Repression of cyclin D1 expression is necessary for the maintenance of cell cycle exit in adult mammalian cardiomyocytes

Abstract

The hearts of neonatal mice and adult zebrafish can regenerate after injury through proliferation of preexisting cardiomyocytes. However, adult mammals are not capable of cardiac regeneration because almost all cardiomyocytes exit their cell cycle. Exactly how the cell cycle exit is maintained and how many adult cardiomyocytes have the potential to reenter the cell cycle are unknown. The expression and activation levels of main cyclin-cyclin-dependent kinase (CDK) complexes are extremely low or undetectable at adult stages. The nuclear DNA content of almost all cardiomyocytes is 2C, indicating the cell cycle exit from G1-phase. Here, we induced expression of cyclin D1, which regulates the progression of G1-phase, only in differentiated cardiomyocytes of adult mice. In these cardiomyocytes, S-phase marker-positive cardiomyocytes and the expression of main cyclins and CDKs increased remarkably, although cyclin B1-CDK1 activation was inhibited in an ATM/ATR-independent manner. The phosphorylation pattern of CDK1 and expression pattern of Cdc25 subtypes suggested that a deficiency in the increase in Cdc25 (a and -b), which is required for M-phase entry, inhibited the cyclin B1-CDK1 activation. Finally, analysis of cell cycle distribution patterns showed that >40% of adult mouse cardiomyocytes reentered the cell cycle by the induction of cyclin D1. The cell cycle of these binucleated cardiomyocytes was arrested before M-phase, and many mononucleated cardiomyocytes entered endoreplication. These data indicate that silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit and suggest a mechanism that involves inhibition of M-phase entry.

Keywords: Cardiac Muscle; Cell Cycle; Cyclin D1; Gene Silencing; Regeneration.

FIGURE 1.

CM-specific induction of cyclin D1 expression in adult CCD(+)/MER (+) mouse by Tam administration. A, the constructs of transgenes are shown. CCD (+)/MER (+) mice were generated by intercrossing between CCD (+) and MER (+) mice. MER-Cre-MER localizes from cytoplasm to nuclei in CMs by administration of Tam and excludes the sequences containing the CAT gene with poly(A) signal between loxP sites only in differentiated CMs, resulting in CM-specific induction of cyclin D1 and EGFP expression. B, the bright field (upper panels) and EGFP fluorescence (lower panels) images of the hearts of cyclin D1-induced (CCD (+)/MER (+), Tam (+)) and control (CCD (+)/MER (−), Tam (+), and CCD (+)/MER (+), Tam (−)) mice at 7 d.p.i. are shown. Tam (+) and Tam (−) indicate mice administered Tam and only vehicle, respectively. These images were photographed immediately after thoracotomy. Scale bar, 5 mm. C, cardiac sections of mice at 7 d.p.i. of Tam were immunostained with an antibody against a CM marker, Nkx2.5, using a peroxidase-labeled antibody method (DAB, upper panels), and then the same sections were stained with an antibody against cyclin D1 (red) using an immunofluorescence method (middle panels). These images were merged (lower panels), and double-positive CM nuclei with Nkx2.5 (pseudo-color, green) and exogenous cyclin D1 (red) are visualized in yellow. These double-positive cells were also positive for sarcomeric actin. Scale bar, 100 μm. D, tissue specificity of exogenous cyclin D1 expression. A CCD (+)/MER (+) adult mouse was administered Tam, and equal amounts of extracts from the indicated tissues at 7 d.p.i. were analyzed by Western blot analysis. Whole embryos at E14.5 were used as a positive control. Arrowheads show the positions of positive signals. Exo and End indicate exogenous and endogenous cyclin D1, respectively. E, genotype and Tam specificity of exogenous cyclin D1 induction. Equal amounts of extracts from the cardiac ventricles of the indicated mice at 28 d.p.i. were analyzed by Western blot analysis. Whole embryos at E14.5 were used as a positive control. GAPDH was used as an endogenous control. F, CDK4 activation by cyclin D1 induction. Equal amounts of extracts from the cardiac ventricles of CCD (+)/MER (+) adult mice administered Tam were immunoprecipitated with an antibody against CDK4 at indicated d.p.i. The immunoprecipitates (IP) were analyzed with an in vitro kinase assay using Rb as a substrate. ³²P-Labeled Rb (upper panel), autoradiograph for phosphorylated Rb. Rb (lower panel), Coomassie Brilliant Blue staining images of Rb. Arrowheads show the positions of positive signals.

FIGURE 2.

Increase in proliferation marker-positive CMs by cyclin D1 induction. A, percentages of positive cells for proliferation markers among CMs. Cardiac sections of control adult mice (single hemizygote mice at 7 d.p.i. of Tam) and CCD (+)/MER (+) adult mice at 7 and 14 d.p.i. of Tam were immunostained with antibodies against proliferation markers and a CM marker, Nkx2.5. Percentages of double-positive cells among Nkx2.5 positive cells in the ventricles were calculated and are shown as the means ± S.E. n = 3. *, p < 0.05; **, p < 0.01 versus control mice (Tukey's test after obtaining a significant difference with one-way analysis of variance). These double-positive cells were also positive for sarcomeric actin. B, cardiac sections of the indicated adult mice at 7 d.p.i. of Tam were co-immunostained with antibodies against PCNA (red) and Νkx2.5. The merged images in the left ventricle are shown. The same methods as described in Fig. 1C were used. Double-positive CM nuclei with Nkx2.5 (pseudo-color, green) and PCNA (red) are visualized in yellow. Scale bar, 100 μm. C, cardiac sections of CCD (+)/MER (+) mice at 7 d.p.i. of Tam were coimmunostained with antibodies against cyclin D1 and pH3-S10. Arrowheads show very rare double-positive cardiomyocyte nuclei with cyclin D1 (green, exogenous expression) and pH3-S10 (red) in three independent images in the left ventricles. Insets show the high power views of the pH3-S10-positive nuclei stained with DAPI. These nuclei appear to be at late G₂-phase because nuclear membrane and nucleoli, but not any chromosome condensation, can be seen. Because signals for Nkx2.5 in phospho-H3-S10 positive nuclei are very weak, cyclin D1 was used as a marker for cardiomyocytes. Scale bar, 50 μm.

FIGURE 3.

Expression patterns of cell cycle regulators in hearts of adult CCD (+)/MER (+) mice after administration of Tam. A and C–E, protein expression in the cardiac ventricles was examined by Western blot analysis (A and E, each lane, 50 μg of protein; C, 15 μg of protein; D, 20 μg of protein). Extracts from whole embryos at E10.5 were used as a positive control. Arrowheads show the positions of positive signals. Exo and End represent exogenous and endogenous cyclin D1, respectively. The well known band shifts by phosphorylation are indicated in CDK2 or CDK1. GAPDH was used as an endogenous control. E, GAPDH controls are same as those in A because the same membrane was used. B, real-time RT-PCR analyses were performed in duplicate using cDNA from the cardiac ventricles of CCD (+)/MER (+) adult mice at indicated d.p.i. The relative mRNA levels are presented as the mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.005 versus 0 d.p.i. (Tukey's multiple comparison test after obtaining a significant difference with one-way analysis of variance). The vertical axis is shown as a logarithmic scale.

FIGURE 4.

Inhibition of cyclin B1-CDK1 activation in adult hearts after induction of cyclin D1. A, CDKs bound to cyclin E (CycE-IP), cyclin A (CycA-IP), and cyclin B1 (CycB1-IP) were immunoprecipitated (IP) with corresponding cyclin antibodies from the cardiac ventricles of CCD (+)/MER (+) adult mice at indicated d.p.i. of Tam. Protein patterns of cyclin-CDK complexes were analyzed by Western blot analysis. Arrowheads show the positions of positive signals and phosphorylation forms in CDK1. CDK activities of these complexes were analyzed with an in vitro kinase assay using histone H1 as a substrate. Coomassie Brilliant Blue staining image of histone H1 and autoradiograph indicating phosphorylated histone H1 are shown as HH1 and ³²P-labeled HH1, respectively. Whole embryos at E10.5 were used as a reference for CDK activities. Due to the strong activities, the protein contents of these references used were one-tenth those in CCD (+)/MER (+) mice. NC, negative controls using no immunoprecipitates for in vitro kinase assay. B, activities of CDKs in A are shown as percentages of relative levels to whole embryos at E10.5. C, phosphorylation patterns of CDK1 immunoprecipitated with an anti-cyclin B1 antibody in the ventricles of CCD (+)/MER (+) adult mice at indicated d.p.i. of Tam were analyzed by Western blot analysis. Whole embryos at E10.5 and growing NIH3T3 cells were used as references exhibiting three phosphorylation forms. Due to the strong signals, the protein contents of these references used were one-tenth of those in CCD (+)/MER (+) mice. pCDK1-Tyr-15, antibody for phospho-CDK1-Tyr15. Arrowheads show the positions of positive signals and phosphorylation forms in CDK1. The activities of cyclin B1-CDK1 complexes were analyzed and are shown as A. D, real-time RT-PCR analyses were performed in duplicate using cDNA from the cardiac ventricles of CCD (+)/MER (+) adult mice at the indicated d.p.i. The relative mRNA levels of Cdc25a-c are presented as the mean ± S.E. **, p < 0.01 versus 0 d.p.i. (Tukey's multiple comparison test after obtaining a significant difference with one-way analysis of variance). The vertical axis is shown as a logarithmic scale. E, Cdc25a and -b protein expression in the cardiac ventricles was examined by Western blot analysis (each lane, 20 μg of protein). Extracts from whole embryos at E10.5 were used as a positive control. Arrowheads show the positions of positive signals. GAPDH was used as an endogenous control.

FIGURE 5.

Phosphorylated forms of histone H2AX and Chk1 and expression of p53 are not detected after induction of cyclin D1. Phosphorylated histone H2AX (γ-H2AX), histone H2AX (H2AX) (A), phosphorylated Chk1 (pChk1-S345), Chk1 (B), and p53 (C) were analyzed by Western blot analysis using extracts from the cardiac ventricles of CCD (+)/MER (+) adult mice or other genotyped mice at indicated d.p.i. of Tam. NIH3T3 cells were used as positive (+IR and +Thy) and negative controls (−IR and −Thy) for phosphorylation. +IR, irradiated at 10-gray dose; −IR, no irradiation; +Thy, treated with 2 mm thymidine for 20 h; −Thy, no treatment with thymidine. Whole embryos at E10.5 were also used as a positive control for Chk1 and p53.

FIGURE 6.

Reentry by >40% of CMs to the cell cycle by cyclin D1 induction. Aa, the histograms showed the number of nuclei with various DNA content of total (upper), bi- (middle), and mononucleated (lower) CMs in adult control mice (wild type mice at 7 d.p.i. of Tam) and CCD (+)/MER (+) mice at 7, 14, 28, and 91 d.p.i. of Tam. EdU-positive nuclei are shown in black. Insets show the magnified images. EdU was injected into CCD (+)/MER (+) mice at 5 d.p.i. for analysis at 7 d.p.i. or at both 5 and 7 d.p.i. for analysis on other days. b, a negative control using CCD (+)/MER (−) mice at 14 d.p.i. of Tam. Ba, percentages of nuclei with 2C, 2C-4C, 4C, and >4C in the cell cycle distribution patterns of total and mono- and binucleated CMs in indicated mice in A. b, the percentages of mono- and binucleated CMs in the indicated mice in A. C, examples of single CMs dissociated from ventricles of control and CCD (+)/MER (+) mice at 91 d.p.i. of Tam. Images stained with DAPI (lower panels) were merged with bright field images (upper panels). DAPI signals are shown in blue. Arrowheads and arrows represent various DNA contents (2C, 4C, or 8C) in nuclei of mono- and binucleated CMs, which were determined by measurement of DAPI fluorescence intensity. Scale bar, 50 μm. D, percentages of 2C, 2C-4C, 4C, and >4C populations in bi- and mononucleated CMs in adult control mice (single hemizygote mice) and CCD (+)/MER (+) mice at 14 d.p.i. of Tam (mean ± S.E., n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.005 versus control mice (Student's t test).

FIGURE 7.

p21^Cip1 is not essential for inhibition of the M-phase entry. A, the histograms show the number of nuclei with various DNA contents for bi- (upper) and mononucleated (lower) CMs in CCD (−)/MER (−); p21^Cip1−/− and CCD (+)/MER (+); p21^Cip1−/− mice at 14 d.p.i. of Tam. EdU-positive nuclei are shown in black. Insets show the magnified images. EdU was injected at both 5 and 7 d.p.i. No EdU positive CMs were detected in the CCD (−)/MER (−); p21^Cip1−/− mouse. B, percentages of 2C, 2C-4C, 4C and >4C populations in bi- and mononucleated CMs of mice in A. C, percentages of mono-, bi-, and tri-/tetranucleated CMs of mice in A.