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.     

Myotube Formation on Micro-patterned Glass: Intracellular Organization and Protein Distribution in C2C12 Skeletal Muscle Cells

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. Studies of myocyte differentiation, cell fusion, and muscle repair are limited by an appropriate in vitro muscle cell culture system. We developed a novel cell culture technique [two-dimensional muscle syncytia (2DMS) technique] that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. This technique is based on UV lithography–produced micro-patterned glass on which conventionally cultured C2C12 myoblasts proliferate, align, and fuse to neatly arranged contractile myotubes in parallel arrays. Combining this technique with fluorescent microscopy, we observed alignment of actin filament bundles and a perinuclear distribution of glucose transporter 4 after myotube formation. Newly formed myotubes contained adjacently located MyoD-positive and MyoD-negative nuclei, suggesting fusion of MyoD-positive and MyoD-negative cells. In comparison, the closely related myogenic factor Myf5 did not exhibit this pattern of distribution. Furthermore, cytoplasmic patches of MyoD colocalized with bundles of filamentous actin near myotube nuclei. At later stages of differentiation, all nuclei in the myotubes were MyoD negative. The 2DMS system is thus a useful tool for studies on muscle alignment, differentiation, fusion, and subcellular protein localization. (J Histochem Cytochem 56:881–892, 2008)     

The relationship of polyoma virus-induced tumor (T) antigen to activation of DNA synthesis in rat myotubes

Rat myotubes infected with polyoma virus (PV) introduced into the multinucleated cells by virus-bearing myoblasts at the time of cell fusion incorporate ³H-TdR and exhibit mitotic-type figures. The infected myotubes also produce a viral-specific nuclear antigen, tumor (T) antigen, which appears in groups of adjacent nuclei or in all nuclei of the myotubes. The proportion of myotubes which synthesize DNA, T-antigen and exhibit mitotic-type figures is related to the multiplicity of virus infection.Intact myotubes which are resistant to infection with PV by virus absorption can be infected by microinjection of the virus into the cells. Myotubes thus infected produce T-antigen which appears in multiple nuclei, but do not incorporate ³H-TdR or contain mitotic-type figures. The data suggest that the resistance of myotubes to infection with PV might be due to a change in the cell surface membrane during differentiation so that virus cannot penetrate the cell. The T-antigen apparently has no bearing on the activation of the DNA-synthesizing apparatus in multinucleated muscle cells.     

Linc-smad7 promotes myoblast differentiation and muscle regeneration via sponging miR-125b

Long noncoding RNAs (lncRNAs) are involved in the regulation of skeletal muscle development. In the present study, differentially expressed lncRNAs were identified from RNA-seq data derived from myoblasts and myotubes. We conducted studies to elucidate the function and molecular mechanism of action of Linc-smad7 during skeletal muscle development. Our findings show that Linc-smad7 is upregulated during the early phase of myoblasts differentiation. In in vitro studies, we showed that overexpression of Linc-smad7 promoted the arrest of myoblasts in G1 phase, inhibited DNA replication, and induced myoblast differentiation. Our in vivo studies suggest that Linc-smad7 stimulates skeletal muscle regeneration in cardiotoxin-induced muscle injury. Mechanistically, Linc-smad7 overexpression increased smad7 and IGF2 protein levels. On the contrary, overexpression of miR-125b reduced smad7 and IGF2 protein levels. Results of RNA immunoprecipitation analysis and biotin-labeled miR-125b capture suggest that Linc-smad7 could act as a competing endogenous RNA (ceRNA) for miRNA-125b. Taken together, our findings suggest that the novel noncoding regulator Linc-smad7 regulates skeletal muscle development.     

Rb is required for progression through myogenic differentiation but not maintenance of terminal differentiation

To investigate the requirement for pRb in myogenic differentiation, a floxed Rb allele was deleted either in proliferating myoblasts or after differentiation. Myf5-Cre mice, lacking pRb in myoblasts, died immediately at birth and exhibited high numbers of apoptotic nuclei and an almost complete absence of myofibers. In contrast, MCK-Cre mice, lacking pRb in differentiated fibers, were viable and exhibited a normal muscle phenotype and ability to regenerate. Induction of differentiation of Rb-deficient primary myoblasts resulted in high rates of apoptosis and a total inability to form multinucleated myotubes. Upon induction of differentiation, Rb-deficient myoblasts up-regulated myogenin, an immediate early marker of differentiation, but failed to down-regulate Pax7 and exhibited growth in low serum conditions. Primary myoblasts in which Rb was deleted after expression of differentiated MCK-Cre formed normal multinucleated myotubes that did not enter S-phase in response to serum stimulation. Therefore, Rb plays a crucial role in the switch from proliferation to differentiation rather than maintenance of the terminally differentiated state.     

Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin

Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts induced to fuse. Additionally, we have compared the localization of syncytin with the localization of caveolin-3 and of myogenin, which are also involved in myoblast fusion and maturation. Syncytin was localized to areas of the cell membrane and to filopodial structures connecting myoblasts to each other and to myotubes. Weaker staining was present over intracellular vesicles and tubules. Caveolin-3 was detected in the sarcolemma and in vesicles and tubules in a subset of myoblasts and myotubes. The strongest staining occurred in multinucleated myotubes. Wide-field fluorescence microscopy indicated a partial colocalization of syncytin and caveolin-3 in a subset of myoblasts. Super-resolution microscopy showed such colocalization to occur in the sarcolemma. Myogenin was restricted to nuclei of myoblasts and myotubes and the strongest staining occurred in multinucleated myotubes. Syncytin staining was observed in both myogenin-positive and myogenin-negative cells. Antisense treatment downmodulated syncytin-1 expression and inhibited myoblast cell fusions. Importantly, syncytin-1 antisense significantly decreased the frequency of multinucleated myotubes demonstrating that the treatment inhibited secondary myoblast fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma.     

Isolation of myosin-synthesizing polysomes from cultures of embryonic chicken myoblasts before fusion.

Mononucleated myoblasts and multinucleated myotubes were obtained by culturing embryonic chicken skeletal muscle cells. Comparison of total polysomes isolated from these mononucleated and multinucleated cell cultures by density gradient centrifugation and electron microscopy revealed that mononucleated myoblasts contain polysomes similar to those contained by multinucleated myotubes and large enough to synthesize the 200,000-dalton subunit of myosin. When placed in an in vitro protein-synthesizing assay containing [³H]leucine, total polysomes from both mononucleated and multinucleated myogenic cultures were active in synthesizing polypeptides indistinguishable from myosin heavy chains as detected by measurement of radioactivity in slices through the myosin band on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Fractionation of total polysomes on sucrose density gradients showed that myosin-synthesizing polysomes from mononucleated myoblasts may be slightly smaller than myosin-synthesizing polysomes from myotubes. Multinucleated myotubes contain approximately two times more myosin-synthesizing polysomes per unit of DNA than mononucleated myoblasts, and the proportion of total polysomes constituted by myosin polysomes is only 1.2 times higher in multinucleated myotubes than it is in mononucleated myoblasts. The results of this study suggest that mononucleated myoblasts contain significant amounts of myosin messenger RNA before the burst of myosin synthesis that accompanies muscle differentiation and that a portion of this messenger RNA is associated with ribosomes to form polysomes that will actively translate myosin heavy chains in an in vitro protein-synthesizing assay.     

Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program

When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C(2)C(12) cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C(2)C(12) myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C(2)C(12) myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C(2)C(12) myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.     

Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis

During terminal differentiation of skeletal myoblasts, cells fuse to form postmitotic multinucleated myotubes that cannot reinitiate DNA synthesis. Here we investigated the temporal relationships among these events during in vitro differentiation of C2C12 myoblasts. Cells expressing myogenin, a marker for the entry of myoblasts into the differentiation pathway, were detected first during myogenesis, followed by the appearance of mononucleated cells expressing both myogenin and the cell cycle inhibitor p21. Although expression of both proteins was sustained in mitogen-restimulated myocytes, 5- bromodeoxyuridine incorporation experiments in serum-starved cultures revealed that myogenin-positive cells remained capable of replicating DNA. In contrast, subsequent expression of p21 in differentiating myoblasts correlated with the establishment of the postmitotic state. Later during myogenesis, postmitotic (p21-positive) mononucleated myoblasts activated the expression of the muscle structural protein myosin heavy chain, and then fused to form multinucleated myotubes. Thus, despite the asynchrony in the commitment to differentiation, skeletal myogenesis is a highly ordered process of temporally separable events that begins with myogenin expression, followed by p21 induction and cell cycle arrest, then phenotypic differentiation, and finally, cell fusion.     

Myoblasts from affected and non-affected FSHD muscles exhibit morphological differentiation defects

Facioscapulohumeral dystrophy (FSHD) is a muscular hereditary disease with a prevalence of 1 in 20,000 caused by a partial deletion of a subtelomeric repeat array on chromosome 4q. However, very little is known about the pathogenesis as well as the molecular and biochemical changes linked to the progressive muscle degeneration observed in these patients. Several studies have investigated possible pathophysiological pathways in FSHD myoblasts and mature muscle cells but some of these reports were apparently in contradiction. The discrepancy between these studies may be explained by differences between the sources of myoblasts. Therefore, we decided to thoroughly analyze affected and unaffected muscles from patients with FSHD in terms of vulnerability to oxidative stress, differentiation capacity and morphological abnormalities. We have established a panel of primary myoblast cell cultures from patients affected with FSHD and matched healthy individuals. Our results show that primary myoblasts are more susceptible to an induced oxidative stress than control myoblasts. Moreover, we demonstrate that both types of FSHD primary myoblasts differentiate into multi-nucleated myotubes, which present morphological abnormalities. Whereas control myoblasts fuse to form branched myotubes with aligned nuclei, FSHD myoblasts fuse to form either thin and branched myotubes with aligned nuclei or large myotubes with random nuclei distribution. In conclusion, we postulate that these abnormalities could be responsible for muscle weakness in patients with FSHD and provide an important marker for FSHD myoblasts.     

In vitro and in vivo evaluation of L‐lactide/ε‐caprolactone copolymer scaffold to support myoblast growth and differentiation

Skeletal muscle regeneration involves the activation of satellite cells to myoblasts, followed by their proliferation and fusion to form multinucleated myotubes and myofibers. The potential of in vitro proliferated myoblasts to treat various diseases and tissue defects can be exploited using tissue‐engineering principles. With an aim to develop a biocompatible and biodegradable scaffold that supports myoblast growth and differentiation, we have developed a porous sponge with 70/30 L ‐lactide/ε‐caprolactone copolymer (PLC) using a phase inversion combined with particulate leaching method. Degradation studies indicated that the sponge retained its structural integrity for 5 months in vitro and had undergone complete biodegradation within 9 months in vivo. The sponge supported human myoblasts attachment and its proliferation. Myoblasts seeded on the PLC sponge differentiated and fused in vitro to form myotubes expressing myosin heavy chain. Histological and molecular analyses of the PLC scaffolds seeded with green fluorescent protein‐labeled human myoblasts and implanted ectopically under the skin in SCID mice demonstrated the presence of multinucleated myotubes expressing human muscle‐specific markers. Our results suggest that PLC sponges loaded with myoblasts can be used for skeletal muscle engineering or for inducing muscle repair. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Inhibition of miR-214 expression represses proliferation and differentiation of C2C12 myoblasts

MicroRNAs (miRNAs) are small non-coding RNAs that participate in diverse biological processes including skeletal muscle development. MiR-214 is an miRNA that is differentially expressed in porcine embryonic muscle and adult skeletal muscle, suggesting that miR-214 may be related to embryonic myogenesis. In this study, the myoblast cell line C2C12 was used for functional analysis of miR-214 in vitro. The results showed that miR-214 was expressed both in myoblasts and in myotubes and was upregulated during differentiation. After treatment with an miR-214 inhibitor and culturing in differentiation medium, myoblast differentiation was repressed, as indicated by the significant downregulation of expression of the myogenic markers myogenin and myosin heavy chain (MyHC). Interestingly, myoblast proliferation was also repressed when cells were transfected with an miR-214 inhibitor and cultured in growth medium by real-time proliferation assay and cell cycle analysis. Our results showed that miR-214 regulates both proliferation and differentiation of myoblasts depending on the conditions.     

The disappearance of a cyclin-like protein and the appearance of statin is correlated with the onset of differentiation during myogenesis in vitro

We have used monoclonal antibodies to statin (S-44) and a cyclin-like protein (S-132) to examine the distribution of these two antigens in proliferating and in nonproliferating populations of cells. We have found that this cyclin-like protein is present in proliferating fibroblasts, whereas statin is absent from these same cell populations; in contrast, in senescent populations of fibroblasts the cyclin-like antigen disappears and statin labeling of nuclei appears. During myogenesis in rat muscle cell cultures, S-132 labeling is present in proliferating myoblasts and disappears after cells fuse and differentiate as multinucleated myotubes. In contrast, statin is absent from proliferating myoblasts, but appears when these cells become postmitotic and begin to differentiate. Similar results were seen during chick myogenesis. We have also found similar results during serum-starvation-induced differentiation in neuroblastoma cells. These results indicate that the cyclin-like protein disappears and statin appears upon commitment to differentiation in vitro, and the presence or the absence of these proteins appears to provide cellular markers for the transition from the proliferative to the nonproliferative state during differentiation.     

Hyaluronic acid bonded to cell culture surfaces inhibits the program of myogenesis

Primary isolates of chick leg muscle myoblasts cultured on hyaluronic acid substrates have been examined by transmission electron microscopy for evidence of myoblast fusion and subsequent differentiation. Even though these cells form close contacts, no evidence of multinucleated myotubes is found in these cultures. Two-dimensional SDS-polyacrylamide gel electrophoresis shows that the muscle macromolecular biosynthetic program is not initiated in these hyaluronic acid fusion-blocked cells. Further, these fusion-blocked myoblasts continue replicating while cultured on hyaluronic acid surfaces. The inhibition of both fusion and the myogenic expressional program is reversed by replating these myoblasts onto a denatured collagen (gelatin) substrate; both the synthesis of muscle-specific proteins and the formation of multinucleated myotubes are observed when these subcultured cells are introduced onto gelatin substrates. These observations indicate that the hyaluronic acid inhibition of fusion is not permanent and is manifested in a way different from other fusion blockers in that hyaluronic acid inhibits both fusion and the myogenic expressional program.     

Position-dependent nuclear accumulation of the retinoblastoma (RB) protein during in vitro myogenesis

The expression of the retinoblastoma (RB) protein has been studied during in vitro muscle differentiation by immunofluorescence staining with three different antibodies against RB protein. Proliferating mononucleate L6 rat myoblasts showed a low level of expression. As cells began to enter a nonreplicating G0 state, the cell population became heterogeneous. Some nonreplicating cells showed a high level of expression. Nuclei at the two ends of myotubes were strongly positive, whereas centrally located nuclei showed low RB expression. Overexpression of the human RB protein in rat L6 myotubes from a Semliki forest virus (SFV)-based, transient expression vector produced a similar picture. Terminally located nuclei expressed human RB at a much higher level than did the centrally located nuclei. The results suggest that individual nuclei with a multinucleated syncytium may undergo position-dependent specialization. © 1993 Wiley-Liss, Inc.     

Overexpression of nPKC theta is permissive for myogenic differentiation

Although protein kinase C (PKC) has been shown to participate in skeletal myogenic differentiation, the functions of individual isoforms of PKC in myogenesis have not been completely elucidated. These studies focused on the role of nPKC straight theta, an isoform of the PKC family whose expression has been shown to be regulated by commitment to the myogenic lineage, myogenic differentiation and innervation. We used the myogenic cell line C(2)C(12) as a tissue culture model system to explore the role of nPKC straight theta in the formation of multinucleated myotubes. We examined endogenous levels of nPKC straight theta in C(2)C(12) cells and showed that it is expressed at low levels in myoblasts compared to mouse skeletal muscle and that expression is maintained in myotubes. We overexpressed nPKC straight theta in C(2)C(12) myoblasts and examined the ability of overexpressing cells to differentiate into myotubes. Using an nPKC straight theta - green fluorescent protein (GFP) chimera to detect transfected myoblasts, we showed that overexpressed nPKC straight theta-GFP translocates to the plasma membrane in response to phorbol ester treatment of myoblast cultures in situ. nPKC straight theta-GFP was found to be completely extracted into the detergent-soluble fraction of cell lysates and was stably expressed throughout the extent of differentiation into myotubes. No difference was seen in the ability of myoblasts either overexpressing nPKC straight theta - GFP or GFP alone to form myotubes. These studies demonstrate that overexpression of nPKC straight theta does not interfere with fusion of myoblasts into myotubes suggesting that nPKC straight theta activity is not inhibitory for myogenesis. These studies also demonstrate a method for transfecting myoblasts and identifying differentiated cells that overexpress nPKC straight theta-GFP for investigating the function of nPKC straight theta in living myotubes.