Myocyte Enhancer Factor 2C and Nkx2–5 Up-regulate Each Other’s Expression and Initiate Cardiomyogenesis in P19 Cells

The Nkx2–5 homeodomain protein plays a key role in cardiomyogenesis. Ectopic expression in frog and zebrafish embryos results in an enlarged myocardium; however, expression of Nkx2–5 in fibroblasts was not able to trigger the development of beating cardiac muscle. In order to examine the ability of Nkx2–5 to modulate endogenous cardiac specific gene expression in cells undergoing early stages of differentiation, P19 cell lines overexpressing Nkx2–5 were differentiated in the absence of Me 2 SO. Nkx2–5 expression induced cardiomyogenesis in these cultures aggregated without Me 2 SO. During differentiation into cardiac muscle, Nkx2–5 expression resulted in the activation of myocyte enhancer factor 2C (MEF2C), but not MEF2A, -B, or -D. In order to compare the abilities of Nkx2–5 and MEF2C to induce cellular differentiation, P19 cells overexpressing MEF2C were aggregated in the absence of Me 2 SO. Similar to Nkx2–5, MEF2C expression initiated cardiomyogenesis, resulting in the up-regulation of Brachyury T, bone morphogenetic protein-4, Nkx2–5, GATA-4, cardiac α-actin, and myosin heavy chain expression. These findings indicate the presence of a positive regulatory network between Nkx2–5 and MEF2C and show that both factors can direct early stages of cell differentiation into a cardiomyogenic pathway.