Muscle-specific Genes Research Articles

Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD.

ABSTRACTPurpose:RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs), also known as small activating RNAs (saRNAs). Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI) is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs) into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD) into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI)–a condition primarily resulted from urethral sphincter deficiency.

Potential gene regulatory role for cyclin D3 in muscle cells.

Cyclin D3 is important for muscle development and regeneration, and is involved in post-mitotic arrest of muscle cells. Cyclin D3 also has cell-cycle-independent functions such as regulation of specific genes in other tissues. Ectopic expression of cyclin D3 in myoblasts, where it is normally undetectable, promotes muscle gene expression and faster differentiation kinetics upon serum depletion. In the present study, we investigated the mechanistic role of cyclin D3 in muscle gene regulation. We initially showed by mutational analysis that a stable and functional cyclin D3 was required for promoting muscle differentiation. Using chromatin immunoprecipitation assays, we demonstrated that expression of cyclin D3 in undifferentiated myoblasts altered histone epigenetic marks at promoters of muscle-specific genes like MyoD, Pax7, myogenin and muscle creatine kinase but not non-muscle genes. Cyclin D3 expression also reduced the mRNA levels of certain epigenetic modifier genes. Our data suggest that epigenetic modulation of muscle-specific genes in cyclin-D3-expressing myoblasts may be responsible for faster differentiation kinetics upon serum depletion. Our results have implications for a regulatory role for cyclin D3 in muscle-specific gene activation.

Kelch Repeat and BTB Domain Containing Protein 5 (Kbtbd5) Regulates Skeletal Muscle Myogenesis through the E2F1-DP1 Complex

We have previously isolated a muscle-specific Kelch gene, Kelch repeat and BTB domain containing protein 5 (Kbtbd5)/Kelch-like protein 40 (Klhl40). In this report, we identified DP1 as a direct interacting factor for Kbtbd5 using a yeast two-hybrid screen and in vitro binding assays. Our studies demonstrate that Kbtbd5 interacts and regulates the cytoplasmic localization of DP1. GST pulldown assays demonstrate that the dimerization domain of DP1 interacts with all three of the Kbtbd5 domains. We further show that Kbtbd5 promotes the ubiquitination and degradation of DP1, thereby inhibiting E2F1-DP1 activity. To investigate the in vivo function of Kbtbd5, we used gene disruption technology and engineered Kbtbd5 null mice. Targeted deletion of Kbtbd5 resulted in postnatal lethality. Histological studies reveal that the Kbtbd5 null mice have smaller muscle fibers, a disorganized sarcomeric structure, increased extracellular matrix, and decreased numbers of mitochondria compared with wild-type controls. RNA sequencing and quantitative PCR analyses demonstrate the up-regulation of E2F1 target apoptotic genes (Bnip3 and p53inp1) in Kbtbd5 null skeletal muscle. Consistent with these observations, the cellular apoptosis in Kbtbd5 null mice was increased. Breeding of Kbtbd5 null mouse into the E2F1 null background rescues the lethal phenotype of the Kbtbd5 null mice but not the growth defect. The expression of Bnip3 and p53inp1 in Kbtbd5 mutant skeletal muscle are also restored to control levels in the E2F1 null background. In summary, our studies demonstrate that Kbtbd5 regulates skeletal muscle myogenesis through the regulation of E2F1-DP1 activity.

Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

MicroRNAs modulated by local mIGF-1 expression in mdx dystrophic mice.

Duchenne muscular dystrophy (DMD) is a X-linked genetic disease in which the absence of dystrophin leads to progressive lethal skeletal muscle degeneration. It has been demonstrated that among genes which are important for proper muscle development and function, micro-RNAs (miRNAs) play a crucial role. Moreover, altered levels of miRNAs were found in several muscular disorders, including DMD. A specific group of miRNAs, whose expression depends on dystrophin levels and whose deregulation explains several DMD pathogenetic traits, has been identified. Here, we addressed whether the anabolic activity of mIGF-1 on dystrophic muscle is associated with modulation of microRNAs expression. We demonstrated that some microRNAs are strictly linked to the dystrophin expression and are not modulated by mIGF-1 expression. In contrast, local expression of mIGF-1 promotes the modulation of other microRNAs, such as miR-206 and miR-24, along with the modulation of muscle specific genes, which are associated with maturation of regenerating fibers and with the stabilization of the differentiated muscle phenotype. These data suggest that mIGF-1, modifying the expression of some of the active players of muscle homeostasis, is able, even in absence of dystrophin expression, to activate circuitries that confer robustness to dystrophic muscle.

583. Nonionic Block Copolymers Guide Macrophages To Transfect Skeletal Muscle Cells Upon Direct i. m. Injections

Purpose: Skeletal muscle offers a clinically feasible site for i. m. delivery of genes for therapeutic proteins expression and prophylactic DNA vaccine purposes. Though much research has been focused on development of cationic lipids and polymers for encapsulating plasmid DNA (pDNA) for in vitro transfections, direct i. m. injection of pDNA still results in higher muscle transfection levels. In comparison to existing technologies such as viral vectors there still is a need to further improve muscle transfection levels with pDNA injections. Non-ionic block copolymers, Pluronic P85 and SP1017 (mixture of L61 and F127), when mixed with pDNA, enhance skeletal muscle transfection in euthymic (BALB/c and C57BL/6) mouse and not athymic (BALB/c nu/nu) mouse. Therefore, we studied role of immune cells in muscle transfection.Methods and Results: pDNA alone or an admix of pDNA+Pluronic were injected into tibialis anterior (TA) muscle in presence or absence of local inflammation to demonstrate the effect of immune cells in muscle transfection. Local-inflammation drastically enhanced DNA, RNA and gene expression levels when pDNA was co-delivered with P85 or SP1017 and not pDNA alone. Pluronics enhanced in vitro gene expression in MPs to upto 4 orders in magnitude and not muscle cells. Moreover, adoptive transfer of ex vivo transfected MPs with GFP DNA to ischemic mice resulted in GFP expression in muscle fibers along with MPs. Therefore, in vitro muscle inflammation model was developed by co-culturing DNA transfected macrophages (MPs) with myotubes (MTs) to see the effect of P85 on gene transfer to MTs. Treatment of co-culture (pDNA transfected MPs with MTs) with P85 resulted in increase in total and muscle specific gene expression ca. 30 and 120 times respectively, confirming pDNA transfer from MPs to MTs. Moreover, in vivo depletion of MPs resulted in significant decrease in gene expression confirming the role of MPs in Pluronic based muscle transfection. Finally, pre-injection with P85 on day 1.5 and not day 5 at subsequent sites of DNA injections, recruited MPs to the site of injections and further enhanced the gene expression levels of muscles injected with pDNA+P85 and not pDNA alone. View Large Image | Download PowerPoint SlideConclusions: Pluronic block copolymers utilize immune cells, specifically MPs, to deliver genes to otherwise very hard to transfect skeletal muscle fibers and hence constitute a novel class of biopharmaceuticals or a platform technology for in vivo gene delivery.

Subcutaneous fat transplantation alleviates diet-induced glucose intolerance and inflammation in mice.

Adipose tissue (AT) distribution is a major determinant of mortality and morbidity in obesity. In mice, intra-abdominal transplantation of subcutaneous AT (SAT) protects against glucose intolerance and insulin resistance (IR), but the underlying mechanisms are not well understood. We investigated changes in adipokines, tissue-specific glucose uptake, gene expression and systemic inflammation in male C57BL6/J mice implanted intra-abdominally with either inguinal SAT or epididymal visceral AT (VAT) and fed a high-fat diet (HFD) for up to 17 weeks. Glucose tolerance was improved in mice receiving SAT after 6 weeks, and this was not attributable to differences in adiposity, tissue-specific glucose uptake, or plasma leptin or adiponectin concentrations. Instead, SAT transplantation prevented HFD-induced hepatic triacylglycerol accumulation and normalised the expression of hepatic gluconeogenic enzymes. Grafted fat displayed a significant increase in glucose uptake and unexpectedly, an induction of skeletal muscle-specific gene expression. Mice receiving subcutaneous fat also displayed a marked reduction in the plasma concentrations of several proinflammatory cytokines (TNF-α, IL-17, IL-12p70, monocyte chemoattractant protein-1 [MCP-1] and macrophage inflammatory protein-1β [ΜIP-1β]), compared with sham-operated mice. Plasma IL-17 and MIP-1β concentrations were reduced from as early as 4 weeks after transplantation, and differences in plasma TNF-α and IL-17 concentrations predicted glucose tolerance and insulinaemia in the entire cohort of mice (n = 40). In contrast, mice receiving visceral fat transplants were glucose intolerant, with increased hepatic triacylglycerol content and elevated plasma IL-6 concentrations. Intra-abdominal transplantation of subcutaneous fat reverses HFD-induced glucose intolerance, hepatic triacylglycerol accumulation and systemic inflammation in mice.

Characterization of RING Finger/SPRY Domain Containing 1 (Rspry1) and NEFA‐interacting Nuclear Protein 30 (Nip30) Transcriptional Regulation

Muscle atrophy results from a range of conditions, including immobilization, spinal cord damage, inflammation and aging. An investigation designed to identify changes in gene expression in skeletal muscle following denervation revealed a set of genes that show differential expression patterns in response to neurogenic atrophy. Nip30 and Rspry1, two genes not previously characterized in muscle, were found to be transcriptionally induced during muscle wasting in mice and protein expression was confirmed in C2C12 cells by western blot. In order to characterize the transcriptional regulation of Nip30 and Rspry1, a fragment of the promoter region of each gene was cloned, fused to a reporter gene, and transfected into muscle cells in combination with expression plasmids for Muscle RING Finger 1 (MuRF1) and myogenic regulatory factors (MRFs). The MuRF1 protein is an E3 ubiquitin ligase that is induced under most atrophic conditions and is believed to promote protein degradation; however, data presented in this study suggests that MuRF1 may also regulate the transcriptional activity of genes that are differentially expressed following neurogenic atrophy. Myogenic regulatory factors (MRFs) are a class of E-box binding proteins that regulate muscle-specific gene expression. The results of this study demonstrate that MuRF1 and MRFs cooperatively repress Rspry1 and Nip30 reporter activity. Furthermore, mutation of a conserved E-box element in the shared regulatory region of Nip30 and Rspry1 eliminated the MuRF1 and MRF cooperative repression of these genes. Identifying novel atrophy-induced genes and characterizing their role in the atrophy cascade may help further our understanding of the molecular mechanisms of muscle wasting.

Preliminary study of genetic diversity and population structure of the Relict Gull Larus relictus (Charadriiformes Laridae) using mitochondrial and nuclear genes

The genetic diversity and the population history of the Relict Gull (Larus relictus) were studied based on the sequences of two mitochondrial genes (Cyt b and ND2) and one nuclear gene (the third intron of the Z-linked muscle-specific kinase gene [MUSK]) from 47 individuals. The results showed that the population expresses very low genetic diversity while lacking large geographical population. Demographic history analysis suggested that population expansion might have been recent and that the average estimated expansion time of L. relictus is from 0.09 to 0.23 Ma BP. Whether the other three breeding subpopulations are involved in the Ordos breeding subpopulation, require further study with more effective molecular markers. The species is threatened by habitat loss and fragmentation, and actions to protect it are suggested to improve the population fragmentation of breeding.

Molecular characterization of four genes highly expressed during megalopa stage in Chinese mitten crab, Eriocheir sinensis

The molecular markers to distinguish different larval stages have various applications in ecological studies. Using the differential display RT-PCR technique, we isolated and characterized four genes, which are expressed predominantly in the megalopa stage of Eriocheir sinensis. The four genes include two cuticular proteins with different domain organization (Ers-CP15 and Ers-CP34) and two skeletal-muscle-specific genes (Ers-SCP and Ers-ActinSK1). The two cuticular protein genes were expressed predominantly in the epidermis and their expression level was significantly higher in the megalopa stage (about 7.0-folds) than it was in the zoea stage. However, their high transcriptional level in zoea IV suggested that these two cuticular protein genes may not be a useful target to discriminate megalopa from zoea. Ers-SCP encoded the invertebratespecific sarcoplasmic calcium binding protein and Ers-ActinSK1 gene encoded the crustacean skeletal muscle actin. Expressions of these two genes were detected only in muscular tissues; leg muscle, claw muscle and thoracic muscle. This suggests that the increased transcription levels of two muscle-specific genes during the megalopa stage are mainly due to increased muscular tissues. Among its three isoforms, Ers-SCPa displayed the highest difference (22.4-folds) between megalopa and zoea suggesting Ers-SCPa is the most reliable marker to distinguish megalopa from zoea. Although Ers-SCPc and Ers-ActinSK1 also showed similar expression profiles to Ers-SCPa and Ers-SCPb, differences in their expression levels were not as high as Ers-SCPa and Ers-SCPb.

Setdb1 is required for myogenic differentiation of C2C12 myoblast cells via maintenance of MyoD expression.

Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification. Functions of Setdb1 during development have been extensively studied in embryonic and mesenchymal stem cells as well as neurogenic progenitor cells. But the role of Sedtdb1 in myogenic differentiation remains unknown. In this study, we report that Setdb1 is required for myogenic potential of C2C12 myoblast cells through maintaining the expressions of MyoD and muscle-specific genes. We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on. Gene expression profiling analysis using oligonucleotide micro-array and RNA-Seq technologies demonstrated that depletion of Setdb1 results in downregulation of MyoD as well as the components of muscle fiber in proliferating C2C12 cells. In addition, exogenous expression of MyoD reversed transcriptional repression of MyoD promoter-driven lucif-erase reporter by Setdb1 shRNA and rescued myogenic differentiation of C2C12 myoblast cells depleted of endogenous Setdb1. Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

In vivo generation of a mature and functional artificial skeletal muscle.

Extensive loss of skeletal muscle tissue results in mutilations and severe loss of function. In vitro-generated artificial muscles undergo necrosis when transplanted in vivo before host angiogenesis may provide oxygen for fibre survival. Here, we report a novel strategy based upon the use of mouse or human mesoangioblasts encapsulated inside PEG-fibrinogen hydrogel. Once engineered to express placental-derived growth factor, mesoangioblasts attract host vessels and nerves, contributing to in vivo survival and maturation of newly formed myofibres. When the graft was implanted underneath the skin on the surface of the tibialis anterior, mature and aligned myofibres formed within several weeks as a complete and functional extra muscle. Moreover, replacing the ablated tibialis anterior with PEG-fibrinogen-embedded mesoangioblasts also resulted in an artificial muscle very similar to a normal tibialis anterior. This strategy opens the possibility for patient-specific muscle creation for a large number of pathological conditions involving muscle tissue wasting.

Conditional deletion of the relaxin receptor gene in cells of smooth muscle lineage affects lower reproductive tract in pregnant mice.

Relaxin hormone secreted into the circulation during pregnancy was discovered through its effects on pubic symphysis relaxation and parturition. Genetic inactivation of the relaxin gene or its cognate relaxin family peptide receptor 1 (RXFP1) in mice caused failure of parturition and mammary nipple enlargement, as well as increased collagen fiber density in the cervix and vagina. However, the relaxin effect on discrete cells and tissues has yet to be determined. Using transgenic mice with a knockin LacZ reporter in the Rxfp1 allele, we showed strong expression of this gene in vaginal and cervical stromal cells, as well as pubic ligament cells. We produced a floxed Rxfp1 allele that was used in combination with the Tagln-cre transgene to generate mice with a smooth muscle-specific gene knockout. In pregnant females, the ROSA26 reporter activated by Tagln-cre was detected in smooth muscle cells of the cervix, vagina, uterine artery, and in cells of the pubic symphysis. In late pregnant females with conditional gene ablation, the length of pubic symphysis was significantly reduced compared with wild-type or heterozygous Rxfp1(+/-) females. Denser collagen content was revealed by Masson trichrome staining in reproductive tract organs, uterine artery, and pubic symphysis. The cervical and vaginal epithelium was less developed than in heterozygous or wild-type females, although nipple size was normal and the dams were able to nurse their pups. In summary, our data indicate that relaxin/RXFP1 signaling in smooth muscle cells is important for normal collagen turnover and relaxation of the pubic symphysis during pregnancy.

Mir181A Targets the 3'UTR of MG29, a Muscle-Specific Synaptophysin Family Gene, for Down-Regulation of MG29 Expression in Dystrophic Skeletal Muscle

MG29 is a muscle-specific member of the synaptophysin family protein that participates in controlling the maturation and development of transverse tubule (TT) structure and the maintenance of intracellular Ca2+ signaling in skeletal muscle. Genetic ablation of MG29 leads to defective formation of the TT network in skeletal muscle, which resembles the abnormal TT structure observed in dystrophic muscles. We found that MG29 protein is drastically reduced in mouse models with muscular dystrophy (e.g. mdx and dysferlin-/-), and such reduced MG29 expression and disrupted TT structure were also observed in human patient biopsies with muscular dystrophy. The mg29 gene contains a unique 3’ untranslated region (UTR) with potential binding sites for microRNA (miRNA) or RNA-stabilizing factors. Mutagenesis studies revealed that miR-181a could target a region in the 3′UTR that is highly conserved between mouse and human mg29 genes to exhibit posttranscriptional control of MG29 expression in skeletal muscle. Overexpression of anti-sense miR-181a by microinjection into mouse flexor digitorum brevis fibers significantly increased the expression of endogenous MG29. Studies from other investigators have shown that miR-181a was elevated in human patients with muscular dystrophy. These data suggest an intriguing possibility that elevated miR-181a may be a contributing factor for the reduced MG29 expression under dystrophic conditions. Further understanding of the post-transcriptional regulation by microRNA of MG29 expression can provide more insights into the therapeutic targets of muscular dystrophy.

Molecular Analysis of a Recurrent Sarcoma Identifies a Mutation in FAF1.

A patient presented with a recurrent sarcoma (diagnosed as leiomyosarcoma) 12 years after the removal of an initial cancer (diagnosed as extracompartmental osteosarcoma) distally on the same limb. Following surgery, the sarcoma and unaffected muscle and bone were subjected to measurements of DNA exome sequence, RNA and protein expression, and transcription factor binding. The investigation provided corroboration of the diagnosis leiomyosarcoma, as the major upregulations in this tumor comprise muscle-specific gene products and calcium-regulating molecules (calcium is an important second messenger in smooth muscle cells). A likely culprit for the disease is the point mutation S181G in FAF1, which may cause a loss of apoptotic function consecutive to transforming DNA damage. The RNA levels of genes for drug transport and metabolism were extensively skewed in the tumor tissue as compared to muscle and bone. The results suggest that the tumor represents a recurrence of a dormant metastasis from an originally misdiagnosed neoplasm. A loss of FAF1 function could cause constitutive WNT pathway activity (consistent with the downstream inductions of IGF2BP1 and E2F1 in this cancer). While the study has informed on drug transport and drug metabolism pharmacogenetics, it has fallen short of identifying a suitable target for molecular therapy.

Mesenchymal Stem Cells Suppress Muscle Atrophy Induced by Hindlimb Suspension

Objective: The protective effects of human mesenchymal stem cells (MSCs) isolated from different sources (bone marrow, adipose tissue and umbilical cord) on skeletal muscle regeneration were investigated using a hindlimb suspension (HS)-induced muscle atrophy rodent model. Methods: Female SD rats were randomly assigned to three groups: control, hindlimb suspension (HS), and hindlimb suspension and reloaded (HR). After induction of muscle atrophy for two weeks, MSCs (1 x 106 cells) were injected into the soleus muscle in the HS and HR groups. Two weeks later, the changes of muscle mass, crosssectional area (CSA), muscle-specific gene expression, lactate accumulation and muscle atrophy signaling pathwayrelated proteins were compared. Results: In the MSCs-injected groups, there was an increase in soleus muscle mass, CSA and expression of skeletal muscle actin and desmin, as well as a decrease in lactate accumulation. All MSCs play an effective role in improvement of the muscle state from the muscle atrophy condition. Especially, HR groups accompanied by normal activity after MSCs injection showed synergic effect on recovery of atrophic muscle. Furthermore, treatment with the MSCs activated the PI3K/AKT pathway known as major signaling of muscle atrophy, consequently reducing expression of the muscle-specific RING finger protein-1 (MuRF-1) and atrophy F-box (MAFbx/Atrogin-1). Conclusions: The results can serve as fundamental data toward the development of new medical strategies for the rapid rehabilitation and regeneration of atrophied muscles.

Alternative Splicing of MEF2C pre-mRNA Controls Its Activity in Normal Myogenesis and Promotes Tumorigenicity in Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Many cellular disruptions contribute to the progression of this pediatric cancer, including aberrant alternative splicing. The MEF2 family of transcription factors regulates many developmental programs, including myogenesis. MEF2 gene transcripts are subject to alternate splicing to generate protein isoforms with divergent functions. We found that MEF2Cα1 was the ubiquitously expressed isoform that exhibited no myogenic activity and that MEF2Cα2, the muscle-specific MEF2C isoform, was required for efficient differentiation. We showed that exon α in MEF2C was aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 highly down-regulated in RMS cells compared with normal myoblasts. Compared with MEF2Cα2, MEF2Cα1 interacted more strongly with and recruited HDAC5 to myogenic gene promoters to repress muscle-specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increased myogenic activity and promoted differentiation in RMS cells. We also identified a serine protein kinase, SRPK3, that was down-regulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage-independent growth of RMS cells. Together, our findings indicate that the alternative splicing of MEF2C plays an important role in normal myogenesis and RMS development. An improved understanding of alternative splicing events in RMS cells will potentially reveal novel therapeutic targets for RMS treatment.

New insights into the epigenetic control of satellite cells.

Epigenetics finely tunes gene expression at a functional level without modifying the DNA sequence, thereby contributing to the complexity of genomic regulation. Satellite cells (SCs) are adult muscle stem cells that are important for skeletal post-natal muscle growth, homeostasis and repair. The understanding of the epigenome of SCs at different stages and of the multiple layers of the post-transcriptional regulation of gene expression is constantly expanding. Dynamic interactions between different epigenetic mechanisms regulate the appropriate timing of muscle-specific gene expression and influence the lineage fate of SCs. In this review, we report and discuss the recent literature about the epigenetic control of SCs during the myogenic process from activation to proliferation and from their commitment to a muscle cell fate to their differentiation and fusion to myotubes. We describe how the coordinated activities of the histone methyltransferase families Polycomb group (PcG), which represses the expression of developmentally regulated genes, and Trithorax group, which antagonizes the repressive activity of the PcG, regulate myogenesis by restricting gene expression in a time-dependent manner during each step of the process. We discuss how histone acetylation and deacetylation occurs in specific loci throughout SC differentiation to enable the time-dependent transcription of specific genes. Moreover, we describe the multiple roles of microRNA, an additional epigenetic mechanism, in regulating gene expression in SCs, by repressing or enhancing gene transcription or translation during each step of myogenesis. The importance of these epigenetic pathways in modulating SC activation and differentiation renders them as promising targets for disease interventions. Understanding the most recent findings regarding the epigenetic mechanisms that regulate SC behavior is useful from the perspective of pharmacological manipulation for improving muscle regeneration and for promoting muscle homeostasis under pathological conditions.

Fetal heart extract facilitates the differentiation of human umbilical cord blood-derived mesenchymal stem cells into heart muscle precursor cells.

Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) are a promising stem cell source with the potential to modulate the immune system as well as the capacity to differentiate into osteoblasts, chondrocytes, and adipocytes. In previous publications, UCB-MSCs have been successfully differentiated into cardiomyocytes. This study aimed to improve the efficacy of differentiation of UCB-MSCs into cardiomyocytes by combining 5-azacytidine (Aza) with mouse fetal heart extract (HE) in the induction medium. UCB-MSCs were isolated from umbilical cord blood according to a published protocol. Murine fetal hearts were used to produce fetal HE using a rapid freeze-thaw procedure. MSCs at the 3rd to 5th passage were differentiated into cardiomyocytes in two kinds of induction medium: complete culture medium plus Aza (Aza group) and complete culture medium plus Aza and fetal HE (Aza+HE group). The results showed that the cells in both kinds of induction medium exhibited the phenotype of cardiomyocytes. At the transcriptional level, the cells expressed a number of cardiac muscle-specific genes such as Nkx2.5, Gata 4, Mef2c, HCN2, hBNP, α-Ca, cTnT, Desmin, and β-MHC on day 27 in the Aza group and on day 18 in the Aza+HE group. At the translational level, sarcomic α-actin was expressed on day 27 in the Aza group and day 18 in the Aza+HE group. Although they expressed specific genes and proteins of cardiac muscle cells, the induced cells in both groups did not contract and beat spontaneously. These properties are similar to properties of heart muscle precursor cells in vivo. These results demonstrated that the fetal HE facilitates the differentiation process of human UCB-MSCs into heart muscle precursor cells.

Chemopreventive effect of Korean Angelica root extract on TRAMP carcinogenesis and integrative "omic" profiling of affected neuroendocrine carcinomas.

Angelica gigas Nakai (AGN) root ethanol extract exerts anti-cancer activity in several allograft and xenograft models. Here we examined its chemopreventive efficacy through gavage administration against primary carcinogenesis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Male C57BL/6 TRAMP mice and wild type littermates were given a daily gavage (5 mg/mouse, Monday-Friday) of AGN or vehicle, beginning at 8 wk of age (WOA). All mice were terminated at 24 WOA, unless earlier euthanasia was necessitated by large tumors. Whereas AGN-treated TRAMP mice decreased dorsolateral prostate lesion growth by 30% (P = 0.009), they developed fewer and smaller neuroendocrine-carcinomas (NE-Ca) (0.12 g/mouse) than vehicle-treated counterparts (0.81 g/mouse, P = 0.037). We analyzed the proteome and transcriptome of banked NE-Ca to gain molecular insights. Angiogenesis-antibody array detected a substantial reduction in AGN-treated NE-Ca of basic fibroblast growth factor (FGF2), an angiogenesis stimulator. iTRAQ proteomics plus data mining suggested changes of genes upstream and downstream of FGF2 functionally consistent with AGN inhibiting FGF2/FGFR1 signaling at different levels of the transduction cascade. Moreover, AGN upregulated mRNA of genes related to immune responses, restored expression of many tumor suppressor genes, and prostate function and muscle differentiation genes. On the other hand, AGN down-regulated mRNA of genes related to neuron signaling, oncofetal antigens, inflammation, and mast cells, Wnt signaling, embryonic morphogenesis, biosynthesis, cell adhesion, motility, invasion, and angiogenesis. These changes suggest not only multiple cancer cell targeting actions of AGN but also impact on the tumor microenvironments such as angiogenesis, inflammation, and immune surveillance.