The inhibitory effect of adult cardiomyocyte cytoplasmic extract is reversible. (A and B) Immobilized S phase nuclei were incubated with increasing amounts (total protein in μg) of S phase H9c2 cytoplasmic extracts (S phase) in the absence or presence of adult cardiomyocyte cytoplasmic extract (Adult) as indicated. (C and D) S phase nuclei were incubated with adult cardiomyocyte cytoplasmic extracts (60 μg of total protein) and S phase cytoplasmic extract (45 μg of total protein) in the presence or absence of increasing amounts of recombinant PCNA (10, 50, and 100 ng) as indicated. (A and C) Quantitative analysis (n = 3). Data are means + standard errors of the means (error bars). (B and D) Representative examples of the anti-PCNA immunofluorescence analysis, which was performed as described for Fig. 1.

Proteasome inhibitors, but not inhibition of transcription or translation, can abolish the effect of adult cardiomyocyte cytoplasmic extract on PCNA protein levels. Immobilized S phase nuclei were incubated with cytoplasmic extracts from S phase H9c2 cells (S phase) and/or from adult cardiomyocytes in the absence or presence of the transcriptional inhibitor actinomycin D (Act D), the translational inhibitor cycloheximide (CHX) or proteasome inhibitors MG 132 and lactacystin as indicated. (A) Quantitative analysis (n = 3). Data are means + standard errors of the means (error bars). (B) Representative examples of the anti-PCNA immunofluorescence analysis, which was performed as described for Fig. 1.

Recombinant p21 is sufficient to restore the inhibitory effect of p21-depleted adult extract. (A) Biological activity of recombinant proteins. Cdk2 was immunoprecipitated from S phase H9c2 cells and kinase assays were performed in the absence (−) or presence of increasing amounts (0.02, 0.04, 0.2, and 2 μg) of GST, GST-p21, or GST-p27 using histone H1 as a substrate. Quantifications are shown above and are normalized for signal of Coomassie staining. (B) Immunofluorescence analysis. S phase nuclei were incubated with untreated or p21-depleted adult cardiomyocyte cytoplasmic extracts and/or S phase cytoplasmic extract (60 μg of total protein) in the presence or absence of increasing amounts (0.01, 0.1, and 1 μg) of GST-p21, 1 μg of GST, or 1 μg of GST-p27 as indicated. Representative examples of the anti-PCNA immunofluorescence analysis are shown, which were performed as described for Fig. 1. (C) Quantitative analysis (n = 3) of experiments described for panel B. Data are means + standard errors of the means (error bars).

p21 regulates PCNA expression in adult cardiomyocytes. (A) Adult cardiomyocytes were costained for actin (red) and myomesin (green) before and after serum stimulation. Nuclear DNA was stained with DAPI. (B) Immunoblot analysis. Cytoplasmic extract of untreated or serum-stimulated adult cardiomyocytes (25 μg of total protein) was resolved by SDS-12% PAGE and immunoblotted with antibodies specific for p21 (C-19) and p27 (C-19). As positive control, total cellular extracts of Ad-p21- or Ad-p27-infected neonatal cardiomyocytes were used (58). (C) Immunofluorescence analysis. Before and after serum stimulation cardiomyocytes were costained with anti-p21 (H-164), anti-p27 (C-19), and anti-PCNA (PC10) (all green) together with antimyomesin or antiactin antibodies (red). Nuclear DNA was stained with DAPI (blue). (D) Induction of kinase activities. Cell lysates (250 μg of total protein) of adult cardiomyocytes before (white bars) and after (black bars) serum stimulation were prepared, and cdk2, cyclin A, and cyclin E were immunoprecipitated with anti-cdk2 (M2), anti-cyclin A (C-19), and anti-cyclin E (M-20) antibodies. Kinase activities of the resulting immune complexes were measured using histone H1 as substrate. (E) Quantitative analysis. Immobilized nuclei of S phase H9c2 cells were incubated with cytoplasmic extracts (60 μg of total protein) from untreated or serum-stimulated adult cardiomyocytes (adult) or S phase cells (S phase) as indicated. Immunofluorescence analysis was performed as described for Fig. 1 and experiments were quantitatively analyzed thereafter (n = 3). Data are means + standard errors of the means (error bars). (F) Immunofluorescence analysis of PCNA after ectopic expression of p21 and p27 or β-Gal in cardiomyocytes and serum stimulation. Before serum stimulation cardiomyocytes were infected with Ad-p21, Ad-p27, or Ad-β-Gal. After serum stimulation cardiomyocytes were costained with anti-PCNA (PC10) (green) together with antiactin antibodies (red). Nuclear DNA was stained with DAPI (blue).

Adult cardiomyocyte cytoplasmic extract negatively influences the PCNA protein level in S phase nuclei. (A and B) Immobilized nuclei of H9c2 cells or primary cardiac nonmyocytes synchronized in S phase were incubated with cytoplasmic extracts (60 μg of total protein) from adult cardiomyocytes (Adult) and/or S phase cells (S phase) before or after heat inactivation (Heat) (3 min at 100°C) as indicated. Before (control) and after a 2-h incubation, nuclei were stained with monoclonal anti-PCNA antibody (PC10) or with FITC-coupled streptavidin against biotin-16-dUTP. Nuclear DNA was stained with DAPI, and experiments were quantitatively analyzed thereafter. Data are means + standard errors of the means (error bars) of three independent experiments.

The effect of adult cardiomyocyte cytoplasmic extract on PCNA protein levels cannot be mimicked by transcriptional, translational, or cdk2 inhibitors. Immobilized S phase nuclei were incubated with S phase H9c2 cytoplasmic extracts (S phase) in the absence or presence of the transcriptional inhibitor actinomycin D (Act D), the translational inhibitor cycloheximide (CHX), or the cdk2 inhibitor roscovitine. (A) Quantitative analysis (n = 3). Data are means + standard errors of the means (error bars). (B) Representative examples of the anti-PCNA immunofluorescence analysis, which was performed as described for Fig. 1.

Immunodepletion of p21 but not of p27 abolishes the effect of adult cardiomyocyte cytoplasmic extract on PCNA protein levels. (A) Immunodepletion of adult cardiomyocyte cytoplasmic extract was confirmed by immunoblot analysis. p21 and p27 were immunoprecipitated with specific antibodies for p21 (H 164) and p27 (C-19). Extracts were then resolved by SDS-12% PAGE (25 μg of total protein) and immunoblotted with anti-p21 (C-19) and anti-p27 (C-19) antibodies. As positive control, total cellular extracts of Ad-p21- or Ad-p27-infected neonatal cardiomyocytes were used (58). (B) S phase nuclei were incubated with untreated or depleted extracts and/or S phase cytoplasmic extract (60 μg of total protein) as indicated. Representative examples of the anti-PCNA immunofluorescence analysis, which was performed as described for Fig. 1, are shown. (C) Quantitative analysis (n = 3). Data are means + standard errors of the means (error bars).

Adult cardiomyocyte cytoplasmic extract contains factors acting synergistically with p21 in the control of PCNA expression. (A) Immunofluorescence analysis. S phase nuclei were incubated with untreated or p21-depleted adult cardiomyocyte cytoplasmic extracts or S phase cytoplasmic extract (60 μg of total protein) in the presence or absence of increasing amounts of recombinant proteins (0.01, 0.02, 0.1, and 1 μg) as indicated. Representative examples of the anti-PCNA immunofluorescence analysis are shown, which were performed as described for Fig. 1. (B) Quantitative analysis (n = 3) of experiments described for panel A. Data are means + standard errors of the means(error bars). (C) Expression profiles of p21 and PCNA in neonatal cardiomyocytes. Expression was determined by immunocytological staining. Cardiomyocytes were costained with anti-p21 (H-164) and anti-PCNA (PC10) together with antisarcomere (antitropomyosin or antiactin) antibodies (red). Nuclear DNA was stained with DAPI (blue). (D) Immobilized nuclei of H9c2 cells synchronized in S phase were incubated with cytoplasmic (cyt) or nuclear (nuc) extracts from adult (adult) or neonatal (neo) cardiomyocytes as described in Fig. 1. Data are means + standard errors of the means (error bars) of three independent experiments.

p21 regulates PCNA expression in adult cardiomyocytes in vivo. (A) Immunoblot analysis. Whole extracts of wild-type and p21 knockout hearts (80 μg of total protein) were resolved by SDS-10% PAGE and immunoblotted with antibodies specific for PCNA (PC10) and actin (A-2066). Western blot results were quantitated by Quantity One (Bio-Rad, Hercules, Calif.), and the signal for PCNA was normalized by actin staining. (B) Immunofluorescence analysis. Tissue sections of wild-type or p21 knockout hearts were costained with anti-PCNA rabbit PAb (FL-261, green) and anti-GATA-4 goat PAb (C-20, red, cardiac specific). Nuclear DNA was stained with DAPI (blue). Arrows mark examples for nuclei of cardiomyocytes that are anti-PCNA positive.