Stimulation of Cardiomyocyte Proliferation Is Dependent on Species and Level of Maturation

Figures

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  FIGURE 1. Stimulation of cardiomyocyte DNA synthesis. (A) Graphical overview of experimental outline. shRNAs are p21shRNA or scrambled shRNA adenoviruses. Other conditions are growth factors that were added on Day 2. (B) Representative images of EdU stained (red) cardiomyocytes (green) in response to various treatments to induce cardiomyocyte proliferation (green: Troponin T, red: EdU, blue: DAPI, scale bar equals 50 µm) (C) Percentage of EdU positive cardiomyocyte nuclei relative to total cardiomyocyte nuclei. ***p < 0.001 vs respective controls, n = 12 for each group except p21 shRNA (n = 11) and TT-10 (n = 10). (D) Representative images of pHH3 staining (green: Troponin T, red: pHH3, blue: DAPI, scale bar equals 50 µm) (E) Percentage of pHH3-positive cardiomyocyte nuclei relative to total cardiomyocyte nuclei. ***p < 0.001 vs respective control, n = 12 for each group except p21 shRNA (n = 11) and TT-10 (n = 10).

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  FIGURE 2. Live-cell imaging platform to quantify cardiomyocyte proliferation. (A) Schematic representation of Troponin T promoter driven Histone H2B-mCherry expressing adenovirus (B) Example of cardiomyocyte nuclear division (C) Live cell imaging mediated measurement of cardiomyocyte mitosis. Cell growth percentage is measured as number of cardiomyocyte nuclei at days 1–3 relative to day 0. ***p < 0.001 vs respective control, n = 12 for each group.

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  FIGURE 3. Effect of proliferative stimuli on mouse E13.5 cardiomyocytes. (A) Experimental outline. E13.5 mouse cardiomyocytes were isolated and plated on Day 0, 36 h later siRNAs and 48 h later proliferative stimuli were added. Edu was added along with proliferative stimuli and was present for the last 48 h of culture. (B) Representative images of E13.5 murine cardiomyocytes stained for TnnT (green), EdU (Red) and DAPI (blue) (scalebar = 50 µm) (C) Quantification of Fold Change in relative EdU + CM nuclei (n = 6 for each group, **p < 0.01, ***p < 0.001) (D) Quantification of EdU + polyploid (4n+) nuclei based on DAPI histograms (n = 6, *p < 0.05, ***p < 0.001) (E) Representative image of Aurkb + cardiomyocyte in the process of undergoing cytokinesis (F) Abundance of Aurkb + CMs relative to total number of CMs after treatments (n = 6 for each group, *p < 0.05) (G) Percentage of binucleated CMs at the end of culturing period (n = 6 for each group).

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  FIGURE 4. Effect of proliferative stimuli on mouse E19.5 cardiomyocytes. (A) Experimental outline. E19.5 mouse cardiomyocytes were isolated and plated on Day 0, 36 h later siRNAs and 48 h later proliferative stimuli were added. Edu was added along with proliferative stimuli and was present for the last 48 h of culture. (B) Representative images of embryonic cardiomyocytes stained for TnnT (green), EdU (Red) and DAPI (blue) (scalebar = 50 µm) (C) Quantification of Fold Change in relative EdU + CM nuclei (n = 6 for each group, *p < 0.05, ***p < 0.001) (D) Quantification of EdU + polyploid (4n+) nuclei based on DAPI histograms (n = 6, *p < 0.05, ***p < 0.001) (E) Representative image of Aurkb + cardiomyocyte in the process of undergoing cytokinesis (F) Abundance of Aurkb + CMs relative to total number of CMs after treatments (n = 6 for each group) (G) Percentage of binucleated CMs at the end of culturing period (n = 6 for each group).

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  FIGURE 5. Effect of proliferative stimuli on neonatal mouse cardiomyocytes. (A) Experimental outline. Neonatal mouse cardiomyocytes were isolated and plated on Day 0, 36 h later siRNAs and 48 h later proliferative stimuli were added. Edu was added along with proliferative stimuli and was present for the last 48h of culture. (B) Representative images of neonatal mouse cardiomyocytes stained for TnnT (green), EdU (Red) and DAPI (blue) (scalebar = 50 µm) (C) Quantification of Fold Change in relative EdU + CM nuclei (n = 6 for each group, *p < 0.05, ***p < 0.001) (D) Quantification of EdU + polyploid (4n+) nuclei based on DAPI histograms (n = 6, *p < 0.05, ***p < 0.001) (E) Representative image of Aurkb + cardiomyocyte in the process of undergoing cytokinesis (F) Abundance of Aurkb + CMs relative to total number of CMs after treatments (n = 6 for each group) (G) Percentage of binucleated CMs at the end of culturing period (n = 6 for each group).

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  FIGURE 6. Effect of proliferative stimuli on adult mouse cardiomyocytes. (A) Schematic overview of the experimental outline. (B) Representative images of wild type and p21KO adult cardiomyocytes. Troponi T (green), EdU (red), DAPI (blue), scale bar equals 100 µm (C) Quantification of EdU + adult mouse cardiomyocytes 24 h after treatments. ***p < 0.001. (n = 9 for each group).

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  FIGURE 7. Effect of proliferative stimuli on human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) (A) Schematic overview of the experiment involving immature hiPSC-CMs. (B) Representative images of EdU + immature hiPSC-CMs after each treatment. Troponin T (green), EdU (red), DAPI (blue), scale bar equals 50 µm (n = 6, **p < 0.01, ***p < 0.001) (C) Quantification of EdU + immature hiPSC-CMs 48 h after treatment. *p < 0.05 ***p < 0.001 (D) Schematic overview of the experiment involving mature hiPSC-CMs. (E) Representative images of EdU + mature hiPSC-CMs after each treatment. Troponin T (green), EdU (red), DAPI (blue), scale bar equals 50 µm (F) Quantification of EdU + mature hiPSC-CMs 48 h after treatment. *p < 0.05 ***p < 0.001 (n = 6 for each group).