(A) Progressive cardiac dysfunction and dilatation after cardiomyocyte-restricted ablation of Eed. w, weeks. (B) Nppa mRNA level in WT and CKO hearts at the indicated ages. w, weeks. (C, D) Cardiac fibrosis was evident by Mason Trichrome staining at 2 months of age. Fraction of myocardial area occupied by fibrotic tissue (blue staining) was quantified using ImageJ. Bar = 50 µm. (E, F) Immunofluorescence for cardiomyocyte marker TNNI3 and cardiomyocyte membrane marker WGA, and quantification of cell size from WGA-stained cardiomyocyte outlines (f). Bar = 50 µm. (G) Immunostaining for TNNI3 and H3K27me3. Isolated adult cardiomyocytes were >95% pure and EedCKO CMs had little H3K27me3 signal. Bar = 50 µm. (H) PCR of genomic DNA from purified CMs using primers that amplify unexcised floxed DNA (233 bp product) or Cre-excised DNA (453 bp product). In CKO-purified CMs, unexcised floxed DNA was not detected, consistent with highly efficient Cre-mediated gene inactivation, as well as high purity of dissociated CMs. (I) RNA-seq track view showing deletion of floxed exons 3–6 of Eed (red box). (J,K) Genome browser view ofH3K27me3 and H3K27ac ChIP-seq signals on Myh6 (J) and Vim (K) loci in purified adult cardiomyocytes. (L) EED enrichment on downstream genes was measured by ChIP-qPCR in P5 heart ventricle apex. Numbers following gene names indicate the number of nucleotides between the probed amplicon and the TSS. (M) Box and scatter plots of H3K27me3 at TSS ±500 bp of EED target genes in four quantiles of WT H3K27me3 intensity. (N) Aggregation plots of H3K27me3 ChIP-seq signals near the TSS of genes upregulated, downregulated, or unchanged between WT and EEDCKO. O. H3K27me3 enrichment was measured by ChIP-qPCR on target genes using adult cardiomyocytes isolated from WT and EEDCKO hearts. *p<0.05; **p<0.01; ***p<0.001 by ANOVA with Dunnett’s post-hoc test using Eedfl/+::Myh6-Cre– as the control group (A), by Welch’s t-test (B,D,F,N,O), or by Wilcoxon-Mann-Whitney test (M).