Decreased cardiomyocyte proliferation in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts.

<p>Microtome sections of paraffin-embedded hearts were prepared from <i>Nkx2.5-Cre/Foxm1^fl/fl</i> and control mice at E14.5 (A–D, J–K), E17.5 (E–F) and P11 (G–H) and stained for proliferation markers phospho-histone 3 (PH3) (A–B, E–H, J–K) and Ki-67 (C–D). The percent of PH3-positive cardiomyocytes was significantly lower in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts compared to control at E14.5 and E17.5, though proliferation was unaltered in the lung and in endocardial cells (I). Proliferating cardiomyocytes were more prevalent in control (J) than in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts (K) as evidenced by colocalization of PH3-positive nuclei with α-actinin. Consistent with decreased proliferation in <i>Nkx2.5-Cre/Foxm1^fl/fl</i> hearts, there was decreased mRNA expression of the cell cycle regulators Cdc25B, Cyclin B₁, Plk-1 and nMyc as determined by qRT-PCR (M). There was no change in the expression of Cyclin D₁ or cMyc. Also consistent with decreased proliferation, there was increased mRNA expression of the cell cycle inhibitor p21^cip1. Gene expression was determined using whole heart RNA and normalized to β-actin mRNA. Decreased gene expression of Foxm1, Cyclin B₁, and increased p21^cip1 expression translated to changes in protein levels as demonstrated by Western blot analysis (L). β-actin was used as a loading control. Significant differences (p<0.05) were indicated by asterisk. “N” values were represented by boxes inside bars. Scale bars represent 50 µm in A–H and J–K and 10 µm in insets E–H.