Role of metalloprotease disintegrin ADAM12 in determination of quiescent reserve cells during myogenic differentiation in vitro

Skeletal myoblasts grown in vitro and induced to differentiate either form differentiated multinucleated myotubes or give rise to quiescent, undifferentiated "reserve cells" that share several characteristics with muscle satellite cells. The mechanism of determination of reserve cells is poorly understood. We find that the expression level of the metalloprotease disintegrin ADAM12 is much higher in proliferating C2C12 myoblasts and in reserve cells than in myotubes. Inhibition of ADAM12 expression in differentiating C2C12 cultures by small interfering RNA is accompanied by lower expression levels of both quiescence markers (retinoblastoma-related protein p130 and cell cycle inhibitor p27) and differentiation markers (myogenin and integrin alpha7A isoform). Overexpression of ADAM12 in C2C12 cells under conditions that promote cell cycle progression leads to upregulation of p130 and p27, cell cycle arrest, and downregulation of MyoD. Thus, enhanced expression of ADAM12 induces a quiescence-like phenotype and does not stimulate differentiation. We also show that the region extending from the disintegrin to the transmembrane domain of ADAM12 and containing cell adhesion activity as well as the cytoplasmic domain of ADAM12 are required for ADAM12-mediated cell cycle arrest, while the metalloprotease domain is not essential. Our results suggest that ADAM12-mediated adhesion and/or signaling may play a role in determination of the pool of reserve cells during myoblast differentiation.     

Formation and characterization of spontaneously formed heterokaryons between quail myoblasts and 3T3-L1 preadipocytes: correlation between differential plasticity and degree of differentiation

Skeletal muscle cells and adipose cells have a close relationship in developmental lineage. Our previous study has shown that the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells (preadipocytes) normally differentiated into myotubes, whereas the heterokaryons between myoblasts and differentiated 3T3-L1 cells (adipocytes) failed myogenic differentiation. These results suggest differences between preadipocytes and adipocytes. The purpose of this study was to clarify whether preadipocytes have flexibility in differentiation before terminal adipose differentiation. Presumptive quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and mouse 3T3-L1 cells (either preadipocytes or adipocytes) were co-cultured for 48 h under conditions allowing myogenic differentiation. On co-culture between myoblasts and undifferentiated 3T3-L1 cells, heterokaryotic myotubes formed spontaneously, but not on co-culture with differentiated 3T3-L1 cells. In addition, the heterokaryotic myotubes expressed mouse myogenin derived from the 3T3-L1 cell gene. Our previous study indicated that the fusion sensitivity of differentiating myoblasts change with decreasing cholesterol of the cell membrane during myoblast fusion. Thus we compared the level of membrane cholesterol between undifferentiated and differentiated 3T3-L1 cells. The result showed that the level of membrane cholesterol in 3T3-L1 cells increases during adipose differentiation. Corresponding to the increase in membrane cholesterol content, differentiated 3T3-L1 cells had lower sensitivity to HVJ (Sendai virus)-mediated cell fusion than undifferentiated 3T3-L1 cells. This study demonstrated that 3T3-L1 cells at an undifferentiated state have a capacity for spontaneous fusion with differentiating myoblasts following myogenic differentiation, and that the capacity is lost after terminal adipose differentiation.     

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.     

Requirement of the transmembrane semaphorin Sema4C for myogenic differentiation

Semaphorins constitute a large family of signaling proteins that contribute to axonal guidance. Here we demonstrate that the transmembrane semaphorin Sema4C is up-regulated both in the early stage of differentiation of C2C12 mouse skeletal myoblasts into myotubes and during injury-induced muscle regeneration in vivo. Depletion of Sema4C in C2C12 cells resulted in marked attenuation of myotube formation. A fusion protein containing the extracellular Sema domain and a peptide corresponding to the intracellular COOH-terminal region of Sema4C each inhibited the differentiation of C2C12 cells. These findings indicate that Sema4C-mediated interaction among myoblasts plays an important role in terminal myogenic differentiation.     

c-Myb Inhibits Myoblast Fusion

Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3′ untranslated region (3′ UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3′ UTR of c-Myb was also important because the expression of c-Myb coding region with its 3′ UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3′ UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.     

Presence and Importance of Connexin43 During Myogenesis

We analyzed the expression of connexin(Cx)43 in proliferating and differentiating C₂C₁₂cells and in myoblasts obtained from newborn mice. Cx43 was present in both cell types and under both conditions. The functional role of gap junctional communication (GJC) during terminal differentiation was evaluated in C₂C₁₂myoblasts in the presence or absence of the gap junction blocker 18β-glycyrrhetinic acid (β-GA). Differentiation was temporally analyzed through myogenin expression, activity of creatine kinase (CK), and yield of multinucleated cells. In cells treated with β-GA, the CK activity and myotube formation were reversibly blocked. While in control cultures positive myogenin expression was seen in cell clusters, in β-GA treated cultures the myogenin immunoreactivity was detected in few, preferentially sparse cells. The role of Cx43 during terminal differentiation was evaluated in cultures of myoblasts obtained from Cx43^Cre-ER(T)/fltransgenic mice. Inducible deletion of Cx43 was obtained upon activation of Cre-ER(T) via 4-OH-tamoxifen applications. Cx43 deletion led to a drastic decrease in myogenin expression at 24 h of differentiation as compared to myoblasts from control mice. Our results indicate that Cx43-containing gap junctions are required for normal skeletal muscle terminal differentiation. These channels might provide a pathway for the intercellular transfer of signals involved in myogenesis.     

Inhibition of Notch signaling induces myotube hypertrophy by recruiting a subpopulation of reserve cells

During muscle differentiation, a population of quiescent undifferentiated myoblasts (reserve cells) emerges among mature muscle cells. However, the molecular mechanisms underlying such cell segregation and the characterization of this subpopulation of myoblasts remain to be determined. Notch is known to control the behavior and fate of murine muscle stem cells. In this study, we examined the role of Notch in myoblast segregation. We showed that inhibition of Notch activity by either overexpressing Numb or by using a pharmacological gamma-secretase inhibitor (DAPT) enhanced differentiation of murine and human myoblasts. This effect was not restricted to in vitro culture systems since DAPT-treated zebrafish embryos also showed increased differentiation. Using C2.7 myoblasts as a model, we showed that inhibition of Notch induced myotube hypertrophy by recruiting reserve cells that do not normally fuse. We further showed that endogenous Notch-signaling components were differentially expressed and activated in reserve cells with respect to Notch 1 and CD34 expression. We identified CD34 negative reserve cells as the subpopulation of myoblasts recruited to fuse into myotubes during differentiation in response to Notch inhibition. Therefore, we showed here that the activation of Notch 1 is important to maintain a subpopulation of CD34 negative reserve cells in an undifferentiated state.     

Evidence on the participation of protein kinase C alpha in the proliferation of cultured myoblasts.

There is evidence involving protein kinase C (PKC) in the signal transduction pathways that regulate the differentiation of myoblasts into mature multinucleated muscle cells (myotubes). In order to obtain information on the possible role of individual PKC isozymes in myogenesis, in the present work we investigated the differential expression of PKC isoforms alpha, beta, delta, epsilon, and zeta during muscle cell development in vitro. Chick embryo myoblasts cultured from 1 to 6 days were used as experimental model. Morphological characterization and measurement of specific biochemical parameters in cultures, e.g., DNA synthesis, creatine kinase activity, and myosin levels, revealed a typical muscle cell developmental pattern consisting of an initial proliferation of myoblasts followed by their differentiation into myotubes. PKC activity was high at the proliferation stage, decreased as myoblasts elongated and fused, and increased again in differentiated myotubes. In proliferating myoblasts, the PKC inhibitors calphostin C and bisindolylmaleimide I decreased DNA synthesis whereas in myoblasts undergoing differentiation they exerted the opposite effect, suggesting that PKC plays a role at both stages of myogenesis. Western blot analysis of changes in the expression of PKC isoforms during muscle cell development showed high levels of PKC alpha in the proliferating phase which markedly decreased as myoblasts differentiated. Treatment with TPA of proliferative myoblasts inhibited DNA synthesis and selectively down-regulated PKC alpha, suggesting that this isozyme may have an important role in maintaining myoblast proliferation. On the other hand, an increase in the expression of PKC beta, delta, and epsilon was detected during myogenesis, suggesting that one or more of these isoforms may participate in the differentiation process of myoblasts.     

Irreversible reduction in potassium fluxes accompanies terminal differentiation of human myoblasts to myotubes

Potassium and sodium fluxes believed to be important in the cellular response to serum and growth factors have not been widely investigated in cells which have undergone terminal differentiation. In this study we have analyzed two main K+ transport systems--the ouabain-sensitive Na+/K+ pump and the bumetanide-sensitive transporter--in human muscle in vitro at two developmental stages: proliferating myoblasts and differentiated myotubes. Myoblast differentiation to myotubes was accompanied by a marked decrease in both the ouabain-sensitive and the bumetanide-sensitive K+ (Rb+) influxes. The addition of serum to the terminally differentiated myotubes had no effect on these K+ transporters. However, serum addition to serum-deprived, undifferentiated myoblasts produced a marked stimulation of these K+ fluxes. The bumetanide-sensitive K+ transporter in human myoblasts and myotubes has the following properties: (1) It carries 30% and 40% of the total K+ influx in myoblasts and myotubes, respectively. (2) It performs net efflux of K+ in the undifferentiated myoblasts and zero net flux (self-exchange) in terminally differentiated myotubes. (3) It is dependent on extracellular Na+ and Cl- in addition to K+. (4) In myoblasts, the Km value for K+ is 1.36 mM, similar to the Km for K+ of the Na+/K+ pump. (5) It is resistant to ouabain (up to 2 mM) and sensitive to furosemide (K0.5 = 5 X 10(-6) M) and bumetanide (K0.5 = 10(-7) M). These data indicate that following terminal differentiation of proliferating myoblasts to mitotically inactive myotubes there is an irreversible reduction of K+ fluxes with a change in the net flux of K+ carried by the bumetanide-sensitive transporter.     

Coexpression of α-sarcomeric actin, α-smooth muscle actin and desmin during myogenesis in rat and mouse embryos I. Skeletal muscle

Expression of vimentin, desmin, alpha-sarcomeric and alpha-smooth muscle actins in embryonic tissues of rat and mice was examined using an immunohistochemical approach. The results showed a similarity in the expression of desmin and alpha-actin isoforms (alpha-sr and alpha-sm) in skeletal muscle cells during murine feto-embryonic development. In the two species, coexpression of alpha-sr and alpha-sm actins has been observed in cardiomyoblasts, myotomal myoblasts and myotubes. The intensity of alpha-sm actin expression decreased during the terminal steps of myogenesis and disappeared completely in mature cardiomyocytes and myofibres. Desmin was expressed in all prefusion myoblasts (type 1 and 2 myoblasts), myotubes, and in myofibres. The appearance of desmin in myoblasts of somites preceded by a few hours the expression of the alpha-actins (alpha-sr and alpha-sm). Our study on vimentin expression, limited to rat embryos, revealed that somite premyoblasts expressed only vimentin, type 1 myoblasts expressed vimentin and desmin, and type 2 myoblasts (rhabdomyoblasts) expressed desmin and alpha-actins (alpha-sr and alpha-sm). Our study demonstrates the resemblance between feto-embryonic myogenesis and myogenic neoplastic differentiation: desmin appears before the alpha-actins in embryonic myoblasts, and can be considered as a marker of an initial step in myogenic differentiation. alpha-sm actin, considered as a striated muscle cell feto-embryonic actin, is expressed transiently in skeletal myoblasts and cardiomyoblasts during development and reappears during neoplastic transformation of skeletal muscle.     

Inhibitory effect of p53 on mitochondrial content and function during adipogenesis

The p53 protein is known as a guardian of the genome and is involved in energy metabolism. Since the metabolic system is uniquely regulated in each tissue, we have anticipated that p53 also would play differential roles in each tissue. In this study, we focused on the functions of p53 in white adipose tissue (adipocytes) and skeletal muscle (myotubes), which are important peripheral tissues involved in energy metabolism. We found that in 3T3-L1 preadipocytes, but not in C2C12 myoblasts, p53 stabilization or overexpression downregulates the expression of Ppargc1a, a master regulator of mitochondrial biogenesis. Next, by using p53-knockdown C2C12 myotubes or 3T3-L1 preadipocytes, we further examined the relationship between p53 and mitochondrial regulation. In C2C12 myoblasts, p53 knockdown did not significantly affect Ppargc1a expression and mtDNA, but did suppress differentiation to myotubes, as previously reported. However, in 3T3-L1 preadipocytes and mouse embryonic fibroblasts, p53 downregulation enhanced both differentiation into adipocytes and mitochondrial DNA content. Furthermore, p53-depleted 3T3-L1 cells showed increase in mitochondrial proteins and enhancement of both Citrate Synthase and Complex IV activities during adipogenesis. These results show that p53 differentially regulates cell differentiation and mitochondrial biogenesis between adipocytes and myotubes, and provide evidence that p53 is an inhibitory factor of mitochondrial regulation in adipocyte lineage.     

Up-regulation of the vitamin C transporter SVCT2 upon differentiation and depolarization of myotubes

In addition to its role as a strong antioxidant, vitamin C regulates the differentiation of several cell lineages. In vertebrate skeletal muscle, the vitamin C transporter SVCT2 is preferentially expressed in slow muscle fibers. To gain insights into the possible involvement of intracellular vitamin C on early myogenesis, we investigated the regulation of SVCT2 expression in cultures of chick fetal myoblasts. SVCT2 expression increases in cultures of both, slow and fast muscle-derived myoblasts, as they fuse to form mainly fast myotubes. Interestingly, we found that SVCT2 could be positively modulated by potassium-induced depolarization of myotubes. These findings suggest that SVCT2-mediated uptake of vitamin C could play diverse roles on skeletal muscle development and physiology.     

Dependence of myoblast fusion on a cortical actin wall and nonmuscle myosin IIA

Cell-cell fusion is a fundamental cellular process that is essential for development as well as fertilization. Myoblast fusion to form multinucleated skeletal muscle myotubes is a well studied, yet incompletely understood example of cell-cell fusion that is essential for formation of contractile skeletal muscle tissue. Studies in this report identify several novel cytoskeletal events essential to an early phase of myoblast fusion among cultured murine myoblasts. During myoblast pairing and alignment, cortical actin filaments organize into a dense actin wall structure that parallels and extends the length of the plasma membrane of the bipolar, aligned cells. As fusion progresses, gaps appear within the actin wall at sites of vesicle accumulation, the vesicles pair across the aligned myoblasts, cell-cell contacts and fusion pores form. Inhibition of nonmuscle myosin IIA (NM-MHC-IIA) motor activity prevents formation of this cortical actin wall, as well as the appearance of vesicles at a membrane proximal location, and myoblast fusion. These results suggest that early formation of a subplasmalemmal actin wall during myoblast alignment is a critical event for myoblast fusion that supports bipolar membrane alignment and temporally regulates trafficking of vesicles to the nascent fusion sites during skeletal muscle myoblast differentiation.     

Transforming Growth Factor-��-activated Kinase 1 Is an Essential Regulator of Myogenic Differentiation

Satellite cells/myoblasts account for the majority of muscle regenerative potential in response to injury and muscular adaptation to exercise. Although the ability to influence this process would provide valuable benefits for treating a variety of patients suffering from muscle loss, the regulatory mechanisms of myogenesis are not completely understood. We have tested the hypothesis that transforming growth factor-β-activated kinase 1 (TAK1) is an important regulator of skeletal muscle formation. TAK1 is expressed in proliferating C2C12 myoblasts, and its levels are reduced upon differentiation of myoblasts into myotubes. In vivo, TAK1 is predominantly expressed in developing skeletal muscle of young mice. However, the expression of TAK1 was significantly up-regulated in regenerating skeletal muscle of adult mice. Overexpression of a dominant negative mutant of TAK1 or knockdown of TAK1 inhibited the proliferation and differentiation of C2C12 myoblasts. TAK1 was required for the expression of myogenic regulatory factors in differentiating myoblasts. Genetic ablation of TAK1 also inhibited the MyoD-driven transformation of mouse embryonic fibroblasts into myotubes. Inhibition of TAK1 suppressed the differentiation-associated activation of p38 mitogen-activated protein kinase (MAPK) and Akt kinase. Overexpression of a constitutively active mutant of MAPK kinase 6 (MKK6, an upstream activator of p38 MAPK) but not constitutive active Akt restored the myogenic differentiation in TAK1-deficient mouse embryonic fibroblasts. Insulin growth factor 1-induced myogenic differentiation was also found to involve TAK1. Collectively, our results suggest that TAK1 is an important upstream regulator of skeletal muscle cell differentiation.