TITLE:
Antibody to MyoD or Myogenin Decreases Acetylcholine Receptor Clustering in C2C12 Myotube Culture
AUTHORS:
Matthew K. Ball, David H. Campbell, Kelly Ezell, Jessica B. Henley, Paul R. Standley, Wade A. Grow
KEYWORDS:
Agrin; Acetylcholine Receptor; MyoD; Myogenin; C2C12
JOURNAL NAME:
CellBio,
Vol.2 No.3,
September
18,
2013
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.