Deletion of Drosophila muscle LIM protein decreases flight muscle stiffness and power generation

Muscle LIM protein (MLP) can be found at the Z-disk of sarcomeres where it is hypothesized to be involved in sensing muscle stretch. Loss of murine MLP results in dilated cardiomyopathy, and mutations in human MLP lead to cardiac hypertrophy, indicating a critical role for MLP in maintaining normal cardiac function. Loss of MLP in Drosophila (mlp84B) also leads to muscle dysfunction, providing a model system to examine MLP's mechanism of action. Mlp84B-null flies that survive to adulthood are not able to fly or beat their wings. Transgenic expression of the mlp84B gene in the Mlp84B-null background rescues flight ability and restores wing beating ability. Mechanical analysis of skinned flight muscle fibers showed a 30% decrease in oscillatory power production and a slight increase in the frequency at which maximum power is generated for fibers lacking Mlp84B compared with rescued fibers. Mlp84B-null muscle fibers displayed a 25% decrease in passive, active, and rigor stiffness compared with rescued fibers, but no significant decrease in isometric tension generation was observed. Muscle ultrastructure of Mlp84B-null muscle fibers is grossly normal; however, the null fibers have a slight decrease, 11%, in thick filament number per unit cross-sectional area. Our data indicate that MLP contributes to muscle stiffness and is necessary for maximum work and power generation.     

Developmental regulation of the Drosophila tropomyosin I (TmI) gene is controlled by a muscle activator enhancer region that contains multiple cis-elements and binding sites for multiple proteins

Developmental gene regulation in vertebrate somatic muscles involves the cooperative interaction of MEF2 (myocyte-specific enhancer-binding factor 2) and members of the b-HLH (basic helix-loop-helix) family of myogenic factors. Until recently, however, nothing was known about the factors that control the developmental regulation of muscle genes during embryogenesis in Drosophila. The Drosophila Tropomyosin I (TmI) gene contains a proximal and distal muscle enhancer within the first intron that regulates its expression in embryonic/larval and adult muscles. We have recently shown that the 355-bp proximal enhancer contains a binding site for the Drosophila homologue of vertebrate MEF2 and that MEF2 acts cooperatively with a basal level muscle activator region to direct high level muscle expression in transgenic flies. The 92-bp muscle activator region, however, does not contain any consensus E-box (CANNTG) binding site sequences for b-HLH myogenic factors, suggesting the MEF2 may interact with other factors to regulate muscle genes in Drosophila. In this study we have used mutation analysis and germ-line transformation to analyze the cis-acting elements within the muscle activator region that regulate its expression in transgenic flies. We have identified a 71-bp region that is sufficient for low basal level temporal- and muscle-specific expression in the embryo, larva, and adult. Substitution mutations within the muscle activator region have identified several cis-element regions spanning 60-bp that are required for either full or partial muscle activator function. An analysis of proteins that bind to this region by gel mobility shift assay and copper nuclease footprinting has allowed us to identify the sites in this region at which multiple proteins complex and interact. We propose that these cis-elements and the proteins that they bind regulate muscle activator function and together with MEF2 are capable of regulating high level muscle expression. Dev. Genet. 20:297–306, 1997. © 1997 Wiley-Liss, Inc.     

Synergistic up-regulation of muscle LIM protein expression in C2C12 and NIH3T3 cells by myogenin and MEF2C

Although the role of muscle LIM protein (MLP, also known as CRP3), a LIM-only protein of LIM domain-containing protein family, is well-characterized, the mechanism by which the MLP gene expresses remains unclear. Herein, we demonstrate that myogenin and myocyte enhancer factor 2C (MEF2C) cooperate in activating the MLP gene in myogenesis. RT-PCR, real-time PCR and Western blotting showed that overexpression of myogenin or myogenin plus MEF2C led to induction of the MLP gene in differentiating C2C12 and NIH3T3 fibroblasts. By contrary, knocking-down of myogenin by RNA interference (RNAi) suppressed MLP expression in differentiating C2C12. Deletion and reporter enzyme assay revealed that the promoter activity was determined largely by the region extending from −260 to −173, which containing three E-box (CANNTG motif) candidates. Site-directed mutagenesis demonstrated that the E-box at position −186 to −180 was crucial for activating the promoter by myogenin. Furthermore, MEF2C could enhance myogenin-mediated activation of the promoter. In addition, chromatin immunoprecipitation (ChIP) and re-ChIP showed that myogenin and MEF2C were associated with the activated MLP promoter. Together, these results suggest that myogenin and MEF2C cooperate in the MLP gene activation. The linking of the MLP gene activation with myogenin and MEF2C may facilitate myogenin-mediated differentiation of striated muscle.     

Mechano growth factor (MGF) promotes proliferation and inhibits differentiation of porcine satellite cells (PSCs) by down-regulation of key myogenic transcriptional factors

Porcine satellite cells represent an ideal model system for studying the cellular and molecular basis regulating myogenic stem cell proliferation and differentiation and for exploring the experimental conditions for myoblast transplantation. Here, we investigated the effects of mechano growth factor (MGF), a spliced variant of the IGF-1 gene, on porcine satellite cells. We show that MGF potently stimulated proliferation while inhibited differentiation of porcine satellite cells. MGF-treatment acutely down-regulates the expression of myogenic determination factor (MyoD) and the cyclin-dependent kinase inhibitor p21. MGF-treatment also markedly reduced the overall expression of cyclin B1 and key factors of the myogenic regulatory and myocyte enhancer families, including Myogenein and MEF2A. Taken together, the gene expression data from MGF-treated porcine satellite cells are in favor of a molecular model in which MGF inhibits porcine satellite cell differentiation by down-regulating either the activity or expression of MyoD, which, in turn, suppresses the expression of key genes required for cell cycle progression and differentiation, such as p21, Myogenin, and MEF2. Overall, our findings are in support of the previous suggestion that MGF may be used in vivo and in vitro to promote proliferation of myogenic stem cells to prevent and treat age-related muscle degenerative diseases.