Targeting Cardiomyocyte PCNA and POLD1 Prevents Pathologic Myocardial Hypertrophy

Abstract

Background: Activation of cell cycle regulatory pathways has been detected during pathological cardiomyocyte growth. However, it has remained unclear whether DNA synthesis pathways play a direct role in cardiomyocyte hypertrophy. We previously discovered in a mouse model of hypertrophic cardiomyopathy that there was increased DNA synthesis, which led to cardiomyocyte endoreplication and replication stress-induced DNA damage. We hypothesized that targeting cardiomyocyte endoreplication pathways could reduce pathological myocardial hypertrophy.

Methods: We utilized murine models of hypertrophic cardiomyopathy secondary to mutations in cardiac Mybpc3 (myosin-binding protein C3)^-/- or Myh6 (myosin heavy chain 6)^R404Q and transverse aortic constriction as a model of pressure overload cardiomyocyte hypertrophy. We manipulated in vivo p21 (cyclin dependent kinase inhibitor 1) protein levels using transgenic mouse models or viral transduction. Cardiomyocyte endoreplication was assessed using flow cytometry and immunohistochemistry of cardiomyocyte nuclei. We also utilized proteomics, proximity ligation assays, and human-induced pluripotent stem cell-derived cardiomyocytes.

Results: We discovered that p21 protein peaked during the early stages of hypertrophic growth in both murine hypertrophic cardiomyopathy models and a pressure overload hypertrophy model. Using genetic manipulation of p21 expression, we discovered that cardiomyocyte endoreplication and hypertrophic growth were negatively correlated with p21 expression. Mechanistically, we discovered that p21 bound to PCNA (proliferating cell nuclear antigen), which led to a reduction of PCNA binding to POLD1 (DNA polymerase delta 1). Directly targeting PCNA or POLD1 prevented cardiomyocyte DNA synthesis and hypertrophic cardiomyocyte growth. Cardiomyocyte-selective overexpression of p21 using an adeno-associated virus vector reduced long-term pathological left ventricular hypertrophy and improved diastolic function in a preclinical murine model of hypertrophic cardiomyopathy (Myh6^R404Q).

Conclusions: Our results demonstrate that PCNA-POLD1-mediated cardiomyocyte endoreplication drives hypertrophic cardiomyocyte growth, and p21 serves as a negative regulator of this process. Targeting these pathways demonstrates therapeutic potential in preventing pathological myocardial hypertrophy.

Keywords: cardiomyopathy, hypertrophic; disease models, animal; endoreduplication; heart failure; hypertrophy; myocytes, cardiac; polyploidy.

Figure 1.

Cardiomyocyte p21 (cyclin-dependent kinase inhibitor 1) is increased in Mybpc3 (myosin-binding protein C3)^−/− mice during the early stages of hypertrophic cardiomyocyte growth. A, Representative immunoblot of p21 (Santa Cruz Biotechnology [SCBT], sc-6246) from control (Ctl, wild-type C57BL/6) and Mybpc3^−/− left ventricular (LV) tissue at postnatal day (P) 2, P7, P25, P60, and P180. Total protein stain (TPS) was used as the loading Ctl. B, Immunoblot of p21 (SCBT, sc-6246) and β-actin in Ctl and Mybpc3^−/− LV tissue at P2, P7, P25, P60, and P180. C, Quantification of p21 protein expression normalized to β-actin in Ctl (n=4) and Mybpc3^−/− (n=4) LV tissue at P2, P7, P25, P60, and P180. p21 protein expressions are shown relative to Ctl of each time point. D, Measurement of p21 gene expression (Cdkn1a [cyclin-dependent kinase inhibitor 1a]) from Ctl and Mybpc3^−/− LV tissue at P7 (n=3), P25 (n=4), and P180 (n=3). Cdkn1a expression was normalized to Rpl32 (ribosomal protein L32) expression. Fold changes are shown relative to the Ctl gene expression at each time point. E and F, Immunoblot and quantification of p21 (eBioscience, 14-6715-81) from cardiomyocyte (CM) and noncardiomyocyte (non-CM) populations from Ctl and Mybpc3^−/− myocardial tissue at P25 (n=4). Immunoblot of Sar. α-actinin (sarcomeric α-actinin) was used to check the CM purity. TPS was used as the loading Ctl. Fold changes are shown relative to Ctl CMs. G and H, Immunoblot and quantification of p21 (eBioscience, 14-6715-81) from cytosolic and nuclear protein fractions from Ctl and Mybpc3^−/− myocardial tissue at P25 (n=5). Immunoblot of GAPDH and lamin B1 were used as loading Ctl. Fold changes are shown relative to Ctl for each fraction. All results are shown as mean±SEM. The Mann-Whitney U test is used for C, D, F, and H.

Figure 2.

p21 (cyclin-dependent kinase inhibitor 1) deficiency increases hypertrophic remodeling in Mybpc3 (myosin-binding protein C3)^−/− mice. A, Schematic of Cdkn1a (cyclin-dependent kinase inhibitor 1a)^−/−Mybpc3^−/− murine model generated by crossing a Cdkn1a^−/− mouse with a Mybpc3^−/− mouse. B, Western blot of p21 (Santa Cruz Biotechnology [SCBT], sc-6246) from control (Ctl, wild-type C57BL/6), Mybpc3^−/−, and Cdkn1a^−/−Mybpc3^−/− left ventricular (LV) tissue at postnatal day (P) 25. Immunoblot of β-actin used as a loading Ctl. C, Immunofluorescence staining of p21 (eBioscience, 14-6715-81; red) in Ctl, Mybpc3^−/−, and Cdkn1a^−/−Mybpc3^−/− myocardial tissue at P25. Cardiomyocytes were identified with PCM1 (pericentriolar material 1; green) and nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 20 µm. D, Hematoxylin and eosin–stained cross-sections of Ctl, Mybpc3^−/−, and Cdkn1a^−/−Mybpc3^−/− myocardial tissue at P25. Scale bars, 1 mm. E, Heart weight (HW) and (F) heart weight to tibia length ratio (HW/TL) of Ctl (n=10), Cdkn1a^−/− (n=5), Mybpc3^−/− (n=10), and Cdkn1a^−/−Mybpc3^−/− (n=11) mice at P25. Echocardiography assessment of (G) interventricular septal thickness at end-diastole (IVSd), (H) left ventricular posterior wall thickness at end-diastole (LVPWd), (I) left ventricular internal diameter at end-diastole (LVIDd), and (J) left ventricular fractional shortening (FS) in Ctl, Cdkn1a^−/−, Mybpc3^−/−, and Cdkn1a^−/−Mybpc3^−/− hearts at P25 and P180 (n=5–7). K, Representative wheat germ agglutinin (WGA) staining (green) with nuclei labeled by DAPI (blue) from Ctl, Cdkn1a^−/−, Mybpc3^−/−, and Cdkn1a^−/−Mybpc3^−/− myocardial tissue at P25 and P180. Scale bars, 50 µm. L, Cardiomyocyte cross-sectional area at P25 and P180 of Ctl, Cdkn1a^−/−, Mybpc3^−/−, and Cdkn1a^−/−Mybpc3^−/− mice as assessed by WGA staining of left ventricular cross-sections (n=5). Minimum 100 cardiomyocytes (CMs)/sample. All results are shown as mean±SEM. Two-way ANOVA with the Tukey multiple comparison test is used for E through J and L. ANOVA factor: Cdkn1a^−/− and Mybpc3^−/−. Normality was assumed based on the central limit theorem for L.

Figure 3.

Overexpression of cardiomyocyte p21 (cyclin-dependent kinase inhibitor 1) reduces cardiomyocyte hypertrophy in Mybpc3 (myosin-binding protein C3)^−/− mice. A, Schematic of the Cdkn1a (cyclin-dependent kinase inhibitor 1a)^SUPERMybpc3^−/−Myh6Cre (Tg(Myh6-cre)1Jmk) murine model generated by crossing a Cdkn1a^SUPERMyh6Cre mouse with a Mybpc3^−/− mouse. B, Immunoblot for p21 (Santa Cruz Biotechnology [SCBT], sc-6246) identified tdTomato-tagged p21 (p21-tdT) and endogenous p21 (p21-endo) from control (Ctl, wild-type C57BL/6), Mybpc3^−/−Myh6Cre, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre left ventricular (LV) tissue at postnatal day (P) 25. Western blot of β-actin used as a loading Ctl. C, Immunofluorescence staining for tdTomato protein (tdTomato; red) in Ctl, Mybpc3^−/−Myh6Cre, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre myocardial tissue at P25. Cardiomyocytes were identified with sarcomeric α-actinin (green) and nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 5 µm. D, Hematoxylin and eosin–stained cross-sections of Ctl, Mybpc3^−/−Myh6Cre, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre myocardial tissue at P25. Scale bars, 1 mm. E, Heart weight (HW) and (F) heart weight to tibia length ratio (HW/TL) of Ctl (n=7), Cdkn1a^SUPERMyh6Cre (n=6), Mybpc3^−/−Myh6Cre (n=7), and Cdkn1a^SUPERMybpc3^−/−Myh6Cre (n=9) at P25. Echocardiography assessment of (G) interventricular septal thickness at end-diastole (IVSd), (H) left ventricular posterior wall thickness at end-diastole (LVPWd), (I) left ventricular internal diameter at end-diastole (LVIDd), and (J) left ventricular fractional shortening (FS) in Ctl, Cdkn1a^SUPERMyh6Cre, Mybpc3^−/−Myh6Cre, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre hearts at P25 and P180 (n=5–9). K, Representative wheat germ agglutinin (WGA) staining (green) with nuclei labeled by DAPI (blue) from Ctl, Mybpc3^−/−Myh6Cre, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre myocardial tissue at P25. Scale bars, 50 µm. L, Cardiomyocyte cross-sectional area at P25 of Ctl, Mybpc3^−/−Myh6Cre, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre myocardial tissue as assessed by WGA staining of left ventricle cross-sections (n=5). Minimum 100 cardiomyocytes (CMs)/sample. M, Quantification of total CMs per left ventricular (LV) wall section in Ctl, Cdkn1a^−/−, Mybpc3^−/−, Cdkn1a^−/−Mybpc3^−/−, Cdkn1a^SUPERMyh6Cre, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre hearts (n=4–5). All results are shown as mean±SEM. Two-way ANOVA with the Tukey multiple comparison test is used for E through J and M. ANOVA factor: Cdkn1a^−/−, Cdkn1a^SUPERMyh6Cre, and Mybpc3^−/−Myh6Cre. One-way ANOVA with the Tukey multiple comparison test is used for L. Normality was assumed based on the central theorem for L and M.

Figure 4.

p21 (cyclin-dependent kinase inhibitor 1) negatively regulates cardiomyocyte polyploidy in Mybpc3 (myosin-binding protein C3)^−/− mice. A, Schematic of 5-ethynyl-2′-deoxyuridine (EdU) injection in mice from postnatal day (P) 21 to P25. B, Representative click-EdU (green) and PCM1 (pericentriolar material 1; red) staining with nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue) and (C) quantification of EdU-positive cardiomyocytes (CMs) from control (Ctl, wild-type C57BL/6; n=5), Cdkn1a (cyclin-dependent kinase inhibitor 1a)^−/− (n=5), Mybpc3^−/− (n=6), Cdkn1a^−/−Mybpc3^−/− (n=6), and Cdkn1a^SUPERMybpc3^−/−Myh6Cre (Tg(Myh6-cre)1Jmk) (n=5) myocardial tissue. Minimum 100 nuclei/sample. Arrow indicates cardiomyocyte nuclei, and arrowhead indicates noncardiomyocyte nuclei. Scale bars, 25 µm. D, Schematic representation of left ventricular tissue nuclei isolation followed by PCM1 labeling and Hoechst staining followed by flow cytometry (FC) analysis. E, Cardiomyocyte nuclear ploidy from Ctl, Cdkn1a^−/−, Mybpc3^−/−, Cdkn1a^−/−Mybpc3^−/−, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre nuclei by FC analysis. F, Quantitative representation of nuclear ploidy through FC analysis shown as percentage of diploid (2n) and greater than diploid (>2n) from Ctl, Cdkn1a^−/−, Mybpc3^−/−, Cdkn1a^−/−Mybpc3^−/−, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre tissue samples (n=5). Minimum 30000 nuclei/sample. ANOVA factor: genotype and ploidy. G, Linear regression analysis of the relationship between ploidy measured by FC and DNA content measured by immunohistochemistry (IHC) in Ctl, Mybpc3^−/−, and Cdkn1a^−/−Mybpc3^−/− left ventricular (LV) tissue samples. H, Determination of cardiomyocyte ploidy (DNA content) using immunofluorescence staining by measuring DAPI intensity from Ctl, Cdkn1a^−/−, Mybpc3^−/−, Cdkn1a^−/−Mybpc3^−/−, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre cardiomyocyte nuclei. Minimum 100 nuclei/sample (n=5). All results are shown as mean±SEM. Two-way ANOVA with the Tukey multiple comparison test is used for C, F, and H. ANOVA factor: Cdkn1a^−/−, Mybpc3^−/−, and Cdkn1a^SUPERMyh6Cre. The simple linear regression analysis is used for G. Normality was assumed based on the central limit theorem for C, F, and H.

Figure 5.

p21 (cyclin-dependent kinase inhibitor 1) is a negative regulator of DNA synthesis and hypertrophic growth in human cardiomyocytes. A, Representative images of human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) stimulated with 10% serum (serum+) for 24 hours followed by wheat germ agglutinin (WGA) staining (red), immunocytochemistry for sarcomeric α-actinin (green), and nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 25 µm. B, Quantification of cardiomyocyte (CM) area (µm²) from control (serum [−]) and serum-stimulated (serum [+]; n=4). Minimum 100 CMs/sample. C, Representative immunofluorescence staining of OP-Puromycin-peptide complexes (green) and sarcomeric α-actinin (red) with nuclei labeled by DAPI (blue) and (D) quantification of OP-Puromycin-peptide complexes per 100 nuclei in hiPSC-CMs stimulated with or without serum (n=4). Scale bars, 25 µm. Minimum 100 nuclei/sample. E, Measurement of NPPA and NPPB gene expressions from hiPSC-CMs stimulated with or without serum (n=4). The genes of interest were normalized to GAPDH expression. Fold changes are shown relative to control gene expression. F, Representative click 5-ethynyl-2′-deoxyuridine (EdU; green) staining and (G) quantification of EdU-positive CM nuclei from serum (−) and serum (+) hiPSC-CMs. Cardiomyocytes were identified with sarcomeric α-actinin (red) and nuclei labeled by DAPI (blue; n=4). Scale bars, 25 µm. Minimum 100 nuclei/sample. H, Relative quantification of cardiomyocyte DNA content normalized to serum (−) by measuring nuclear DAPI intensity in hiPSC-CMs stimulated with or without serum (n=4). Minimum 100 nuclei/sample. I, Quantification of total CMs per well in hiPSC-CMs stimulated with or without serum (n=4). J, Knockdown of p21 in hiPSC-CMs using small interfering RNA (siRNA) targeting CDKN1A or scrambled control siRNA (Ctl). Western blots were then performed for p21 (Santa Cruz Biotechnology [SCBT], sc-6246) and GAPDH (n=2–3). K, Representative immunofluorescence staining and (L) quantification of Ki67 (proliferation marker protein Ki67) (red) from control and CDKN1A siRNA knockdown hiPSC-CMs stimulated with or without serum (n=4). Scale bars, 50 µm. Minimum 100 nuclei/sample. M, Representative WGA (red) staining with nuclei labeled by DAPI (Blue) from control and CDKN1A siRNA knockdown hiPSC-CMs stimulated with or without serum. Scale bars, 70 µm. N, Relative quantification of cardiomyocyte DNA content (relative to Ctl siRNA without serum) and (O) quantification of cardiomyocyte area in control and CDKN1A siRNA knockdown hiPSC-CMs stimulated with or without serum (n=4). Minimum 100 CMs/sample. All results are shown as mean±SEM. The unpaired Student t test is used for B and G through I. The Welch t test is used for D. The Mann-Whitney U test is used for E. Two-way ANOVA with the Tukey multiple comparison test is used for L, N, and O. ANOVA factor: siRNA and serum. Normality was assumed based on the central limit theorem for B, D, G, H, I, L, N, and O.

Figure 6.

p21 (cyclin-dependent kinase inhibitor 1) and PCNA (proliferating cell nuclear antigen) protein interactions are increased in cardiomyocyte hypertrophy. A, Schematic of p21 coimmunoprecipitation from control (Ctl, wild-type C57BL/6) and Mybpc3 (myosin-binding protein C3)^−/− left ventricular (LV) lysates followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. List of p21-bound cell cycle regulatory proteins detected in Mybpc3^−/− LV tissue samples after proteomic analysis. Representative images from in situ proximity ligation assay (PLA) for (B) p21 and PCNA protein complexes, (C) p21 and CDK (cyclin-dependent kinase) 1 protein complexes, and (D) p21 and CDK2 protein complexes in Ctl and Mybpc3^−/− left ventricular tissue. Protein complexes are red with nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 10 µm. E, Quantification of protein complexes per 100 nuclei in Ctl and Mybpc3^−/− myocardial tissue (n=4). Minimum of 100 nuclei/sample (p21 antibody Millipore, MABE1816). F, Coimmunoprecipitation of p21 from left ventricular tissue lysate from Ctl and Mybpc3^−/− mice and immunoblotting for PCNA and p21 (Santa Cruz Biotechnology [SCBT], sc-6246; upper). Whole myocardial tissue lysate immunoblotting for PCNA, p21, and β-actin as input (lower). G, Representative PLA images and (H) quantification of p21 and PCNA protein complexes in Ctl, Mybpc3^−/−Myh6Cre (Tg(Myh6-cre)1Jmk), Cdkn1a (cyclin-dependent kinase inhibitor 1a)^−/−Mybpc3^−/−, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre myocardial tissue (n=5). Positive p21 and PCNA complexes are red, with nuclei counterstained with DAPI (blue). Scale bars, 10 µm. Minimum of 100 nuclei/sample. I, Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) were exposed to either scrambled Ctl small interfering RNA (siRNA) or PCNA targeting siRNA. Quantification of PCNA protein was assessed by immunoblot, and β-actin was used as a loading (Ctl siRNA n=5; PCNA siRNA n=6). J, Representative immunofluorescence staining and (K) quantification of Ki67 (Marker Of Proliferation Ki-67) (red) from Ctl and PCNA siRNA knockdown hiPSC-CMs stimulated with or without serum (n=4). Cardiomyocytes (CMs) were identified with sarcomeric α-actinin (green), and nuclei were labeled by DAPI (blue). Scale bars, 50 µm. Minimum 100 nuclei/sample. ANOVA factor: siRNA and serum. L, Representative staining of wheat germ agglutinin (WGA; red) with nuclei labeled by DAPI (blue) from Ctl and PCNA siRNA knockdown hiPSC-CMs stimulated with or without serum. Scale bars, 70 µm. M, Relative quantification of cardiomyocyte DNA content (relative to Ctl siRNA without serum) and (N) quantification of cardiomyocyte area in Ctl and PCNA siRNA knockdown hiPSC-CMs stimulated with or without serum (n=4). Minimum 100 CMs/sample. ANOVA factor: siRNA and serum. All results are shown as mean±SEM. The Welch t test is used for E. The Mann-Whitney U test is used for H and I. Two-way ANOVA with the Tukey multiple comparison test is used for K, M, and N. Normality was assumed based on the central limit theorem for E, K, M, and N.

Figure 7.

p21 (cyclin-dependent kinase inhibitor 1) inhibits PCNA (proliferating cell nuclear antigen)-POLD1 (DNA polymerase delta 1) interactions to negatively regulate cardiomyocyte endoreplication and hypertrophy. A, Quantification of p21 and PCNA protein complexes and (B) PCNA and POLD1 protein complexes in control (Ctl, wild-type C57BL/6) and Mybpc3 (myosin-binding protein C3)^−/− left ventricular tissue at postnatal day (P) 7 and P25 (n=4). Minimum of 100 nuclei/sample. ANOVA factor: genotype and age. C, Representative in situ proximity ligation assay (PLA) images and (D) quantification of PCNA and POLD1 complexes in Ctl, Mybpc3^−/−, Cdkn1a (cyclin-dependent kinase inhibitor 1a)^−/−Mybpc3^−/−, and Cdkn1a^SUPERMybpc3^−/−Myh6Cre (Tg(Myh6-cre)1Jmk) left ventricular tissue at P25 (n=4). Positive PCNA and POLD1 complexes are red and nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 10 µm. Minimum of 100 nuclei/sample. E, Representative in situ PLA images and (F) quantification of PCNA and POLD1 complexes from Ctl and CDKN1A small interfering RNA (siRNA) knockdown human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) stimulated with or without serum (n=4). Positive PCNA and POLD1 complexes are red, with nuclei counterstained with DAPI (blue). Scale bars, 10 µm. Minimum of 50 nuclei/sample. ANOVA factor: siRNA and serum. G, Representative immunofluorescence staining and (H) quantification of Ki67 (proliferation marker protein Ki-67) (red) from hiPSC-CMs treated with either vehicle or POLD inhib. (Zelpolib, POLD inhibitor, 10 µM) for 24 hours (n=7). Cardiomyocytes (CMs) were identified with sarcomeric α-actinin (green) and nuclei labeled by DAPI (blue). Scale bars, 50 µm. Minimum 100 nuclei/sample. I, Representative staining of wheat germ agglutinin (WGA; red) with nuclei labeled by DAPI (Blue) from vehicle Ctl and Zelpolib (10 µM)–exposed hiPSC-CMs stimulated with or without serum. Scale bars, 70 µm. J, Quantification of cardiomyocyte DNA content (relative to vehicle Ctl without serum), (K) cardiomyocyte area, and (L) OP-Puromycin-peptide complexes in vehicle Ctl and Zelpolib (10 µM)–exposed hiPSC-CMs stimulated with or without serum (n=4). Minimum 100 nuclei/sample. ANOVA factor: drug and serum. M, Measurement of NPPA and NPPB gene expressions in vehicle Ctl (n=4), Zelpolib alone (n=3), serum alone (n=5), and Zelpolib plus serum (n=5)–treated hiPSC-CMs. The genes of interest were normalized to GAPDH expression. Fold changes are shown relative to the Ctl gene expression. All results are shown as mean±SEM. Two-way ANOVA with the Tukey multiple comparison test is used for A, B, D, F, and J through L. The Welch t test is used for H. The Mann-Whitney U test is used for M. Normality was assumed based on the central limit theorem for A, B, D, F, H, J, K, and L.

Figure 8.

Targeting cardiomyocyte endoreplication pathways can prevent the development of myocardial hypertrophy and diastolic dysfunction in Myh6 (myosin heavy chain 6)^R404Q/WT mice. A, Representative click 5-ethynyl-2′-deoxyuridine (EdU; green) and PCM1 (pericentriolar material 1; red) staining with nuclei labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue) from control (Ctl, wild-type C57BL/6) and Myh6^R404Q/WT at postnatal day (P) 10. Mice were injected with EdU for 3 consecutive days (P7–P9) and euthanized at P10. Arrow indicates cardiomyocyte nuclei, and arrowhead indicates noncardiomyocyte nuclei. Scale bars, 25 µm. B, Quantification of EdU-positive cardiomyocytes (CMs) from Ctl (n=6) and Myh6^R404Q/WT (n=5) myocardial tissue. Minimum of 100 nuclei/sample. C, Determination of cardiomyocyte DNA content (relative to Ctl) by measuring DAPI intensity from Ctl and Myh6^R404Q/WT (n=5) mice at P25. Minimum of 100 nuclei/sample. D, Echocardiography assessment of left ventricular posterior wall thickness at end-diastole (LVPWd) from Ctl and Myh6^R404Q/WT mice at P25 (n=5). E, Immunoblot and (F) relative quantification of p21 protein expression (relative to Ctl; Santa Cruz Biotechnology [SCBT], sc-6246) from Ctl (n=4) and Myh6^R404Q/WT (n=5) myocardial tissue at P7. β-Actin was used as loading Ctl. G, Representative proximity ligation assay (PLA) images and (H) quantification of PCNA and POLD1 (DNA polymerase delta 1) complexes from Ctl (n=4), Myh6^R404Q/WT (n=5), and Cdkn1a (cyclin-dependent kinase inhibitor 1a)^−/−Myh6^R404Q/WT (n=5) myocardial tissue at P7. Cardiomyocytes were identified with PCM1 (green). Scale bars, 10 µm. Minimum of 50 nuclei/sample. I, Schematic of cardiomyocyte p21 overexpression strategy using cardiomyocytes-selective promoter (cTnT [cardiac troponin T]) and adeno-associated virus (AAV) 9 in Myh6^R404Q/WT animals. Echocardiography assessment of (J) interventricular septal thickness at end-diastole (IVSd), (K) left ventricular posterior wall thickness at end-diastole (LVPWd), (L) left ventricular internal diameter at end-diastole (LVIDd), (M) fractional shortening (FS), (N) isovolumic relaxation time (IVRT), and (O) early transmitral valve flow velocity to early mitral annulus tissue velocity ratio (E/e’) in Ctl, Myh6^R404Q/WT, Cdkn1a^−/−Myh6^R404Q/WT, AAV9-cTnT-GFP (green flourescent protein)–injected Myh6^R404Q/WT, and AAV9-cTnT-Cdkn1a-injected Myh6^R404Q/WT animals at 3 and 8 months (n=4–7). P, Heart weight to tibia length ratio (HW/TL) at 8 months from the groups outlined above (n=5–7). Q, Graphic abstract, created in BioRender. All results are shown as mean±SEM. The unpaired Student t test is used for B through D. The Mann-Whitney U test is used for F. One-way ANOVA with the Tukey multiple comparison test is used for H. Two-way ANOVA with the Tukey multiple comparison test is used for J through P. ANOVA factor: AAV and genotype. Normality was assumed based on the central limit theorem for B, C, and H.