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.     

Peroxisome proliferator-activated receptor β/δ induces myogenesis by modulating myostatin activity

Classically, peroxisome proliferator-activated receptor β/δ (PPARβ/δ) function was thought to be restricted to enhancing adipocyte differentiation and development of adipose-like cells from other lineages. However, recent studies have revealed a critical role for PPARβ/δ during skeletal muscle growth and regeneration. Although PPARβ/δ has been implicated in regulating myogenesis, little is presently known about the role and, for that matter, the mechanism(s) of action of PPARβ/δ in regulating postnatal myogenesis. Here we report for the first time, using a PPARβ/δ-specific ligand (L165041) and the PPARβ/δ-null mouse model, that PPARβ/δ enhances postnatal myogenesis through increasing both myoblast proliferation and differentiation. In addition, we have identified Gasp-1 (growth and differentiation factor-associated serum protein-1) as a novel downstream target of PPARβ/δ in skeletal muscle. In agreement, reduced Gasp-1 expression was detected in PPARβ/δ-null mice muscle tissue. We further report that a functional PPAR-responsive element within the 1.5-kb proximal Gasp-1 promoter region is critical for PPARβ/δ regulation of Gasp-1. Gasp-1 has been reported to bind to and inhibit the activity of myostatin; consistent with this, we found that enhanced secretion of Gasp-1, increased Gasp-1 myostatin interaction and significantly reduced myostatin activity upon L165041-mediated activation of PPARβ/δ. Moreover, we analyzed the ability of hGASP-1 to regulate myogenesis independently of PPARβ/δ activation. The results revealed that hGASP-1 protein treatment enhances myoblast proliferation and differentiation, whereas silencing of hGASP-1 results in defective myogenesis. Taken together these data revealed that PPARβ/δ is a positive regulator of skeletal muscle myogenesis, which functions through negatively modulating myostatin activity via a mechanism involving Gasp-1.     

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.     

Myostatin gene silenced by RNAi show a zebrafish giant phenotype

Myostatin is a member of the transforming growth factor-beta (TGF-beta) family that functions as a negative regulator of skeletal muscle development and growth. Recently, it has been reported that the transgenic zebrafish expressing myostatin prodomain exhibited an increased number of fiber in skeletal muscle. Other novel results suggest that myostatin plays a mayor role during myogenesis, apart from inhibition of proliferation as well as differentiation. We have investigated the ability of double-stranded RNA (dsRNA) to inhibit myostatin function in the zebrafish. By microinjection dsRNA, corresponding to biologically active C-terminal domain from aminoacid 268 to end codon of tilapia myostatin protein, we produced an increased body mass in treated fish. The dsRNA injection in early development stage in zebrafish produced hyperplasia or hypertrophy. In addition, the interference of gene function showed a strong dependence on the amount of dsRNA.     

Human myostatin negatively regulates human myoblast growth and differentiation

Myostatin, a member of the transforming growth factor-β superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the γ-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway.     

MicroRNA-27a promotes myoblast proliferation by targeting myostatin

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3'UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.     

MicroRNAs in skeletal and cardiac muscle development

MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs, which are approximately 22 nucleotides in length. miRNAs negatively regulate gene expression by translational repression and target mRNA degradation. It has become clear that miRNAs are involved in many biological processes, including development, differentiation, proliferation, and apoptosis. Interestingly, many miRNAs are expressed in a tissue-specific manner and several miRNAs are specifically expressed in cardiac and skeletal muscles. In this review, we focus on those miRNAs that have been shown to be involved in muscle development. Compelling evidences have demonstrated that muscle miRNAs play an important role in the regulation of muscle proliferation and differentiation processes. However, it appears that miRNAs are not essential for early myogenesis and muscle specification. Importantly, dysregulation of miRNAs has been linked to muscle-related diseases, such as cardiac hypertrophy. A mutation resulting in a gain-of-function miRNA target site in the myostatin gene leads to down regulation of the targeted protein in Texel sheep. miRNAs therefore are a new class of regulators of muscle biology and they might become novel therapeutic targets in muscle-related human diseases.     

Myostatin induces degradation of sarcomeric proteins through a Smad3 signaling mechanism during skeletal muscle wasting

Ubiquitination-mediated proteolysis is a hallmark of skeletal muscle wasting manifested in response to negative growth factors, including myostatin. Thus, the characterization of signaling mechanisms that induce the ubiquitination of intracellular and sarcomeric proteins during skeletal muscle wasting is of great importance. We have recently characterized myostatin as a potent negative regulator of myogenesis and further demonstrated that elevated levels of myostatin in circulation results in the up-regulation of the muscle-specific E3 ligases, Atrogin-1 and muscle ring finger protein 1 (MuRF1). However, the exact signaling mechanisms by which myostatin regulates the expression of Atrogin-1 and MuRF1, as well as the proteins targeted for degradation in response to excess myostatin, remain to be elucidated. In this report, we have demonstrated that myostatin signals through Smad3 (mothers against decapentaplegic homolog 3) to activate forkhead box O1 and Atrogin-1 expression, which further promotes the ubiquitination and subsequent proteasome-mediated degradation of critical sarcomeric proteins. Smad3 signaling was dispensable for myostatin-dependent overexpression of MuRF1. Although down-regulation of Atrogin-1 expression rescued approximately 80% of sarcomeric protein loss induced by myostatin, only about 20% rescue was seen when MuRF1 was silenced, implicating that Atrogin-1 is the predominant E3 ligase through which myostatin manifests skeletal muscle wasting. Furthermore, we have highlighted that Atrogin-1 not only associates with myosin heavy and light chain, but it also ubiquitinates these sarcomeric proteins. Based on presented data we propose a model whereby myostatin induces skeletal muscle wasting through targeting sarcomeric proteins via Smad3-mediated up-regulation of Atrogin-1 and forkhead box O1.     

MiR-206在骨骼肌发育中的功能

微RNA(microRNA,miRNA)是一类在分子进化中十分保守的非编码RNA,长度约22个核苷酸,一般情况下它在转录后水平抑制基因表达。miRNA在细胞增殖、分化、凋亡等诸多生理过程中发挥着重要作用。有些miRNA具有组织特异性表达,其中miR-206是目前发现的唯一在骨骼肌中特异表达的miRNA,它在调节骨骼肌发生过程中扮演重要角色。miR-206表达异常与一些肌肉相关疾病如肌肉营养不良、肌萎缩性侧索硬化症等有关。此外,在Texel羊中,myostatin基因的一个点突变就产生了一个miR-206和miR-1的靶点,抑制了myostain基因的表达,从而产生了双肌表型。因此,miR-206有可能成为治疗肌肉相关疾病和畜禽改良育种的重要候选分子。     

Expression of basic fibroblast growth factor results in the decrease of myostatin mRNA in murine C2C12 myoblasts

During the development and regeneration of skeletal muscle,many growth factors,such asbasic fibroblast growth factor (bFGF,FGF-2) and myostatin,have been shown to play regulating roles.bFGF contributes to promote proliferation and to inhibit differentiation of skeletal muscle,whereas myostatinplays a series of contrasting roles.In order to elucidate whether the expression of bFGF has any relationshipwith the expression of myostatin in skeletal muscle cells,we constructed a eukaryotic expression vector forthe expression of exogenous bFGF in murine C2C12 myoblasts.Quantitative RT-PCR assays indicated thatwith the increase of the expression of exogenous bFGF gene,the expression of endogenous myostatin genewas suppressed at mRNA level and protein level.     

Relationships between transforming growth factor-beta1, myostatin, and decorin: implications for skeletal muscle fibrosis

Recent studies have shown that myostatin, first identified as a negative regulator of skeletal muscle growth, may also be involved in the formation of fibrosis within skeletal muscle. In this study, we further explored the potential role of myostatin in skeletal muscle fibrosis, as well as its interaction with both transforming growth factor-beta1 and decorin. We discovered that myostatin stimulated fibroblast proliferation in vitro and induced its differentiation into myofibroblasts. We further found that transforming growth factor-beta1 stimulated myostatin expression, and conversely, myostatin stimulated transforming growth factor-beta1 secretion in C2C12 myoblasts. Decorin, a small leucine-rich proteoglycan, was found to neutralize the effects of myostatin in both fibroblasts and myoblasts. Moreover, decorin up-regulated the expression of follistatin, an antagonist of myostatin. The results of in vivo experiments showed that myostatin knock-out mice developed significantly less fibrosis and displayed better skeletal muscle regeneration when compared with wild-type mice at 2 and 4 weeks following gastrocnemius muscle laceration injury. In wild-type mice, we found that transforming growth factor-beta1 and myostatin co-localize in myofibers in the early stages of injury. Recombinant myostatin protein stimulated myofibers to express transforming growth factor-beta1 in skeletal muscles at early time points following injection. In summary, these findings define a fibrogenic property of myostatin and suggest the existence of co-regulatory relationships between transforming growth factor-beta1, myostatin, and decorin.     

Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation

Regeneration of muscle fibers that are lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell-derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compare transgenic mice that overexpress necdin in skeletal muscle with both wild-type and necdin null mice. After muscle injury the necdin null mice show a considerable defect in muscle healing, whereas mice that overexpress necdin show a substantial increase in myofiber regeneration. We also find that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Collectively, these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration.     

MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.