Isolation and analysis of cardiac myocytes Isolation of ACMs was performed by collagenase/protease digestion as previously described (Mitra and Morad, 1985; Pott et al., 2005). 9–13- wk-old mice were i.p. injected with heparin (2,000 U/mouse). 20 min after heparin injection, the heart was removed from the chest, cannulated via the ascending aorta, and mounted on a Langendorff perfusion appa- ratus. The heart was perfused for 4 min with myocyte buffer at 37°C. Then, the perfusion was switched to an enzyme solution containing 1.0 mg/ml collagenase type 2 (Worthington Biochemical Corp.), 0.1 mg/ml protease XIV (Sigma-Aldrich), and 0.015 mg/ml DNase I grade 2 (Roche). After 2 min of perfusion, the enzyme solution was supplemented with 40 µl of 100 mM CaCl2, and perfusion was continued for another 8 min. After atria and aorta were removed, the ventricles were cut into two pieces and disaggregated with forceps (No. 5/45; Dumont) in 5 ml enzyme solu- tion. The resulting tissue cell suspension was transferred to a 15-ml conical tube and incubated for 3–5 min to allow for further digestion. Digestion was stopped by adding 5 ml of stopping buffer (myocyte buffer plus 2.5% BSA and 0.1 mM CaCl2). After isolation, the dissociated cells were col- lected in modified Tyrode’s solution containing the following (in mmol/ liter): 136 NaCl, 5.4 KCl, 10 Hepes, 1.0 MgCl2, 0.33 NaH2PO4, 0.2 CaCl2, 10 dextrose, and 10 2,3-butanedione monoxime, pH 7.4, with NaOH. The cells were pelleted at 18 g for 2 min, resuspended in plating medium, and plated on 10 µg/ml Laminin-coated flasks (Invitrogen) or chamber slides to allow attachment. After 2 h, serum-containing plating media were switched to culture media (Kabaeva et al., 2008). The culture media were prepared by supplementing MEM (Invitrogen) with 1% peni- cillin-streptomycin-glutamine (PSG; Invitrogen), 1% insulin-transferrin- selenium (ITS; Sigma-Aldrich), 4 mM NaHCO3, 10 mM Hepes, 0.2% BSA, and 25 µM blebbistatin (Toronto Research Chemicals). Culture me- dia were changed every day. Plating media were supplemented with 5% FBS (Invitrogen). To measure ACM size, the phase-contrast images of iso- lated ACM were acquired using a microscope (Axiovert 200; Carl Zeiss), and cell size was calculated using ImageJ (National Institutes of Health). 100 ACMs per heart were examined for cell size. Fetal mouse ventricular myocytes and NRVMs were isolated as previously described (Ueno et al., 1988). Fetal mouse cardiac myocytes were prepared from ventricles of E15.5 mice embryos, and NRVMs were prepared from the ventricles of 2-d-old Sprague-Dawley rats. In brief, cells were isolated by four cycles of enzymatic digestion with gentle mechanical disaggregation. Cells were dissociated at 37°C and 40 rpm in Ca2+- and Mg2+-free PBS supplemented with 0.1% trypsin, 0.1% collagenase, and 0.025% DNAase (Worthing- ton Biochemical Corp.). After the final cycle of digestion, the dissociated cells were pooled and purified by density centrifugation through a discon- tinuous Percoll gradient (GE Healthcare). Cells were then resuspended in culture medium (DME: M199 = 4:1, 1% PSG, and 1% ITS) containing 10% neonatal calf serum (Invitrogen) and plated on polystyrene culture dishes treated with 0.1% gelatin (ICN Biochemicals, Inc.). After 12–16 h, NRVMs were maintained in culture in serum-free medium. Histological analysis and BrdU studies For histology, hearts were fixed overnight in 10% buffered formalin and then routinely processed. To quantify DNA synthesis, sham or TAC was performed 1 wk after TAM treatment. 5 d after surgery, mice were injected with 50 mg/kg BrdU every 12 h for 2 d. The animals were euthanized, and their hearts were recovered 4 h after the last injection. For in vitro stud- ies, NRVMs were transfected with HP1- siRNA or negative control siRNA. After a further 48 h in serum-free media, cultures were stimulated with 20% FBS in the presence of 10 µm BrdU labeling reagent. Then, cells were fixed and subjected to immunostaining. Protein analyses Heart tissue, isolated embryonic cardiac myocytes, or ACMs were used to obtain nuclear extracts. Heart tissues were homogenized in homogeniza- tion buffer (250 mM sucrose, 10 mM Tris, pH 7.4, 1 mM EDTA, 0.5% NP- 40, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor cocktail). Isolated cardiac myocytes were resuspended in homogenization buffer and centri- fuged at 1,000 g for 10 min at 4°C. Pellets were resuspended in 0.3 ml homogenization buffer, layered on 1 M sucrose, and recentrifuged at 1,600 g for 5 min at 4°C. The nuclear protein was extracted with sonica- tion in EBC250 buffer (25 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1 mM NaF, 1 mM Na3VO4, 0.5% NP-40, and protease inhibitor). Equal amounts of nuclear protein (5–50 µg) were separated on SDS-PAGE gels, trans- ferred to polyvinylidene fluoride membrane, and hybridized using an ap- propriate primary antibody and HRP-conjugated secondary antibody for subsequent detection by ECL. For IP, 1 mg nuclear extract from an adult 420 JCB • VOLUME 194 • NUMBER 3 • 2011 mouse heart was incubated with 5 µg anti-Rb (G3-245; BD) at 4°C over- night, and precipitates were subjected to Western blot analysis. Antibodies to Rb (G3-245; BD), p130 (C-20; Santa Cruz Biotechnol- ogy, Inc.), H3K9/14Ac (06-599; Millipore), H3K27Ac (07–360; Millipore), H3K9me2 (ab7312; Abcam), H3K9me3 (ab8898; Abcam), H3K27me3 (07–449; Millipore), phosphor-Histone (Ser10, 04–817; Millipore), Ezh1 (ab64850; Abcam), Ezh2 (39639; Active Motif), G9a (ab31874; Abcam), Suv39h1 (MG44; Millipore), HP1- (MAB3450; Millipore), phos- phor-Histone (Ser10; Millipore), BrdU (ab6326; Abcam), tubulin (T8660; Sigma-Aldrich), Laminin B (Santa Cruz Biotechnology, Inc.), and H3 (05–928; Millipore) were purchased commercially. Anti-H3K18Ac and -p107 were provided by S.K. Kurdistani (University of California, Los Angeles, Los Angeles, CA). Immunostaining and confocal analysis Isolated cardiac myocyte cells were fixed with 4% PFA. Paraffin-embedded tissue sections or fixed cells were immunostained following standard proto- cols. Antibodies to BrdU (ab6326), HP1- (15.19s2; Millipore), HP1- (MAB3448; Millipore), HP1- (MAB3450), troponin C (E-7; Santa Cruz Biotechnology, Inc.), troponin I (H-170; Santa Cruz Biotechnology, Inc.), and sarcomeric MHCs (MF20; Developmental Studies Hybridoma Bank) were purchased commercially. Phalloidin and secondary antibodies were purchased from Invitrogen. Nuclei were visualized by DAPI staining. For calculation of hetero- chromatin percentages, >100 nuclei per heart were examined by two in- dependent blinded observers. Images were acquired with a microscope (Axiovert 200) equipped with imaging software (AxioVision 4.6; Carl Zeiss) at room temperature. A charge-coupled device camera (CoolSnap fx; Photometrics) was used for immunofluorescence staining, and a micros- copy camera (AxioCam MRc; Carl Zeiss) was used for immunohistochem- istry or immunocytochemistry staining. For confocal analysis, images were acquired with a true confocal scanner laser-scanning microscope system (SP2 AOBS; Leica) with 40× 1.3 and 63× 1.4 oil immersion ob- jectives at room temperature. All images within the same panel were pro- cessed similarly using Photoshop (version 6.0; Adobe). siRNA and RNA analysis ACMs were transfected with 50 nM HP1- siRNA or negative control siRNA (QIAGEN) by using Lipofectamine RNAiMAX (QIAGEN). 24 h after transfection, cells were stimulated with 20% FBS for 24 h. For RT-PCR, total RNA was isolated from cells or tissue using TRI Reagent (Sigma-Aldrich). First-strand cDNA was generated from 1 µg of total RNA by using a re- verse transcription kit (Omniscript; QIAGEN). Real-time quantitative PCR was conducted using the ABI PRISM 7700 sequence detection system (Taq- man; Applied Biosystems). PCR amplicons were detected by fluorescent detection of SYBR green (QuantiTect SYBR green PCR kit; QIAGEN). The siRNA oligonucleotide sequences used are as follows: HP1-, 5-GGC- UUUUCUGAGGAGCACATT-3 (sense) and 5-UGUGCUCCUCAAGA- AAAGCCTT-3 (antisense); HP1-, 5-GGAAGCCAAUGUCAAGUGCTT-3 (sense) and 5-GCACUUGACAUUGGCUUCCTT-3 (antisense); and HP1-, 5-GCAGCGGAGAGUUAAUGUUTT-3 (sense) and 5-AACAUUAACU- CUCCGCUGCTG-3 (antisense). The Suv39h1 oligonucleotide sequences were previously published (Ait-Si-Ali et al., 2004). NS siRNA (QIAGEN) was used as a negative control. ChIP Isolated cardiac myocytes were fixed with 1% formaldehyde for 10 min at room temperature, and then 0.125 M glycine was added and incubated for 5 min at room temperature. To release the nuclei, cells were suspended in Mg-nonionic (NI)–NP-40 buffer (15 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, 300 mM sucrose, and 1% NP-40) and incubated on ice for 20 min. Nuclei from the NP-40–lysed cells were spun down and washed in Ca-NI buffer (15 mM Tris-HCl, 1 mM CaCl2, pH 7.5, 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, and 300 mM su- crose). After they were spun down, pellets were resuspended in Ca-NI buf- fer containing 25 µg/ml Staphylococcus aureus nuclease (Roche) and incu- bated on ice for 30 min. After centrifugation at 3,500 rpm for 5 min at 4°C, cell pellets were lysed by sonication on ice with nuclear lysis buffer (50 mM Tris-Cl, pH 8.1, 10 mM EDTA, 1% SDS, and protease inhibitors). After centrifugation at 13,000 rpm for 10 min at 4°C, the lysates were di- luted 10 times with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 167 mM NaCl, and protease inhibi- tor). After preclearing with 30 µl Protein A/G Dynabeads (Invitrogen), pri- mary antibodies specific to H3K9me3 (Millipore), H3K27me3 (Millipore), and HP1- (MAB3450) were added and incubated overnight at 4°C.