Pharmacological or genetic inhibition of LTCC promotes cardiomyocyte proliferation through inhibition of calcineurin activity

Figures

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  Fig. 1. Drug screen targeting proteins involved in CM Ca2+ cycling revealed that inhibition of LTCC enhances CM cell cycle activity in hCOs. a Representative images of hCOs treated with DMSO (vehicle) or nifedipine (1, 3 or 10 µM) for 48 h and stained against α-actinin (green), Hoechst (blue), and Ki-67 (red) (Scale bar 20 µm). b Bar graphs representing the percentage of CMs positive for Ki-67 (n = 9–14 hCOs from 2-3 experiments). c Representative images of hCOs treated with DMSO (vehicle) or nifedipine (3 µM) for 48 h and stained against α-actinin (green), Hoechst (blue), and Ki-67 (red) (Scale bar 20 µm). d Bar graphs representing the percentage of CMs positive for PHH3 (n = 13–14 hCOs from 4 experiments e Relative CM nuclei count based on NKX2.5 positive immunostaining (n = 24–25 hCOs from 3 experiments). f Schematic of the 7-day experiment with continuous (7-day) or 48 H (recovery) treatment with 3 µM nifedipine. g Overall cell cycle activity after continuous treatment or recovery. (Ctrl, DMSO; Rec, recovery; Cont, continuous) (n = 17–22 hCOs from 3 to 4 experiments). The relative data is normalized to DMSO-treated hCOs. Data are presented as mean ± SD with individual data points…

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  Fig. 2. Single Cell RNAseq from P1 NMCM demonstrated RRAD is highly expressed in spontaneously proliferating CMs and CMs that respond to cell cycle induction. a UMAP plots displaying global gene expression for all single CMs sequenced 24 h post LacZ (control) or 4 F transduction (n = 11482, 10301 respectively). b All cells show high expression of the cardiac markers Tnnt2, Tnnc1, and Myh6, indicating a pure CM population. c P1 NMCM that showed high expression of mitotic/cytokinesis genes (Ki-67, Aurora Kinase B and A, Top2, Ccna2, Pcna, E2f1, Cdc20, Plk1, and Anln). d P1 NMCM showed high expression of Cd36, a marker for CMs that are more likely to respond to cell cycle stimulation. e Re-clustering based on the expression of the mitotic genes and the expression of Cd36 generated 3 clusters - the spontaneously proliferating CMs, primed CMs, and non-cell cycle active CMs f RRAD expression colocalized within the spontaneous proliferating CMs and the primed CMs.

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  Fig. 3. RRAD overexpression in NMCM P7 promotes cell cycle induction. a Representative images of NMCM P7 transduced with LacZ or RRAD adenovirus and stained against troponin-T (green), PHH3 (red), and Dapi (blue) (Scale bar 100 µm). b Quantification of the percentage PHH3 positive CM nuclei (n = 8). c Quantification of CM counts (n = 8). d Representative images of NMCM P7 transduced with LacZ or RRAD adenovirus and stained against troponin-T (green), Aurora Kinase B (red), and Dapi (blue) (Scale bar 50 µm) arrows referes to the nucleous at different stage of cell cycle. e Quantification of the percentage Aurora Kinase B positive CM nuclei (n = 4). Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ****p < 0.0001.

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  Fig. 4. RRAD overexpression in human heart slices promotes cell cycle induction and induces more CMs to enter the cell cycle in response to cell cycle stimuli. a Representative images of human heart slices transfected with LacZ or RRAD adenovirus for 24 h, then transfected with CDK4 and CCND (2 F) for 48 h and stained against troponin-T (green), PHH3 (red) and Dapi (blue) (Scale bar 20 µm). b–c Quantification of the percentage PHH3 positive CM nuclei (n = 4–6 individual slices). d Representative images of human heart slices transduced with LacZ or RRAD adenovirus for 24 h, then transduced with CDK4 and CCND (2 F) for 48 h and stained against troponin-T (green), Aurora Kinase B (red), and Dapi (blue) (Scale bar 20 µm). e, f Quantification of the percentage of Aurora Kinase B positive CM nuclei (n = 8). Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 5. Complete CM division occurs in MADM mice following RRAD overexpression. a Schematic diagram of the experimental design for MADM mice injection with LacZ, RRAD, CDK4/CCND (2 F), or RRAD/2 F. b Representative images show single-colored CMs of MADM mice hearts treated with LacZ, RRAD, CDK4/CCND (2 F), or RRAD/2 F (Scale bar= 20 μm). c Quantification of the percentage of the single-colored CMs to the total labeled CMs in LacZ or RRAD treated mice (n = 8), d 2 F or 2 F + RRAD treated mice (n = 8). Data are presented as mean ± SD. *p < 0.05.

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  Fig. 6. Inhibition of LTCC activity modulates the calcineurin activity. NMCM P7 transduced with LacZ or RRAD adenovirus for 72 h. a Quantification of the calcineurin phosphatase activity (n = 3). b Fold change analysis of the mRNA expression of (Rcan1) (n = 4). c Fold change analysis of the mRNA expression of (Rcan1) in NMCM P7 transduced with ShRNA-control-IL (Ctrl) or ShRNA-RRAD-IL (RRAD KD) for 24 h and then treated with vehicle or 0.5 mM nifedipine for 72 h (n = 3). d Fold change analysis of the mRNA expression of (Rcan1) in MADM mice transduced with LacZ or RRAD Adenovirus (n = 8). e Immunostaining against troponin-T (Green), DAPI (Blue), Hoxb13 (Red) in NMCM P7 transduced with LacZ or RRAD adenovirus for 72 h. (scale bar 100 μm). f Bar graph represents the percentage of Hoxb13 positive CM nuclei (n = 4). g NMCM P7 transduced with ShRNA-control-IL (Ctrl) or ShRNA-RRAD-IL (RRAD KD) for 24 h and then treated with vehicle or 0.5 mM nifedipine for 72 h and stained against troponin-T (Green), DAPI (Blue), Hoxb13 (Red) (scale bar 100 μm). h Bar graph represents the percentage of Hoxb13 positive CM nuclei (n = 3).

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  Fig. 7. RRAD/2 F combination improves cardiac function and reduces scar size post MI. a schematic diagram of the experimental design. Echocardiography assessment of b, c EF%, d, e FS%, f, g delta EF, h, i delta FS (4 weeks post virus injection- 1 week post MI). (n = 5–6 mice per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, error bars indicate S.D.). j Representative images of mice hearts were stained with Masson’s trichrome stain (healthy myocardium stains red and fibrotic tissue stains blue) at the end of the experiment (scale bar= 2 mm). k, l The scar size quantification as a percentage of total heart tissue (n = 6 mice per group, 12–13 heart sections per animal, *p < 0.05, error bars indicate S.D.). (delta function = cardiac function 4 weeks post virus injection- cardiac function before virus injection (1 week Post MI)).

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  Fig. 8. LTCC inhibition by RRAD or nifedipine induced CM cell cycle by modulating calcineurin activity. Schematic diagram of the mechanism by which LTCC inhibition by RRAD or nifedipine promotes CM proliferation.