Normal mouse or rabbit IgG was used as a control. After IP, 60 µl Protein A/G Dynabeads was added and incubated for 3 h. Precipitates were washed with dialysis buffer (2 mM EDTA and 50 mM Tris-Cl, pH 8.0) twice, IP wash buffer (100 mM Tris-Cl, pH 8.0, 500 mM LiCl, 1% NP-40, and 1% deoxycholic acid) four times, and Tris-EDTA buffer twice. The com- plexes were eluted with 1% SDS and 0.1 M NaHCO3. The eluates were incubated overnight at 65°C to reverse the formaldehyde cross-linking. After proteinase K treatment, DNA was extracted by phenol/chloroform/iso- amyl alcohol and precipitated by ethanol. PCR was performed using Plati- num Taq polymerase (Invitrogen). The primers used are as follows: cdc2, 5-GGTAAAGCTCCCGGGATCCGCCAAT-3 (sense) and 5-GTGGACT- GTCACTTTGGTGGCTGGC-3 (antisense); cyclin E1, 5-ACGCCCGTA- AAAGAACACGCCC-3 (sense) and 5-CCGGCTTCGAGCGGGACA- TTTA-3 (antisense); E2F1, 5-AATGGAGGAGGCGTTCG-3 (sense) and 5-CCGCTGCCTGCAAAGT-3 (antisense); PLK, 5-AGTTTCAGTTCTAG- AAGGCCGGGT-3 (sense) and 5-ATTGCCTGGGAAACCAGGAAC- GAA-3 (antisense); and Myc, 5-AAACTTTGCCCATTGCAGCG-3 (sense) and 5-GTTTCCAACGCCCAAAGGAA-3 (antisense). Real-time quantitative PCR Real-time quantitative PCR was conducted using the ABI PRISM 7700 se- quence detection system. PCR amplicons were detected by fluorescent de- tection of SYBR green (QuantiTect SYBR green PCR kit). Error bars represent the SEM from three replicates. TLVM Freshly isolated ACMs were initially cultured in M199 medium supplemented with 5 mM creatine, 5 mM taurine, 2 mM l -carnitine, 1% PSG, 8% FBS, 25 µM blebbistatin, and 10 µM cytosine -d -arabinofuranoside. After 48 h, blebbistatin was removed. After another 3 d, cytosine -d -arabinofuranoside was removed. Cells were infected with an adenovirus expressing a fusion construct of histone 2B–GFP under control of the chicken troponin T promoter (Ad–chicken troponin T–H2B-GFP; provided by F. del Monte, Beth Israel Deaconess Medical Center, Boston, MA) on day 7. 2 d after infection, TLVM was performed using an inverted microscope equipped with a phase- contrast temperature-controlled motorized stage (DMIRE2; Leica). The 12.5- cm2 flask with an air-permeable cap was maintained on the stage at 37°C in 95% air/5% CO2 saturated with water, similar conditions to a regular CO2 incubator. The bright-field and spin disc laser confocal images of car- diomyocyte cells were acquired at 37°C using a charge-coupled device camera (Hamamatsu Photonics) at 200× magnification using Volocity soft- ware (PerkinElmer) and collected at 15-min intervals. Patch-clamp methods ACMs were patch clamped in the whole-cell configuration of the patch- clamp technique in the current clamp mode (Xie et al., 2009). Patch pi- pettes (2–4-M resistance) were filled with internal solution containing (mM) 110 K-aspartate, 30 KCl, 5 NaCl, 10 Hepes, 0.1 EGTA, 5 MgATP, 5 creatine phosphate, and 0.1 cAMP, pH 7.2, adjusted with KOH. Myo- cytes were superfused with standard Tyrode’s solution containing (mM) 136 NaCl, 5.4 KCl, 0.33 Na2PO4, 1.8 CaCl2, 1 MgCl2, 10 glucose, and 10 Hepes, pH 7.4, adjusted with NaOH. All chemicals and reagents were purchased from Sigma-Aldrich. To induce ACM contraction, action poten- tials were elicited with trains of 20 square pulses (for 2 ms at 2–4 nA) at 300-, 500-, or 1,000-ms pacing cycle lengths. Voltage signals were moni- tored with a patch-clamp amplifier (Axopatch 200A; Molecular Devices) controlled by a personal computer using an acquisition board (Digidata 1200; Molecular Devices) driven by pCLAMP software (version 9.0; Molecular Devices). All experiments were performed at 37°C. ACM contraction analysis Patch-clamped ACMs were simultaneously imaged during pacing using a high-speed charge-coupled device–based camera (128 × 128 pixels; Cas- cade 128+; Photometrics) at 290 frames per second. The acquired video image data were then processed using Imaging Workbench software (ver- sion 6.0; INDEC BioSystems). ACM lengths were measured and analyzed using ImageJ software, and the percentage of change in myocyte length was calculated as a percentage for one minus the ratio of myocyte length at peak contraction over the length at rest. Contraction lengths were averaged over the 20 contractions evoked by the stimulus train for each myocyte. Statistical analysis All data are presented as the mean ± SEM. Results were compared by analysis of variance and Fisher’s protected least significant difference tests with a significance of P < 0.05. Online supplemental material Fig. S1 shows heterochromatin formation in ACMs and is related to Fig. 1. Fig. S2 shows that cardiac myocytes from adult MCM;RbL/L;p130+/+, MCM+;RbL/L;p130+/+, MCM;RbL/L;p130/, and MCM+;RbL/L;p130/ mice differentiate normally before TAM treatment and is related to Fig. 3. Fig. S3 shows characterization of IDKO cardiac myocytes and is related to Fig. 3. Fig. S4 shows that proliferation-promoting gene expressions were up-regulated after depletion of Rb and p130 in ACMs; however, levels of H3K9me3 and H3K27me3 were unchanged. Fig. S4 shows up-regula- tion of proliferation-promoting gene expression after depletion of Rb and p130 in ACMs and is related to Fig. 4. Fig. S5 shows characterization of cardiac growth in IDKO hearts and is related to Fig. 5. Video 1 shows that Rb- and p130-null ACMs complete cytokinesis in vitro and is related to Fig. 5. Online supplemental material is available at http://www.jcb .org/cgi/content/full/jcb.201012049/DC1. We would like to thank Nicole Petrochuk, Rui Zhang, and Roberto Ferrari for technical assistance and Michael Schneider for helpful discussion and reading of the manuscript. We are grateful to Dr. Siavash K. Kurdistani for antibodies and Dr. Federica del Monte for Ad–chicken troponin T–H2B-GFP. This work was supported by National Institutes of Health (NIH) grants (HL70748 and HL080111) and Laubisch and Cardiovascular Development funds to W.R. MacLellan and Larry Hillblom Foundation (2007-D-003-NET) and NIH (DK077967) grants to P.C. Butler. 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