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.     

敲减DTX4表达抑制C2C12细胞成肌分化

DTX4（Deltex 4 homolog）蛋白属于Deltex家族成员|Deltex家族是Notch信号通路的调节因子. 已知Notch信号通路在成肌分化中发挥重要作用. 然而,DTX4是否参与调控肌肉发育尚未有报道. 本研究探索DTX4对成肌分化的影响及作用机制. 实时定量PCR和蛋白质印迹分析揭示,伴随小鼠C2C12成肌细胞（myoblast）分化为肌管（myotube）过程,成肌分化标志蛋白肌球蛋白重链（myosin heavy-chain,MyHC）、肌细胞生成素(myogenin)表达逐渐升高,DTX4 mRNA及蛋白质表达水平也逐渐升高. 通过顺序专一的siRNA敲减DTX4表达后,C2C12成肌细胞肌管面积和肌管融合指数明显减少|MyHC、肌细胞生成素蛋白表达水平明显降低|但ERK信号通路未见明显变化.上述结果表明,敲减DTX4表达抑制C2C12细胞成肌分化.我们的结果提示,DTX4可能参与C2C12细胞成肌分化.     

Simvastatin Inhibits Glucose Metabolism and Legumain Activity in Human Myotubes

Simvastatin, a HMG-CoA reductase inhibitor, is prescribed worldwide to patients with hypercholesterolemia. Although simvastatin is well tolerated, side effects like myotoxicity are reported. The mechanism for statin-induced myotoxicity is still poorly understood. Reports have suggested impaired mitochondrial dysfunction as a contributor to the observed myotoxicity. In this regard, we wanted to study the effects of simvastatin on glucose metabolism and the activity of legumain, a cysteine protease. Legumain, being the only known asparaginyl endopeptidase, has caspase-like properties and is described to be involved in apoptosis. Recent evidences indicate a regulatory role of both glucose and statins on cysteine proteases in monocytes. Satellite cells were isolated from the Musculus obliquus internus abdominis of healthy human donors, proliferated and differentiated into polynuclear myotubes. Simvastatin with or without mevalonolactone, farnesyl pyrophosphate or geranylgeranyl pyrophosphate were introduced on day 5 of differentiation. After 48 h, cells were either harvested for immunoblotting, ELISA, cell viability assay, confocal imaging or enzyme activity analysis, or placed in a fuel handling system with [¹⁴C]glucose or [³H]deoxyglucose for uptake and oxidation studies. A dose-dependent decrease in both glucose uptake and oxidation were observed in mature myotubes after exposure to simvastatin in concentrations not influencing cell viability. In addition, simvastatin caused a decrease in maturation and activity of legumain. Dysregulation of glucose metabolism and decreased legumain activity by simvastatin points out new knowledge about the effects of statins on skeletal muscle, and may contribute to the understanding of the myotoxicity observed by statins.     

Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G₀/G₁ phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21^Cip1 and p27^Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death.     

Pannexin 1 and Pannexin 3 Channels Regulate Skeletal Muscle Myoblast Proliferation and Differentiation

Pannexins constitute a family of three glycoproteins (Panx1, -2, and -3) forming single membrane channels. Recent work demonstrated that Panx1 is expressed in skeletal muscle and involved in the potentiation of contraction. However, Panxs functions in skeletal muscle cell differentiation, and proliferation had yet to be assessed. We show here that Panx1 and Panx3, but not Panx2, are present in human and rodent skeletal muscle, and their various species are differentially expressed in fetal versus adult human skeletal muscle tissue. Panx1 levels were very low in undifferentiated human primary skeletal muscle cells and myoblasts (HSMM) but increased drastically during differentiation and became the main Panx expressed in differentiated cells. Using HSMM, we found that Panx1 expression promotes this process, whereas it was impaired in the presence of probenecid or carbenoxolone. As for Panx3, its lower molecular weight species were prominent in adult skeletal muscle but very low in the fetal tissue and in undifferentiated skeletal muscle cells and myoblasts. Its overexpression (∼43-kDa species) induced HSMM differentiation and also inhibited their proliferation. On the other hand, a ∼70-kDa immunoreactive species of Panx3, likely glycosylated, sialylated, and phosphorylated, was highly expressed in proliferative myoblasts but strikingly down-regulated during their differentiation. Reduction of its endogenous expression using two Panx3 shRNAs significantly inhibited HSMM proliferation without triggering their differentiation. In summary, our results demonstrate that Panx1 and Panx3 are co-expressed in human skeletal muscle myoblasts and play a pivotal role in dictating the proliferation and differentiation status of these cells.     

Secreted Nucleobindin-2 Inhibits 3T3-L1 Adipocyte Differentiation

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.     

Quantal Division and a Postmitotic State in Myoblast Differentiation

The reversible arrest of myoblast differentiation by ethidium bromide (EB) has been used to examine the nature of the transition from the proliferative state to terminal differentiation resulting in fusion into muscle fibers. If EB is introduced at the time that myoblasts are shifted to medium that induces fusion, all apparent cytodifferentiation is suspended. When such EB arrested myoblasts are released from EB inhibition they fuse without reentering the cell cycle. If EB arrested myoblasts are released into proliferation promoting medium rather than medium that induces fusion they neither fuse nor proliferate. In this case they remain quiescent in the proliferating medium for an extended period, however, if these myoblasts are subsequently shifted to medium that induces fusion, they fuse without reentering the cell cycle. Apparently the myoblasts have become postmitotic and competent to fuse into muscle fibers during their initial exposure to fusion inducing medium, even though cytodifferentiation has been blocked. Exposure of these postmitotic fusion competent myoblasts to proliferation promoting medium does not stimulate them to reenter the cell cycle but does prevent fusion into muscle fibers. These results are most consistent with a quantal division model of myoblast differentiation rather than a gradual transition from the proliferative state to a state in which fusion occurs.     

Controlling the Orientation and Synaptic Differentiation of Myotubes with Micropatterned Substrates

Micropatterned poly(dimethylsiloxane) substrates fabricated by soft lithography led to large-scale orientation of myoblasts in culture, thereby controlling the orientation of the myotubes they formed. Fusion occurred on many chemically identical surfaces in which varying structures were arranged in square or hexagonal lattices, but only a subset of patterned surfaces yielded aligned myotubes. Remarkably, on some substrates, large populations of myotubes oriented at a reproducible acute angle to the lattice of patterned features. A simple geometrical model predicts the angle and extent of orientation based on maximizing the contact area between the myoblasts and patterned topographic surfaces. Micropatterned substrates also provided short-range cues that influenced higher-order functions such as the localization of focal adhesions and accumulation of postsynaptic acetylcholine receptors. Our results represent what we believe is a new approach for musculoskeletal tissue engineering, and our model sheds light on mechanisms of myotube alignment in vivo.     

CEP2 Attenuates Myoblast Differentiation But Does Not Affect Proliferation

CEP2 (CDC42EP2) is a member of the CDC42 subfamily that belongs to the Rho family. The Rho family plays an important role in a variety of cellular processes including skeletal myogenesis. Here, we find the expression of CEP2 increased significantly during C2C12 myogenesis. Overexpression of CEP2 could attenuate myoblast differentiation, while knockdown of CEP2 by siRNA results in enhancing myogenesis. Furthermore, we demonstrate for the first time that CEP2 attenuates myoblast differentiation via suppression of muscle regulatory factors (MRFs) rather than influencing myoblast proliferation. These results indicate that CEP2 acts as a repressor during myogenesis, which provides new insights into the role of CEP2 in muscle development.     

Cdo Interacts with APPL1 and Activates AKT in Myoblast Differentiation

Cell–cell interactions between muscle precursors are required for myogenic differentiation; however, underlying mechanisms are largely unknown. Promyogenic cell surface protein Cdo functions as a component of multiprotein complexes containing other cell adhesion molecules, Boc, Neogenin and N-cadherin, and mediates some of signals triggered by cell–cell interactions between muscle precursors. Cdo activates p38MAPK via interaction with two scaffold proteins JLP and Bnip-2 to promote myogenesis. p38MAPK and Akt signaling are required for myogenic differentiation and activation of both signaling pathways is crucial for efficient myogenic differentiation. We report here that APPL1, an interacting partner of Akt, forms complexes with Cdo and Boc in differentiating myoblasts. Both Cdo and APPL1 are required for efficient Akt activation during myoblast differentiation. The defective differentiation of Cdo-depleted cells is fully rescued by overexpression of a constitutively active form of Akt, whereas overexpression of APPL1 fails to do so. Taken together, Cdo activates Akt through association with APPL1 during myoblast differentiation, and this complex likely mediates some of the promyogenic effect of cell–cell interaction. The promyogenic function of Cdo involves a coordinated activation of p38MAPK and Akt via association with scaffold proteins, JLP and Bnip-2 for p38MAPK and APPL1 for Akt.     

RNAi Screen Reveals Potentially Novel Roles of Cytokines in Myoblast Differentiation

Cytokines are cell-secreted signaling molecules that modulate various cellular functions, with the best-characterized roles in immune responses. The expression of numerous cytokines in skeletal muscle tissues and muscle cells has been reported, but their function in skeletal myogenesis, the formation of skeletal muscle, has been largely underexplored. To systematically examine the potential roles of cytokines in skeletal myogenesis, we undertook an RNAi screen of 134 mouse cytokine genes for their involvement in the differentiation of C2C12 myoblasts. Our results have uncovered 29 cytokines as strong candidates for novel myogenic regulators, potentially conferring positive and negative regulation at distinct stages of myogenesis. These candidates represent a diverse collection of cytokine families, including interleukins, TNF-related factors, and chemokines. Our findings suggest the fundamental importance of cytokines in the cell-autonomous regulation of myoblast differentiation, and may facilitate future identification of novel therapeutic targets for improving muscle regeneration and growth in health and diseases.     

不同代次牛成肌细胞的诱导分化及相关基因的表达分析

利用初生荷斯坦牛的成肌细胞,在不同代次以含体积分数为2%马血清的DMEM进行诱导分化,在之后0、2、4、6、8、10 d观察细胞的形态变化并收集细胞提取总RNA,检测成肌相关基因的表达情况,为进一步研究牛肌肉发生过程及相关基因的表达调控提供依据。同时利用实时荧光定量PCR分别检测肌性相关基因MyoD(生肌决定因子)、MyoG(肌细胞生成素)以及非肌性相关基因A-FABP(脂肪细胞型脂肪酸结合蛋白)表达水平的变化。研究表明：①各代细胞在诱导培养4 d后开始有肌管形成,其后越来越多,8 d时达高峰。②成肌细胞向成熟肌细胞分化过程中,MyoD、MyoG、A-FABP基因都呈先上升然后下降的趋势。③高代次成肌细胞比低代次细胞增殖慢,而且在诱导分化后,肌管的数目明显变少; MyoD、MyoG、A-FABP随着代次的升高而下降。故推断MyoD、MyoG以及A-FABP基因会在成肌分化开始后的不同阶段被激活,从而发挥不同的调控作用,而且随着传代次数的增加成肌细胞的分化能力减弱。     

Stac3 Is a Novel Regulator of Skeletal Muscle Development in Mice

The goal of this study was to identify novel factors that mediate skeletal muscle development or function. We began the study by searching the gene expression databases for genes that have no known functions but are preferentially expressed in skeletal muscle. This search led to the identification of the Src homology three (SH3) domain and cysteine rich (C1) domain 3 (Stac3) gene. We experimentally confirmed that Stac3 mRNA was predominantly expressed in skeletal muscle. We determined if Stac3 plays a role in skeletal muscle development or function by generating Stac3 knockout mice. All Stac3 homozygous mutant mice were found dead at birth, were never seen move, and had a curved body and dropping forelimbs. These mice had marked abnormalities in skeletal muscles throughout the body, including central location of myonuclei, decreased number but increased cross-sectional area of myofibers, decreased number and size of myofibrils, disarrayed myofibrils, and streaming Z-lines. These phenotypes demonstrate that the Stac3 gene plays a critical role in skeletal muscle development and function in mice.