Quiescent Myoblasts Research Articles

Tissue Engineering Applied to Skeletal Muscle: Strategies and Perspectives.

Muscle tissue is formed by elongated and contractile cells with specific morphofunctional characteristics. Thus, it is divided into three basic types: smooth muscle tissue, cardiac striated muscle tissue and skeletal striated muscle tissue. The striated skeletal muscle tissue presents high plasticity, regeneration and growth capacity due to the presence of satellite cells, quiescent myoblasts that are activated in case of injury to the tissue and originate new muscle fibers when they differentiate. In more severe deficiencies or injuries there is a loss of their regenerative capacity, thus compromising the body's functionality at different levels. Tissue engineering studies the development of biomaterials capable of stimulating the recovery of cellular activity in injured body tissues, as well as the activity of cells with muscle differentiation potential in injury repair. However, the need for three-dimensional re-assembly in a complex organization makes it difficult to mimic this tissue and fully regenerate it for the sake of precise and effective movements. Thus, this article aims to provide a narrative review of tissue engineering strategies applied to the regeneration of skeletal muscle, in a critical evaluation of research, whether aimed at injury or atrophies such as spinal muscular atrophy.

MRNP granule proteins Fmrp and Dcp1a differentially regulate mRNP complexes to contribute to control of muscle stem cell quiescence and activation

BackgroundDuring skeletal muscle regeneration, satellite stem cells use distinct pathways to repair damaged myofibers or to self-renew by returning to quiescence. Cellular/mitotic quiescence employs mechanisms that promote a poised or primed state, including altered RNA turnover and translational repression. Here, we investigate the role of mRNP granule proteins Fragile X Mental Retardation Protein (Fmrp) and Decapping protein 1a (Dcp1a) in muscle stem cell quiescence and differentiation.MethodsUsing isolated single muscle fibers from adult mice, we established differential enrichment of mRNP granule proteins including Fmrp and Dcp1a in muscle stem cells vs. myofibers. We investigated muscle tissue homeostasis in adult Fmr1-/- mice, analyzing myofiber cross-sectional area in vivo and satellite cell proliferation ex vivo. We explored the molecular mechanisms of Dcp1a and Fmrp function in quiescence, proliferation and differentiation in a C2C12 culture model. Here, we used polysome profiling, imaging and RNA/protein expression analysis to establish the abundance and assembly status of mRNP granule proteins in different cellular states, and the phenotype of knockdown cells.ResultsQuiescent muscle satellite cells are enriched for puncta containing the translational repressor Fmrp, but not the mRNA decay factor Dcp1a. MuSC isolated from Fmr1-/- mice exhibit defective proliferation, and mature myofibers show reduced cross-sectional area, suggesting a role for Fmrp in muscle homeostasis. Expression and organization of Fmrp and Dcp1a varies during primary MuSC activation on myofibers, with Fmrp puncta prominent in quiescence, but Dcp1a puncta appearing during activation/proliferation. This reciprocal expression of Fmrp and Dcp1a puncta is recapitulated in a C2C12 culture model of quiescence and activation: consistent with its role as a translational repressor, Fmrp is enriched in non-translating mRNP complexes abundant in quiescent myoblasts; Dcp1a puncta are lost in quiescence, suggesting stabilized and repressed transcripts. The function of each protein differs during proliferation; whereas Fmrp knockdown led to decreased proliferation and lower cyclin expression, Dcp1a knockdown led to increased cell proliferation and higher cyclin expression. However, knockdown of either Fmrp or Dcp1a led to compromised differentiation. We also observed cross-regulation of decay versus storage mRNP granules; knockdown of Fmrp enhances accumulation of Dcp1a puncta, whereas knockdown of Dcp1a leads to increased Fmrp in puncta.ConclusionsTaken together, our results provide evidence that the balance of mRNA turnover versus utilization is specific for distinct cellular states.

Effect of PGC1‐α Overexpression on Cardiotoxin‐Induced Damage and Repair of Human Myotubes

Introduction Metabolic disease alters many biochemical and morphological characteristics of skeletal muscle, including reductions in fiber integrity and the regenerative response following muscle damage. In obesity and diabetes, skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) is reduced and may contribute to impairments in inflammatory and metabolic signaling. PGC-1α is important in regulating skeletal muscle cellular energy metabolism, mitochondrial biogenesis, and myofiber integrity. The purpose of the present study was to examine if PGC-1α overexpression (OE) reduces cardiotoxin (CTX)-induced skeletal muscle damage and improves regeneration after CTX treatment in human myotubes. Methods Differentiated primary, human myotubes were transfected with adenovirus containing PGC-1α (PGC-AV) or green fluorescent protein (GFP). At 48 h following transfection, myotubes were treated with 1.0 μM CTX or vehicle control (CTL) for 1 h. Cells were allowed to recover in skeletal muscle growth media for 3 days. Cells were isolated for analysis immediately (0D) and 3 days (3D) following CTX or CTL administration. Significance was established at P ≤ 0.05. Results Under control conditions, PGC-AV increased myotube metabolic activity compared to GFP and CTX administration lowered metabolic activity assessed by MTT (GFP-CTL = 1.00 au; GFP-CTX= 0.51 au; PGC-CTL = 1.65 au; PGC-CTX= 1.13 au). CTX administration reduced the fusion index (FI) similarly in PGC-AV and GFP groups (GFP-CTL = 27.8%; GFP-CTX= 21.9%; PGC-CTL = 26.9%; PGC-CTX= 20.5%). At time 0D, both PGC-AV and CTX lowered myotube numbers (GFP-CTL = 13.5 tubes; GFP-CTX= 9.5 tubes; PGC-CTL = 7.3 tubes; PGC-CTX = 6.8 tubes). PGC-AV tended (P < 0.10) to reduce CTX-induced myotube damage as measured by media content of lactate dehydrogenase (LDH) (GFP-CTL = 1.0 au; GFP-CTX= 1.5 au; PGC-CTL control = 1.0 au; PGC-CTX= 1.2 au). At 3D, PGC-AV treatment lowered %Edu+ nuclei (GFP-CTL = 31.3%; GFP-CTX= 34.4%; PGC-CTL =25.5%; PGC-CTX= 20.3%) and MyoD mRNA expression (GFP-CTL = 1.0 au; GFP-CTX= 1.0 au; PGC-CTL control = 0.2 au; PGC-CTX= 0.3 au) indicating a reduction in proliferation and an increase in quiescent myoblasts. Conclusion Overexpression of PGC-1α may limit CTX induced damage, but may also blunt the proliferation of myoblasts following CTX treatment.

The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts

BackgroundReversible cell cycle arrest (quiescence/G0) is characteristic of adult stem cells and is actively controlled at multiple levels. Quiescent cells also extend a primary cilium, which functions as a signaling hub. Primary cilia have been shown to be important in multiple developmental processes, and are implicated in numerous developmental disorders. Although the association of the cilium with G0 is established, the role of the cilium in the control of the quiescence program is still poorly understood.ResultsPrimary cilia are dynamically regulated across different states of cell cycle exit in skeletal muscle myoblasts: quiescent myoblasts elaborate a primary cilium in vivo and in vitro, but terminally differentiated myofibers do not. Myoblasts where ciliogenesis is ablated using RNAi against a key ciliary assembly protein (IFT88) can exit the cell cycle but display an altered quiescence program and impaired self-renewal. Specifically, the G0 transcriptome in IFT88 knockdown cells is aberrantly enriched for G2/M regulators, suggesting a focused repression of this network by the cilium. Cilium-ablated cells also exhibit features of activation including enhanced activity of Wnt and mitogen signaling and elevated protein synthesis via inactivation of the translational repressor 4E-BP1.ConclusionsTaken together, our results show that the primary cilium integrates and dampens proliferative signaling, represses translation and G2/M genes, and is integral to the establishment of the quiescence program.

Distinguishing States of Arrest: Genome-Wide Descriptions of Cellular Quiescence Using ChIP-Seq and RNA-Seq Analysis.

Regenerative potential in adult stem cells is closely associated with the establishment of-and exit from-a temporary state of quiescence. Emerging evidence not only provides a rationale for the link between lineage determination programs and cell cycle regulation but also highlights the understanding of quiescence as an actively maintained cellular program, encompassing networks and mechanisms beyond mitotic inactivity or metabolic restriction. Interrogating the quiescent genome and transcriptome using deep-sequencing technologies offers an unprecedented view of the global mechanisms governing this reversibly arrested cellular state and its importance for cell identity. While many efforts have identified and isolated pure target stem cell populations from a variety of adult tissues, there is a growing appreciation that their isolation from the stem cell niche in vivo leads to activation and loss of hallmarks of quiescence. Thus, in vitro models that recapitulate the dynamic reversibly arrested stem cell state in culture and lend themselves to comparison with the activated or differentiated state are useful templates for genome-wide analysis of the quiescence network.In this chapter, we describe the methods that can be adopted for whole genome epigenomic and transcriptomic analysis of cells derived from one such established culture model where mouse myoblasts are triggered to enter or exit quiescence as homogeneous populations. The ability to synchronize myoblasts in G0 permits insights into the genome in "deep quiescence." The culture methods for generating large populations of quiescent myoblasts in either 2D or 3D culture formats are described in detail in a previous chapter in this series (Arora et al. Methods Mol Biol 1556:283-302, 2017). Among the attractive features of this model are that genes isolated from quiescent myoblasts in culture mark satellite cells in vivo (Sachidanandan et al., J Cell Sci 115:2701-2712, 2002) providing a validation of its approximation of the molecular state of true stem cells. Here, we provide our working protocols for ChIP-seq and RNA-seq analysis, focusing on those experimental elements that require standardization for optimal analysis of chromatin and RNA from quiescent myoblasts, and permitting useful and revealing comparisons with proliferating myoblasts or differentiated myotubes.

Fad24, a Positive Regulator of Adipogenesis, Is Required for S Phase Re-entry of C2C12 Myoblasts Arrested in G0 Phase and Involved in p27(Kip1) Expression at the Protein Level.

Factor for adipocyte differentiation 24 (fad24) is a positive regulator of adipogenesis. We previously found that human fad24 is abundantly expressed in skeletal muscle. However, the function of fad24 in skeletal muscle remains largely unknown. Because skeletal muscle is a highly regenerative tissue, we focused on the function of fad24 in skeletal muscle regeneration. In this paper, we investigated the role of fad24 in the cell cycle re-entry of quiescent C2C12 myoblasts-mimicked satellite cells. The expression levels of fad24 and histone acetyltransferase binding to ORC1 (hbo1), a FAD24-interacting factor, were elevated at the early phase of the regeneration process in response to cardiotoxin-induced muscle injury. The knockdown of fad24 inhibited the proliferation of quiescent myoblasts, whereas fad24 knockdown did not affect differentiation. S phase entry following serum activation is abrogated by fad24 knockdown in quiescent cells. Furthermore, fad24 knockdown cells show a marked accumulation of p27(Kip1) protein. These results suggest that fad24 may have an important role in the S phase re-entry of quiescent C2C12 cells through the regulation of p27(Kip1) at the protein level.

Identification of PRDM2 regulated genes in quiescent C2C12 myoblasts.

Quiescent stem cells contribute to tissue homeostasis and repair in adult mammals. We identified a tumor suppressor PRDM2, as an epigenetic regulator induced in quiescent muscle stem cells as well as in cultured quiescent myoblasts. To delineate the functions of PRDM2 in muscle cells, we compared the gene expression profiles of control and PRDM2 knockdown myoblasts in growing, differentiating and quiescent conditions (GEO accession number: GSE 58676). To identify the direct targets of PRDM2 and the promoters co-associated with H3K9me2 (mark catalyzed by PRDM2), ChIP-Chip analysis was performed (GSE58748). In this report we discuss in detail the methodology used to identify PRDM2 regulated genes and classify them into potential direct and indirect targets.

Inducing myoblast re-entry into the cell cycle: a potential mechanism for laser-enhanced skeletal muscle regeneration

This study investigated the effect of low-level laser irradiation (LLLI) on the cell cycle and proliferative activity of cultured myoblasts, and sought to elucidate the possible cellular mechanism by which LLLI promotes the regeneration of skeletal muscle in vivo. Primary myoblasts isolated from rat hindlegs were irradiated with helium-neon laser light at different energy densities. Distributions of cell-cycle subpopulations and the expression of cell-cycle regulatory proteins in myoblasts were assessed using flow cytometric analysis and western blot assay. It was found that laser irradiation stimulated cell-cycle entry; induced the expression of cyclin A and cyclin D; and increased cell proliferation index and bromodeoxyuridine incorporation as compared to the unirradiated control cells, indicating LLLI augmented the number of proliferative myoblasts in the S phase and G2/M phase of the cell cycle. These results suggest that LLLI at certain fluxes and wavelengths could activate quiescent myoblasts, leading to cell division and facilitating new myofiber formation. This could contribute to the improvement of skeletal muscle regeneration following trauma and myopathic diseases.

Expression analysis of Gli1 and Gli2 in different tissues and muscle-derived cells of Qinchuan cattle.

The Hedgehog (Hh) signaling pathway regulates the differentiation of many kinds of cells and plays a critical role in many embryonic and postnatal developmental processes. Gli1 and Gli2 are two transcription factors of the Hh signaling pathway. In this study, we used quantitative real-time polymerase chain reaction to detect the relative expression of Gli1 and Gli2 in 13 tissues from three two-year-old purebred Qinchuan cattle, as well as in different cell populations derived from muscle and different stages of myogenic differentiation of myoblasts. The expression levels of Gli1 and Gli2 in muscle were the lowest of the 13 tissues (P < 0.05), and they declined predominantly from preplate (pp)1 to pp6 cells. However, the expression of Gli2 was elevated during myogenic differentiation until the 6th day. We speculated that Hh signaling was negatively activated in myocytes and quiescent myoblasts. The increased expression of Gli1 and Gli2 in the early days of myogenic differentiation suggested that Hh signaling would be activated when the quiescent bovine myoblast was stimulated to initiate myogenic differentiation.

Stimulative Effects of Low Intensity He-Ne Laser Irradiation on the Proliferative Potential and Cell-Cycle Progression of Myoblasts in Culture

Low intensity laser irradiation (LILI) was found to promote the regeneration of skeletal musclein vivobut the cellular mechanisms are not fully understood. Myoblasts, normally quiescent and inactivated in adult skeletal muscle, are a type of myogenic progenitor cells and considered as the major candidates responsible for muscle regeneration. The aim of the present study was to study the effect of LILI on the growth potential and cell-cycle progression of the cultured myoblasts. Primary myoblasts isolated from rat hind legs were cultured in nutrient-deficient medium for 36 hours and then irradiated by helium-neon laser at a certain energy density. Immunohistochemical and flow cytometric analysis revealed that laser irradiation could increase the expression of cellular proliferation marker and the amount of cell subpopulations in the proliferative phase as compared with the nonirradiated control group. Meanwhile, the expressions of cell-cycle regulatory proteins in the laser-treated myoblasts were markedly upregulated as compared to the unirradiated cells, indicating that LILI could promote the reentry of quiescent myoblasts into the cell division cycle. These results suggest that LILI at certain fluences could promote their proliferation, thus contributing to the skeletal muscle regeneration following trauma and myopathic diseases.

Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest

Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.

Regulation of Muscle Satellite Cell Activation and Chemotaxis by Angiotensin II

The role of angiotensin II (Ang II) in skeletal muscle is poorly understood. We report that pharmacological inhibition of Ang II signaling or ablation of the AT1a receptor significantly impaired skeletal muscle growth following myotrauma, in vivo, likely due to impaired satellite cell activation and chemotaxis. In vitro experiments demonstrated that Ang II treatment activated quiescent myoblasts as evidenced by the upregulation of myogenic regulatory factors, increased number of β-gal+, Myf5-LacZ myoblasts and the acquisition of cellular motility. Furthermore, exogenous treatment with Ang II significantly increased the chemotactic capacity of C2C12 and primary cells while AT1a−/− myoblasts demonstrated a severe impairment in basal migration and were not responsive to Ang II treatment. Additionally, Ang II interacted with myoblasts in a paracrine-mediated fashion as 4 h of cyclic mechanical stimulation resulted in Ang II-induced migration of cocultured myoblasts. Ang II-induced chemotaxis appeared to be regulated by multiple mechanisms including reorganization of the actin cytoskeleton and augmentation of MMP2 activity. Collectively, these results highlight a novel role for Ang II and ACE inhibitors in the regulation of skeletal muscle growth and satellite cell function.

MLL5, a trithorax homolog, indirectly regulates H3K4 methylation, represses cyclin A2 expression, and promotes myogenic differentiation

Most cells in adult tissues are nondividing. In skeletal muscle, differentiated myofibers have exited the cell cycle permanently, whereas satellite stem cells withdraw transiently, returning to active proliferation to repair damaged myofibers. We have examined the epigenetic mechanisms operating in conditional quiescence by analyzing the function of a predicted chromatin regulator mixed lineage leukemia 5 (MLL5) in a culture model of reversible arrest. MLL5 is induced in quiescent myoblasts and regulates both the cell cycle and differentiation via a hierarchy of chromatin and transcriptional regulators. Knocking down MLL5 delays entry of quiescent myoblasts into S phase, but hastens S-phase completion. Cyclin A2 (CycA) mRNA is no longer restricted to S phase, but is induced throughout G(0)/G(1), with activation of the cell cycle regulated element (CCRE) in the CycA promoter. Overexpressed MLL5 physically associates with the CCRE and impairs its activity. MLL5 also regulates CycA indirectly: Cux, an activator of CycA promoter and S phase is induced in RNAi cells, and Brm/Brg1, CCRE-binding repressors that promote differentiation are repressed. In knockdown cells, H3K4 methylation at the CCRE is reduced, reflecting quantitative global changes in methylation. MLL5 appears to lack intrinsic histone methyl transferase activity, but regulates expression of histone-modifying enzymes LSD1 and SET7/9, suggesting an indirect mechanism. Finally, expression of muscle regulators Pax7, Myf5, and myogenin is impaired in MLL5 knockdown cells, which are profoundly differentiation defective. Collectively, our results suggest that MLL5 plays an integral role in novel chromatin regulatory mechanisms that suppress inappropriate expression of S-phase-promoting genes and maintain expression of determination genes in quiescent cells.

Lamin A N-terminal phosphorylation is associated with myoblast activation: impairment in Emery–Dreifuss muscular dystrophy

Background: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery–Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown....

004.Control of skeletal muscle cell proliferation and differentiation

Skeletal muscle is formed by mononucleated precursor cells (myoblasts) that cease cell proliferation to start differentiation; this results in fusion between the myoblasts to form multinucleated cells (myotubes) that continue to differentiate (and fuse with more muscle cells) and mature into myofibres. Myogenesis has been widely used as a model to study in vitro factors controlling cell proliferation and differentiation. Condition in vitro may not reflect what happens in the more complex in vivo environment. Some of the key issues are what activates quiescent myoblasts in mature skeletal muscle in vivo, and what controls the switch between proliferation and differentiation? The role of the matrix, and molecules such as MyoD, p53, NFAT and IGF-1 will be considered.

M-Calpain implication in cell cycle during muscle precursor cell activation

Milli-calpain, a member of the ubiquitous cysteine protease family, is known to control late events of cell–cell fusion in skeletal muscle tissue through its involvement in cell membrane and cytoskeleton component reorganization. In this report, we describe the characterization of m-calpain compartmentalization and activation during the initial steps of muscle precursor cell recruitment and differentiation. By immunofluorescence analysis, we show that m-calpain is present throughout the cell cycle in the nucleus of proliferating myoblast C2 cells. However, when myoblasts enter a quiescent/G0 stage, m-calpain staining is detected only in the cytoplasm. Moreover, comparison of healthy and injured muscle shows distinct m-calpain localization in satellite stem cells. Indeed, m-calpain is not found in quiescent satellite cells, but following muscle injury, when satellite cells start to proliferate, m-calpain appears in the nucleus. To determine the implication of m-calpain during the cell cycle progression, quiescent myoblasts were forced to re-enter the cell cycle in the presence or not of the specific calpain inhibitor MDL 28170. We demonstrate that this calpain inhibitor blocks the cell cycle, prevents accumulation of MyoD in the G1 phase and enhances Myf5 expression. These data support an important new role for m-calpain in the control of muscle precursor cell activation and thus suggest its possible implication during the initial events of muscle regeneration.

Distinct changes in intranuclear lamin A/C organization during myoblast differentiation.

Intranuclear lamin foci or speckles have been observed in various cell types. In order to explore the possibility of changes in internal lamin organization during muscle differentiation, we have examined the appearance of A-type lamin speckles that associate with RNA splicing factor speckles in C2C12 myoblasts and myotubes. Lamin speckles were observed in dividing myoblasts but disappeared early during the course of differentiation in postmitotic myocytes, and were absent in myotubes and muscle fibers. However, no changes were seen in the typical peripheral organization of lamins A/C or B1 or in RNA splicing factor speckles. Lamin speckles were also absent in quiescent myoblasts but reappeared as cells were reactivated to enter the cell cycle. These changes were not observed in other quiescent cell types. Immunoblot analysis indicated that the abundance and migration of lamins A and C was not altered in differentiated myoblasts. When myotube or quiescent myoblast nuclei were extracted with nucleases and detergent, a uniformly stained internal lamina was revealed, indicating that lamins A/C were antigenically masked in these cells, probably owing to structural reorganization of the lamina during differentiation or quiescence. Our results suggest that muscle cell differentiation is accompanied by regulated rearrangements in the organization of the A-type lamins.

Tumor Necrosis Factor-α and Basic Fibroblast Growth Factor Differentially Inhibit the Insulin-like Growth Factor-I Induced Expression of Myogenin in C2C12 Myoblasts

Tumor necrosis factor-α (TNF-α) plays a role in several disease states such as sepsis, cachexia, and non-insulin-dependent diabetes. TNF-α interferes with insulin signaling and inhibits differentiation-specific gene expression in adipose tissue and skeletal muscle. We have examined the mechanisms by which TNF-α, in comparison to basic fibroblast growth factor (bFGF), inhibits the insulin-like growth factor-I (IGF-I)-induced differentiation of C2C12 myoblasts. Adhesion of quiescent, suspended myoblasts to collagen in high concentrations of IGF-I (10 nM) induced these cells to proliferate during the initial 24 h postplating and in so doing transiently inhibited the expression of myogenin, an essential transcription factor controlling myoblast differentiation. Low doses of IGF-I (1 nM) were minimally mitogenic and enhanced muscle-specific gene expression. Quiescent myoblasts treated with bFGF in combination with IGF-I did not express myogenin, but expressed proliferating cell nuclear antigen and underwent DNA synthesis. In contrast, TNF-α in the presence or absence of 1 nM IGF-I, did not stimulate DNA synthesis in myoblasts. However, TNF-α inhibited myogenin mRNA and protein expression. Expression of the cyclin-dependent kinase inhibitor p21 correlated with myogenin expression and myoblast differentiation, but not with growth arrest. These results indicate that both TNF-α and bFGF inhibit myogenin expression but differentially influence myoblast proliferation.

Uncoupling of p21 induction and MyoD activation results in the failure of irreversible cell cycle arrest in doxorubicin-treated myocytes.

Doxorubicin (Dox, Adriamicin), a potent broad spectrum anthracycline anticancer drug, selectively inhibits muscle specific gene expression in cardiac cells in vivo and prevents terminal differentiation of skeletal muscle cells in vitro. By inducing the expression of the helix-loop-helix (HLH) transcriptional inhibitor ld2, Dox represses the myogenic function of the MyoD family of muscle regulatory factors (MRFs). In many cell types, terminal differentiation is coupled to an irreversible exit from the cell cycle and MyoD plays a critical role in the permanent cell cycle arrest of differentiating myocytes by upregulating the cyclin dependent kinase inhibitor (cdki) p21. Here, we correlate Dox effects on cell cycle with changes of E2F/DP complexes and activity in differentiating C2C12 myocytes. In Dox-treated quiescent myoblasts, which fail to differentiate into myotubes under permissive culture conditions, serum re-stimulation induces cyclin/cdk re-association on the E2F/DP complexes and this correlates with an evident increase in E2F/DP driven transcription and re-entry of myoblasts into the cell cycle. Despite Dox ability to activate the DNA-damage dependent p53/p21 pathway, when induced in the absence of MyoD or other MRFs, p21 fails to maintain the postmitotic state in Dox-treated myocytes induced to differentiate. Thus, uncoupling p21 induction and MyoD activity results in a serum-reversible cell cycle arrest, indicating that MRF specific activation of cdki(s) is required for permanent cell cycle arrest in differentiating muscle cells.

MyoD prevents cyclinA/cdk2 containing E2F complexes formation in terminally differentiated myocytes.

Withdrawal from the cell cycle of differentiating myocytes is regulated by the myogenic basic helix-loop-helix (bHLH) protein MyoD and the pocket proteins pRb, p107 and pRb2/p130. Downstream effectors of 'pocket' proteins are the components of the E2F family of transcription factors, which regulate the G1/S-phase transition. We analysed by EMSA the composition of E2F complexes in cycling, quiescent undifferentiated and differentiated C2C12 skeletal muscle cells. An E2F complex containing mainly E2F4 and pRb2/p130 (E2F-G0/G1 complex) appears when DNA synthesis arrests, replacing the cyclinA/cdk2 containing E2F complex of proliferating myoblasts (E2F-G1/S complex). Serum stimulation reinduces DNA synthesis and the re-appearance of E2F-G1/S complexes in quiescent myoblasts but not in differentiated C2C12 myotubes. In differentiating C2C12 cells, E2F complexes switch and DNA synthesis in response to serum are prevented when MyoD DNA binding activity and the cdks inhibitor MyoD downstream effector p21 are induced. Thus, during myogenic differentiation, formation of E2F4 and pRb2/p130 containing complexes is an early event, but not enough on its own to prevent the reactivation of DNA synthesis. Using a subclone of C3H10T1/2 mouse fibroblasts stably expressing Estrogen Receptor-MyoD (ER-MyoD) chimerae, we found that estrogen directed MyoD activation prevents the reassociation of cyclinA/cdk2 to the E2F4 containing complex following serum stimulation and this correlates with suppression of E2F activity and the inability of cells to re-enter the cell cycle. Our data indicate that, in differentiating myocytes, one mechanism through which MyoD induces permanent cell cycle arrest involves p21 upregulation and suppression of the proliferation-associated cdks-containing E2F complexes formation.