SIRT3, a Mitochondrial NAD+-Dependent Deacetylase,Is Involved in the Regulation of Myoblast Differentiation

Sirtuin 3 (SIRT3), one of the seven mammalian sirtuins, is a mitochondrial NAD⁺-dependent deacetylase known to control key metabolic pathways. SIRT3 deacetylases and activates a large number of mitochondrial enzymes involved in the respiratory chain, in ATP production, and in both the citric acid and urea cycles. We have previously shown that the regulation of myoblast differentiation is tightly linked to mitochondrial activity. Since SIRT3 modulates mitochondrial activity, we decide to address its role during myoblast differentiation. For this purpose, we first investigated the expression of endogenous SIRT3 during C2C12 myoblast differentiation. We further studied the impact of SIRT3 silencing on both the myogenic potential and the mitochondrial activity of C2C12 cells. We showed that SIRT3 protein expression peaked at the onset of myoblast differentiation. The inhibition of SIRT3 expression mediated by the stable integration of SIRT3 short inhibitory RNA (SIRT3shRNA) in C2C12 myoblasts, resulted in: 1) abrogation of terminal differentiation - as evidenced by a marked decrease in the myoblast fusion index and a significant reduction of Myogenin, MyoD, Sirtuin 1 and Troponin T protein expression - restored upon MyoD overexpression; 2) a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and citrate synthase protein expression reflecting an alteration of mitochondrial density; and 3) an increased production of reactive oxygen species (ROS) mirrored by the decreased activity of manganese superoxide dismutase (MnSOD). Altogether our data demonstrate that SIRT3 mainly regulates myoblast differentiation via its influence on mitochondrial activity.     

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.     

Inhibition of desmin expression blocks myoblast fusion and interferes with the myogenic regulators MyoD and myogenin

The muscle-specific intermediate filament protein, desmin, is one of the earliest myogenic markers whose functional role during myogenic commitment and differentiation is unknown. Sequence comparison of the presently isolated and fully characterized mouse desmin cDNA clones revealed a single domain of polypeptide similarity between desmin and the basic and helix-loop-helix region of members of the myoD family myogenic regulators. This further substantiated the need to search for the function of desmin. Constructs designed to express anti-sense desmin RNA were used to obtain stably transfected C2C12 myoblast cell lines. Several lines were obtained where expression of the anti-sense desmin RNA inhibited the expression of desmin RNA and protein down to basal levels. As a consequence, the differentiation of these myoblasts was blocked; complete inhibition of myoblast fusion and myotube formation was observed. Rescue of the normal phenotype was achieved either by spontaneous revertants, or by overexpression of the desmin sense RNA in the defective cell lines. In several of the cell lines obtained, inhibition of desmin expression was followed by differential inhibition of the myogenic regulators myoD and/or myogenin, depending on the stage and extent of desmin inhibition in these cells. These data suggested that myogenesis is modulated by at least more than one pathway and desmin, which so far was believed to be merely an architectural protein, seems to play a key role in this process.     

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E₂ (PGE₂), a bioactive lipid mediator in various physiological or pathological conditions. PGE₂ is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE₂ signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE₂ or specific agonists. All four PGE₂ receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE₂ and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE₂ (17-PT PGE₂) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE₂ signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.     

Mitochondrial activity regulates myoblast differentiation by control of c-Myc expression

We have previously shown that mitochondrial activity is an important regulator of myoblast differentiation, partly through processes targeting myogenin expression. Here, we investigated the possible involvement of c-myc in these processes. Inhibition of mitochondrial activity by chloramphenicol abrogated the decrease in c-myc mRNA and protein levels occurring at the onset of terminal differentiation. Conversely, stimulation of mitochondrial activity by overexpression of the T3 mitochondrial receptor (p43) down-regulated c-myc expression. In addition, c-myc overexpression mimicked the influence of mitochondrial activity inhibition on myoblast differentiation. Moreover, like chloramphenicol, c-myc overexpression strongly inhibited the myogenic influence of p43 overexpression. These data suggest that c-Myc is an important target of mitochondrial activity involved in the myogenic influence of the organelle. Lastly, we found that chloramphenicol influence is negatively related to the frequency of post-mitotic myoblasts in the culture at the onset of treatment, and cell cycle analyses demonstrated that the frequency of myoblasts in G0-G1 phase at cell confluence is increased by p43 overexpression and decreased by chloramphenicol or c-myc overexpression. These results suggest that irreversible myoblast withdrawal from the cell cycle is a target of mitochondrial activity by control of c-Myc expression.     

P43-dependent mitochondrial activity regulates myoblast differentiation and slow myosin isoform expression by control of Calcineurin expression

We have previously shown that mitochondrial protein synthesis regulates myoblast differentiation, partly through the control of c-Myc expression, a cellular oncogene regulating myogenin expression and myoblast withdrawal from the cell cycle. In this study we provide evidence of the involvement of Calcineurin in this regulation. In C2C12 myoblasts, inhibition of mitochondrial protein synthesis by chloramphenicol decreases Calcineurin expression. Conversely, stimulation of this process by overexpressing the T3 mitochondrial receptor (p43) increases Calcineurin expression. Moreover, expression of a constitutively active Calcineurin (ΔCN) stimulates myoblast differentiation, whereas a Calcineurin antisense has the opposite effect. Lastly, ΔCN expression or stimulation of mitochondrial protein synthesis specifically increases slow myosin heavy chain expression. In conclusion, these data clearly suggest that, partly via Calcineurin expression, mitochondrial protein synthesis is involved in muscle development through the control of myoblast differentiation and probably the acquisition of the contractile and metabolic phenotype of muscle fibres.     

Insulin receptor substrate protein 53kDa (IRSp53) is a negative regulator of myogenic differentiation

Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell-extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes.     

Changes in mitochondrial reactive oxygen species synthesis during differentiation of skeletal muscle cells

Myogenesis is accompanied by an intensive metabolic remodeling. We investigated the mitochondrial reactive oxygen species (ROS) generation at different levels of skeletal muscle differentiation: in C2C12 myoblasts, in C2C12 myotubes and in adult mouse skeletal muscle. Differentiation was accompanied by an increase in mitochondrial content and respiratory chain activity. The detected ROS production levels correlated with mitochondrial content, being the lowest in the myoblasts. Unlike the adult skeletal muscle, myoblast ROS production was significantly stimulated by the complex I inhibitor rotenone. Our results show that mitochondria are an important ROS source in skeletal muscle cells. The substantial changes in mitochondrial ROS synthesis during skeletal muscle differentiation can be explained by intensive bioenergetic remodeling.     

Notch Pathway Activation Contributes to Inhibition of C2C12 Myoblast Differentiation by Ethanol

The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.     

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.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

Macrophage migration inhibitory factor in the regulation of myoblast proliferation and differentiation

Obesity is documented to be a state of chronic mild inflammation associated with increased macrophage infiltration into adipose tissue and liver and skeletal muscle. As a pleiotropic inflammatory mediator, macrophage migration inhibitory factor (MIF) is associated with metabolic disease, so MIF may signal molecular links between adipocytes and myocytes. MIF expression was modified during myoblast differentiation, but the role of MIF during this process is unclear. C2C12 cells were transfected with MIF to investigate their role during differentiation. MIF expression attenuated C2C12 differentiation. It did not change proliferation, but downregulated cyclin D1 and CDK4, causing cell accumulation in the G1 phase. p21 protein was increased significantly and MyoD, MyoG, and p21 mRNA also increased significantly in the C2C12 cells treated with ISO-1, suggesting that inhibition of MIF promotes differentiation. MIF inhibits the myoblast differentiation by affecting the cell cycle progression, but does not affect proliferation.