Genetic Deficiency of p38α Reveals its Critical Role in Myoblast Cell Cycle Exit:The p38α-JNK Connection

The regulation of skeletal muscle formation (myogenesis) is essential for normal development as well as in pathological conditions such as muscular dystrophies and inflammatory myopathies. Findings published over the past years have established a key role for the p38 MAP kinase signaling pathway in the control of muscle gene expression and myotube formation. However, the relative contribution of the four p38 MAP kinases (p38α, p38β, p38γ and p38δ) to this process was unknown. We have recently demonstrated that myoblasts lacking p38α, but not those lacking p38β or p38δ, were unable to differentiate and form multinucleated myotubes, while p38γ-deficient myoblasts exhibited an attenuated fusion capacity. Defective myogenesis in the absence of p38α was attributed to delayed cell cycle exit and continuous proliferation in differentiation-promoting conditions, caused by enhanced activation of the JNK/cJun pathway. We discuss these findings in the context of the emerging crosstalk of p38 and JNK signaling pathways in controlling cell growth and differentiation.     

Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells

Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle.     

E and F alpha series prostaglandins in developing muscles

Prostaglandins are known to affect myoblast proliferation and fusion in vitro and are putative regulators of in vivo myogenesis. The levels of E and F alpha series prostaglandins in the thigh muscles of chicken embryos were measured by radioimmunoassays and correlated with indicators of muscle development. Just prior to the onset of secondary myogenesis, the amounts of PGE1, PGE2 and PGF1 alpha plus PGF2 alpha per mg of protein were high. In temporal association with myotube formation, the amount of PGE1 and PGE2 per mg of protein decreased. PGF alpha levels also fell, but at a slower rate than observed with the E series prostaglandins. The decreases in the amounts of prostaglandins per mg protein appeared to be due to a decline in the total amount of prostaglandin within each muscle. These observations are consistent with prostaglandins being one of the factors that controls in vivo muscle formation.     

Knockdown of hypoxia-inducible factor-1alpha by siRNA inhibits C2C12 myoblast differentiation

We analyzed the role of Hypoxia-inducible factor (HIF)-1alpha in myoblast differentiation by examining the expression and regulation of HIF-1alpha in proliferating and differentiating C2C12 myoblast, and by knocking down HIF-1alpha of C2C12 myoblasts with small interfering RNA (siRNA), given that HIF-1alpha has been shown to be involved in differentiative process in non-muscle tissues. Although HIF-1alpha mRNA was constantly expressed in C2C12 myoblasts both under growth and differentiating phase, HIF-1alpha protein was hardly detectable in the growth phase but became detectable only during myogenic differentiation even under normoxia. During early stage of C2C12 myogenesis, HIF-1alpha accumulated in the nuclei of myogenin-positive myoblasts. The inhibition of proteasome in the growth phase led to HIF-1alpha protein accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF-1alpha, suppressed HIF-1alpha accumulation. Therefore, we suggest that the level of HIF-1alpha protein expression is regulated by a proteasome-and chaperon-dependent pathway in C2C12 myoblast. Knockdown of HIF-1alpha effectively blocked myotube formation and myosin heavy chain (MHC) expression. Finally, HIF-1alpha expression in vivo was confirmed in the regenerative muscle tissue of mice after eccentric exercise. We conclude that HIF-1alpha is required for C2C12 myogenesis in vitro, and suggest that HIF-1alpha may have an essential role in regenerative muscle tissue in vivo.     

TIEG1 negatively controls the myoblast pool indispensable for fusion during myogenic differentiation of C2C12 cells

The transforming growth factor (TGF)-β inducible early gene (TIEG)-1 is implicated in the control of cell proliferation, differentiation, and apoptosis in some cell types. Since TIEG1 functioning may be associated with TGF-β, a suppressor of myogenesis, TIEG1 is also likely to be involved in myogenesis. Therefore, we investigated the function of TIEG1 during myogenic differentiation in vitro using the murine myoblasts cell line, C2C12. TIEG1 expression increased during differentiation of C2C12 cells. Constitutive expression of TIEG1 reduced survival and decreased myotube formation. Conversely, knocking down TIEG1 expression increased the number of viable cells during differentiation, and accelerated myoblast fusion into multinucleated myotubes. However, expression of the myogenic differentiation marker, myogenin, remained unaffected by TIEG1 knockdown. The mechanism underlying these events was investigated by focusing on the regulation of myoblast numbers after induction of differentiation. The knockdown of TIEG1 led to changes in cell cycle status and inhibition of apoptosis during the initial stages of differentiation. Microarray and real-time PCR analyses showed that the regulators of cell cycle progression were highly expressed in TIEG1 knockdown cells. Therefore, TIEG1 is a negative regulator of the myoblast pool that causes inhibition of myotube formation during myogenic differentiation.     

Neuregulin stimulates myogenic differentiation in an autocrine manner.

During myogenesis, mononucleated myoblasts form multinucleated myotubes by membrane fusion. Efficiency of this intercellular process can be maximized by a simultaneous progress, with a time window, of other neighboring myoblasts in the differentiation program. This phenomenon has been described as the community effect. It proposes the existence of a molecule that acts as a differentiation-inducing signal to a group of identical cells. Here, we show that neuregulin is a strong candidate for this molecule in myoblast differentiation. The expression of neuregulin increased rapidly but transiently at early stage of differentiation of rat L6 cells. Neuregulin showed a potent differentiation-promoting activity in membrane fusion and expression of myosin heavy chain. The antibodies raised against neuregulin and its cognate receptor ErbB3, which were capable of neutralizing the signal pathway, inhibited myotube formation and expression of myosin heavy chain in both L6 cells and primary rat myoblasts. The progress of differentiation was mostly halted after the expression of myogenin and cell cycle arrest. These results suggest that the activation of an autocrine signaling of neuregulin may provide a basic mechanism for the community effect observed in the differentiation of the embryonic muscle cells.     

Merosin and laminin in myogenesis; specific requirement for merosin in myotube stability and survival

Laminin (laminin-1; alpha 1-beta 1-gamma 1) is known to promote myoblast proliferation, fusion, and myotube formation. Merosin (laminin- 2 and -4; alpha 2-beta 1/beta 2-gamma 1) is the predominant laminin variant in skeletal muscle basement membranes; genetic defects affecting its structure or expression are the causes of some types of congenital muscular dystrophy. However, the precise nature of the functions of merosin in muscle remain unknown. We have developed an in vitro system that exploits human RD and mouse C2C12 myoblastic cell lines and their clonal variants to study the roles of merosin and laminin in myogenesis. In the parental cells, which fuse efficiently to multinucleated myotubes, merosin expression is upregulated as a function of differentiation while laminin expression is downregulated. Cells from fusion-deficient clones do not express either protein, but laminin or merosin added to the culture medium induced their fusion. Clonal variants which fuse, but form unstable myotubes, express laminin but not merosin. Exogenous merosin converted these myotubes to a stable phenotype, while laminin had no effect. Myotube instability was corrected most efficiently by transfection of the merosin-deficient cells with the merosin alpha 2 chain cDNA. Finally, merosin appears to promote myotube stability by preventing apoptosis. Hence, these studies identify novel biological functions for merosin in myoblast fusion and muscle cell survival; furthermore, these explain some of the pathogenic events observed in congenital muscular dystrophy caused by merosin deficiency and provide in vitro models to further investigate the molecular mechanisms of this disease.     

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.     

Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation

It has recently been established that exosomes can mediate intercellular cross-talk under normal and pathological conditions through the transfer of specific miRNAs. As muscle cells secrete exosomes, we addressed the question of whether skeletal muscle (SkM) exosomes contained specific miRNAs, and whether they could act as “endocrine signals” during myogenesis. We compared the miRNA repertoires found in exosomes released from C2C12 myoblasts and myotubes and found that 171 and 182 miRNAs were exported into exosomes from myoblasts and myotubes, respectively. Interestingly, some miRNAs were expressed at higher levels in exosomes than in their donor cells and vice versa, indicating a selectivity in the incorporation of miRNAs into exosomes. Moreover miRNAs from C2C12 exosomes were regulated during myogenesis. The predicted target genes of regulated exosomal miRNAs are mainly involved in the control of important signaling pathways for muscle cell differentiation (e.g., Wnt signaling pathway). We demonstrated that exosomes from myotubes can transfer small RNAs (C. elegans miRNAs and siRNA) into myoblasts. Moreover, we present evidence that exosome miRNAs secreted by myotubes are functionally able to silence Sirt1 in myoblasts. As Sirt1 regulates muscle gene expression and differentiation, our results show that myotube–exosome miRNAs could contribute to the commitment of myoblasts in the process of differentiation. Until now, myokines in muscle cell secretome provided a conceptual basis for communication between muscles. Here, we show that miRNA exosomal transfer would be a powerful means by which gene expression is orchestrated to regulate SkM metabolic homeostasis.     

The vitamin C transporter SVCT2 is down-regulated during postnatal development of slow skeletal muscles

Vitamin C plays key roles in cell homeostasis, acting as a potent antioxidant as well as a positive modulator of cell differentiation. In skeletal muscle, the vitamin C/sodium co-transporter SVCT2 is preferentially expressed in oxidative slow fibers. Besides, SVCT2 is up-regulated upon the early fusion of primary myoblasts. However, our knowledge of the postnatal expression profile of SVCT2 remains scarce. Here we have analyzed the expression of SVCT2 during postnatal development of the chicken slow anterior and fast posterior latissimus dorsi muscles, ranging from day 7 to adulthood. SVCT2 expression is consistently higher in the slow than in the fast muscle at all stages. After hatching, SVCT2 expression is significantly down-regulated in the anterior latissimus dorsi, which nevertheless maintains a robust slow phenotype. Taking advantage of the C2C12 cell line to recapitulate myogenesis, we confirmed that SVCT2 is expressed in a biphasic fashion, reaching maximal levels upon early myoblasts fusion and decreasing during myotube growth. Together, these findings suggest that the dynamic expression levels of SVCT2 could be relevant for different features of skeletal muscle physiology, such as muscle cell formation, growth and activity.     

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti-genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.     

Differential roles of HIC-5 isoforms in the regulation of cell death and myotube formation during myogenesis

Hic-5 is a LIM-Only member of the paxillin superfamily of focal adhesion proteins. It has been shown to regulate a range of biological processes including: senescence, tumorigenesis, steroid hormone action, integrin signaling, differentiation, and apoptosis. To better understand the roles of Hic-5 during development, we initiated a detailed analysis of Hic-5 expression and function in C(2)C(12) myoblasts, a well-established model for myogenesis. We have found that: (1) myoblasts express at least 6 distinct Hic-5 isoforms; (2) the two predominant isoforms, Hic-5alpha and Hic-5beta, are differentially expressed during myogenesis; (3) any experimentally induced change in Hic-5 expression results in a substantial increase in apoptosis during differentiation; (4) ectopic expression of Hic-5alpha is permissive to differentiation while expression of either Hic-5beta or antisense Hic-5 blocks myoblast fusion but not chemodifferentiation; (5) Hic-5 localizes to focal adhesions in C(2)C(12) myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (6) alterations in Hic-5 expression interfere with the normal dynamics of laminin expression; and (7) ectopic laminin, but not fibronectin, can rescue the Hic-5-induced blockade of myoblast survival and differentiation. Our data demonstrate differential roles for individual Hic-5 isoforms during myogenesis and support the hypothesis that Hic-5 mediates these effects via integrin signaling.