Progesterone, via yes‐associated protein, promotes cardiomyocyte proliferation and cardiac repair

Figures

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  Figure 1. Serum progesterone concentration declines after birth and is associated with decrease in CM proliferation. A,B, Evaluation of CM proliferation by Ki67 and PH3 immunostaining at E16, P1, P7 and P14 hearts. Representative images with z‐stacking are shown in A1 and B1, and quantification of percentages of Ki67+ and PH3+ CMs is shown in A2 and B2, respectively (n = 5). Scale bars are 20 µm. cTnT indicates cardiac troponin T. C, Serum concentrations of progesterone in mice at E16, P1, P7 and P14 detected using ELISA kit (n = 5‐8). *** P < .001 vs E16; ### P < .001 vs P1; &&& P < .001 vs P7. CM, cardiomyocyte

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  Figure 2. Progesterone supplementation promotes postnatal CM proliferation in vivo. A, Overview of the experimental set‐up of the daily intraperitoneal injection of progesterone (8 mg/kg) or control vehicle (corn oil) from P1 to P6. Hearts were harvested at P7. B‐D, CM proliferation was evaluated by Ki67, PH3 and Aurora B immunostaining. Representative images with z‐stacking are shown in B1, C1 and D1, and quantification of percentages of Ki67+, PH3+ or Aurora B+ CMs is shown in B2, C2 and D2, respectively (n = 6). Scale bars are 20 µm. E, Representative images (E1) and quantification (E2) of the cell size of CMs (labelled by cTnT and pan‐cadherin to show intact cardiomyocytes) isolated from P7 heart. Eighty to one hundred cells were randomly selected per heart (n = 6). Scale bars are 20 µm. F, Heart weight and body weight (HW/BW) ratio (n = 10‐11). G, The mRNA expression of cell cycle activators in mouse hearts was analysed by real‐time PCR (n = 5). CM, cardiomyocyte

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  Figure 3. Progesterone promotes CM proliferation in vitro in a progesterone receptor‐dependent manner. Cultured P7 CMs were treated with control vehicle (DMSO) or progesterone (10−7 M), alone or in combination with the progesterone receptor inhibitor RU486 (10−6 M). CM proliferation was analysed 24 h after progesterone stimulation. A‐C, CM proliferation was evaluated by Ki67, PH3 and Aurora B immunostaining. Representative images are shown in A1, B1 and C1, and quantification of percentages of Ki67+, PH3+ or Aurora B+ CMs is shown in A2, B2 and C2, respectively. (3000‐5000 cells were randomly selected and analysed for each group, and four independent experiments were conducted). Scale bars are 40 µm. D, The mRNA expression of cell cycle activators in P7 CMs was analysed by quantitative PCR (n = 5). (** P < .01, *** P < .001) vs Control; (## P < .01, ### P < .001) vs Progesterone. CM, cardiomyocyte

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  Figure 4. RNA‐Seq profiles of P7 CMs treated with or without progesterone. A, Heatmap of genes whose expression was significantly different between control and progesterone‐treated CMs. Selected genes including YAP and cell cycle genes are indicated. The colour chart indicates fold change of expression using a log2 scale. B, KEGG pathway analysis of up‐regulated genes. Significantly enriched pathways of up‐regulated genes are shown. C, Selection strategy of cardiac regeneration regulators involved in progesterone‐induced CM proliferation from RNA‐Seq profiling. CM, cardiomyocyte; YAP, yes‐associated protein

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  Figure 5. Progesterone promotes CM proliferation through up‐regulation of YAP expression. Cultured P7 CMs were treated with control (vehicle, DMSO) or progesterone (10−7 M), alone or in combination with RU486 (A‐C) or YAP siRNAs (D‐F). A, Proliferation regulator mRNA expressions of CM were analysed by quantitative PCR (n = 4). B, Representative immunoblots (B1) and quantification (B2) of YAP protein expression were analysed by Western blotting (GAPDH was used as internal control) (n = 4). C, The mRNA expression of YAP target genes ANKRD1, CYR61 and CTGF was analysed by quantitative real‐time PCR (n = 4). D‐F, CM proliferation was evaluated by Ki67, PH3 and Aurora B immunostaining. Representative images are shown in D1, E1 and F1, and quantification of percentages of Ki67+, PH3+ or Aurora B+ CMs is shown in D2, E2 and F2, respectively (3000‐5000 cells were randomly selected and analysed for each group) (n = 4). Scale bars are 40 µm (** P < .01, *** P < .001) vs Control; (## P < .01, ### P < .001) vs Progesterone. CM, cardiomyocyte; YAP, yes‐associated protein

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  Figure 6. Progesterone promotes YAP expression by progesterone receptor‐mediated activation of YAP promoter in CM. A, YAP luciferase reporter activity assay. The firefly luciferase/Renilla luciferase ratio in cell lysates was calculated and expressed as relative luciferase activity (P < .0001 vs Control, n = 5). B, Binding of progesterone receptor on YAP promoter was detected by ChIP‐qPCR. DNA/protein complexes of cells treated with or without progesterone were captured by progesterone receptor antibody, and IgG was used as negative control. The immunoprecipitates were further analysed by quantitative PCR (qPCR) using three pairs of primers for YAP gene promoter (n = 4). *** P < .001 vs IgG; ### P < .001 vs Control. C, EMSA assay. Nuclear extracts of NRVMs were incubated with DyLight 800‐labelled DNA oligonucleotide probes containing YAP promoter fragments to show the binding of probe with the corresponding protein in the nuclear extracts; 200‐fold unlabelled probe as competitor was taken as negative control; progesterone receptor antibody was used to show the mobility super‐shift. CM, cardiomyocyte; YAP, yes‐associated protein

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  Figure 7. Progesterone promotes adult CM proliferation and improves cardiac function after MI. A, Adult mice were subjected to MI by ligation of the left anterior descending coronary artery and intraperitoneally injected daily with progesterone (8 mg/kg) or control vehicle (corn oil) from day 2 to day 35 post‐MI. B‐D, Hearts were harvested at day 7 post‐MI; CM proliferation in the peri‐infarcted area was evaluated by Ki67, PH3 and Aurora B staining in CMs. Representative images with z‐stacking and quantification of percentages of those markers are shown (more than 5000 cells were randomly selected in each heart, and 6 mice were analysed in each group). Scale bars are 20 µm. E,F, Echocardiographic (Echo) analyses of the effect of progesterone on cardiac function were performed 28 days after MI. Quantitative analysis of left ventricular ejection fraction (EF) and left ventricular fraction shortening (FS) is shown (n = 11‐12). G, Hearts were harvested at day 35 post‐MI. Representative images (G1) and quantification (G2) of fibrotic scar of heart sections were analysed using Masson’s trichrome staining (n = 8). Scale bars are 1 mm. CM, cardiomyocyte; MI, myocardial infarction

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  Figure 8. Progesterone promotes CM proliferation by transcriptional activation of YAP. Schematic diagram proposes that progesterone promotes CM proliferation by transcriptional activation of YAP and thereafter enhancing expression of cell cycle genes. CM, cardiomyocyte; YAP, yes‐associated protein