CellBio  Vol.2 No.3 , September 2013
Antibody to MyoD or Myogenin Decreases Acetylcholine Receptor Clustering in C2C12 Myotube Culture
Abstract: Skeletal muscle development is influenced by myogenic regulatory factors, including the expression of MyoD and myogenin. Our objective was to use the C2C12 cell culture model to test the hypothesis that both MyoD and myogenin were required for agrin-induced acetylcholine receptor (AChR) clustering and the fusion of myoblasts into myotubes. We induced fusion of myoblasts into myotubes by switching from growth medium (GM) to differentiation medium (DM). During myotube formation AChRs cluster spontaneously, but treatment with motor neuron derived agrin increases clustering of AChRs and other postsynaptic components of the neuromuscular synapse. We examined the normal expression pattern of MyoD and myogenin in C2C12 cell culture using immunofluorescence. MyoD was highly expressed while myoblasts were in GM, but expression declined within 72 hours after cell cultures were switched to DM. Myogenin expression was low in GM, but increased when cell cultures were switched to DM. Next we used antibodies to decrease MyoD and/or myogenin function. Fluorescence microscopy images were captured and then analyzed to assess agrin-induced AChR clustering with or without antibody treatment. Finally we calculated the proportion of nuclei in myotubes and myoblasts by creating digital overlays of phase contrast and DAPI stained microscopy images. This allowed the comparison of myotube formation with or without antibody treatment. We report that antibody to either MyoD or myogenin decreases the frequency of agrin-induced AChR clustering without affecting myotube formation. We conclude that agrin-induced AChR clustering requires both MyoD and myogenin.
Cite this paper: K. Ball, M. , H. Campbell, D. , Ezell, K. , B. Henley, J. , R. Standley, P. and A. Grow, W. (2013) Antibody to MyoD or Myogenin Decreases Acetylcholine Receptor Clustering in C2C12 Myotube Culture. CellBio, 2, 138-148. doi: 10.4236/cellbio.2013.23016.

[1]   J. Piette, J. L. Bessereau, M. Huchet and J. P. Changeux, “Two Adjacent MyoD1-Binding Sites Regulate Expres- sion of the AChR Alpha-Subunit Gene,” Nature, Vol. 345, No. 6273, 1990, pp. 353-355. doi:10.1038/345353a0

[2]   B. P. Gilmour, G. R. Fanger, C. Newton, E. M. Evans and P. D. Gardner, “Multiple Binding Sites for Myogenic Regulatory Factors Are Required for Expression of the AChR Gamma-Subunit Gene,” The Journal of Biological Chemistry, Vol. 266, No. 30, 1991, pp. 19871-19874.

[3]   M. Numberger, I. Durr, W. Kues, M. Koenen and V. Witzemann, “Different Mechanisms Regulate Muscle-Specific AChR Gamma- and Epsilon-Subunit Gene Expression,” The EMBO Journal, Vol. 10, No. 10, 1991, pp. 2957-2964.

[4]   C. A. Prody and J. P. Merlie, “A Developmental and Tissue-Specific Enhancer in the Mouse Skeletal Muscle AChR Alpha-Subunit Gene Regulated by Myogenic Factors,” The Journal of Biological Chemistry, Vol. 266, No. 23, 1991, pp. 22588-22596.

[5]   A. M. Simon and S. J. Burden, “An E Box Mediates Activation and Repression of the AChR Delta-Subunit Gene during Myogenesis,” Molecular and Cellular Biology, Vol. 13, No. 9, 1993, pp. 5133-5140.

[6]   T. Braun, M. A. Rudnicki, H. H. Arnold and R. Jaenisch, “Targeted Inactivation of the Muscle Regulatory Gene Myf-5 Results in Abnormal Rib Development and Perinatal Death,” Cell, Vol. 71, No. 3, 1992, pp. 369-382. doi:10.1016/0092-8674(92)90507-9

[7]   M. A. Rudnicki, T. Braun, S. Hinuma and R. Jaenisch, “Inactivation of MyoD in Mice Leads to Up-Regulation of the Myogenic HLH Gene Myf-5 and Results in Apparently Normal Muscle Development,” Cell, Vol. 71, No., 1992, pp. 383-390. doi:10.1016/0092-8674(92)90508-A

[8]   M. A. Rudnicki, P. N. Schnegelsberg, R. H. Stead, T. Braun, H. H. Arnold and R. Jaenisch, “MyoD or Myf-5 Is Required for the Formation of Skeletal Muscle,” Cell, Vol. 75, No. 7, 1993, pp. 1351-1359. doi:10.1016/0092-8674(93)90621-V

[9]   P. Hasty, A. Bradley, J. H. Morris, D. G. Edmondson, J. M. Venuti, E. N. Olson and W. H. Klein, “Muscle Deficiency and Neonatal Death in Mice with a Targeted Mutation in the Myogenin Gene,” Nature, Vol. 364, No. 6437, 1993, pp. 501-506. doi:10.1038/364501a0

[10]   Y. Nabeshima, K. Hanaoka, M. Hayasaka, E. Esumi, S. Li, I. Nonaka and Y. Nabeshima, “Myogenin Gene Disruption Results in Perinatal Lethality Because of Severe Muscle Defect,” Nature, Vol. 364, No. 6437, 1993, pp. 532-535. doi:10.1038/364532a0

[11]   S. J. Rhodes and S. F. Konieczny, “Identification of MRF4: A New Member of the Muscle Regulatory Factor Gene Family,” Genes & Development, Vol. 3, No. 12b, 1989, pp. 2050-2061. doi:10.1101/gad.3.12b.2050

[12]   E. Bober, G. E. Lyons, T. Braun, G. Cossu, M. Buckingham and H. H. Arnold, “The Muscle Regulatory Gene, Myf-6, Has a Biphasic Pattern of Expression during Early Mouse Development,” The Journal of Cell Biology, Vol. 113, No. 6, 1991, pp. 1255-1265. doi:10.1083/jcb.113.6.1255

[13]   T. J. Hinterberger, D. A. Sassoon, S. J. Rhodes and S. F. Konieczny, “Expression of the Muscle Regulatory Factor MRF4 during Somite and Skeletal Myofiber Development,” Developmental Biology, Vol. 147, No. 1, 1991, pp. 144-156. doi:10.1016/S0012-1606(05)80014-4

[14]   A. Buonanno, L. Apone, M. I. Morasso, R. Beers, H. R. Brenner and R. Eftimie, “The MyoD Family of Myogenic Factors Is Regulated by Electrical Activity: Isolation and Characterization of a Mouse Myf-5 cDNA,” Nucleic Acids Research, Vol. 20, No. 3, 1992, pp. 539-544. doi:10.1093/nar/20.3.539

[15]   K. Hannon, C. K. Smith, K. R. Bales and R. F. Santerre, “Temporal and Quantitative Analysis of Myogenic Regulatory and Growth Factor Gene Expression in the Developing Mouse Embryo,” Developmental Biology, Vol. 151, No. 1, 1992, pp. 137-144. doi:10.1016/0012-1606(92)90221-2

[16]   J. K. Yoon, E. N. Olson, H. H. Arnold and B. J. Wold, “Different MRF4 Knockout Alleles Differentially Disrupt Myf-5 Expression: Cis-Regulatory Interactions at the MRF4/Myf-5 Locus,” Developmental Biology, Vol. 188, No. 2, 1997, pp. 349-362. doi:10.1006/dbio.1997.8670

[17]   J. M. Venuti, J. H. Morris, J. L. Vivian, E. N. Olson and W. H. Klein, “Myogenin Is Required for Late but Not early Aspects Of Myogenesis during Mouse Development,” Journal of Cell Biology, Vol. 128, No. 4, 1995, pp. 563-576. doi:10.1083/jcb.128.4.563

[18]   A. Blais, M. Tsikitis, D. Acosta-Alvear, R. Sharan, T. Kluger and B. D. Dynlacht, “An Initial Blueprint for Myogenic Differentiation,” Genes & Development, Vol. 19, No. 5, 2005, pp. 553-569. doi:10.1101/gad.1281105

[19]   D. Yaffe and O. Saxel, “Serial Passaging and Differentiation of Myogenic Cells Isolated from Dystrophic Mouse Muscle,” Nature, Vol. 270, No. 5639, 1977, pp. 725-727. doi:10.1038/270725a0

[20]   H. M. Blau, G. K. Pavlath, E. C. Hardeman, C. P. Chiu, L. Silberstein, S. G. Webster, S. C. Miller and C. Webster, “Plasticity of the Differentiated State,” Science, Vol. 230, No. 4727, 1985, pp. 758-766. doi:10.1126/science.2414846

[21]   D. M. Fambrough, “Control of Acetylcholine Receptors in Skeletal Muscle,” Physiological Reviews, Vol. 59, No. 1, 1979, pp. 165-227.

[22]   H. C. Fertuck and M. M. Salpeter, “Quantitation of Junctional and Extrajunctional Acetylcholine Receptors by Electron Microscope Autoradiography after 125I-Alpha-Bungarotoxin Binding at Mouse Neuromuscular Junctions,” Journal of Cell Biology, Vol. 69, No. 1, 1976, pp. 144-158. doi:10.1083/jcb.69.1.144

[23]   C. Fuhrer, J. E. Sugiyama, R. G. Taylor and Z. W. Hall, “Association of Muscle-Specific Kinase MuSK with the Acetylcholine Receptor in Mammalian Muscle,” The EMBO Journal, Vol. 16, No. 16, 1997, pp. 4951-4960. doi:10.1093/emboj/16.16.4951

[24]   W. J. La Rochelle and S. C. Froehner, “Determination of the Tissue Distributions and Relative Concentrations of the Postsynaptic 43 kDa Protein and the Acetylcholine Receptor in Torpedo,” Journal of Cell Biology, Vol. 261, No. 12, 1986, pp. 5270-5274.

[25]   P. G. Noakes, W. D. Phillips, T. A. Hanley, J. R. Sanes and J. P. Merlie, “43 k Protein and Acetylcholine Receptors Colocalize during the Initial Stages of Neuromuscular Synapse Formation in Vivo,” Developmental Biology, Vol. 155, No. 1, 1993, pp. 275-280. doi:10.1006/dbio.1993.1025

[26]   D. C. Bowen, J. S. Park, S. Bodine, J. L. Stark, D. M. Valenzuela, T. N. Stitt, G. D. Yancopoulos, R. M. Lindsay, D. J. Glass and P. S. DiStefano, “Localization and Regulation of MuSK at the Neuromuscular Junction,” Developmental Biology, Vol. 199, No. 2, 1998, pp. 309-319. doi:10.1006/dbio.1998.8936

[27]   M. Gautam, T. M. DeChiara, D. J. Glass, G. D. Yancopoulos and J. R. Sanes, “Distinct Phenotypes of Mutant Mice Lacking agrin, MuSK, or rapsyn,” Brain Research. Developmental Brain Research, Vol. 114, No. 2, 1999, pp. 171-178. doi:10.1016/S0165-3806(99)00013-9

[28]   D. J. Glass, D. C. Bowen, T. N. Stitt, C. Radziejewski, J. Bruno, T. E. Ryan, D. R. Gies, S. Shah, K. Mattsson, S. J. Burden, P. S. DeStefano, D. M. Valenzuela, T. M. DeChiara and G. D. Yancopolous, “Agrin Acts via a MuSK Receptor Complex,” Cell, Vol. 85, No. 4, 1996, pp. 513-523. doi:10.1016/S0092-8674(00)81252-0

[29]   J. E. Sugiyama, D. J. Glass, G. D. Yancopoulos and Z. W. Hall, “Laminin-Induced Acetylcholine Receptor Clustering: An Alternative Pathway,” Journal of Cell Biology, Vol. 139, No. 1, 1997, pp. 181-191. doi:10.1083/jcb.139.1.181

[30]   M. Gautam, P. G. Noakes, J. Mudd, M. Nichol, G. C. Chu, J. R. Sanes and J. P. Merlie, “Failure of Postsynaptic Specialization to Develop at Neuromuscular Junctions of Rapsyn-Deficient Mice,” Nature, Vol. 377, No. 6546, 1995, pp. 232-236. doi:10.1038/377232a0

[31]   H. Tang, Z. Sun and D. Goldman, “CaM Kinase II-Dependent Suppression of Nicotinic Acetylcholine Receptor Delta-Subunit Promoter Activity,” Journal of Biological Chemistry, Vol. 276, No. 28, 2001, pp. 26057-26065. doi:10.1074/jbc.M101670200

[32]   H. Tang, P. Macpherson, L. S. Argetsinger, D. Cieslak, S. T. Suhr, C. Carter-Su and D. Goldman, “CaM Kinase II-Dependent Phosphorylation of Myogenin Contributes to Activity-Dependent Suppression of nAChR Gene Expression in Developing Rat Myotubes,” Cell Signal, Vol. 16, No. 5, 2004, pp. 551-563. doi:10.1016/j.cellsig.2003.09.006

[33]   E. W. Godfrey, R. M. Nitkin, B. G. Wallace, L. L. Rubin and U. J. McMahan, “Components of Torpedo Electric Organ and Muscle That Cause Aggregation of Acetylcholine Receptors on Cultured Muscle Cells,” Journal of Cell Biology, Vol. 99, No. 2, 1984, pp. 615-627. doi:10.1083/jcb.99.2.615

[34]   R. M. Nitkin, M. A. Smith, C. Magill, J. R. Fallon, Y. M. Yao, B. G. Wallace and U. J. McMahan, “Identification of Agrin, a Synaptic Organizing Protein from Torpedo Electric Organ,” Journal of Cell Biology, Vol. 105, No. 6, 1987, pp. 2471-2478. doi:10.1083/jcb.105.6.2471

[35]   U. J. McMahan, “The Agrin Hypothesis,” Cold Spring Harbor Symposia on Quantitative Biology, Vol. 55, 1990, pp. 407-418. doi:10.1101/SQB.1990.055.01.041

[36]   M. A. Bowe and J. R. Fallon, “The Role of Agrin in Synapse Formation,” Annual Review of Neuroscience, Vol. 18, 1995, pp. 443-462. doi:10.1146/

[37]   N. Kim, A. L. Stiegler, T. O. Cameron, P. T. Hallock, A. M. Gomez, J. H. Huang, S. R. Hubbard, M. L. Dustin and S. J. Burden, “LRP4 Is a Receptor for Agrin and Forms a Complex with MuSK,” Cell, Vol. 135, No. 2, 2008, pp. 334-342. doi:10.1016/j.cell.2008.10.002

[38]   B. Zhang, S. Luo, Q. Wang, T. Suzuki, W. C. Xiong and L. Mei, “LRP4 Serves as a Co-Receptor of Agrin,” Neuron, Vol. 60, No. 2, 2008, pp. 285-297. doi:10.1016/j.neuron.2008.10.006

[39]   M. Ferns, M. Deiner and Z. Hall, “Agrin-Induced Acetylcholine Receptor Clustering in Mammalian Muscle Requires Tyrosine Phosphorylation,” The Journal of Cell Biology, Vol. 132, No. 5, 1996, pp. 937-944. doi:10.1083/jcb.132.5.937

[40]   B. G. Wallace, Z. Qu and R. L. Huganir, “Agrin Induces Phosphorylation of the Nicotinic Acetylcholine Receptor,” Neuron, Vol. 6, No. 6, 1991, pp. 869-878. doi:10.1016/0896-6273(91)90227-Q

[41]   W. A. Grow and H. Gordon, “Acetylcholine Receptors Are Required for Postsynaptic Aggregation Driven by the Agrin Signalling Pathway,” European Journal of Neuroscience, Vol. 12, No. 2, 2000, pp. 467-472. doi:10.1046/j.1460-9568.2000.00923.x

[42]   P. C. D. Macpherson, D. Cieslak and D. Goldman, “My- ogenin-Dependent nAChR Clustering in Aneural Myotubes,” Molecular and Cellular Neuroscience, Vol. 31, No. 4, 2006, pp. 649-660. doi:10.1016/j.mcn.2005.12.005

[43]   P. Ravdin and D. Axelrod, “Fluorescent Tetramethyl Rhodamine Derivatives of Alpha-Bungarotoxin: Preparation, Separation, and Characterization,” Analytical Biochemistry, Vol. 80, No. 2, 1977, pp. 585-592. doi:10.1016/0003-2697(77)90682-0

[44]   D. H. Campbell, M. Hicks, W. Grow and P. Standley, “Improved Quantification of Acetylcholine Receptor (AChR) Clustering in C2C12 Myotubes Using an Objective Computational Algorithm,” Arizona Imaging & Microscopy Society Conferenc 2011. .

[45]   T. A. Rando and H. M. Blau, “Primary Mouse Myoblast Purification, Characterization, and Transplantation for Cell-Mediated Gene Therapy,” The Journal of Cell Biology, Vol. 125, No. 6, 1994, pp. 1275-1287. doi:10.1083/jcb.125.6.1275

[46]   T. J. Miller and W. A. Grow, “Mercury Decreases the Frequency of Induced But Not Spontaneous Clustering of Acetylcholine Receptors,” Cell and Tissue Research, Vol. 316, No. 2, 2004, pp. 211-219. doi:10.1007/s00441-004-0878-6

[47]   B. W. Steffens, L. M. Batia, C. J. Baarson, C.-K. C. Choi and W. A. Grow, “The Pesticide Methoxychlor Decreases Myotube Formation in Cell Culture by Slowing Myoblast Proliferation,” Toxicology in Vitro, Vol. 21, No. 5, 2007, pp. 770-781. doi:10.1016/j.tiv.2007.01.007

[48]   D. B. Owen, K. T. Chamberlain, S. Shishido and W. A. Grow, “Ethanol Decreases Agrin-Induced Acetylcholine Receptor Clustering in C2C12 Myotube Culture,” Toxicology in Vitro, Vol. 24, No. 2, 2010, pp. 645-651. doi:10.1016/j.tiv.2009.09.020

[49]   W. Zhang, A. S. Coldefy, S. R. Hubbard and S. J. Burden, “Agrin Binds to the N-Terminal Region of LRP4 Protein and Stimulates Association between LRP4 and the First Immunoglobulin-Like Domain in Muscle-Specific Kinase (MuSK),” The Journal of Biological Chemistry, Vol. 286, 2011, pp. 40624-40630. doi:10.1074/jbc.M111.279307

[50]   Y. Cao, R. M. Kumar, B. H. Penn, C. A. Berkes, C. Kooperberg, L. A. Boyer, R. A. Young and S. J. Tapscott, “Global and Gene-Specific Analyses Show Distinct Roles for Myod and Myog at a Common Set of Promoters,” The EMBO Journal, Vol. 25, 2006, pp. 502-511. doi:10.1038/sj.emboj.7600958

[51]   K. Gundersen, I. Rabben, B. J. Klocke and J. P. Merlie, “Overexpression of Myogenin in Muscles of Transgenic Mice: Interaction with Id-1, Negative Crossregulation of Myogenic Factors, and Induction of Extrasynaptic Acetylcholine Receptor Expression,” Molecular and Cellular Biology, Vol. 15, No. 12, 1995, pp. 7127-7134.