Antibody to MyoD or Myogenin Decreases Acetylcholine Receptor Clustering in C2C12 Myotube Culture

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.