The Effect of Ethanol on the Differentiation of C2C12 Mouse Myogenic Cells.
Abstract: The loss
of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell
differentiation into myotubes. In order to examine this possibility, the effects of
alcohol on the mouse C2C12 cell line, an established model of myogenic cell
differentiation, was measured through the use of a custom high content
immunocytochemical assay. Exposure to ethanol caused shorter ... read moreand thinner myotubes to
form and inhibited the myogenic fusion index (MFI) (p<0.05). Ethanol reduced levels
of the muscle regulatory factors (MRFs), Myf5, MyoD and myogenin (p<0.05) during
differentiation. Transcriptional profiling over three days identified genes
significantly altered by alcohol during differentiation compared with control treated
cells and MyoD was found to be significantly associated with ethanol driven C2C12
differentiation (p=8.7X10-4 ). The microarray also predicted activation of the Notch
signaling pathway (p=4.35X10-3). Notch signaling has been shown to inhibit muscle
differentiation. The Notch effectors, Hes-1 and Hey-1 were consistently up-regulated by
alcohol treatment and ethanol increased activity of the proximal Hes-1 gene promoter
which contains the CBF-1/RBP-Jk response element (p<0.05). Blocking Notch signaling
with a gamma-secretase inhibitor (GSI) abrogated the ethanol induced increase in Hes-1
and partly restored MyoD levels (p<0.05). The GSI completely restored the myogenic
fusion index (MFI) in C2C12 cells differentiating in the presence of ethanol and modest
improvements were seen in all other cellular parameters measured in the
immunocytochemical assay (p<0.05). Signaling through Notch1 is required to maintain
the satellite cell pool in post-natal myogenesis. Ethanol reduced levels of activated
Notch1 (N1-ICD) during C2C12 differentiation (p<0.05) and tended to decrease the
number of quiescent CD34- cells which are reported to express Notch 1 (p=0.08). My
results suggest that ethanol inhibits C2C12 differentiation by reducing MyoD, blocks
myoblast fusion events by activating Notch signaling and decreases Notch1 to attenuate
maintenance of the satellite cell pool.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Genetics.
Advisor: Gordon Huggins.
Committee: Philip Hinds, Pamela Yelick, and Claire Moore.
Keywords: Genetics, Molecular biology, and Cellular biology.read less