PARP1 Promotes Heart Regeneration and Cardiomyocyte Proliferation

Figures

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  Figure 1. PARP1 expression is restricted in the developing heart. (A) Representative immunostaining images of pH3+ (red) cardiomyocyte number in C57BL/6J WT mice heart at E15.5, P1, P7, and P21. Scale bars, 10μm. (B) Statistical analysis of pH3+ cardiomyocytes in C57BL/6J WT mice with heart development (n=6 per group). ***P < 0.001 by one-way ANOVA with Bonferroni's multiple comparisons test. (C) Representative western blot images of PAR and GAPDH protein expression from E15, P1, P7, and P21 C57BL/6J WT mice heart. (D) Statistical analysis of PAR protein expression with heart development (n=3). *P < 0.05, **P < 0.01 by one-way ANOVA with Bonferroni's multiple comparisons test. (E) Representative western blot images of PARP1, PARP2, and GAPDH protein expression from E15.5, P1, P7, and P21 mice hearts. (F) Statistical analysis of PARP1 and PARP2 protein expression with heart development (n=3). *P < 0.05 by one-way ANOVA with Bonferroni's multiple comparisons test.

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  Figure 2. Knockout of PARP1 inhibits cardiac regeneration in neonatal mice. (A) Representative western blot images and statistical analysis of PARP1 protein expression in WT mice and PKO mice. GAPDH was used as a loading control (n=6 per group). ***P < 0.001 by unpaired Student's t-test. (B) Statistical analysis of ejection fraction (EF) and fractional shortening (FS) of echocardiography in WT mice and PKO mice at P28 (n=5-6 per group). **P < 0.01 by unpaired Student's t-test. (C and D) WGA staining and statistical analysis of WGA staining and HW/BW ratio in WT and PKO mice at P7 (n=6 per group). Scale bars, 10μm. **P < 0.01 by unpaired Student's t-test. (E and F) Representative immunostaining images and statistical analysis of pH3+ (red) cardiomyocyte number in WT and PKO mice at P7 (n=5 per group). Scale bars, 10μm. **P < 0.01 by unpaired Student's t-test. (G and H) WGA staining and statistical analysis of WGA staining and HW/BW ratio in WT and PKO mice at P28 (n=6 per group). Scale bars, 10μm. **P < 0.01, ***P < 0.001 by unpaired Student's t-test. (I and J) Representative immunostaining images and statistical analysis of pH3+ (red) cardiomyocyte number in WT and PKO mice at P28 (n=4-6…

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  Figure 3. PARP1 overexpression promotes heart regeneration in neonatal mice. (A) Representative western blot and statistical analysis of PARP1 protein expression in control mice and PARP1CKI mice. GAPDH was used as a loading control (n=7 per group). ***P < 0.001 by unpaired Student's t-test. (B) Statistical analysis of EF and FS of echocardiography at P28 in control mice and PARP1CKI mice (n=4 per group). (C and D) WGA staining and statistical analysis of WGA staining and HW/BW ratio in control mice and PARP1CKI mice at P7 (n=5-6 per group), **P < 0.01 by unpaired Student's t-test. (E and F) Representative immunostaining images and statistical analysis of pH3+ (red) cardiomyocyte number in control mice and PARP1CKI mice at P7 (n=4 per group). Scale bars, 10μm. ***P < 0.001 by unpaired Student's t-test. (G and H) WGA staining and statistical analysis of WGA staining and HW/BW ratio in control mice and PARP1CKI mice at P28 (n=4-7 per group). **P < 0.01 by unpaired Student's t-test. (I and J) Representative immunostaining images and statistical analysis of pH3+ (red) cardiomyocyte number in control mice and PARP1CKI mice at P28 (n=4 per group). Scale bars, 10μm. ***P < 0.001 by unpaired…

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  Figure 4. PARP1 controls cardiomyocyte proliferation in vitro. (A) Representative western blot images and statistical analysis of PARP1 protein expression in NRCMs infected with Ad-Ctrl or Ad-PARP1. GAPDH was used as a loading control (n=6). ***P < 0.001 by unpaired Student's t-test. (B-E) Representative immunostaining images and statistical analysis of EdU+, Ki67+, Aurora B+, and pH3+ (red) cardiomyocyte number in Ad-Ctrl or Ad-PARP1 infected NRCMs (n=5-6). Scale bars, 10 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student's t-test. (F) Representative western blot images and statistical analysis of PARP1 protein expression in NRCMs transfected with si-Ctrl or si-PARP1. GAPDH was used as a loading control (n=6). ***P < 0.001 by unpaired Student's t-test. (G-J) Representative immunostaining images and statistical analysis of EdU+, Ki67+, Aurora B+, and pH3+(red) cardiomyocyte number in si-Ctrl or si-PARP1 transfected NRCMs (n=5-7). Scale bars, 10 μm. *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student's t-test.

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  Figure 5. PARP1 directly interacts with and poly(ADP-ribosyl)ates HSP90AB1 in cardiomyocytes. (A) Co-IP assay displayed the interaction of PARP1 and HSP90AB1 in NRCMs. (B) Co-IP assay showed the poly(ADP-ribosyl)ation of HSP90AB1 in NRCMs. (C and D) Representative images of BiFC and FRET assay showing the interaction of PARP1 and HSP90AB1. Scale bars, 10 μm (C) and 20 μm (D). (E) GST-pull down assay showed the interaction of PARP1-E domain and PARP1-E domain deletion mutant with HSP90AB1. (F) GST-pull down assay showed the interaction of HSP90AB1-δ domain (Left) and HSP90AB1-δ domain deletion mutant (Right) with PARP1. (G and H) Co-IP assay showed the poly(ADP-ribosyl)ation level of HSP90AB1 in NRCMs transinfected with Ad-Ctrl or Ad-PARP1 and in nuclear extracts from WT and PKO mice. (I) Co-IP assay showed the poly(ADP-ribosyl)ation level of HSP90AB1 in nuclear extracts from WT or PKO mice heart incubated with active DNA and NAD+, respectively. (J) Co-IP assay displayed the poly(ADP-ribosyl)ation level of HSP90AB1 in HSP90AB1 and HSP90AB1mut purified protein incubated with active DNA and NAD+.

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  Figure 6. PARP1 controls cardiomyocyte proliferation through HSP90AB1-CDC37 functional complex formation. (A and B) Co-IP assay showed the binding between HSP90AB1 and CDC37 in NRCMs infected with or without Ad-PARP1. (C and D) Co-IP assay showed the binding between HSP90AB1 and CDC37 in NRCMs infected with or without si-PARP1. (E and F) Representative western blot images and statistical analysis of protein expression of p-AKT/AKT, p-ERK /ERK, CDK4, PARP1, and GAPDH in NRCMs infected with Ad-Ctrl and Ad-PARP1 (n=3-6). *P < 0.05, ***P < 0.001, ***P < 0.001 by unpaired Student's t-test. (G and H) Representative western blot images and statistical analysis of protein expression of p-AKT/AKT, p-ERK /ERK, CDK4, PARP1, and GAPDH in NRCMs transfected with si-Ctrl and si-PARP1 (n=3-6). *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student's t-test. (I) Co-IP assay showed the interaction between HSP90AB1, CDC37, and CDK4 in NRCMs infected with or without Ad-PARP1. (J) Statistical analysis of CDK4 kinase activity in NRCMs infected with Ad-Ctrl and Ad-PARP1 at different time points (n=3). *P < 0.05 by two-way ANOVA with Bonferroni test. (K and L) Representative immunostaining images and statistical…

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  Figure 7. PARP1 improves cardiac repair and cardiomyocyte proliferation in adult mice after MI. Representative western blot images and statistical analysis of PARP1 protein expression in C57BL/6J WT mice heart intramyocardially injected with AAV9-Ctrl and AAV9-PARP1. GAPDH was used as a loading control (n=6 per group). *P< 0.05 by unpaired Student's t-test. (B and C) Masson staining images and statistical analysis of scar size in AAV9-Ctrl and AAV9-PARP1 mice post MI (n=5-6 per group). Scale bars, 1mm. ***P< 0.001 by unpaired Student's t-test. (D and E) Representative images and statistical analysis of EF and FS of echocardiography post-MI and 28 dpi in AAV9-Ctrl and AAV9-PARP1 mice (n=10 per group). **P< 0.01 by unpaired Student's t-test. (F and G) Representative immunostaining images and statistical analysis of BrdU+ and Ki67+ (white) cardiomyocytes in AAV9-Ctrl mice and AAV9-PARP1 mice at 28dpi (n=5-7 per group). Scale bars, 10μm. **P< 0.01 by ANOVA followed by the Bonferroni test.