Figure 2. Rb and p130 have overlapping roles in the establishment of heterochromatin and H3K27me3 in differentiating cardiac myocytes. (A–C) Con- focal microscopy was performed on myocardial sections from adult mice with the indicated genotypes (red, troponin C; green, indicated H3K9 or H3K27 modification; blue, DAPI). (A and B) Formation of H3K9me3-enriched heterochromatic foci was disrupted specifically in ACMs from CRbL/L;p130/ mice. Panels e–h are enlarged images of the boxed areas in panels a–d. Bars, 5 µm. (C) H3K27me3 levels were dramatically decreased in CRbL/L;p130/ myocardium compared with CRb+/+;p130+/+ myocardium. Bar, 25 µm. stopping TAM to avoid any nonspecific (NS) effects related to treatment (Koitabashi et al., 2009). Fetal cardiac genes, includ- ing ANF, -MHC, and skeletal -actin, were up-regulated spe- cifically in IDKO hearts, whereas the cardiac genes (such as cardiac -actin and -MHC) that were predominantly ex- pressed in the ACMs remained unchanged (Fig. 3 B). IDKO ACMs reexpressed a panel of fetal cardiac-specific genes, in- cluding a 3.7-fold increase in ANF, a threefold increase of -MHC, and a 2.3-fold increase of skeletal actin (Fig. S3 A). Histological examination of IDKO myocardium revealed large, hyperchromatic nuclei, consistent with cycling cells (Fig. 3 C). There was no increase in myocardial fibrosis or apoptosis ob- served in IDKO hearts (Fig. S3, B and C). Although normal at the baseline, IDKO mice developed left ventricle dilation and systolic dysfunction after TAM treatment (Table I). This con- tractile defect was intrinsic to the myocytes themselves, as fractional shortening of isolated IDKO ACMs was reduced (Fig. S3 D). Epigenetic regulation of cardiac proliferation • Sdek et al. 411