Negative Regulator Of Myogenesis Research Articles

UCHL1 Negatively Regulates Key Factors and Signals Involved in Skeletal Muscle Myogenesis

Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme that was originally discovered in neurons, is also expressed in skeletal muscle, but its functions remain to be fully understood. Skeletal muscle injury is prevalent in trauma and surgical procedures, and skeletal muscle ischemia-reperfusion (IR) injury is a common and significant public health problem. Myogenesis is a critical process involved in embryonic development, growth, and regeneration following skeletal muscle injury. In this study we report that UCHL1 negatively affects some key factors that are involved in myogenesis and mitophagy, both during muscle development as well as during the regeneration process following IR injury; we hypothesize that UCHL1 is a negative regulator of myogenesis. First, we observed that UCHL1 knockdown in C2C12 myoblasts resulted in increased differentiation and myotube formation. Furthermore, western blot showed that UCHL1 KD consistently upregulated myogenin and MyoD protein levels, key proteins involved in myogenesis, at multiple time points throughout differentiation of the myotubes. Consistent with this in vitro result, skeletal muscle specific knockout (smKO) of UCHL1 increased muscle fiber size in both 1- and 2-month-old mice (p=1.77x10−17, p=6.51x10−13). Additionally, 1- and 2- month-old UCHL1 smKO mice also exhibited increased protein levels of various myosin heavy chain isoforms. To induce IR injury, a tourniquet was placed on the hindlimb for 90 minutes and then removed, allowing for reperfusion for 3-12 days with the contralateral hindlimb serving as a control. Following skeletal muscle IR injury, myogenin and MyoD protein expression was upregulated in injured muscle from UCHL1 smKO mice. Functionally, UCHL1 smKO mice had increased muscle performance after injury compared to control mice when subjected to in situ contractile testing. Interestingly, several mitophagy markers including Parkin and DRP1 were upregulated in IR injured muscle from UCHL1 smKO mice compared to WT mice (p=0.0003, p=0.029). This data suggests that skeletal muscle UCHL1 may function as a negative regulator of myogenesis, both during growth, and repair following injury, possibly by decreasing mitochondrial activity. This work was funded by NIH grant R01HL147105. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The key role of myostatin b in somatic growth in fishes derived from distant hybridization.

The basic mechanism of heterosis has not been systematically and completely characterized. In previous studies, we obtained three economically important fishes that exhibit rapid growth, WR (WCC ♀ × RCC ♂), WR-II (WR ♀ × WCC ♂), and WR-III (WR-II ♀ × 4nAU ♂), through distant hybridization. However, the mechanism underlying this rapid growth remains unclear. In this study, we found that WR, WR-II, and WR-III showed muscle hypertrophy and higher muscle protein and fat contents compared with their parent species (RCC and WCC). Candidate genes responsible for this rapid growth were then obtained through an analysis of 12 muscle transcriptomes. Notably, the mRNA level of mstnb (myostatin b), which is a negative regulator of myogenesis, was significantly reduced in WR, WR-II, and WR-III compared with the parent species. To verify the function of mstnb, a mstnb-deficient mutant RCC line was generated using the CRISPR-Cas9 technique. The average body weight of mstnb-deficient RCC at 12 months of age was significantly increased by 29.57% compared with that in wild-type siblings. Moreover, the area and number of muscle fibers were significantly increased in mstnb-deficient RCC, indicating hypertrophy and hyperplasia. Furthermore, the muscle protein and fat contents were significantly increased in mstnb-deficient RCC. The molecular regulatory mechanism of mstnb was then revealed by transcription profiling, which showed that genes related to myogenesis (myod, myog, and myf5), protein synthesis (PI3K-AKT-mTOR), and lipogenesis (pparγ and fabp3) were highly activated in hybrid fishes and mstnb-deficient RCC. This study revealed that low expression or deficiency of mstnb regulates somatic growth by promoting myogenesis, protein synthesis, and lipogenesis in hybrid fishes and mstnb-deficient RCC, which provides evidence for the molecular mechanism of heterosis via distant hybridization.

A Novel Nanobody Directed against Ovine Myostatin to Enhance Muscle Growth in Mouse.

Simple SummaryMyostatin (MSTN) is a negative regulator of myogenesis, and various strategies have been used to improve livestock production by inhibiting MSTN. In this study, we developed a single-variable domain of the heavy chain antibody-recombinant MSTN nanobody (RMN) against MSTN. The selected RMN was expressed, purified, and assessed for its cytotoxicity, affinity, specificity, and the ability to inhibit MSTN. The results demonstrated that RMN could specifically detect and bind MSTN, further inhibit myostatin activity, as well as enhance muscle growth in mice.Myostatin (MSTN) is a member of the transforming growth factor beta superfamily and is a negative regulator of myogenesis. It has been shown to function by controlling the proliferation of myoblasts. MSTN inhibition is considered as a promising treatment for promoting animal growth in livestock. Nanobodies, a special antibody discovered in camel, have arisen as an alternative to conventional antibodies and have shown great potential when used as tools in different biotechnology fields, such as diagnostics and therapy. In this study, we examined the effect of MSTN inhibition by RMN on the muscle growth of mice. The results showed that RMN could specifically detect and bind MSTN, as well as inhibit MSTN activity. A significant increase in skeletal muscle mass was observed after intramuscular injection of RMN into mice. Enhanced muscle growth occurred because of myofiber hypertrophy. These results offer a promising approach to enhance muscle growth that warrants further investigation in domestic animals.

Disruption of mstna and mstnb gene through CRISPR/Cas9 leads to elevated muscle mass in blunt snout bream (Megalobrama amblycephala)

Myostatin (mstn) belongs to the TGF-β family, which is mainly involved in the negative regulation of myogenesis in vertebrates. Upon implementation of several gene-editing techniques (ZFN, TALEN, RNAi and CRISPR/Cas9), disruption of mstn gene locus in several animal species has been shown to result in elevated muscle growth. As there are numerous reports on gene editing of mstn in animals (common carp, yellow catfish, channel catfish, etc.), we aimed to study the complete disruption of mstn in economy-farmed fish. Here, we analyzed and characterized mstna and mstnb in blunt snout bream. In blunt snout bream, both mstna and mstnb peptides were found to possess unique structural characteristics of the TGF-β family. We designed three target sites (targets 1, 2, and 3) to generate knock out mutants within the mature mRNA sequence of mstna or mstnb that were located in exon 1, exon 3, and exon 3, respectively. The fertilized eggs (1–2 cell stage) were injected with 1 nL mixture of gRNA (100 pg) and Cas9 protein (900 pg). The mutation efficiency of mstna targets 1, 2, and 3 was 5.1%, 8.2%, and 10.0% with mutation types 5, 4, and 12, respectively. Similarly, the mutation efficiency of mstnb targets 1, 2, and 3 was 15.3%, 14.7%, and 26.0% with mutation types 2, 3, and 5, respectively. At the age of six months, the body weight, height, and thickness of both mstna- (mstna+/−) and mstnb- (mstnb+/−) deficient fish were found to be increased significantly (p < .01) when compared to those of wild-type control fish. Moreover, the average muscle fiber sizes of both mstna+/− and mstnb+/− fish were significantly larger (p < .01) than that of control fish. Our results provide valuable data associated with the regulation of muscle development and growth in blunt snout bream. These results also provide a basis and reference for the application of CRISPR/Cas9 (robust and efficient gene-engineering tool) in other aquaculture fish species.

Distal-less homeobox 3, a negative regulator of myogenesis, is downregulated by microRNA-133.

Distal-less homeobox 3 (Dlx3), a member of the Dlx family of homeobox proteins, is a transcriptional activator of runt-related transcription factor 2 (Runx2) during osteogenic differentiation. It has been demonstrated that forced expression of Runx2 induces an osteogenic program and ectopic calcification in muscles. Therefore, it would be reasonable to predict that Dlx3 also affects myogenic differentiation. The relationship between Dlx3 and myogenesis, however, remains poorly understood. Therefore, in this study, the role and regulation of Dlx3 during myogenic differentiation were investigated. Expression level of Dlx3 was downregulated in human mesenchymal stem cells (MSCs), mouse MSCs, and C2C12 cells cultured in myogenic medium. Dlx3 level was inversely correlated with myogenic differentiation 1 and the muscle-specific microRNA, microRNA-133 (miR-133). The expression level of Runx2 was closely regulated by Dlx3 even under myogenic conditions. Overexpression of Dlx3 markedly downregulated expression levels of myogenic transcription factors and myotube formation in C2C12 cells, whereas Dlx3 knockdown enhanced myogenic differentiation. The Dlx3 3'-untranslated region (3'-UTR) has two potential binding sites for miR-133. Luciferase reporter assays demonstrated that Dlx3 is a direct target of miR-133a and miR-133b, and that the two target sites are redundantly active. Taken together, these results suggest that Dlx3 is a negative regulator of myogenic differentiation and that miR-133a and miR-133b enhance myogenic differentiation, partly through inhibition of Dlx3 expression via direct targeting of the Dlx3 3'-UTR.

Antisense Oligonucleotide Targeting of 3'-UTR of mRNA for Expression Knockdown.

With the recent conditional approval of an antisense oligonucleotide (AON) that restores the reading frame of DMD transcript in a subset of Duchenne muscular dystrophy patients, it has been established that AONs sharing similar chemistry have clear clinical potential. Genetic diseases, such as facioscapulohumeral dystrophy (FSHD), can be the result of gain-of-function mutations. Since mRNA processing in terms of termination of transcription, its transport from the nucleus to the cytoplasm, its stability and translation efficiency are dependent on key 3'UTR elements, it follows that targeting theseelements with AONs have the potential to induce gene silencing. Aberrant expression of the Double homeobox 4 (DUX4) transcription factor and the downstream consequences of such expression is the hallmark of FSHD. Here we describe the bioinformatic strategies behind the design of AONs targeting polyadenylation signals and the methodologies relevant to their in vitro screening for efficacy and safety, including analysis of expression at the transcript and protein level of the specific target and downstream genes, and measurement of the effect on the fusion index of myotubes. The targeting of permissive DUX4 and MSTN are used as examples. MSTN encodes for myostatin, a negative regulator of myogenesis; the downregulation of MSTN expression has the potential to address the muscular atrophy associated with muscular dystrophies, sarcopenia, cancer and acquired immunodeficiency syndrome.

Viral infection upregulates myostatin promoter activity in orange-spotted grouper (Epinephelus coioides).

Myostatin is a negative regulator of myogenesis and has been suggested to be an important factor in the development of muscle wasting during viral infection. The objective of this study was to characterize the main regulatory element of the grouper myostatin promoter and to study changes in promoter activity due to viral stimulation. In vitro and in vivo experiments indicated that the E-box E6 is a positive cis-and trans-regulation motif, and an essential binding site for MyoD. In contrast, the E-box E5 is a dominant negative cis-regulatory. The characteristics of grouper myostatin promoter are similar in regulation of muscle growth to that of other species, but mainly through specific regulatory elements. According to these results, we conducted a study to investigate the effect of viral infection on myostatin promoter activity and its regulation. The nervous necrosis virus (NNV) treatment significantly induced myostatin promoter activity. The present study is the first report describing that specific myostatin motifs regulate promoter activity and response to viral infection.

A Non‐Canonical Function of Leucyl tRNA Synthetase Negatively Regulates Skeletal Myogenesis

Aside from its house‐keeping function in protein synthesis, leucyl tRNA synthetase (LeuRS) has a non‐canonical function in regulating cell growth via the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway by sensing amino acid availability. mTOR also governs skeletal myogenesis, but its signaling network is distinct from that in cell growth regulation. We set out to investigate a potential role of LeuRS in skeletal myogenesis. Knockdown of LeuRS enhanced myoblast differentiation in vitro, whereas overexpression of LeuRS inhibited myogenesis. In a mouse model of injury‐induced adult muscle regeneration, local knockdown of LeuRS enhanced regeneration. Hence, LeuRS acts as a negative regulator of myogenesis both in vitro and in vivo. However, inhibition of the translational function of LeuRS by leucinol severely impaired myogenesis, consistent with the notion that protein synthesis is required for the myogenic process. These observations suggest a non‐translation role for LeuRS in suppressing myogenesis. Indeed, we found leucine‐binding but not tRNA charging activity of LeuRS to be necessary for its overexpression effect on myogenesis, suggesting that LeuRS may act as a leucine sensor in its function to ensure homeostasis of myogenesis. To probe the mechanism underlying this non‐canonical function of LeuRS, we examined the involvement of Rag GTPases, mediators of LeuRS activation of mTORC1 in cell growth. Constitutively active Rag reversed the effect of LeuRS knockdown on myogenesis and, conversely, Rag knockdown overcame the inhibitory effect of LeuRS overexpression. Rag knockdown also enhanced muscle regeneration in vivo, mirroring the effect of LeuRS knockdown. Taken together, our findings reveal for the first time a function for LeuRS in modulating the homeostasis of myogenesis. This new LeuRS function is mediated by a Rag‐mTORC1 pathway that negatively regulates the myogenic IRS1‐PI3K‐Akt pathway.Support or Funding InformationKeck Foundation, NIH/NIAMS R01AR048914

Myogenic differentiation depends on the interplay of Grb2 and N-WASP

Myogenesis requires a well-coordinated withdrawal from cell cycle, morphological changes and cell fusion mediated by actin cytoskeleton. Grb2 is an adaptor protein whose central SH2 domain binds to phosphorylated tyrosine residues of activated receptors and activates intracellular signaling pathway, while its N-terminal and C-terminal SH3 domains bind to proline rich proteins such as N-WASP (Neural-Wiskott Aldrich Syndrome Protein). We found that the expression of Grb2 was increased at the beginning of differentiation and remained constant during differentiation in C2C12 myoblasts. Knocking down endogenous Grb2 expression caused a significant increase in the fusion index and expression of MyHC, a terminal differentiation marker when compared with the control. Over expression of Grb2 in C2C12 (C2C12Grb2-Myc) reduced myotube formation and expression of MyHC. Similarly over expression of Grb2P49L-Myc (N-terminal SH3 domain mutant) or Grb2R86K-Myc (SH2 domain mutant) inhibited myogenic differentiation of C2C12 cells. However, the expression of Grb2P206L-Myc (C-terminal SH3 domain mutant) did not inhibit myotube formation and expression of MyHC. This suggests that the C-terminal SH3 domain of Grb2 is critical for the inhibition of myogenic differentiation. The C2C12Grb2-Myc cells have reduced phalloidin staining at late stages of differentiation. Expression of N-WASP in C2C12Grb2-Myc cells rescued the myogenic defect and increased phalloidin staining (increased F-actin) in these cells. Thus our results suggest that Grb2 is a negative regulator of myogenesis and reduces myogenic differentiation by inhibiting actin polymerization/remodeling through its C-terminal SH3 domain.

Dnmt1 regulates the myogenic lineage specification of muscle stem cells.

DNA methylation is an important epigenetic mark that regulates gene expression. Dnmt1 plays an important role in maintaining DNA methylation patterns on daughter DNA strands. Studies have shed light into the functional role of Dnmt1 regulation in the hematopoietic and epidermal systems. Here we show that Dnmt1 is required for myogenesis. Loss of Dnmt1 results in reduced expression of myogenic genes and defects in myogenic differentiation. We have utilized a conditional knockout mouse approach to examine the functional consequences of Dnmt1 depletion specifically in the developing muscle. These mice were born runted, with smaller body weights, and reduced ability to form myotubes in vitro. We show that expression of Id-1, a negative regulator of myogenesis, is enhanced in Dnmt1-deficient cultures, leading to enhanced transdifferentiation of myoblasts toward the osteogenic lineage. Thus, these studies demonstrate that Dnmt1 influences cellular identity and determines lineage fidelity.

Depletion of Myostatin b Promotes Somatic Growth and Lipid Metabolism in Zebrafish.

Myostatin (MSTN) is a negative regulator of myogenesis in vertebrates. Depletion of mstn resulted in elevated muscle growth in several animal species. However, the report on the complete ablation of mstn in teleost fish has not yet become available. In this study, two independent mstnb-deficient mutant lines in zebrafish were generated with the TALENs technique. In the mstnb-deficient zebrafish, enhanced muscle growth with muscle fiber hyperplasia was achieved. Beginning at the adult stage (80 days postfertilization), the mstnb-deficient zebrafish exhibited increased circumferences and body weights compared with the wild-type sibling control fish. Although the overall total lipid/body weight ratios remained similar between the mstnb-deficient zebrafish and the control fish, the distribution of lipids was altered. The size of the visceral adipose tissues became smaller while more lipids accumulated in skeletal muscle in the mstnb-deficient zebrafish than in the wild-type control fish. Based on the transcriptional expression profiles, our results revealed that lipid metabolism, including lipolysis and lipogenesis processes, was highly activated in the mstnb-deficient zebrafish, which indicated the transition of energy metabolism from protein-dependent to lipid-dependent in mstnb-deficient zebrafish. Our mstnb-deficient model could be valuable in understanding not only the growth trait regulation in teleosts but also the mechanisms of teleost energy metabolism.

Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future.

Dual roles of palladin protein in in vitro myogenesis: inhibition of early induction but promotion of myotube maturation.

Palladin is a microfilament-associated phosphoprotein whose function in skeletal muscle has rarely been studied. Therefore, we investigate whether myogenesis is influenced by the depletion of palladin expression known to interfere with the actin cytoskeleton dynamic required for skeletal muscle differentiation. The inhibition of palladin in C2C12 myoblasts leads to precocious myogenic differentiation with a concomitant reduction in cell apoptosis. This premature myogenesis is caused, in part, by an accelerated induction of p21, myogenin, and myosin heavy chain, suggesting that palladin acts as a negative regulator in early differentiation phases. Paradoxically, palladin-knockdown myoblasts are unable to differentiate terminally, despite their ability to perform some initial steps of differentiation. Cells with attenuated palladin expression form thinner myotubes with fewer myonuclei compared to those of the control. It is noteworthy that a negative regulator of myogenesis, myostatin, is activated in palladin-deficient myotubes, suggesting the palladin-mediated impairment of late-stage myogenesis. Additionally, overexpression of 140-kDa palladin inhibits myoblast differentiation while 200-kDa and 90-kDa palladin-overexpressed cells display an enhanced differentiation rate. Together, our data suggest that palladin might have both positive and negative roles in maintaining the proper skeletal myogenic differentiation in vitro.

Abstract PR02: Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is composed of skeletal myoblast-like cells that have lost the capacity to terminally differentiate. This suggests that RMS cells may contain a factor that blocks normal muscle differentiation. Because cell cycle arrest is coupled to muscle differentiation, identifying putative negative regulators of differentiation could lead to novel therapeutic approaches aimed at fostering terminal differentiation. To gain insight into the events that normally trigger the initial phase of muscle differentiation, we carried out a high content cell-based screen using detection of Myogenin, an early marker of muscle differentiation, as a readout that we measured by automated fluorescence microscopy. We focused on identifying kinases that hinder this developmental transition because of the potential to develop pharmacological inhibitors of “hit” kinases. Using an siRNA library targeting over 600 kinases, we identified 56 as putative negative regulators of myogenic differentiation: 43 with and 19 without PD332991, a Cdk4/6 inhibitor included as a sensitizer. Network analysis showed that 47% of the hits identified without the sensitizer, including SRC family kinases Src and Fyn, were just one interaction node away from MyoD- or Mef2-family myogenic regulatory factors. Although some of the hit kinases were previously implicated in myogenesis, many others were not. Further, 9 (16%) of the hits are causally linked with cancer, based on KEGG and COSMIC databases. Pathway analyses highlighted certain cancer-associated pathways, like EPHA/Ephrin B signaling, the MAPK kinase pathway, de novo pyrimidine synthesis, and mTOR signaling. Among the hits relevant to RMS, Mtor was particularly interesting because this gene encodes a kinase regarded to positively regulate skeletal muscle differentiation. We confirmed our screen findings that Mtor blocks muscle differentiation by showing that its knockdown increases the transcription of a panel of muscle-specific genes in an established myoblast cell line and primary mouse myoblasts. Induction of muscle genes by Mtor knockdown correlated with G0/G1 cell cycle arrest that normally accompanies differentiation. Rapamycin mimicked the effects of Mtor knockdown on muscle gene expression and cell proliferation, implying a role for mTorc1. Finally, preliminary analysis of RMS gene expression data demonstrated that higher expression of muscle differentiation genes correlates with improved survival. These data highlight the potential for mTOR inhibitors to foster the expression of muscle specific genes, pushing the myoblast-like tumor cells to a more differentiated state. Altering their biology in this way may improve outcome. On-going efforts are exploring the mechanisms acting downstream of Mtor to impede muscle differentiation and directly evaluating pro-differentiation effects of mTOR inhibition in RMS models. This abstract is also presented as Poster A1. Citation Format: Raphael A. Wilson, Jing Liu, Lin Xu, Yanbin Zheng, Stephen X. Skapek. Negative regulation of myogenesis by Mtor: A pathway toward differentiation therapy in rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR02.

Grb10 Deletion Enhances Muscle Cell Proliferation, Differentiation and GLUT4 Plasma Membrane Translocation

Grb10 is an intracellular adaptor protein which binds directly to several growth factor receptors, including those for insulin and insulin-like growth factor receptor-1 (IGF-1), and negatively regulates their actions. Grb10-ablated (Grb10(-/-) ) mice exhibit improved whole body glucose homeostasis and an increase in muscle mass associated specifically with an increase in myofiber number. This suggests that Grb10 may act as a negative regulator of myogenesis. In this study, we investigated in vitro, the molecular mechanisms underlying the increase in muscle mass and the improved glucose metabolism. Primary muscle cells isolated from Grb10(-/-) mice exhibited increased rates of proliferation and differentiation compared to primary cells isolated from wild-type mice. The improved proliferation capacity was associated with an enhanced phosphorylation of Akt and ERK in the basal state and changes in the expression of key cell cycle progression markers involved in regulating transition of cells from the G1 to S phase (e.g., retinoblastoma (Rb) and p21). The absence of Grb10 also promoted a faster transition to a myogenin positive, differentiated state. Glucose uptake was higher in Grb10(-/-) primary myotubes in the basal state and was associated with enhanced insulin signaling and an increase in GLUT4 translocation to the plasma membrane. These data demonstrate an important role for Grb10 as a link between muscle growth and metabolism with therapeutic implications for diseases, such as muscle wasting and type 2 diabetes.

Insulin and LiCl Synergistically Rescue Myogenic Differentiation of FoxO1 Over-Expressed Myoblasts

Most recent studies reported that FoxO1 transcription factor was a negative regulator of myogenesis under serum withdrawal condition, a situation not actually found in vivo. Therefore, the role of FoxO1 in myogenesis should be re-examined under more physiologically relevant conditions. Here we found that FoxO1 was preferentially localized to nucleus in proliferating (PMB) and confluent myoblasts (CMB) and its nuclear exclusion was a prerequisite for formation of multinucleated myotubes (MT). The nuclear shuttling of FoxO1 in PMB could be prevented by leptomycin B and we further found that cytoplasmic accumulation of FoxO1 in myotubes was caused by the blockade of its nuclear import. Although over-expression of wildtype FoxO1 in C2C12 myoblasts significantly blocked their myogenic differentiation under serum withdrawal condition, application of insulin and LiCl, an activator of Wnt signaling pathway, to these cells successfully rescued their myogenic differentiation and generated myotubes with larger diameters. Interestingly, insulin treatment significantly reduced FoxO1 level and also delayed nuclear re-accumulation of FoxO1 triggered by mitogen deprivation. We further found that FoxO1 directly repressed the promoter activity of myogenic genes and this repression can be relieved by insulin and LiCl treatment. These results suggest that FoxO1 inhibits myogenesis in serum withdrawal condition but turns into a hypertrophy potentiator when other myogenic signals, such as Wnt and insulin, are available.

CARP, a Myostatin-downregulated Gene in CFM Cells, Is a Novel Essential Positive Regulator of Myogenesis

Myostatin, a member of the TGF-β superfamily, has been shown to act as a negative regulator of myogenesis. Although its role in myogenesis has been clearly documented through genetic analysis, few gene cascades that respond to myostatin signaling and regulate myogenesis have been characterized, especially in avian species. In a previous study, we screened for such genes in chicken fetal myoblasts (CFMs) using the differential display PCR method and found that cardiac ankyrin repeat protein (CARP) was downregulated by myostatin and specifically expressed in chicken skeletal muscle. However, little is known about the potential functions of CARP in chicken skeletal myogenesis. In this study, the expression patterns of chicken CARP and the possible function of this gene in skeletal muscle growth were characterized. Our data showed that CARP was predominantly expressed in postnatal skeletal muscle, and its expression increased during myogenic differentiation in CFM cells. When CARP was overexpressed, CFM cell growth was enhanced by accelerating the cell cycle at the G1 to S phase transition and increasing cyclin D1 expression. CARP knockdown had the opposite effect: while myoblasts underwent differentiation, knockdown of CARP expression induced extensive cell death, suppressed the formation of myotubes, and markedly decreased the expression of differentiation-related genes such as myosin heavy chain (MHC), myoD, and caveolin-3. Our findings indicate that CARP may play a key role in the myostatin signaling cascade that governs chicken skeletal myogenesis through promoting proliferation and avoiding apoptosis during CFM cell differentiation.

Knockdown of endogenous myostatin promotes sheep myoblast proliferation

Myostatin (MSTN), is a known negative regulator of myogenesis. Silencing of the function of MSTN could result in increasing muscle mass in mice. To determine the function of endogenous MSTN expression on proliferation of sheep myoblasts, a short-hairpin RNA-targeting sheep MSTN was constructed into lentiviral vector to silence endogenous MSTN expression. We demonstrated that silencing of endogenous MSTN gene with up to approximately 73.3% reduction by short hairpin RNA (shRNA) resulted in significant increase (overall 28.3%) of proliferation of primary ovine myoblasts. The upregulation of proliferation was accompanied by the decrease expression of MyoD (-37.6%, p = 0.025), myogenin (-33.1%, p = 0.049), p21 (-49.3%, p = 0.046), and Smad3 (-50.0%, p = 0.007). Silencing of myostatin using shRNA may provide a feasible approach to improve meat productivity in farm animals.

In vitro silencing of myostatin gene by shRNAs in chicken embryonic myoblast cells

RNA interference represents one of the potential mechanisms of regulation of gene expression. Selective downregulation of myostatin (MSTN), a member of transforming growth factor-β (TGF-β) superfamily and a negative regulator of myogenesis, has been demonstrated to enhance skeletal muscle growth. In this study, we studied short hairpin RNA (shRNA)-induced myostatin gene silencing in chicken embryonic myoblast cells using seven different shRNA-expressing constructs by reverse transcription-quantitative real time PCR (RT-qPCR). Myostatin-silencing efficiency of all shRNA constructs were first evaluated in human embryonic kidney cell line 293T (HEK293T) cells, where we observed 30-75.6% reduction in myostatin expression, followed by chicken embryo myoblast cells that revealed up to 55% reduction in myostatin expression along with upregulation of MyoD by 4.65-folds. Consistent with the earlier observations, the transfection of cells with plasmids led to significant increase in interferon responsive genes OAS1 and IFN β (2-112-folds), independent of myostatin silencing in both HEK293T and chicken embryonic myoblast cells. Our study suggests that apart from shRNA sequences, cell type-specific factors may play a significant role in determining the knockdown efficiency of shRNAs.

Myostatin inhibits proliferation of human urethral rhabdosphincter satellite cells

Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of myogenesis in skeletal muscle. We examined the effect of myostatin and myostatin inhibition by an antagonistic agent, follistatin, on growth of human urethral rhabdosphincter satellite cells (muscle stem cells) to develop a new strategy for treatment of stress urinary incontinence. Rhabdosphincter satellite cells were cultured and selected by magnetic affinity cell sorting using an anti-neural cell adhesion molecule antibody. The cells were transfected with simian virus-40 antigen to extend their lifespan. A cell proliferation assay, a cell cycle analysis and an investigation of signal transduction were carried out. The autocrine action of endogenous myostatin by western blotting, real-time reverse transcription polymerase chain reaction and immunoneutralization using an anti-myostatin antibody was also evaluated. Selectively cultured cells expressed markers of striated muscles and successfully differentiated into myotubes. Myostatin inhibited proliferation of these cells through Smad2 phosphorylation and cell cycle arrest. Inhibitory effects of myostatin were reversed by addition of follistatin. However, rhabdosphincter satellite cells did not appear to use autocrine secretion of myostatin to regulate their proliferation. Inhibition of myostatin function might be a useful pathway in the development of novel strategies for stimulating rhabdosphincter cells regeneration to treat stress urinary incontinence.