Table I. Assessment of left ventricular size and function Parameter TAM HR (bpm) EF (%) FS (%) LVID;d (mm) LVID;s (mm) LVPW;d (mm) LVPW;s (mm) + 477 ± 6 56.13 ± 2.02 28.88 ± 1.29 3.87 ± 0.07 2.74 ± 0.08 0.63 ± 0.03 0.89 ± 0.02 + 473 ± 14 50.40 ± 2.65 25.32 ± 1.59 3.98 ± 0.15 2.98 ± 0.16 0.65 ± 0.14 0.89 ± 0.03 + 475 ± 16 56.39 ± 3.32 29.13 ± 2.31 3.76 ± 0.02 2.67 ± 0.08 0.67 ± 0.02 0.89 ± 0.02 + 480 ± 12 33.52 ± 2.81a 15.97 ± 1.50a 4.78 ± 0.28a 4.02 ± 0.28a 0.78 ± 0.03a 0.78 ± 0.03a MCM;RbL/L;p130+/+ MCM+;RbL/L;p130+/+ MCM;RbL/L;p130/ MCM+;RbL/L;p130/ 2D echocardiographic analysis of left ventricle function in mice with the indicated genotypes 4 wk after TAM treatment. n = 5 mice per group. Results are presented as the mean ± SEM. HR, heart rate; EF, ejection fraction; FS, fractional shortening; LVID;d/s, left ventricular internal dimension in diastole/systole; LVPW;d/s, left ventricular posterior wall thickness in diastole/systole. aP < 0.05 by analysis of variance for MCM;RbL/L;p130+/+, MCM+;RbL/L;p130+/+, and MCM;RbL/L;p130/ versus MCM+;RbL/L;p130/ littermates. IDKO ACMs spontaneously proliferate To determine the effects of depleting Rb and p130 on cardiac myo- cyte growth and proliferation, we examined heart size and indices of cell cycle progression. When we examined hearts 4 wk after completing the TAM treatment, IDKO hearts displayed a 32% increase in the heart weight to body weight ratio compared with control heart (6.01 ± 0.37 vs. 4.55 ± 0.11 mg/g, P < 0.05; Fig. 5 A), which was directly attributable to an increase in heart weight (Fig. S5 B). To determine whether this increase in heart size was related to hypertrophy or proliferation of ACMs, we measured the cell size of isolated ACMs from control versus IDKO hearts. IDKO ACMs displayed a typical rod-shaped morphology but were 5% smaller than control ACMs (Figs. 5 B and S5 [A, C, and D]), sug- gesting that the increased heart size must be related to ACM prolif- eration. To confirm that ACMs in IDKO hearts were cycling, we determined S phase nuclei at the baseline or after hemodynamic stress. ACMs in the sham (a control surgical procedure similar to TAC)-operated IDKO myocardium demonstrated a 27-fold in- crease in BrdU-positive myocyte nuclei compared with the control myocardium (P < 0.001), which increased to 70-fold after pressure overload (P < 0.001; Fig. 5, C and D). Notably, BrdU staining was only detected in cardiac myocytes that had lost heterochromatin foci (Fig. S5 E). We further assessed the proliferation potential of isolated IDKO ACMs by time-lapse video microscopy (TLVM; Fig. 5 E and Video 1). ACMs from control or IDKO hearts were cultured for 8 d, and cardiac myocyte nuclei were identified by in- fection with an adenovirus expressing a cardiac-specific histone 2B–GFP fusion protein. ACMs lose their typical rod-shaped mor- phology and dedifferentiate when cultured for extended periods of time (Bersell et al., 2009), but this occurred more rapidly in IDKO myocytes. After 8 d in culture, 5.4% of IDKO ACMs completed cytokinesis during the 24-h monitoring period compared with zero control ACMs (P < 0.01), confirming that the proliferative poten- tial of IDKO myocytes was restored. Rb/p130-dependent recruitment of HP1- to E2F-responsive promoters is essential for irreversible silencing of proliferation- promoting genes in ACMs It has been suggested that the ability of Rb to stably repress transcription is related to its capacity to recruit HP1 to target gene promoters, resulting in their incorporation into hetero- chromatin (Nielsen et al., 2001; Narita et al., 2003). All three HP1 family members are expressed in ACMs, although their subnuclear localization differs (Fig. 6 A). HP1- and HP1- localize in both euchromatin and heterochromatin, whereas HP1- is restricted to heterochromatin (Fig. 6 A). To study the role of HP1 family members on irreversible cell cycle exit in postmitotic ACMs, we depleted expression of specific HP1s in primary wild-type (WT) ACMs using siRNA and then stimu- lated the myocytes with serum to induce cell cycle reentry. Cells transfected with NS siRNA were used as a negative con- trol. Each siRNA was able to specifically and effectively knock down expression of the respective HP1 (Fig. 6 B); however, ex- pression of G2/M and cytokinesis genes only increased in HP1- –depleted ACMs (P < 0.05; Fig. 6, B and C). There was no change in the expression of Myc and ANF, which are not stably silenced in ACMs. Because HP1- associates with the Rb pro- tein in ACMs (Fig. 6 D), it is an excellent candidate to mediate the stable silencing of G2/M and cytokinesis genes in ACMs. We further examined the role of HP1- in cardiac myo- cyte cell cycle regulation by determining the effects of deplet- ing HP1- in neonatal rat ventricular myocytes (NRVMs). NRVMs were transfected with NS or HP1- siRNA. After 48 h, cells were collected for Western blot analysis (Fig. 6 E) or stim- ulated with serum in the presence of BrdU (Fig. 6 F). Western blotting demonstrated efficient and specific HP1- knockdown. S phase myocyte nuclei were increased in HP1- siRNA– transfected cells compared with those transfected with NS siRNA (10.11 ± 1.79 vs. 2.34 ± 0.42%, P < 0.001; Fig. 6 G). To deter- mine whether DNA synthesis is followed by cytokinesis in these myocytes, we also quantified total cell number after serum stimu- lation. Depletion of HP1- resulted in a 56% increase in the NRVM cell number (P < 0.05; Fig. 6 H). HP1 recruitment to genomic sites classically occurs by binding to H3K9me3 at specific target genes (Bannister et al., 2001). However, HP1- also interacts with Rb, and H3K9me3 was not altered at G2/M and cytokinesis gene promoters in IDKO ACMs (Fig. 4 E). Thus, we examined whether depleting Rb and p130 might prevent recruitment of HP1-, despite the presence of H3K9me3. ChIP for HP1- was performed on iso- lated control and IDKO ACMs. HP1- was bound to cdc2 and Epigenetic regulation of cardiac proliferation • Sdek et al. 413