state (Fig. 1 A). Although G1/S genes (CycD1 and Myc) were up-regulated in ACMs after pressure overload in vivo, expres- sion of the subset of cell cycle genes that regulates G2/M (CycB1, cdc2, and cdc25C) and cytokinesis (Aurora kinase B and pololike kinase 1 [PLK1]) was not detectable in ACMs and remained unchanged after TAC, whereas pressure overload in ACMs resulted in up-regulation of the ventricular hypertrophy marker atrial natriuretic peptide (also known as atrial natriuretic factor [ANF]; Fig. 1 A). We examined embryonic cardiac myocytes or ACMs for the developmental pattern of histone modifications and the expression of proteins that regulate these posttranslational modifications. H3K9me2/3 and H3K27me3, the histone modifications associated with transcription repres- sion, are increased in ACMs, whereas histone modifications associated with active gene expression (H3K9/14Ac, H3K18Ac, and H3K27Ac) and mitosis (phosphorylation of histone H3S10) are decreased (Fig. 1 B). Consistent with these alterations, H3K9 and H3K27 methyltransferases G9a, Suv39h1, and Ezh1, along with Rb and p130, are up-regulated in ACMs (Fig. 1 C). To determine whether heterochromatin aggregation accu- mulates with cardiac differentiation, we examined DNA in embryonic cardiac myocytes (E15.5) versus ACM nuclei for heterochromatin (Fig. 1 D). DNA within embryonic cardiac myocyte nuclei was homogeneously dispersed with limited DAPI-positive foci indicative of heterochromatin. In contrast, >90% of ACM nuclei demonstrated large, dense DAPI-positive heterochromatin foci. To further characterize these DNA foci, we immunostained cardiac nuclei for euchromatin (H3K9/14Ac) and heterochromatin (H3K9me3) markers (Figs. 1 E and S1 A). H3K9/14Ac was largely excluded from the dense DAPI-positive foci in ACMs, whereas H3K9me3 was concentrated in these DNA foci. H3K9me2 and H3K27me3 staining was found through- out nuclei in both euchromatic and heterochromatic regions, although the levels of these modifications increased dramatically from embryonic cardiac myocytes (E15.5) to ACMs (Fig. 1 E). To determine whether the H3K9me3 or H3K27me3 was associ- ated with E2F-dependent promoters, isolated myocytes from embryonic or adult hearts were cross-linked, and H3K9me3- and H3K27me3-associated promoter sequences were deter- mined by chromatin immunoprecipitation (IP [ChIP]). As shown, H3K9me3 and H3K27me3 were enriched at E2F-dependent G2/M (cdc2) and cytokinesis (PLK1) promoters in ACM com- pared with embryonic cardiac myocytes (Figs. 1 [F and G] and S1 [B and C]). Because H3K9 and H3K27 were also trimethyl- ated at the promoter of Myc, which is up-regulated with hyper- trophic stimuli, it suggests that, if H3K9me3 and H3K27me3 contribute to permanent gene silencing in ACMs, additional factors must be required. Rb and p130 play an overlapping role in the establishment of heterochromatin formation in differentiating cardiac myocytes There is well-documented redundancy in Rb family members (Lee et al., 1996), and our own data have demonstrated that de- fects in cardiac cell cycle control required deletion of both Rb and p130 (MacLellan et al., 2005). We used a cardiac-specific Cre/loxP system to delete Rb specifically in cardiac myocytes at 410 JCB • VOLUME 194 • NUMBER 3 • 2011 a developmental time point at which they were still cycling. The –myosin heavy chain (MHC [-MHC])–driven Cre transgenic mouse we used excised Rb genomic sequences in the perinatal period before cardiac myocytes were fully differentiated (Marino et al., 2000; MacLellan et al., 2005). These cardiac-specific Rb-deficient mice (CRbL/L) were bred to mice with a germline deletion of p130. Deletion of both Rb and p130 in cardiac myo- cytes led to marked defects in differentiation and cell cycle exit, whereas the single deletions were innocuous (MacLellan et al., 2005). To determine the role of Rb and p130 in hetero- chromatin formation in cardiac myocytes, we examined hearts from single or combined Rb and p130 knockout mice. We exam- ined myocardial sections from control (CRb+/+;p130+/+), cardiac- specific Rb-deficient mice (CRbL/L;p130+/+), p130-deficient mice (CRb+/+;p130/), or double Rb;p130-deficient mice (CRbL/L;p130/; Fig. 2 A). Heterochromatin formation was disrupted specifically in CRbL/L;p130/ hearts. No abnormal- ities were seen in single Rb- or p130-deficient hearts (Fig. 2 A). Immunostaining of myocardial sections demonstrated typical heterochromatin aggregates in control myocyte nuclei; however, heterochromatin was absent, and H3K9me3 immunostaining was diffuse in Rb;p130-null myocyte nuclei (Fig. 2 B). H3K27me3 levels were dramatically decreased in CRbL/L;p130/ myocar- dium (Fig. 2 C). Although these results support the notion that Rb and p130 are required for controlling cell cycle exit in the cardiac myocyte, this mouse model cannot distinguish between a primary role for them in initiating terminal differentiation and heterochromatin formation in cardiac myocytes versus a secondary defect related to persistent expression of cell cycle proteins and a persistent immature state. Also, this model could not determine whether Rb and p130 are also required for the maintenance of terminal differentiation and heterochromatin once formed. Creation of cardiac-specific Rb and p130 inducible double knockout (IDKO) mice To determine the role of Rb and p130 more directly on hetero- chromatin and maintenance of the postmitotic state in fully differentiated ACMs, we created inducible, cardiac-specific dou- ble knockout mice. RbL/L;p130/ mice were bred to -MHC– MerCreMer (MCM) mice to generate MCM+;RbL/L;p130/ mice, in which Rb can be selectively and efficiently deleted in ACMs after Cre is activated by tamoxifen (TAM). Adult (8 wk after birth) MCM;RbL/L;p130+/+, MCM+;RbL/L;p130+/+, MCM; RbL/L;p130/, and MCM+;RbL/L;p130/ littermates display normal heart weight to body weight ratio and left ventricle function before TAM treatment. There were no differences in expression of cell cycle genes or histone methylations (Fig. S2, A–C), indicating that neither p130 deletion nor expression of MCM by themselves had any epigenetic effect. Adult MCM;RbL/L;p130+/+, MCM+;RbL/L;p130+/+, MCM;RbL/L; p130/, and MCM+;RbL/L;p130/ littermates were fed with TAM chow for 14 d, resulting in four functional phenotypes: control, Rb-, p130-, and Rb/p130-deficient (IDKO) mice (Fig. 3, A and B). The minor amount of residual Rb expression likely represents the low level expression of Rb in nonmyocytes as well as unrecombined Rb in a minority of ACMs. Unless specifically indicated, analyses were performed 4 wk after