibitor BT2 impairs C2C12 myoblasts proliferation and differentiation.

Abstract Background Black phosphorus (BP) has emerged as a novel class of nanomaterials owing to its unique optical and electronic properties. BP, a two-dimensional (2D) nanomaterial, is a structure where phosphorenes are stacked together in layers by van der Waals interactions. However, although BP nanodots have many advantages, their biosafety and biological effect have not yet been elucidated as compared to the other nanomaterials. Therefore, it is particularly important to assess the cytotoxicity of BP nanodots for exploring their potentials as novel biomaterials. Methods BP nanodots were prepared by exfoliation with a modified ultrasonication-assisted solution method. The physicochemical properties of BP nanodots were characterized by transmission electron microscopy, dynamic light scattering, Raman spectroscopy, and X-ray diffractometry. In addition, the cytotoxicity of BP nanodots against C2C12 myoblasts was evaluated. Moreover, their cell imaging potential was investigated. Results Herein, we concentrated on evaluating the cytotoxicity of BP nanodots and investigating their cell imaging potential. It was revealed that the BP nanodots were cytocompatible at a low concentration, although the cell viability was decreased with increasing BP nanodot concentration. Furthermore, our results demonstrated that the cells took up the BP nanodots, and the BP nanodots exhibited green fluorescence. Conclusions In conclusion, our findings suggest that the BP nanodots have suitable biocompatibility, and are promising candidates as fluorescence probes for biomedical imaging applications.

Skeletal myogenesis is a highly ordered and complex biological process that is mediated by numerous regulatory factors. In previous studies, it has been demonstrated that microRNAs (miRs) and long non-coding RNAs (lncRNAs) serve key roles in skeletal myogenesis. The present study showed that the expression levels of miR-23a-5p showed a dynamic change from decrease to increase during C2C12 myoblast proliferation and differentiation. Functional analysis using 5-ethynyl-2′-deoxyuridine proliferation and Cell Counting Kit-8 detection assays indicated that overexpression of miR-23a-5p significantly promoted C2C12 myoblast proliferation compared with the negative control. In addition, in C2C12 myoblasts transfected with miR-23a-5p mimics, increased expression levels of regulators associated with cell proliferation (Cyclin E, CCND1 and Cyclin B) were observed compared with the negative control. By contrast, overexpression of miR-23a-5p decreased the expression levels of specific-myogenesis factors (MyoD, MyoG and Myf5) and decreased C2C12 myoblast differentiation. Luciferase activity assays indicated that miR-23a-5p suppressed the luciferase activity of lncDum. Further analysis demonstrated that miR-23a-5p not only showed an opposite expression level pattern compared with lncDum, which was first increased and then decreased, but also had an opposite effect on the proliferation and differentiation of C2C12 myoblasts compared with lncDum which inhibited cell proliferation and promoted cell differentiation. Taken together, these results indicated that miR-23a-5p may mediate the proliferation and differentiation of C2C12 myoblasts, which may be involved in lncDum regulation. [ABSTRACT FROM AUTHOR]

To investigate the regulatory effects of anagliptin, a DPP‐IV inhibitor used to treat type 2 diabetes mellitus (T2DM), on myoblast differentiation and mitochondrial biogenesis in C2C12 mouse skeletal muscle cells. C2C12 myoblasts were differentiated into myotubes and then treated with anagliptin (10, 25, and 50 μmol/L) for 24 hours. In C2C12 myotubes, anagliptin treatment was significantly increased the expression of MHC, PGC1α, Sirt‐1, NRF‐1, and TFAM and the phosphorylation of AMPK and ACC in a concentration‐dependent manner. Anagliptin also significantly increased the total ATP levels in the myotubes. These results suggest that anagliptin can help prevent skeletal muscle dysfunction in T2DM by promotion of myoblast differentiation and enhancement of energy production via upregulation of mitochondrial biogenetic factors and activation of the AMPK/ACC signalling pathway. [ABSTRACT FROM AUTHOR]

Osteoporosis and sarcopenia (osteosarcopenia (OS)) are twin-aging diseases. The biochemical crosstalk between muscle and bone seems to play a role in OS. We have previously shown that osteocytes produce soluble factors with beneficial effects on muscle and vice versa. Recently, enhanced FGF9 production was observed in the OmGFP66 osteogenic cell line. To test its role in myogenic differentiation, C2C12 myoblasts were treated with recombinant FGF9. FGF9 as low as 10 ng/mL inhibited myogenic differentiation, suggesting that FGF9 might be a potential inhibitory factor produced from bone cells with effects on muscle cells. FGF9 (10–50 ng/mL) significantly decreased mRNA expression of MyoG and Mhc while increasing the expression of Myostatin. Consistent with the phenotype, RT-qPCR array revealed that FGF9 (10 ng/mL) increased the expression of Icam1 while decreased the expression of Wnt1 and Wnt6 decreased, respectively. FGF9 decreased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and reduced the expression of genes (i.e. Cacna1s, RyR2, Naftc3) directly associated with intracellular Ca2+ homeostasis. Myogenic differentiation in human skeletal muscle cells was similarly inhibited by FGF9 but required higher doses of 200 ng/mL FGF9. FGF9 was also shown to stimulate C2C12 myoblast proliferation. FGF2 and the FGF9 subfamily members FGF16 and FGF20 also inhibited C2C12 myoblast differentiation and enhanced proliferation. Intriguingly, the differentiation inhibition was independent of proliferation enhancement. These findings suggest that FGF9 may modulate myogenesis via a complex signaling mechanism. [ABSTRACT FROM AUTHOR]

Abstract Background The COP9 signalosome (CSN) is a conserved protein complex composed of 8 subunits designated CSN1-CSN8. CSN3 represents the third subunit of the CSN and maintains the integrity of the complex. CSN3 binds to the striated muscle-specific β1D integrin tail, and its subcellular localization is altered in differentiated skeletal muscle cells. However, the role of CSN3 in skeletal muscle differentiation is unknown. The main goal of this study was to identify whether CSN3 participates in myoblast differentiation and the signalling mechanisms involved using C2C12 cells as a skeletal muscle cell model. Methods Small-hairpin (shRNA) was used to knockdown CSN3 in C2C12 cells. Differentiation was evaluated by immunostaining and confocal microscopy. Markers of differentiation, NF-κB signaling and CSN subunits expression, were assessed by immunoblotting and/or immunostaining. Cell proliferation was analysed by cell counting, flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were analyzed by one or two-way analysis of variance (ANOVA) followed by post-hoc testing. Results Transduction of C2C12 cells with two distinct CSN3 shRNAs led to the production of two cells lines expressing 7% of CSN3 protein (shCSN3-Low) and 43% of CSN3 protein (CSN3-Med) compared to controls. Knockdown of CSN3 was accompanied by destabilization of several CSN subunits and increased nuclear NF-κB localization. shCSN3-Med cells expressed less myogenin and formed shorter and thinner myotubes. In contrast, the shCSN3-Low cells expressed higher levels of myogenin prior and during the differentiation and remained mononucleated throughout the differentiation period. Both CSN3 knockdown cell lines failed to express sarcomeric myosin heavy chain (MHC) protein during differentiation. The fusion index was significantly higher in control cells than in shCSN3-Med cells, whereas shCSN3-Low cells showed no cell fusion. Interestingly, CSN3 knockdown cells exhibited a significantly slower growth rate relative to the control cells. Cell cycle analysis revealed that CSN3 knockdowns delayed in S phase and had increased levels of nuclear p21/Cip1 and p27/Kip1. Conclusions This study clarifies the first step toward unrevealing the CSN3/CSN-mediated pathways that controls C2C12 differentiation and proliferation. Further in vivo characterization of CSN/CSN3 may lead to the discovery of novel therapeutic target of skeletal muscle diseases such as muscular dystrophies.

Abstract Seahorse, a syngnathidae fish, is one of the important organisms used in Chinese traditional medicine. Hippocampus abdominalis, a seahorse species successfully cultured in Korea, was validated for use in food by the Ministry of Food and Drug Safety in February 2016; however. the validation was restricted to 50% of the entire composition. Therefore, to use H. abdominalis as a food ingredient, H. abdominalis has to be prepared as a mixture by adding other materials. In this study, the effect of H. abdominalis on muscles was investigated to scientifically verify its potential bioactivity. In addition, the anti-fatigue activity of a mixture comprising H. abdominalis and red ginseng (RG) was evaluated to commercially utilize H. abdominalis in food industry. H. abdominalis was hydrolyzed using Alcalase, a protease, and the effect of H. abdominalis hydrolysate (HH) on the muscles was assessed in C2C12 myoblasts by measuring cell proliferation and glycogen content. In addition, the mixtures comprising HH and RG were prepared at different percentages of RG to HH (20, 30, 40, 50, 60, 70, and 80% RG), and the anti-fatigue activity of these mixtures against oxidative stress was assessed in C2C12 myoblasts. In C2C12 myoblasts, H2O2-induced oxidative stress caused a decrease in viability and physical fatigue-related biomarkers such as glycogen and ATP contents. However, treatment with RG and HH mixtures increased cell viability and the content of fatigue-related biomarkers. In particular, the 80% RG mixture showed an optimum effect on cell viability and ATP synthesis activity. In this study, all results indicated that HH had anti-fatigue activity at concentrations approved for use in food by the law in Korea. Especially, an 80% RG to HH mixture can be used in food for ameliorating fatigue.

Background: Cachexia induced by cancer is a systemic wasting syndrome and it accompanies continuous body weight loss with the exhaustion of skeletal muscle and adipose tissue. Cancer cachexia is not only a problem in itself, but it also reduces the effectiveness of treatments and deteriorates quality of life. However, effective treatments have not been found yet. Although Arctii Fructus (AF) has been studied about several pharmacological effects, there were no reports on its use in cancer cachexia. Methods: To induce cancer cachexia in mice, we inoculated CT-26 cells to BALB/c mice through subcutaneous injection and intraperitoneal injection. To mimic cancer cachexia in vitro, we used conditioned media (CM), which was CT-26 colon cancer cells cultured medium. Results: In in vivo experiments, AF suppressed expression of interleukin (IL)-6 and atrophy of skeletal muscle and adipose tissue. As a result, the administration of AF decreased mortality by preventing weight loss. In adipose tissue, AF decreased expression of uncoupling protein 1 (UCP1) by restoring AMP-activated protein kinase (AMPK) activation. In in vitro model, CM increased muscle degradation factors and decreased adipocytes differentiation factors. However, these tendencies were ameliorated by AF treatment in C2C12 myoblasts and 3T3-L1 cells. Conclusion: Taken together, our study demonstrated that AF could be a therapeutic supplement for patients suffering from cancer cachexia.

Inflammatory conditions caused by cancer, chronic diseases or aging can lead to skeletal muscle atrophy. We identified myogenic compounds from Psoralea corylifolia (PC), a medicinal plant that has been used for the treatment of inflammatory and skin diseases. C2C12 mouse skeletal myoblasts were differentiated in the presence of eight compounds isolated from PC to evaluate their myogenic potential. Among them, corylifol A showed the strongest transactivation of MyoD and increased expression of myogenic markers, such as MyoD, myogenin and myosin heavy chain (MHC). Corylifol A increased the number of multinucleated and MHC-expressing myotubes. We also found that the p38 MAPK signaling pathway is essential for the myogenic action of corylifol A. Atrophic condition was induced by treatment with dexamethasone. Corylifol A protected against dexamethasone-induced myotube loss by increasing the proportion of multinucleated MHC-expressing myotubes compared with dexamethasone-damaged myotubes. Corylifol A reduced the expression of muscle-specific ubiquitin-E3 ligases (MAFbx and MuRF1) and myostatin, while activating Akt. These dual effects of corylifol A, inhibition of catabolic and activation of anabolic pathways, protect myotubes against dexamethasone damage. In summary, corylifol A isolated from P. corylifolia alleviates muscle atrophic condition through activating myoblast differentiation and suppressing muscle degradation in atrophic conditions.

Mechanical stretch may cause myoblasts to either proliferate or undergo apoptosis. Identifying the molecular events that switch the fate of a stretched cell from proliferation to apoptosis is practically important in the field of regenerative medicine. A recent study on vascular smooth muscle cells illustrated that identification of these events may be achieved by addressing the stretch-induced opposite cellular outcomes simultaneously within a single investigation. To define conditions or a model in which both proliferation and apoptosis can be studied at the same time, we exposed in vitro cultured C2C12 myoblasts to a cyclic mechanical stretch regimen of 15% elongation at a stretching frequency of 1 Hz for 0, 2, 4, 6, or 8 h every day, consecutively, for 3 days. Both proliferation and apoptosis were observed. Moreover, as the duration of the stretch was prolonged, cell proliferation increased until it peaked at the optimal stretching duration. Afterwards, apoptosis gradually prevailed. Therefore, we established a model in which stretch-induced cell proliferation and apoptosis can be studied simultaneously. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies of the role of 17β‐estradiol (E2) in myoblast differentiation have produced conflicting data. Therefore, this work aimed to determine the role of E2 on myoblast differentiation and specific myofiber formation. Murine C2C12 myoblasts were cultured in proliferation medium or differentiation medium/10 nM E2. The role of E2 on specific myosin heavy chain (MyHC) or estrogen receptor (ER) expression was examined using real‐time quantitative RT‐PCR (RT‐qPCR). Transcriptome studies of E2 on myoblast differentiation were accomplished by microarray analyses. The expression levels of candidate genes from microarrays and four and a half LIM domains 1 (Fhl1) were detected with RT‐qPCR. E2 in differentiation medium significantly up‐regulated MyHC I expression, but exerted the opposite effects on MyHC II a, MyHC II b, and MyHC II d. Both ER‐α and ER‐β were decreased in differentiated C2C12, and E2 partially restored ER‐β expression. Sixty‐two up‐regulated and 116 down‐regulated genes treated by E2 were identified, and RT‐qPCR validation results showed seven cytoskeletal genes (Myh8, Cenpe, Jak3, Obscn, Ldb3, Mybpc2, Col4a3bp), three genes related to ion channels (Kcnq1, Lrrc26, P2rx3) and Fhl1 transcript 2 were associated with the effects of E2 on myoblast differentiation. These findings suggested E2 helped slow type MyH I fiber formation and impeded fast 2A, 2X/D, and 2B fiber formation. [ABSTRACT FROM AUTHOR]

In this study, we examined the effect of tomatidine on tumor necrosis factor (TNF)-α-induced apoptosis in C2C12 myoblasts. TNF-α treatment increased cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) protein levels in a dose- and time-dependent manner. Pretreatment of cells with 10 μM tomatidine prevented TNF-α-induced apoptosis, caspase 3 cleavage, and PARP cleavage. Cells were treated with 100 ng/mL TNF-α for 24 h, and flow cytometry was utilized to assess apoptosis using annexin-V and 7-aminoactinomycin D. TNF-α up-regulated activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression. This effect was suppressed by pretreatment with tomatidine. Pretreatment with 4-phenylbutyric acid (a chemical chaperone) also inhibited TNF-α-induced cleavage of caspase 3 and PARP and up-regulation of ATF4 and CHOP expression. In addition, tomatidine-mediated inhibition of phosphorylation of c-Jun amino terminal kinase (JNK) attenuated TNF-α-induced cleavage of PARP and caspase 3. However, tomatidine did not affect NF-κB activation in TNF-α-treated C2C12 myoblast cells. Taken together, the present study demonstrates that tomatidine attenuates TNF-α-induced apoptosis through down-regulation of CHOP expression and inhibition of JNK activation. [ABSTRACT FROM AUTHOR]

Background: The COP9 signalosome (CSN) is a conserved protein complex composed of 8 subunits designated CSN1-CSN8. CSN3 represents the third subunit of the CSN and maintains the integrity of the complex. CSN3 binds to the striated muscle-specific β1D integrin tail and its subcellular localization is altered in differentiated skeletal muscle cells. However, the role of CSN3 in skeletal muscle differentiation is unknown. The main goal of this study was to identify whether CSN3 participates in myoblast differentiation and the signalling mechanisms involved using C2C12 cells as a skeletal muscle cell model. Methods: Small-hairpin (shRNA) was used to knockdown CSN3 in C2C12 cells. Differentiation was evaluated by immunostaining and confocal microscopy. Markers of differentiation, NF-κB signaling and CSN subunits expression, were assessed by immunoblotting and/or immunostaining. Cell proliferation was analysed by cell counting, flow cytometry and a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Data were analyzed by one or two-way analysis of variance (ANOVA) followed by post-hoc testing. Results: Transduction of C2C12 cells with two distinct CSN3 shRNAs led to the production of two cells lines expressing 7% of CSN3 protein (shCSN3-Low) and 43% of CSN3 protein (CSN3-Med) compared to controls. Knockdown of CSN3 was accompanied by destabilization of several CSN subunits and increased nuclear NF-κB localization. shCSN3-Med cells expressed less myogenin and formed shorter and thinner myotubes. In contrast, the shCSN3-Low cells expressed higher levels of myogenin prior and during the differentiation and remained mononucleated throughout the differentiation period. Both CSN3 knockdown cell lines failed to express sarcomeric myosin heavy chain (MHC) protein during differentiation. The fusion index was significantly higher in control cells than in shCSN3-Med cells, whereas shCSN3-Low cells showed no cell fusion. Interestingly, CSN3 knockdown cells exhibited a significantly slower growth rate relative to the control cells. Cell cycle analysis revealed that CSN3 knockdowns delayed in S phase and had increased levels of nuclear p21/Cip1 and p27/Kip1. Conclusions: This study clarifies the first step toward unrevealing the CSN3/CSN-mediated pathways that controls C2C12 differentiation and proliferation. Further in vivo characterization of CSN/CSN3 may lead to the discovery of novel therapeutic target of skeletal muscle diseases such as muscular dystrophies. [ABSTRACT FROM AUTHOR]

Large volume deficiencies in skeletal muscle tissue fail to heal with conservative treatments, and improved treatment methods are needed. Tissue engineered scaffolds for skeletal muscle need to mimic the optimal environment for muscle development by providing the proper electric, mechanical, and chemical cues. Electroactive polymers, polymers that change in size or shape in response to an electric field, may be able to provide the optimal environment for muscle growth. In this study, an electroactive polymer made from poly(ethylene glycol) diacrylate (PEGDA) and acrylic acid (AA) is characterized and optimized for movement and biocompatibility. Hydrogel sample thickness, overall polymer concentration, and the ratio of PEGDA to AA were found to significantly impact the actuation response. C2C12 mouse myoblast cells attached and proliferated on hydrogel samples with various ratios of PEGDA to AA. Future experiments will produce hydrogel samples combined with aligned guidance cues in the form of electrospun fibers to provide a favorable environment for muscle development. [ABSTRACT FROM AUTHOR]

Myotubularin related protein 7 (MTMR7) is a key member of the highly conserved myotubularin related proteins (MTMRs) family, which has phosphatase activity. MTMR7 was increased during myoblast differentiation and exhibited high expression level at primary fibers formation stages in pigs. This suggests that MTMR7 may be involved in myogenesis. In our study, we investigated the roles of MTMR7 on proliferation and differentiation of C2C12 myoblasts. Knocking down MTMR7 not only enhanced myoblast early differentiation via altering the expression of Myf5, but also promoted myoblast proliferation through increasing cyclinA2 expression. The improved proliferation capacity was related to the increased phosphorylation of AKT. Taken together, our research demonstrates that MTMR7 plays an important role in proliferation and early differentiation of C2C12 myoblast. [ABSTRACT FROM AUTHOR]

The stable culture of myogenic cells and their differentiation into myotubes in vitro is often hindered by the mechanical destabilisation of the spontaneously contractile neotissue formed, resulting in the complete loss of differentiating myotubes. Electrospun, parallel aligned nylon 6/6 microfibre arrays were use successfully for the culture of C2C12 myoblasts and their differentiation to form mechanically stable, orientated myotubes in vitro. Myoblasts adhered strongly to the parallel fibre array, forming a compact cell sheath across the entire array, aligning individual cells in parallel to the direction of the fibrous substratum. The myogenic potential of C2C12 myoblasts was not impaired and resulted in the formation of elongated myotubes expressing alpha-actinin, adult myosin heavy chain and nicotinic acetylcholine receptors as muscle-specific marker proteins. Newly formed C2C12 myotubes were themselves orientated in parallel to the direction of the underlying fibrous substratum and exhibited a high level of structural integration with the surrounding cells. In contrast, non-woven, non-orientated nylon 6/6 meshes, produced by conventional electrospinning, exhibited greatly reduced levels of C2C12 myoblast attachment and adherent myoblasts did not differentiate into myotubes. In conclusion, parallel microfibre arrays provided a superior microscale topography for the stable maintenance and differentiation of myotubes in vitro.

The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for skeletal muscle repair, notably, via their actions on myogenic precursor cells. However, these interactions during T. spiralis infection have not been characterized. In the present study, the ability of conditioned medium (CM) from J774A.1 macrophages treated with ML-ESPs to influence the differentiation of murine myoblasts, and the mechanisms of this influence, were investigated in vitro . The results showed that the expression of Myogenic Regulatory Factors (MRFs) MyoD and myogenin, myosin heavy chain (MyHC), and the p21 cyclin-dependent kinase inhibitor were reduced in CM treated cells compared to their expression in the control group. These findings indicated that CM inhibited myoblast differentiation. Conversely, CM promoted myoblast proliferation and increased cyclin D1 levels. Taken together, results of our study suggested that CM can indirectly influence myoblast differentiation and proliferation, which provides a new method for the elucidation of the complex mechanisms involved in cell-parasite and cell–cell interactions during T. spiralis infection. [ABSTRACT FROM AUTHOR]

This work investigates the effect of the sequential availability of ZnO nanoparticles, (nanorods of ∼40 nm) loaded within a degradable poly(lactic acid) (PLLA) matrix, in cell differentiation. The system constitutes a dynamic surface, in which nanoparticles are exposed as the polymer matrix degrades. ZnO nanoparticles were loaded into PLLA and the system was measured at different time points to characterise the time evolution of the physicochemical properties, including wettability and thermal properties. The micro and nanostructure were also investigated using AFM, SEM and TEM images. Cellular experiments with C2C12 myoblasts show that cell differentiation was significantly enhanced on ZnO nanoparticles—loaded PLLA, as the polymer degrades and the availability of nanoparticles become more apparent, whereas the release of zinc within the culture medium was negligible. Our results suggest PLLA/ZnO nanocomposites can be used as a dynamic system where nanoparticles are exposed during degradation, activating the material surface and driving cell differentiation. [ABSTRACT FROM AUTHOR]

Tissue-engineered cardiac patch aims at regenerating an infarcted heart by improving cardiac function and providing mechanical support to the diseased myocardium. In order to take advantages of electroactivity, a new synthetic method was developed for the introduction of an electroactive oligoaniline into the backbone of prepared patches. For this purpose, a series of electroactive polyurethane/siloxane films containing aniline tetramer (AT) was prepared through sol-gel reaction of trimethoxysilane functional intermediate polyurethane prepolymers made from castor oil and poly(ethylene glycol). Physicochemical, mechanical, and electrical conductivity of samples were evaluated and the recorded results were correlated to their structural characteristics. The optimized films were proved to be biodegradable and have tensile properties suitable for cardiac patch application. The embedded AT moieties in the backbone of the prepared samples preserved their electroactivity with the electrical conductivity in the range of 10−4 S/cm. The prepared films were compatible with proliferation of C2C12 and had potential for enhancing myotube formation even without external electrical stimulation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 775-787, 2016. [ABSTRACT FROM AUTHOR]