Vitamin C regulates skeletal muscle post-injury regeneration by promoting myoblast proliferation through its direct interaction with the Pax7 protein.

Previous studies have shown that vitamin C (VC), an essential vitamin for the human body, can promote the differentiation of muscle satellite cells (MuSCs) in vitro and play an important role in skeletal muscle post-injury regeneration. However, the molecular mechanism of VC regulating MuSC proliferation has not been elucidated. In this study, the role of VC in promoting MuSC proliferation and its molecular mechanism were explored using cell molecular biology and animal experiments. The results showed that VC accelerates the progress of skeletal muscle post-injury regeneration by promoting MuSC proliferation in vivo. VC can also promote skeletal muscle regeneration in the case of atrophy. Using the C2C12 myoblast murine cell line, we observed that VC also stimulated cell proliferation. In addition, after an in vitro study establishing the occurrence of a physical interaction between VC and Pax7, we observed that VC also upregulated the total and nuclear Pax7 protein levels. This mechanism increased the expression of Myf5 (Myogenic Factor 5), a Pax7 target gene. This study establishes a theoretical foundation for understanding the regulatory mechanisms underlying VC-mediated MuSC proliferation and skeletal muscle regeneration. Moreover, it develops the application of VC in animal muscle nutritional supplements and treatment of skeletal muscle-related diseases.

[Effect of oblique needling at Ashi-point on expression of MyoD1/Pax7 in mice with quadriceps femoris injury].

OBJECTIVE: To observe the effect of oblique needling at Ashi-point on behavior, and cell morphology, myogenic differentiation antigen (MyoD1) and paired box transcription factor Pax7 (Pax7) of quadriceps femoris tissue in quadriceps femoris injured mice. METHODS: A total of 24 C57BL/6 male mice were randomly divided into control, model, shallow insertion and deep insertion groups, with 6 mice in each group. The quadriceps femoris injury model was established by single intramuscular injection of 0.5% bupivacaine (BPVC). Twenty-four hours after modeling, mice of the two acupuncture groups were received oblique needling on the surface or through the muscle belly of quadriceps femoris for once, the oblique needling was lifted and inserted 3 times. The climbing pole test was conducted 24 h after modeling and 24 h after EA. Histopathological changes of quadriceps femoris was observed by H.E. staining. The expressions of MyoD1 and Pax7 were detected by immunohistochemistry. RESULTS: Compared with the control group, the score of climbing pole test was lower (P<0.01), and the expressions of MyoD1 and Pax7 significantly increased (P<0.01) in the model group. After the intervention and compared with the model group, the score of climbing pole test was higher (P<0.01), and the expressions of MyoD1 and Pax7 obviously increased (P<0.01) in the two acupuncture groups. The therapeutic effect of deep insertion group was apparently superior to that of shallow insertion group in up-regulating the climbing pole test score and expressions of MyoD1 and Pax7 (P<0.05, P<0.01). H.E. stain showed large areas of inflammatory infiltration, muscle cells swelling, atrophy, rupture, degeneration and necrosis, different cell sizes and morphologies, enlarged intervals, nuclear aggregation, deep nuclear staining, nuclear pyknosis, and hemorrhage in the model group, which was relatively milder in both needling groups. CONCLUSION: Oblique needling at Ashi-point can effectively promote the benign repair of injured quadriceps muscle and promote the recovery of exercise ability in mice, which may be associated with its effect in up-regulating the expression of MyoD1 and Pax7 protein. The role of deep insertion is superior to that of shallow insertion.

Dietary tributyrin supplementation and submaximal exercise promote activation of equine satellite cells.

Postexercise skeletal muscle repair is dependent on the actions of satellite cells (SCs). The signal(s) responsible for activation of these normally quiescent cells in the horse remain unknown. The objective of the experiment was to determine whether submaximal exercise or tributyrin (TB) supplementation is sufficient to stimulate SC activation. Adult geldings were fed a control diet (n = 6) or a diet containing 0.45% TB (n = 6). After 30 d, the geldings performed a single bout of submaximal exercise. Middle gluteal muscle biopsies and blood were collected on days -1, 1, 3, and 5 relative to exercise. Diet had no effect on any parameter of physical performance. Total RNA isolated from the gluteal muscle of TB fed geldings contained greater (P < 0.05) amounts of myogenin mRNA than controls. Satellite cell isolates from TB supplemented horses had a greater (P = 0.02) percentage of proliferating cell nuclear antigen immunopositive (PCNA+) SC than controls after 48 h in culture. Submaximal exercise was sufficient to increase (P < 0.05) the percentage of PCNA(+) cells in all isolates obtained during recovery period. No change in the amount of gluteal muscle Pax7 mRNA, a lineage marker of SCs, occurred in response to either diet or exercise. Our results indicate that both submaximal exercise and TB prime SCs for activation and cell cycle reentry but are insufficient to cause an increase in Pax7 expression during the recovery period.

A transient protective effect of low-level laser irradiation against disuse-induced atrophy of rats.

Satellite cells, a population of skeletal muscular stem cells, are generally recognized as the main and, possibly, the sole source of postnatal muscle regeneration. Previous studies have revealed the potential of low-level laser (LLL) irradiation in promoting satellite cell proliferation, which, thereby, boosts the recovery of skeletal muscle from atrophy. The purpose of this study is to investigate the beneficial effect of LLL on disuse-induced atrophy. The optimal irradiation condition of LLL (808 nm) enhancing the proliferation of Pax7+ve cells, isolated from tibialis anterior (TA) muscle, was examined and applied on TA muscle of disuse-induced atrophy model of the rats accordingly. Healthy rats were used as the control. On one hand, transiently, LLL was able to postpone the progression of atrophy for 1 week through a reduction of apoptosis in Pax7-veMyoD+ve (myocyte) population. Simultaneously, a significant enhancement was observed in Pax7+veMyoD+ve population; however, most of the increased cells underwent apoptosis since the second week, which suggested an impaired maturation of the population. On the other hand, in normal control rats with LLL irradiation, a significant increase in Pax7+veMyoD+ve cells and a significant decrease of apoptosis were observed. As a result, a strengthened muscle contraction was observed. Our data showed the capability of LLL in postponing the progression of disuse-induced atrophy for the first time. Furthermore, the result of normal rats with LLL irradiation showed the effectiveness of LLL to strengthen muscle contraction in healthy control.

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.

Folic Acid Exposure Rescues Spina Bifida Aperta Phenotypes in Human Induced Pluripotent Stem Cell Model.

Neural tube defects (NTDs) are severe congenital abnormalities, caused by failed closure of neural tube during early embryonic development. Periconceptional folic acid (FA) supplementation greatly reduces the risk of NTDs. However, the molecular mechanisms behind NTDs and the preventive role of FA remain unclear. Here, we use human induced pluripotent stem cells (iPSCs) derived from fetuses with spina bifida aperta (SBA) to study the pathophysiology of NTDs and explore the effects of FA exposure. We report that FA exposure in SBA model is necessary for the proper formation and maturation of neural tube structures and robust differentiation of mesodermal derivatives. Additionally, we show that the folate antagonist methotrexate dramatically affects the formation of neural tube structures and FA partially reverts this aberrant phenotype. In conclusion, we present a novel model for human NTDs and provide evidence that it is a powerful tool to investigate the molecular mechanisms underlying NTDs, test drugs for therapeutic approaches.

Postexercise essential amino acid supplementation amplifies skeletal muscle satellite cell proliferation in older men 24 hours postexercise.

Aged skeletal muscle has an attenuated and delayed ability to proliferate satellite cells in response to resistance exercise. The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is a focal point for cell growth, however, the effect of postexercise mTORC1 activation on human skeletal muscle satellite cell (SC) proliferation is unknown. To test the proliferative capacity of skeletal muscle SC in aging muscle to a potent mTORC1 activator (i.e., EAA; essential amino acids) we recruited older (~72y) men to conduct leg resistance exercise (8setsx10reps) without (-EAA; n = 8) and with (+EAA: n = 11) ingestion of 10 g of EAA 1 h postexercise. Muscle biopsies were taken before exercise (Pre) and 24 h postexercise (Post) for assessment of expression and fiber type-specific Pax7+ SC, Ki67+Pax7+ SC and MyoD+ SC -EAA did not show an increase in Pax7+ satellite cells at Post(P > 0.82). Although statistical significance for an increase in Pax7 +  SC at 24 h post-RE was not observed in +EAA versus -EAA, we observed trends for a treatment difference (P < 0.1). When examining the change from Pre to Post trends were demonstrated (#/myofiber: P = 0.076; and %/myonuclei: P = 0.065) for a greater increase in +EAA versus -EAA Notably, we found an increase SC proliferation in +EAA, but not -EAA with increase in Ki67+ SC and MyoD+ cells (P < 0.05). Ki67+ SC also exhibited a significant group difference Post (P < 0.010). Pax7+ SC in fast twitch myofibers did not change and were not different between groups (P > 0.10). CDK2, MEF2C, RB1 mRNA only increased in +EAA (P < 0.05). Acute muscle satellite cell proliferative capacity may be partially rescued with postexercise EAA ingestion in older men.

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.

Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

[Effect of Electroacupuncture Serum on Proliferation of Cultured Multifidus Muscle Satellite Cells and Expression of Pax-7, MyoD and p-Akt].

OBJECTIVE: To observe the effect of electroacupuncture (EA) serum on proliferation of multifidus muscle sa-tellite cells (SCs) and expression of paired box transcription factor Pax-7, MyoD and protein kinase B (PKB or Akt) proteins of SCs, so as to explore its underlying mechanism in promoting repair of multifidus muscles. METHODS: Thirty-two SD rats were randomly assigned to control, model, EA-Weizhong (BL 40) and EA-Shenshu (BL 23) groups. The multifidus muscle injury (MFMI) model was established by injection of 0.5% bupivacaine hydrochloride (400 µL) into the bilateral L4-L5 paravertebral muscles (4 points, 100 µL for each point). EA stimulation was separately applied to bilateral BL 40 and BL 23 for 20 min, once daily, 4 days altogether. Blood samples of the abdominal artery of rats in the above mentioned 4 groups were separately collected for extracting serum, followed by deactivation and filtration, and then were respectively applied to the Dulbecco's Modified Eagle Media (DMEM) culturing each multifidus muscle SCs of the normal serum, model serum, EA-BL 40 serum and EA-BL 40 serum+LY 294002 (an inhibitor of phosphotidylinsitol-3-kinase, PI 3 K), EA-BL 23 serum and EA-BL 23 serum+LY 294002 groups for ana-lyzing the impact of EA serum on the proliferation state of SCs by Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) methods, respectively. The expression of Pax-7, MyoD and phosphorylated (p)-Akt proteins of the cultured SCs was detected for characterization of SCs by Western blot. RESULTS: Compared with the normal serum group, the proliferation levels (detected by both CCK-8- and EdU) and the expression levels of MyoD and p-Akt proteins of SCs in the model serum group were significantly increased (P<0.05, P<0.01), while in comparison with the model serum group, the proliferation and expression levels of MyoD and p-Akt proteins of SCs were further significantly increased in both EA-BL 23 and EA-BL 40 serum groups (P<0.01, P<0.05), but not in the EA-BL 40 serum+LY 294002 and EA-BL 23 serum+LY 294002 groups (P>0.05), suggesting an involvement of PI 3 K in the proliferation of SCs. No marked differences were found in the proliferation levels between the EA-BL 23 and EA-BL 40 serum groups and in the expression levels of Pax-7 proteins among the 6 serum groups (P>0.05). CONCLUSIONS: Both EA-BL 40 and EA-BL 23 serum can promote proliferation of multifidus muscle SCs, which may contribute to the effect of EA intervention in promoting repair of the injured muscle, partially by way of Akt/PI 3 K signaling.

Whey protein supplementation accelerates satellite cell proliferation during recovery from eccentric exercise.

Human skeletal muscle satellite cells (SCs) are essential for muscle regeneration and remodeling processes in healthy and clinical conditions involving muscle breakdown. However, the potential influence of protein supplementation on post-exercise SC regulation in human skeletal muscle has not been well investigated. In a comparative human study, we investigated the effect of hydrolyzed whey protein supplementation following eccentric exercise on fiber type-specific SC accumulation. Twenty-four young healthy subjects received either hydrolyzed whey protein + carbohydrate (whey, n = 12) or iso-caloric carbohydrate (placebo, n = 12) during post-exercise recovery from 150 maximal unilateral eccentric contractions. Prior to and 24, 48 and 168 h post-exercise, muscle biopsies were obtained from the exercise leg and analyzed for fiber type-specific SC content. Maximal voluntary contraction (MVC) and serum creatine kinase (CK) were evaluated as indices of recovery from muscle damage. In type II fiber-associated SCs, the whey group increased SCs/fiber from 0.05 [0.02; 0.07] to 0.09 [0.06; 0.12] (p < 0.05) and 0.11 [0.06; 0.16] (p < 0.001) at 24 and 48 h, respectively, and exhibited a difference from the placebo group (p < 0.05) at 48 h. The whey group increased SCs/myonuclei from 4 % [2; 5] to 10 % [4; 16] (p < 0.05) at 48 h, whereas the placebo group increased from 5 % [2; 7] to 9 % [3; 16] (p < 0.01) at 168 h. MVC decreased (p < 0.001) and muscle soreness and CK increased (p < 0.001), irrespective of supplementation. In conclusion, whey protein supplementation may accelerate SC proliferation as part of the regeneration or remodeling process after high-intensity eccentric exercise.

Temporal progression of hypothalamic patterning by a dual action of BMP.

In the developing chick hypothalamus, Shh and BMPs are expressed in a spatially overlapping, but temporally consecutive, manner. Here, we demonstrate how the temporal integration of Shh and BMP signalling leads to the late acquisition of Pax7 expression in hypothalamic progenitor cells. Our studies reveal a requirement for a dual action of BMPs: first, the inhibition of GliA function through Gli3 upregulation; and second, activation of a Smad5-dependent BMP pathway. Previous studies have shown a requirement for spatial antagonism of Shh and BMPs in early CNS patterning; here, we propose that neural pattern elaboration can be achieved through a versatile temporal antagonism between Shh and BMPs.

Transcriptional profiling of neuronal differentiation by human embryonal carcinoma stem cells in vitro.

Pluripotent stem cell lines can be induced to differentiate into a range of somatic cell types in response to various stimuli. Such cell-based systems provide powerful tools for the investigation of molecules that modulate cellular development. For instance, the formation of the nervous system is a highly regulated process, controlled by molecular pathways that determine the expression of specific proteins involved in cell differentiation. To begin to decipher this mechanism in humans, we used oligonucleotide microarrays to profile the complex patterns of gene expression during the differentiation of neurons from pluripotent human stem cells. Samples of mRNA were isolated from cultured NTERA2 human embryonal carcinoma stem cells and their retinoic-acid-induced derivatives and were prepared for hybridization on custom microarrays designed to detect the expression of genes primarily associated with the neural lineage. In response to retinoic acid, human NTERA2 cells coordinately regulate the expression of large numbers of neural transcripts simultaneously. Transcriptional profiles of many individual genes aligned closely with expression patterns previously recorded by developing neural cells in vitro and in vivo, demonstrating that cultured human pluripotent stem cells appear to form neurons in a conserved manner. These experiments have produced many new expression data concerning neuronal differentiation from human stem cells in vitro. Of particular interest was the regulated expression of Pax6 and Nkx6.1 mRNA and the absence of Pax7 transcription, indicating that neurons derived from NTERA2 pluripotent stem cells are characteristic of neuroectodermal cells of the ventral phenotype.