Identification of Peucedanum japonicum Thunb. extract components and their protective effects against dexamethasone-induced muscle atrophy.

BACKGROUND: Peucedanum japonicum Thunb. (PJ) is a vegetable widely consumed in East Asia and is known to have anticancer and anti-inflammatory effects. However, the effect of PJ on muscle atrophy remains elusive. PURPOSE: This study aimed to investigate the effect of PJ and its active compound on dexamethasone (DEX)-induced muscle atrophy. METHODS: We performed qualitative and quantitative analysis of PJ using ultra-performance liquid chromatography-mass spectrometry tandem mass spectrometry (UPLC-MS/MS) and high-performance liquid chromatography (HPLC), respectively. The efficacy of PJ and its main compound 4-caffeoylquinic acid (CQA) on muscle atrophy was evaluated in DEX-induced myotube atrophy and DEX-induced muscle atrophy in mouse myoblasts (C2C12) and C57BL/6 mice, in vitro and in vivo, respectively. RESULTS: The UPLC-MS/MS and HPLC data showed that the concentration of 4-CQA in PJ was 18.845 mg/g. PJ and 4-CQA treatments significantly inhibited DEX-induced myotube atrophy by decreasing protein synthesis and glucocorticoid translocation to the nucleus in C2C12 myotubes. In addition, PJ enhanced myogenesis by upregulating myogenin and myogenic differentiation 1 in C2C12 cells. PJ supplementation effectively increased muscle function and mass, downregulated atrogenes, and decreased proteasome activity in C57BL/6 mice. Additionally, PJ effectively decreased the nuclear translocation of forkhead transcription factor 3 alpha by inhibiting glucocorticoid receptor. CONCLUSION: Overall, PJ and its active compound 4-CQA alleviated skeletal muscle atrophy by inhibiting protein degradation. Hence, our findings present PJ as a potential novel pharmaceutical candidate for the treatment of muscle atrophy.

Glutamine supplementation accelerates functional recovery of EDL muscles after injury by modulating the expression of S100 calcium-binding proteins.

The aim of the current study was to investigate the effect of glutamine supplementation on the expression of HSP70 and the calcium-binding proteins from the S100 superfamily in the recovering extensor digitorum longus (EDL) muscle after injury. Two-month-old Wistar rats were subjected to cryolesion of the EDL muscle and then randomly divided into two groups (with or without glutamine supplementation). Starting immediately after the injury, the supplemented group received daily doses of glutamine (1 g/kg/day, via gavage) for 3 and 10 days orally. Then, muscles were subjected to histological, molecular, and functional analysis. Glutamine supplementation induced an increase in myofiber size of regenerating EDL muscles and prevented the decline in maximum tetanic strength of these muscles evaluated 10 days after injury. An accelerated upregulation of myogenin mRNA levels was detected in glutamine-supplemented injured muscles on day 3 post-cryolesion. The HSP70 expression increased only in the injured group supplemented with glutamine for 3 days. The increase in mRNA levels of NF-κB, the pro-inflammatory cytokines IL-1ß and TNF-α, and the calcium-binding proteins S100A8 and S100A9 on day 3 post-cryolesion in EDL muscles was attenuated by glutamine supplementation. In contrast, the decrease in S100A1 mRNA levels in the 3-day-injured EDL muscles was minimized by glutamine supplementation. Overall, our results suggest that glutamine supplementation accelerates the recovery of myofiber size and contractile function after injury by modulating the expression of myogenin, HSP70, NF-κB, pro-inflammatory cytokines, and S100 calcium-binding proteins.

Myogenic Differentiation of iPS Cells Shows Different Efficiency in Simultaneous Comparison of Protocols.

Induced pluripotent stem (iPS) cells constitute a perfect tool to study human embryo development processes such as myogenesis, thanks to their ability to differentiate into three germ layers. Currently, many protocols to obtain myogenic cells have been described in the literature. They differ in many aspects, such as media components, including signaling modulators, feeder layer constituents, and duration of culture. In our study, we compared three different myogenic differentiation protocols to verify, side by side, their efficiency. Protocol I was based on embryonic bodies differentiation induction, ITS addition, and selection with adhesion to collagen I type. Protocol II was based on strong myogenic induction at the embryonic bodies step with BIO, forskolin, and bFGF, whereas cells in Protocol III were cultured in monolayers in three special media, leading to WNT activation and TGF-ß and BMP signaling inhibition. Myogenic induction was confirmed by the hierarchical expression of myogenic regulatory factors MYF5, MYOD, MYF6 and MYOG, as well as the expression of myotubes markers MYH3 and MYH2, in each protocol. Our results revealed that Protocol III is the most efficient in obtaining myogenic cells. Furthermore, our results indicated that CD56 is not a specific marker for the evaluation of myogenic differentiation.

Selenium alleviates the negative effect of heat stress on myogenic differentiation of C2C12 cells with the response of selenogenome.

With the globe warming, heat stress (HS) has frequently affected animal production. Selenium (Se) is an essential trace element for animals and exerts most of its biological functions through selenoproteins. We previously demonstrated that the damage to C2C12 cells by HS accompanied with the response of selenoprotein encoding genes and proteins. The objective of this study was to investigate whether selenium supplementation (sodium selenite, SS and selenomethionine, SeMet) could alleviate the negative effect of heat stress on the differentiation of C2C12 cells, and interpret the potential corresponding selenoproteins response. The differentiated cells were cultured for 4 and 8 days under different condition: at 37 °C, 41.5 °C and 41.5 °C with 0.5 µmol Se/L SS or SeMet, and the HSP70, cell apoptosis, selenoproteins and cell differentiation-related gene or protein were detected. The result showed that HS up-regulated (P < 0.05) mRNA and protein levels of HSP70 and gene expression of AMPKα1 and AMPKα2, and down-regulated (P < 0.05) mRNA or protein levels of MYOGENIN and MYOD. Meanwhile, up to 15 and 17 selenoprotein genes expression were significantly changed response to 4-and 8-days HS challenge, respectively. Relative to the HS group, SS and SeMet supplementation down-regulated the mRNA and protein abundance of HSP70 to different degrees, and partly recovered (P < 0.05) the mRNA or protein abundance of MYOGENIN and MYOD at 4th and 8th day. Especially, 16 and 10 selenoprotein genes expression in cells affected by HS were altered by SS and SeMet supplementation, respectively. Both SS and SeMet supplementation modestly increased (P < 0.05) protein levels of GPX1 and SELENON in cells under HS. In summary, Se supplementation partly alleviated the negative impact of HS on myogenic differentiation of C2C12 cells and the process may associate with the alternation of selenoprotein expression pattern, and SeMet exhibits better effect than SS.

Exploring the ability of low-level laser irradiation to reduce myonecrosis and increase Myogenin transcription after Bothrops jararacussu envenomation.

Envenoming caused by snakebites is a very important neglected tropical disease worldwide. The myotoxic phospholipases present in the bothropic venom disrupt the sarcolemma and compromise the mechanisms of energy production, leading to myonecrosis. Photobiomodulation therapy (PBMT) has been used as an effective tool to treat diverse cases of injuries, such as snake venom-induced myonecrosis. Based on that, the aim of this study was to analyze the effects of PBMT through low-level laser irradiation (904 nm) on the muscle regeneration after the myonecrosis induced by Bothrops jararacussu snake venom (Bjssu) injection, focusing on myogenic regulatory factors expression, such as Pax7, MyoD, and Myogenin (MyoG). Male Swiss mice (Mus musculus), 6-8-week-old, weighing 22 ± 3 g were used. Single sub-lethal Bjssu dose or saline was injected into the right mice gastrocnemius muscle. At 3, 24, 48, and 72 h after injections, mice were submitted to PBMT treatment. When finished the periods of 48 and 72 h, mice were euthanized and the right gastrocnemius were collected for analyses. We observed extensive inflammatory infiltrate in all the groups submitted to Bjssu injections. PBMT was able to reduce the myonecrotic area at 48 and 72 h after envenomation. There was a significant increase of MyoG mRNA expression at 72 h after venom injection. The data suggest that beyond the protective effect promoted by PBMT against Bjssu-induced myonecrosis, the low-level laser irradiation was able to stimulate the satellite cells, thus enhancing the muscle repair by improving myogenic differentiation.

Promotion of myotube differentiation and attenuation of muscle atrophy in murine C2C12 myoblast cells treated with teaghrelin.

This study aimed to investigate the effects of teaghrelin, an active ingredient of Chin-shin oolong tea, on murine C2C12 myoblast cells. Under high serum conditions, teaghrelin inhibited C2C12 cell proliferation, indicating a cell cycle arrest and cessation of proliferative progression. Teaghrelin promoted pro-differentiation of C2C12 cells as evidenced by a progressively elongated morphology, as well as the induction of muscle specific myogenin, myosin heavy chain (MHC), and MyoD. The formation of multinucleated myotubes, and the increase of MHC-positive immunoreactivity within the myotubes, further reflected a complete differentiation and maturation of the contractile skeletal muscle cells induced by teaghrelin. Like ghrelin, teaghrelin attenuated dexamethasone-decreased myotube diameter, indicating its protective effects against skeletal muscle atrophy. Additionally, the expressions of Atrogin-1 and MuRF-1 ubiquitin E3 ligase were reduced. In conclusion, the results highlight a possibility of developing teaghrelin as a functional food for the prevention or therapeutic treatment of disease-associated skeletal muscle atrophy.

Effect of in ovo feeding of ß-hydroxy-ß-methylbutyrate on hatchability, muscle growth and performance in prenatal and posthatch broilers.

BACKGROUND: ß-Hydroxy-ß-methylbutyrate (HMB) is the metabolite of leucine that plays an important role in muscle protein metabolism. The objective of the present study was to determine the effects of in ovo feeding (IOF) of HMB at 7 days of incubation (DOI) via air cell or 18 DOI via amnion on hatchability, muscle growth and performance in prenatal and posthatch broilers. RESULTS: IOF of HMB via air cell at 7 DOI increased hatchability by 4.34% compared with the control (89.67% versus 85.33%). Birds in IOF groups exhibited higher body weight, average daily body weight gain and pectoral muscle percentage. Furthermore, IOF of HMB significantly increased the level of plasma growth hormone, insulin and insulin-like growth factor-1. Chicks hatched from IOF treatment had larger diameters of muscle fiber and higher mitotic activity of satellite cells at early posthatch age. IOF of HMB activated satellite cells by upregulation of mRNA expression of myogenic transcription factors, myogenic differentiation one (MyoD) and myogenin. Chicks hatched from air cell injection group had higher pectoral muscle percentage at 5 d posthatch and greater satellite cell mitotic activity at 7 d posthatch than counterparts from amnion injection group. CONCLUSIONS: IOF of HMB via amnion at 18 DOI or especially via air cell at 7 DOI could be used as an effective approach to enhance hatchability, productive performance and breast muscle yield in broilers. © 2019 Society of Chemical Industry.

The Effect of Hyperbaric Oxygen Treatment on Myoblasts and Muscles After Contusion Injury.

The recommended treatment varies depending on the severity of muscle injuries. The aim of this study was to evaluate the in vitro myoblast proliferation and the in vivo histologic and physiologic effects of hyperbaric oxygen treatment on muscle healing after contusion. Cells from the C2C12 myoblast cell line were exposed to 100% O2 for 25 min then to air for 5 min at 2.5 atmospheres absolute in a hyperbaric chamber for a total treatment duration of 90 min per 48 h at intervals of 2, 4, 6 and 8 days. Cell growth measurements and western blot analysis of myogenin and actin were performed. Then, 18 mice aged 8-10 weeks were used in the muscle contusion model. The histologic and physiologic effects and muscle regeneration after hyperbaric oxygen treatment were evaluated. The myoblast growth rate was significantly higher (p < 0.05) after hyperbaric oxygen treatment. Densitometric evaluation demonstrated a 39% (p < 0.05) and 25% (p < 0.05) increase in myogenin and actin protein levels, respectively, in the cells treated with 1 dose of hyperbaric oxygen. Similarly, the myogenin and actin protein levels increased for samples receiving multiple hyperbaric oxygen treatments when compared with the control. Physiologic evaluation of fast twitch and tetanus strength revealed a significant difference between the control group and the 14-day hyperbaric oxygen group. In conclusion, hyperbaric oxygen treatment increases the myoblast growth rate and myogenin and actin production. Better histologic and physiologic performance were found after hyperbaric oxygen treatment in animal contusion model. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:329-335, 2020.

The molecular effects of electrical stimulation on the muscle components of the urethra of female rats after trauma by vaginal distention.

AIMS: To evaluate the expression of genes and proteins related to the urethral muscles of female rats after trauma by vaginal distention (VD) and after electrical stimulation therapy (EST). METHODS: We compared the urethras of four groups of 20 animals each: control without trauma (C), 7 (recent-trauma) and 30 days (late-trauma) post-VD, and VD-treated with EST. We evaluated the expression of myogenic regulatory factors MYOD1 and myogenin (MYOG); skeletal muscle myosin heavy chain 1, 2, and 3 (MYH1, MYH2, and MYH3); smooth muscle MYH11; and myosin light chain 9 (MYL9). We used real-time quantitative polymerase chain reaction, Western blot analysis, and immunohistochemistry. RESULTS: MYOD1 and MYOG genes were overexpressed in the recent-trauma group compared with the other groups (P < .05). MYH1 and MYH3 genes were upregulated in the recent-trauma group compared with the control and EST groups (P < .05). The MYH2 gene was overexpressed in the late-trauma group (P < .05), while the MYH2 protein was significantly increased in the EST group compared with control, recent-trauma and late-trauma groups by 5-, 3-, and 2.7-fold change, respectively (P < .05). MYL9 and MYH11 messenger RNA were overexpressed in both trauma groups compared with control and EST groups (P < .05). MYH11 protein was not different among the study groups (P > .05). CONCLUSIONS: EST enhances the recovery of the damaged urethral tissue of rats mainly by acting on the striated-muscle components. The MYH2 pathway underlies the positive effects of EST in the external urethral sphincter.

Sinensetin regulates age-related sarcopenia in cultured primary thigh and calf muscle cells.

BACKGROUND: Sarcopenia, the decline of skeletal muscle tissue attributed to primary aging is a major concern in older adults. Flavonoids might have potential benefits by modulating the regulation of satellite cells, thus preventing muscle loss. Sinensetin (SIN), a citrus methylated flavone with anti-inflammatory and anti-proliferative activity, can enhance lipolysis. The objective of the present study was to investigate whether SIN might have sarcopenia-suppressing effect on satellite cells from thigh and calf muscle tissues of young and old rats. METHODS: Primary muscle cells were obtained from thigh and calf tissues of young and old group rats by dissection. Obtained satellite cells were incubated with indicated concentrations of SIN (50 and 100 µM) treated and untreated condition in differentiation medium. Morphological changes of cells were examined using a phase-contrast microscope. Protein expression levels of myoD and myogenin were analyzed by Western blot. Cells treated with or without SIN under differentiation condition were also immunocytochemically stained for myogenin and 4',6-diamidino-2-phenylindole (DAPI). RESULTS: Morphologically, the differentiation extracted satellite cells was found to be more evident in SIN treated group of aged rat's cells than that in SIN untreated group. Expression levels of myoD and myogenin proteins involved in myogenesis were increased upon treatment with SIN. CONCLUSIONS: Collectively, our results indicate that SIN can alleviate age-related sarcopenia by increasing differentiation rate and protein levels of myoD and myogenin.

Cellular and morphological changes with EAA supplementation before and after total knee arthroplasty.

Investigate the underlying cellular basis of muscle atrophy (Placebo) and atrophy reduction (essential amino acid supplementation, EAAs) in total knee arthroplasty (TKA) patients by examining satellite cells and other key histological markers of inflammation, recovery, and fibrosis. Forty-one subjects (53-76 yr) scheduled for TKA were randomized into two groups, ingesting 20 g of EAAs or placebo, twice-daily, for 7 days before TKA and for 6 wk after surgery. A first set of muscle biopsies was obtained from both legs before surgery in the operating room, and patients were randomly assigned and equally allocated to have two additional biopsies at either 1 or 2 wk after surgery. Biopsies were processed for gene expression and immunohistochemistry. Satellite cells were significantly higher in patients ingesting 20 g of essential amino acids twice daily for the 7 days leading up to surgery compared with Placebo (operative leg P = 0.03 for satellite cells/fiber and P = 0.05 for satellite cell proportions for Type I-associated cells and P = 0.05 for satellite cells/fiber for Type II-associated cells.) Myogenic regulatory factor gene expression was different between groups, with the Placebo Group having elevated MyoD expression at 1 wk and EAAs having elevated myogenin expression at 1 wk. M1 macrophages were more prevalent in Placebo than the EAAs Group. IL-6 and TNF-α transcripts were elevated postsurgery in both groups; however, TNF-α declined by 2 wk in the EAAs Group. EAAs starting 7 days before surgery increased satellite cells on the day of surgery and promoted a more favorable inflammatory environment postsurgery.NEW & NOTEWORTHY Clinical studies by our group indicate that the majority of muscle atrophy after total knee arthroplasty (TKA) in older adults occurs rapidly, within the first 2 wks. We have also shown that essential amino acid supplementation (EAAs) before and after TKA mitigates muscle atrophy; however, the mechanisms are unknown. These results suggest that satellite cell numbers are elevated with EAA ingestion before surgery, and after surgery, EAA ingestion positively influences markers of inflammation. Combined, these data may help inform further studies designed to address the accelerated sarcopenia that occurs in older adults after major surgery.

Guanidinoacetic Acid Regulates Myogenic Differentiation and Muscle Growth Through miR-133a-3p and miR-1a-3p Co-mediated Akt/mTOR/S6K Signaling Pathway.

Guanidinoacetic acid (GAA), an amino acid derivative that is endogenous to animal tissues including muscle and nerve, has been reported to enhance muscular performance. MicroRNA (miRNA) is a post-transcriptional regulator that plays a key role in nutrient-mediated myogenesis. However, the effects of GAA on myogenic differentiation and skeletal muscle growth, and the potential regulatory mechanisms of miRNA in these processes have not been elucidated. In this study, we investigated the effects of GAA on proliferation, differentiation, and growth in C2C12 cells and mice. The results showed that GAA markedly inhibited the proliferation of myoblasts, along with the down-regulation of cyclin D1 (CCND1) and cyclin dependent kinase 4 (CDK4) mRNA expression, and the upregulation of cyclin dependent kinase inhibitor 1A (P21) mRNA expression. We also demonstrated that GAA treatment stimulated myogenic differentiation 1 (MyoD) and myogenin (MyoG) mRNA expression, resulting in an increase in the myotube fusion rate. Meanwhile, GAA supplementation promoted myotube growth through increase in total myosin heavy chain (MyHC) protein level, myotubes thickness and gastrocnemius muscle cross-sectional area. Furthermore, small RNA sequencing revealed that a total of eight miRNAs, including miR-133a-3p and miR-1a-3p cluster, showed differential expression after GAA supplementation. To further study the function of miR-133a-3p and miR-1a-3p in GAA-induced skeletal muscle growth, we transfected miR-133a-3p and miR-1a-3p mimics into myotube, which also induced muscle growth. Through bioinformatics and a dual-luciferase reporter system, the target genes of miR-133a-3p and miR-1a-3p were determined. These two miRNAs were shown to modulate the Akt/mTOR/S6K signaling pathway by restraining target gene expression. Taken together, these findings suggest that GAA supplementation can promote myoblast differentiation and skeletal muscle growth through miR-133a-3p- and miR-1a-3p-induced activation of the AKT/mTOR/S6K signaling pathway.

Preclinical characterization of the JAK/STAT inhibitor SGI-1252 on skeletal muscle function, morphology, and satellite cell content.

BACKGROUND: Recent studies have highlighted the JAK/STAT signaling pathway in the regulation of muscle satellite cell behavior. Herein we report preclinical studies designed to characterize the effects of a novel JAK/STAT inhibitor on plantar flexor skeletal muscle function, morphology, and satellite cell content. METHODS: The compound, SGI-1252, was administered orally (400mg/kg) in a 10% dextrose solution to wild type mice (n = 6) 3 times per week for 8 weeks. A control group (n = 6) received only the dextrose solution. RESULTS: SGI-1252 was well tolerated, as animals displayed similar weight gain over the 8-week treatment period. Following treatment, fatigue in the gastrocnemius-soleus-plantaris complex was greater in the SGI-1252 mice during a 300 second tetanic contraction bout (p = 0.035), though both the rate of fatigue and maximal force production were similar. SGI-1252 treated mice had increased type II myofiber cross-sectional area (1434.8 ± 225.4 vs 1754.7 ± 138.5 µm2), along with an increase in wet muscle mass (125.45 ± 5.46 vs 139.6 ± 12.34 mg, p = 0.032) of the gastrocnemius relative to vehicle treated mice. SGI-1252 treatment reduced gastrocnemius STAT3 phosphorylation 53% (94.79 ± 45.9 vs 44.5 ± 6.1 MFI) and significantly increased the concentration of Pax7+ satellite cells (2589.2 ± 105.5 vs 2859.4 ± 177.5 SC/mm3) in the gastrocnemius. SGI-1252 treatment suppressed MyoD (p = 0.013) and Myogenin (p<0.0001) expression in human primary myoblasts, resulting in reduced myogenic differentiation (p = 0.039). CONCLUSIONS: Orally delivered SGI-1252 was well tolerated, attenuates skeletal muscle STAT3 activity, and increases satellite cell content in mouse gastrocnemius muscle, likely by inhibiting myogenic progression.

Dietary tributyrin, an HDAC inhibitor, promotes muscle growth through enhanced terminal differentiation of satellite cells.

Muscle growth and repair rely on two main mechanisms - myonuclear accretion and subsequent protein accumulation. Altering the ability of muscle resident stem cells (satellite cells) to progress through their myogenic lineage can have a profound effect on lifetime muscle growth and repair. The use of the histone deacetylase (HDAC) inhibitor, butyrate, has had positive outcomes on the in vitro promotion of satellite cell myogenesis. In animal models, the use of butyrate has had promising results in treating myopathic conditions as well as improving growth efficiency, but the impact of dietary butyrate on satellite cells and muscle growth has not been elucidated. We investigated the impact of tributyrin, a butyrate prodrug, on satellite cell activity and muscle growth in a piglet model. Satellite cells from tributyrin-treated piglets had altered myogenic potential, and piglets receiving tributyrin had a ~40% increase in DNA:protein ratio after 21 days, indicating the potential for enhanced muscle growth. To assess muscle growth potential, piglets were supplemented tributyrin (0.5%) during either the neonatal phase (d1-d21) and/or the nursery phase (d21-d58) in a 2 × 2 factorial design. Piglets who received tributyrin during the neonatal phase had improved growth performance at the end of the study and had a ~10% larger loin eye area and muscle fiber cross-sectional area. Tributyrin treatment in the nursery phase alone did not have a significant effect on muscle growth or feed efficiency. These findings suggest that tributyrin is a potent promoter of muscle growth via altered satellite cell myogenesis.

Genome-wide survey reveals dynamic effects of folate supplement on DNA methylation and gene expression during C2C12 differentiation.

Folic acid supplements taken during pregnancy can prevent neural tube defects and other developmental abnormalities. Here, we explored the effects of folate supplementation on gene expression and DNA methylation during C2C12 differentiation. Based on the folic acid concentration, this study comprised three groups: low folate (L), normal folate (N), and high-folate supplement (H). Our analyses revealed that differentiation and the mRNA expression of the gene myogenin in C2C12 cell were enhanced by folic acid; however, the overall methylation percentage in myogenin promoter between different treatment groups was not significantly different ( P > 0.05). The results of MeDIP-chip showed that hundreds of differentially methylated regions (DMRs) were identified between every two groups in both promoter and CpG islands, respectively. Genes with DMRs between N and L groups were mainly enriched in the processes of cell differentiation and cell development, whereas those with DMRs between H and N groups were frequently enriched in cellular process/cycle and cell metabolic processes. In addition, correlation analysis between methylation profile and expression profile revealed that some genes were regulated by methylation status directly. Together, these analyses suggest that folate deficiency and supplementation can influence the differentiation, genome-wide DNA methylation level and the expression of myogenesis-related genes including myogenin in the C2C12 cell line.

Epac2a-knockout mice are resistant to dexamethasone-induced skeletal muscle atrophy and short-term cold stress.

Exchange protein directly activated by cAMP (Epac) 2a-knockout (KO) mice exhibit accelerated diet-induced obesity and are resistant to leptin-mediated adipostatic signaling from the hypothalamus to adipose tissue, with sustained food intake. However, the impact of Epac2a deficiency on hypothalamic regulation of sympathetic nervous activity (SNA) has not been elucidated. This study was performed to elucidate the response of Epac2a-KO mice to dexamethasone-induced muscle atrophy and acute cold stress. Compared to age-matched wild-type mice, Epac2a-KO mice showed higher energy expenditures and expression of myogenin and uncoupling protein-1 in skeletal muscle (SM) and brown adipose tissue (BAT), respectively. Epac2a-KO mice exhibited greater endurance to dexamethasone and cold stress. In wild-type mice, exogenous leptin mimicked the responses observed in Epac2a-KO mice. This suggests that leptin-mediated hypothalamic signaling toward SNA appears to be intact in these mice. Hence, the potentiated responses of SM and BAT may be due to their high plasma leptin levels. [BMB Reports 2018; 51(1): 39-44].

(2R,3S,2″R,3″R)-Manniflavanone Protects Proliferating Skeletal Muscle Cells against Oxidative Stress and Stimulates Myotube Formation.

We investigated the antioxidative properties of (2R,3S,2″R,3″R)-manniflavanone (MF) using in vitro assays and examined its effects on myogenesis and lactate-induced oxidative stress in C2C12 cells. MF was purified from Garcinia buchananii stem bark. H2O2 and oxygen radical absorbance capacity assays demonstrated that MF is a powerful antioxidant. This finding was supported by diphenylpicrylhydrazine radical scavenging activity of MF. MF was less cytotoxic to C2C12 cells compared to ascorbic acid and myricetin. Moreover, MF accelerated myotube formation in the differentiated C2C12 cells by up-regulating myogenic proteins such as MyoG and myosin heavy chain. Furthermore, MF rescued late differentiation of myoblast suppressed by lactate treatment and up-regulated the expression levels of Nrf2 in lactate-induced oxidative stress, indicating that MF stimulates antioxidative activity inside C2C12 cells. Collectively, MF is a potent antioxidant with a higher safety profile than ascorbic acid and myricetin. It reduces oxidative stress-induced delaying of skeletal muscle differentiation by scavenging reactive oxygen species and regulating myogenic proteins factors.

(-)-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.

Sex-Specific Muscular Maturation Responses Following Prenatal Exposure to Methylation-Related Micronutrients in Pigs.

Supplementation of micronutrients involved in DNA methylation, particularly during pregnancy, is recommended because of its impacts on human health, but further evidence is needed regarding the effects of over-supplementation and differences between sexes. Here, a porcine model was used to assess effects of maternal supplementation with one-carbon-cycle compounds during prenatal and postnatal stages on offspring muscle development. Sows received either a standard diet (CON) or a standard diet supplemented with folate, B6, B12, methionine, choline, and zinc (MET) throughout gestation. Myogenesis-, growth-, and nutrient utilization-related transcript expression was assessed using quantitative PCR. Organismal phenotype and gene expression effects differed significantly between males and females. Male MET-offspring showed increased fetal weight during late pregnancy but decreased live weight postnatally, with compensatory transcriptional responses comprising myogenic key drivers (Pax7, MyoD1, myogenin). In contrast, female weights were unaffected by diet, and mRNA abundances corresponded to a phenotype of cellular reorganization via FABP3, FABP4, SPP1 and Insulin-like Growth Factor-signaling. These findings in an animal model suggest that supplementation during pregnancy with methylation-related micronutrients can promote sex-specific myogenic maturation processes related to organismal growth and muscle metabolism. The usage of maternal dietary supplements should be more carefully considered regarding its ability to promote fetal and postnatal health.

Nucleoprotein supplementation enhances the recovery of rat soleus mass with reloading after hindlimb unloading-induced atrophy via myonuclei accretion and increased protein synthesis.

Hindlimb unloading results in muscle atrophy and a period of reloading has been shown to partially recover the lost muscle mass. Two of the mechanisms involved in this recovery of muscle mass are the activation of protein synthesis pathways and an increase in myonuclei number. The additional myonuclei are provided by satellite cells that are activated by the mechanical stress associated with the reloading of the muscles and eventually incorporated into the muscle fibers. Amino acid supplementation with exercise also can increase skeletal muscle mass through enhancement of protein synthesis and nucleotide supplements can promote cell cycle activity. Therefore, we hypothesized that nucleoprotein supplementation, a combination of amino acids and nucleotides, would enhance the recovery of muscle mass to a greater extent than reloading alone after a period of unloading. Adult rats were assigned to 4 groups: control, hindlimb unloaded (HU; 14 days), reloaded (5 days) after hindlimb unloading (HUR), and reloaded after hindlimb unloading with nucleoprotein supplementation (HUR + NP). Compared with the HUR group, the HUR + NP group had larger soleus muscles and fiber cross-sectional areas, higher levels of phosphorylated rpS6, and higher numbers of myonuclei and myogenin-positive cells. These results suggest that nucleoprotein supplementation has a synergistic effect with reloading in recovering skeletal muscle properties after a period of unloading via rpS6 activation and satellite cell differentiation and incorporation into the muscle fibers. Therefore, this supplement may be an effective therapeutic regimen to include in rehabilitative strategies for a variety of muscle wasting conditions such as aging, cancer cachexia, muscular dystrophy, bed rest, and cast immobilization.