Expression Of Myogenic Transcription Factors Research Articles

MiR-9-5p promotes myogenic differentiation via the Dlx3/Myf5 axis.

MicroRNAs play an important role in myogenic differentiation, they bind to target genes and regulate muscle formation. We previously found that miR-9-5p, which is related to bone formation, was increased over time during the process of myogenic differentiation. However, the mechanism by which miR-9-5p regulates myogenic differentiation remains largely unknown. In the present study, we first examined myotube formation and miR-9-5p, myogenesis-related genes including Dlx3, Myod1, Mef2c, Desmin, MyoG and Myf5 expression under myogenic induction. Then, we detected the expression of myogenic transcription factors after overexpression or knockdown of miR-9-5p or Dlx3 in the mouse premyoblast cell line C2C12 by qPCR, western blot and myotube formation under myogenic induction. A luciferase assay was performed to confirm the regulatory relationships between not only miR-9-5p and Dlx3 but also Dlx3 and its downstream gene, Myf5, which is an essential transcription factor of myogenic differentiation. The results showed that miR-9-5p promoted myogenic differentiation by increasing myogenic transcription factor expression and promoting myotube formation, but Dlx3 exerted the opposite effect. Moreover, the luciferase assay showed that miR-9-5p bound to the 3’UTR of Dlx3 and downregulated Dlx3 expression. Dlx3 in turn suppressed Myf5 expression by binding to the Myf5 promoter, ultimately inhibiting the process of myogenic differentiation. In conclusion, the miR-9-5p/Dlx3/Myf5 axis is a novel pathway for the regulation of myogenic differentiation, and can be a potential target to treat the diseases related to muscle dysfunction.

The Phosphonate Derivative of C60 Fullerene Induces Differentiation towards the Myogenic Lineage in Human Adipose-Derived Mesenchymal Stem Cells

Inductors of myogenic stem cell differentiation attract attention, as they can be used to treat myodystrophies and post-traumatic injuries. Functionalization of fullerenes makes it possible to obtain water-soluble derivatives with targeted biochemical activity. This study examined the effects of the phosphonate C60 fullerene derivatives on the expression of myogenic transcription factors and myogenic differentiation of human mesenchymal stem cells (MSCs). Uptake of the phosphonate C60 fullerene derivatives in human MSCs, intracellular ROS visualization, superoxide scavenging potential, and the expression of myogenic, adipogenic, and osteogenic differentiation genes were studied. The prolonged MSC incubation (within 7–14 days) with the C60 pentaphoshonate potassium salt promoted their differentiation towards the myogenic lineage. The transcription factors and gene expressions determining myogenic differentiation (MYOD1, MYOG, MYF5, and MRF4) increased, while the expression of osteogenic differentiation factors (BMP2, BMP4, RUNX2, SPP1, and OCN) and adipogenic differentiation factors (CEBPB, LPL, and AP2 (FABP4)) was reduced or did not change. The stimulation of autophagy may be one of the factors contributing to the increased expression of myogenic differentiation genes in MSCs. Autophagy may be caused by intracellular alkalosis and/or short-term intracellular oxidative stress.

Dexamethasone Inhibits the Pro-Angiogenic Potential of Primary Human Myoblasts.

Tissue regeneration depends on the complex processes of angiogenesis, inflammation and wound healing. Regarding muscle tissue, glucocorticoids (GCs) inhibit pro-inflammatory signalling and angiogenesis and lead to muscle atrophy. Our hypothesis is that the synthetic GC dexamethasone (dex) impairs angiogenesis leading to muscle atrophy or inhibited muscle regeneration. Therefore, this study aims to elucidate the effect of dexamethasone on HUVECs under different conditions in mono- and co-culture with myoblasts to evaluate growth behavior and dex impact with regard to muscle atrophy and muscle regeneration. Viability assays, qPCR, immunofluorescence as well as ELISAs were performed on HUVECs, and human primary myoblasts seeded under different culture conditions. Our results show that dex had a higher impact on the tube formation when HUVECs were maintained with VEGF. Gene expression was not influenced by dex and was independent of cells growing in a 2D or 3D matrix. In co-culture CD31 expression was suppressed after incubation with dex and gene expression analysis revealed that dex enhanced expression of myogenic transcription factors, but repressed angiogenic factors. Moreover, dex inhibited the VEGF mediated pro angiogenic effect of myoblasts and inhibited expression of angiogenic inducers in the co-culture model. This is the first study describing a co-culture of human primary myoblast and HUVECs maintained under different conditions. Our results indicate that dex affects angiogenesis via inhibition of VEGF release at least in myoblasts, which could be responsible not only for the development of muscle atrophy after dex administration, but also for inhibition of muscle regeneration after vascular damage.

Transcriptome profile analysis reveals KLHL30 as an essential regulator for myoblast differentiation

Skeletal muscle development is a sophisticated multistep process orchestrated by diverse myogenic transcription factors. Recent studies have suggested that Kelch-like genes play vital roles in muscle disease and myogenesis. However, it is still unclear how Kelch-like genes impact myoblast physiology. Here, through integrative analysis of the mRNA expression profile during chicken primary myoblast and C2C12 differentiation, many differentially expressed genes were found and suggested to be enriched in myoblast differentiation and muscle development. Interestingly, a little-known Kelch-like gene KLHL30 was screened as skeletal muscle-specific gene with essential roles in myogenic differentiation. Transcriptomic data and quantitative PCR analysis indicated that the expression of KLHL30 is upregulated under myoblast differentiation state. KLHL30 overexpression upregulated the protein expression of myogenic transcription factors (MYOD, MYOG, MEF2C) and induced myoblast differentiation and myotube formation, while knockdown of KLHL30 caused the opposite effect. Furthermore, KLHL30 was found to significantly decrease the numbers of cells in the S stage and thereby depress myoblast proliferation. Collectively, this study highlights that KLHL30 as a muscle-specific regulator plays essential roles in myoblast proliferation and differentiation.

METTL3 regulates skeletal muscle specific miRNAs at both transcriptional and post-transcriptional levels

METTL3 increasing the mature miRNA levels via N6-Methyladenosine (m6A) modification of primary miRNA (pri-miRNA) transcripts has emerged as an important post-transcriptional regulation of miRNA biogenesis. Our previous studies and others have showed that muscle specific miRNAs are essential for skeletal muscle differentiation. Whether these miRNAs are also regulated by METTL3 is still unclear. Here, we found that m6A motifs were present around most of these miRNAs, which were indeed m6A modified as confirmed by m6A-modified RNA immunoprecipitation (m6A RIP). However, we surprisingly found that these muscle specific miRNAs were repressed instead of increased by METTL3 in C2C12 in vitro differentiation and mouse skeletal muscle regeneration after injury in vivo model. To elucidate the underlined mechanism, we performed reporter assays in 293T cells and validated METTL3 increasing these miRNAs at post-transcriptional level as expected. Furthermore, in myogenic C2C12 cells, we found that METTL3 not only repressed the expression of myogenic transcription factors (TFs) which can enhance the muscle specific miRNAs, but also increased the expression of epigenetic regulators which can repress these miRNAs. Thus, METTL3 could repress the muscle specific miRNAs at transcriptional level indirectly. Taken together, our results demonstrated that skeletal muscle specific miRNAs were repressed by METTL3 and such repression is likely synthesized transcriptional and post-transcriptional regulations.

Ppp1r1b-lncRNA inhibits PRC2 at myogenic regulatory genes to promote cardiac and skeletal muscle development in mouse and human

Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators and play important roles in cardiac development and congenital heart disease. In a previous study, we identified a novel lncRNA, Ppp1r1b, with expression highly correlated with myogenesis. However, the molecular mechanism that underlies Ppp1r1b-lncRNA function in myogenic regulation is unknown. By silencing Ppp1r1b-lncRNA, mouse C2C12 and human skeletal myoblasts failed to develop fully differentiated myotubes. Myogenic differentiation was also impaired in PPP1R1B-lncRNA deficient human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCs-CMs). The expression of myogenic transcription factors, including MyoD, Myogenin, and Tbx5, as well as sarcomere proteins, was significantly suppressed in Ppp1r1b-lncRNA inhibited myoblast cells and neonatal mouse heart. Histone modification analysis revealed increased H3K27 tri-methylation at MyoD1 and Myogenin promoters in GapmeR treated C2C12 cells. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and chromatin isolation by RNA purification (ChIRP) assay revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic transcription factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Therefore, our results suggest that Ppp1r1b-lncRNA promotes myogenic differentiation through competing for PRC2 binding with chromatin of myogenic master regulators during heart and skeletal muscle development.

Rhabdomyosarcomas: structural distribution and analysis of an immunohistochemical profile

To qualitatively and quantitatively assess the expression of myogenic transcription factors on a large sample, to identify potential phenotypic differences, and to estimate the distribution and frequency of aberrant markers, such as ALK, PAX5, WT1, PCK, CAM5.2, SIX1, and Synaptophysin. The investigation included 202 tumor tissue samples. Five tissue microarrays were assembled from the obtained material for subsequent histological and immunohistochemical studies. Embryonal RMS (ERMS) was diagnosed in 103 cases; alveolar RMS (ARMS) was detected in 80; spindle-cell/sclerosing RMS (SRMS) was found in 16 cases; epithelioid RMS (EpiRMS) was diagnosed in 2 patients. The expression of Myogenin and MyoD1 was detected in all the examined RMS tissue samples. ARMS was more characterized by staining at 1+ and 2+ intensities; at the same time, more than 50% of ERMS, SRMS, and EpiRMS cases showed staining at 1+ intensity. ALK expression was investigated using the D5F3 and p80 clones. The D5F3 clone displayed a higher staining intensity than the p80 clone (p<0.05). The expression of PAX5 was observed in 13 of 75 ARMS cases. That of WT1 and SIX1 was found in all RMS groups. The morphological diagnosis of RMS requires a careful assessment of all of the above factors, especially taking into account the variability in the expression of myogenic transcription factors and the high level of phenotypic aberration.

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

β-Hydroxy-β-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. 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 5d posthatch and greater satellite cell mitotic activity at 7d posthatch than counterparts from amnion injection group. 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.

Abstract A25: Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition

Abstract PAX-fusion negative rhabdomyosarcoma (RMS) arises from skeletal muscle precursors that have failed to differentiate normally despite the expression of the myogenic master transcription factor, MYOD1. The cure rate for relapsed or refractory fusion negative RMS is poor despite aggressive multi-modality treatment. Novel treatment approaches such as the use of targeted therapies including those that induce skeletal muscle differentiation might improve overall survival for patients with fusion negative RMS. Genetic studies have shown that the most common single nucleotide variant in fusion negative RMS is an oncogenic change in one of the RAS isoforms, namely NRAS, HRAS or KRAS. In this study, we hypothesized that targeting aberrant RAS activity releases the differentiation block in fusion negative RMS and sought to unravel the underlying epigenetic mechanisms through which RAS signaling drives oncogenic transcription in RMS. To achieve this goal, we combined high-throughput drug screening with biochemical, RNAseq and ChIPseq assays across a panel of RMS cell lines driven by oncogenic RAS mutations. Critically, we demonstrated that expression of oncogenic RAS was necessary for survival of these RAS-mutated RMS cells. In addition, overexpression of mutant RAS isoforms in C2C12 myoblasts inhibited myogenic differentiation induced by low-serum conditions. This differentiation block was mediated primarily by engagement of the RAF-MEK-ERK MAP kinase pathway. In corroboration with these observations, an unbiased screen of the ability of small molecules to impact cell viability demonstrated that inhibitors of the MAP kinase pathway were the most potently selective class of molecules for RAS-mutated RMS. In particular, trametinib, an allosteric, non-ATP competitive inhibitor of MEK1/2, was the most consistently potent MEK inhibitor in RAS-mutated RMS cell lines. Trametinib treatment induced G1 arrest and skeletal muscle differentiation in RAS-mutated RMS cell lines. Trametinib also slowed tumor growth and prolonged survival in xenograft models of RAS-mutated RMS. To determine the mechanism by which MEK inhibition induced skeletal muscle differentiation in RAS-mutated RMS, we analyzed changes in gene expression, transcription factor deposition and histone modification in RMS cells treated with trametinib. Trametinib treatment increased expression of myogenic transcription factors, such as MYOG and MEF2C, and decreased expression of transcription factors important for proliferation, such as MYC and ID3, in RAS-mutated RMS cells. ChIPseq experiments demonstrated that this transcriptional reprogramming was driven in part by changes in the active enhancer landscape, since H3K27ac deposition at MYH3, TTNT2 and other muscle-specific loci increased with trametinib treatment. Both MYC and MYOD1 bound the active enhancers induced by trametinib treatment in RAS-mutated RMS, despite an overall decrease in MYC expression. Finally, we found significant ERK2 deposition on the MYOG promoter in the untreated cells. ERK2 is known to recruit the Polycomb repressive machinery at developmental loci in embryonic stem cells and therefore aberrant ERK2 activity may facilitate repression of MYOG expression in RAS-mutated RMS. In summary, our data support a model of RAS-driven RMS in which aberrant ERK activity drives tumor cell proliferation, in part through increased expression and stability of MYC, and prevents myogenic differentiation, in this case through alterations in the enhancer landscape and interactions with the Polycomb repressive machinery. Future work is aimed at identifying rational combinations of trametinib and direct epigenetic modulators that synergistically drive RAS-mutated RMS differentiation with the goal of providing measurable clinical benefit in relapsed or refractory RAS-mutated RMS. Citation Format: Marielle E. Yohe, Berkley E. Gryder, Jack F. Shern, Young K. Song, Hongling Liao, Hsein-Chao Chou, Sivasish Sindiri, Arnulfo Mendoza, Xiaohu Zhang, Rajarashi Guha, Diana C. Haines, James P. Madigan, Jun S. Wei, Marc Ferrer, Craig J. Thomas, Javed Khan. Reprogramming RAS-driven rhabdomyosarcoma via MEK inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A25.

Investigation of the Expression of Myogenic Transcription Factors, microRNAs and Muscle-Specific E3 Ubiquitin Ligases in the Medial Gastrocnemius and Soleus Muscles following Peripheral Nerve Injury.

Despite surgical innovation, the sensory and motor outcome after a peripheral nerve injury remains incomplete. One contributing factor to the poor outcome is prolonged denervation of the target organ, leading to apoptosis of both mature myofibres and satellite cells with subsequent replacement of the muscle tissue with fibrotic scar and adipose tissue. In this study, we investigated the expression of myogenic transcription factors, muscle specific microRNAs and muscle-specific E3 ubiquitin ligases at several time points following denervation in two different muscles, the gastrocnemius (containing predominantly fast type fibres) and soleus (slow type) muscles, since these molecules may influence the degree of atrophy following denervation. Both muscles exhibited significant atrophy (compared with the contra-lateral sides) at 7 days following either a nerve transection or crush injury. In the crush model, the soleus muscle showed significantly increased muscle weights at days 14 and 28 which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. This study provides further insights regarding the intracellular regulatory molecules that generate and maintain distinct patterns of gene expression in different fibre types following peripheral nerve injury.

Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis.

The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, β1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

Epidermal Growth Factor Receptor Down-Regulation Triggers Human Myoblast Differentiation

Initiation of human myoblast differentiation requires a negative shift (hyperpolarization) of the resting potential of myoblasts that depends on the activation of Kir2.1 potassium channels. These channels are regulated by a tyrosine phosphorylation. Using human primary myoblast culture, we investigated a possible role of various receptor tyrosine kinases in the induction of the differentiation process. We found that Epidermal Growth Factor Receptor (EGFR) is a key regulator of myoblast differentiation. EGFR activity is down-regulated during early human myoblast differentiation, and this event is required for normal differentiation to take place. Furthermore, EGFR silencing in proliferation conditions was able to trigger the differentiation program. This occurs through an increase of Kir2.1 channel activity that, via a rise of store-operated Ca2+ entry, leads to the expression of myogenic transcription factors and muscle specific proteins (Myogenin, Myocyte Enhancer Factor 2 (MEF2), Myosin Heavy Chain (MyHC)). Finally, blocking myoblast cell cycle in proliferation conditions using a cdk4 inhibitor greatly decreased myoblast proliferation but was not able, on its own, to promote myoblast differentiation. Taken together, these results show that EGFR down-regulation is an early event that is required for the induction of myoblast differentiation.

Local Applications of Myostatin-siRNA with Atelocollagen Increase Skeletal Muscle Mass and Recovery of Muscle Function

BackgroundGrowing evidence suggests that small-interfering RNA (siRNA) can promote gene silencing in mammalian cells without induction of interferon synthesis or nonspecific gene suppression. Recently, a number of highly specific siRNAs targeted against disease-causing or disease-promoting genes have been developed. In this study, we evaluate the effectiveness of atelocollagen (ATCOL)-mediated application of siRNA targeting myostatin (Mst), a negative regulator of skeletal muscle growth, into skeletal muscles of muscular dystrophy model mice.Methods and FindingsWe injected a nanoparticle complex containing myostatin-siRNA and ATCOL (Mst-siRNA/ATCOL) into the masseter muscles of mutant caveolin-3 transgenic (mCAV-3Tg) mice, an animal model for muscular dystrophy. Scrambled (scr) -siRNA/ATCOL complex was injected into the contralateral muscles as a control. Two weeks after injection, the masseter muscles were dissected for histometric analyses. To investigate changes in masseter muscle activity by local administration of Mst-siRNA/ATCOL complex, mouse masseter electromyography (EMG) was measured throughout the experimental period via telemetry. After local application of the Mst-siRNA/ATCOL complex, masseter muscles were enlarged, while no significant change was observed on the contralateral side. Histological analysis showed that myofibrils of masseter muscles treated with the Mst-siRNA/ATCOL complex were significantly larger than those of the control side. Real-time PCR analysis revealed a significant downregulation of Mst expression in the treated masseters of mCAV-3Tg mice. In addition, expression of myogenic transcription factors was upregulated in the Mst-siRNA-treated masseter muscle, while expression of adipogenic transcription factors was significantly downregulated. EMG results indicate that masseter muscle activity in mCAV-3Tg mice was increased by local administration of the Mst-siRNA/ATCOL complex.ConclusionThese data suggest local administration of Mst-siRNA/ATCOL complex could lead to skeletal muscle hypertrophy and recovery of motor disability in mCAV-3Tg mice. Therefore, ATCOL-mediated application of siRNA is a potential tool for therapeutic use in muscular atrophy diseases.

Analysis of myogenic and endothelial progenitor cells in muscle biopsies of juvenile dermatomyositis

Aims: Juvenile dermatomyositis (JDM) is a rare immune-mediated microangiopathy presenting with skin lesions and proximal muscle weakness. Muscle biopsies of JDM are characterized by a reduction of endomysial capillaries and perifascicular atrophy of muscle fibers. In response to injury, normal muscle tissue has a high potential to regenerate by a process of activation, proliferation, and differentiation of satellite cells accompanied by neovascularization of regenerated muscle fibers. Little is known about muscle repair in JDM. The aim of the present study was to analyze expression of myogenic transcription factors involved in the regulation of myogenesis as well as the frequency of interstitial endothelial progenitor cells in muscle biopsies from JDM patients.

Magnetic targeting of human peripheral blood CD133+ cells for skeletal muscle regeneration.

Skeletal muscle injuries often leave lasting functional damage or pain. Muscle injuries are routinely treated conservatively, but the most effective treatment to promote the repair of injured muscles has not yet been established. Our previous report demonstrated that human peripheral blood-derived CD133(+) cell transplantation to rat skeletal muscle injury models inhibited fibrosis and enhanced myogenesis after injury. However, the acquisition of a sufficient number of cells remains the limitation for clinical application, as the CD133(+) population is rare in human blood. In this study, we applied a magnetic cell targeting system to accumulate transplanted cells in the muscle injury site and to enhance the regenerative effects of CD133(+) cell transplantation, focusing on the fact that CD133(+) cells are labeled with a magnetic bead for isolation. For the magnetic cell targeting, the magnet field generator was set up to adjust the peak of the magnetic gradient to the injury site of the tibialis anterior muscle, and 1×10(4) human peripheral blood CD133(+) cells were locally injected into the injury site. This cell number is 10% of that used in the previous study. In another group, the same number of CD133(+) cells was injected without magnetic force. The CD133(+) cells transplanted with the magnetic force were more accumulated in the muscle injury site compared with the CD133(+) cells transplanted without the magnetic force. In addition, the transplantation of CD133(+) cells under the magnetic control inhibited fibrous scar formation and promoted angiogenesis and myogenesis, and also upregulated the mRNA expression of myogenic transcription factors, including Pax7, MyoD1 and Myogenin. However, the transplantation of CD133(+) cells without the magnetic force failed to demonstrate these effects. Thus, our magnetic cell targeting system enables transplantation of a limited number of CD133(+) cells to promote the repair of skeletal muscle injury.

Enriching Satellite Cells with α2δ1 Promotes Differentiation

Cell transplants into skeletal muscle of patients with muscular dystrophy are limited by donor cell attachment, migration, and survival in the host tissue. In animal models, despite HLA matching and a reduction of the host's immune response, few donor cells are retained in the host muscle. Enriching cells for a surface marker that enhances ability of the cell to attach, migrate, and survive will result in improved cell retention. The purpose of this study was to determine whether α2δ1-enriched primary satellite cells were better candidates for cell transplant than satellite cells without this surface marker. The α2δ1 subunit is part of the L-type calcium channel but appears earlier than the other subunits. We isolated satellite cells from the hind limb muscles of neonatal mice and separated four subpopulations of cells based on the presence or absence of α2δ1 and a marker of quiescence, CD34, by fluorescence activated cell sort (FACS). Satellite cells enriched with α2δ1 survived in heat deactivated media past day 19, while there was no evidence of attachment or survival of cells without α2δ1. In addition, cells that were positive for α2δ1 and negative for CD34 demonstrated the most robust myogenesis out of all four subpopulations. Enhanced myogenesis of this subpopulation was determined by morphology, the pattern of expression of myogenic transcription factors, and the development of excitation-contraction coupling as demonstrated by the presence of L-type calcium currents and calcium transients. As the data relate to differentiation and survival, these results suggest that cells enriched with α2δ1 and without CD34 will demonstrate greater cell retention and force generation after satellite cell transplant, which is a strong candidate for therapy in muscular dystrophy.Supported by MDA

In ovo administration of rhIGF-1 to duck eggs affects the expression of myogenic transcription factors and muscle mass during late embryo development

In ovo administration of IGF-1 to poultry eggs has effective roles on post hatching muscle development. However, the secondary muscle development stages at the late embryo development stage are important for muscle fiber formation and differentiation. To investigate the roles of in ovo administration of IGF-1 on duck secondary muscle development, we injected rhIGF-1 into duck eggs in hatching at day 12. After administration on days 18, 21, 24, and 27 in hatching (E18d, E21d, E24d, and E27d, respectively), muscle samples were isolated, and the muscle tissue weight, muscle fiber parameters, and myoblast proliferation rate in leg and breast muscle were analyzed. Additionally, the expression levels of the transcription factors MyoG and MRF4 were detected using qPCR. Results show that embryo body weight and muscle fiber parameters, including muscle fiber diameter (MFD) and the number of myofibers per unit area, are upregulated in IGF-1-treated groups. Moreover, the transcription factors MyoG and MRF4 are expressed at higher levels in the experimental groups compared with the control groups. These results suggest that in ovo administration of IGF-1 to poultry eggs can mediate the expression of MyoG and MRF4, induce myoblast proliferation, and finally influence muscle development during the secondary muscle development stages.

Cardiac interstitial cells express GATA4 and control dedifferentiation and cell cycle re-entry of adult cardiomyocytes

Interstitial cells of the adult rat heart were characterized with respect to i) expression of cardiac markers of commitment and differentiation, ii) myogenic potential in vitro and iii) ability to modulate cardiomyocyte differentiation state. We demonstrate for the first time that fibroblasts and a proportion of pericytes in the adult rat heart express the transcription factor GATA4. This appears to be a peculiar property of the heart. Fibroblasts that are also derived from the splanchnopleuric mesoderm, such as those of the gut, or fibroblasts of different embryological origin, such as those of skin and skeletal muscle, lack this property. Of note, a nestin+/GATA4+ putative stem cell population is also detected in the adult heart. GATA4+ cardiac interstitial cells do not display myogenic potential in vitro. However, cardiac fibroblasts, but not skin fibroblasts, stimulate dedifferentiation of adult cardiomyocytes and their re-entry into the cell cycle in vitro, as demonstrated by the high number of cardiomyocytes expressing Ki67, phosphorylated histone H3 (H3P) and incorporating 5-bromodeoxiuridine (BrdU) in the co-cultures. In conclusion, cardiac fibroblasts have peculiar expression of myogenic transcription factors, a property that may have an impact for reprogramming these cells to the myogenic differentiation. In addition, they are able to modulate the behavior of adult cardiomyocytes, a property that may be used to promote dedifferentiation and proliferation of cardiac cells in the damaged myocardium.

Ectopic Expression of Myostatin Induces Atrophy of Adult Skeletal Muscle by Decreasing Muscle Gene Expression

Myostatin is a master regulator of myogenesis and early postnatal skeletal muscle growth. However, myostatin has been also involved in several forms of muscle wasting in adulthood, suggesting a functional role for myostatin in the regulation of skeletal muscle mass in adult. In the present study, localized ectopic expression of myostatin was achieved by gene electrotransfer of a myostatin expression vector into the tibialis anterior muscle of adult Sprague Dawley male rats. The corresponding empty vector was electrotransfected in contralateral muscle. Ectopic myostatin mRNA was abundantly present in muscles electrotransfected with myostatin expression vector, whereas it was undetectable in contralateral muscles. Overexpression of myostatin elicited a significant decrease in muscle mass (10 and 20% reduction 7 and 14 d after gene electrotransfer, respectively), muscle fiber cross-sectional area (15 and 30% reduction 7 and 14 d after gene electrotransfer, respectively), and muscle protein content (20% reduction). No decrease in fiber number was observed. Overexpression of myostatin markedly decreased the expression of muscle structural genes (myosin heavy chain IIb, troponin I, and desmin) and the expression of myogenic transcription factors (MyoD and myogenin). Incidentally, mRNA level of caveolin-3 and peroxisome proliferator activated receptor gamma coactivator-1alpha was also significantly decreased 14 d after myostatin gene electrotransfer. To conclude, our study demonstrates that myostatin-induced muscle atrophy elicits the down-regulation of muscle-specific gene expression. Our observations support an important role for myostatin in muscle atrophy in physiological and physiopathological situations where myostatin expression is induced.

Decreased expression of myogenic transcription factors and myosin heavy chains in Caenorhabditis elegans muscles developed during spaceflight

Decreased expression of myogenic transcription factors and myosin heavy chains in <i>Caenorhabditis elegans</i> muscles developed during spaceflight