Transcriptional and Cell Cycle-mediated Regulation of Myosin Heavy Chain Gene Expression During Muscle Cell Differentiation

The molecular mechanisms regulating the induction of myosin heavy chain (MHC) gene expression during muscle cell differentiation were studied using a MHC cDNA recombinant plasmid. During in vitro L6E9 cell myogenesis, cytoplasmic MHC mRNA content/cell nucleus increases a minimum of 500-fold during the first 6 days of differentiation. Two independent parameters regulating MHC mRNA accumulation were directly measured. (i) Intrinsic (chemical) MHC mRNA stability (t1/2 = 55-60 h) is the same in both myotubes and when first detected in myoblasts. This suggests that the intrinsic stability of the MHC mRNA molecule does not change during myogenesis. (ii) The rate of MHC gene transcription and MHC mRNA synthesis increases approximately 100-fold during myogenesis but is insufficient to account for the entire MHC mRNA accumulation. An additional independent parameter was found to profoundly affect MHC mRNA accumulation. Withdrawal from the cell cycle, as occurs during terminal myogenic differentiation, increases the final accumulation of stable mRNAs, such as MHC, by increasing mRNA effective stability. During L6E9 myogenesis, the transition from mitotically active myoblasts (doubling time = 16 h) to postmitotic myotubes results in a 4-5-fold increase in the effective stability of cytoplasmic MHC mRNA. This cell cycle-mediated effect, combined with the induction of MHC mRNA synthesis, completely accounts for MHC mRNA accumulation. A parallel effect occurs in the total cytoplasmic poly(A)+ mRNA population. The rates of synthesis of each of the two major stability components (t1/2 = 5 and 50 h, respectively) are equally increased 2-3-fold during myogenesis. However, the composition of the cytoplasmic mRNA population changes due to the preferential accumulation of stable mRNAs. We conclude that both transcriptional and cell cycle-mediated regulation of MHC gene expression is necessary, but either alone is not sufficient to produce the differentiated muscle cell phenotype.