Proteomic analysis of neonatal mouse hearts shows PKA functions as a cardiomyocyte replication regulator

Figures

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  Fig. 1. Age-related proteomic changes in neonatal C57BL/6J mouse hearts from day 1 to day 7. A Workflow of the quantitative proteomic analysis of neonatal mouse hearts; (B) and (C) Gaussian distribution of dimethyl labeling ratio. The “significantly changed” threshold was set by μ ± 1.96σ. The “significantly changed” ratios between the P4/P1 groups were > 2.18 or < 0.46, and between the P7/P1 groups > 2.27 or < 0.43; (D) Heatmap of significantly changed proteins. Red and blue reflect high and low expression, respectively; (E) Real-time PCR results of selected genes (n = 3). (F) Western blotting of selected genes (n = 3)

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  Fig. 2. KEGG pathway analysis of significantly changed proteins. A Upregulated proteins enriched in KEGG pathways at day 4; B Downregulated proteins enriched in KEGG pathways at day 4; C Upregulated proteins enriched in KEGG pathways at day 7; D Downregulated proteins enriched in KEGG pathways at day 7

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  Fig. 3. Validation of the cAMP signaling pathway-related proteomics results using PCR and western blotting. A Schematic representation of the cAMP signaling pathway; 7 upregulated genes are highlighted in red. Heatmap showing age-related changes in the expression levels of the seven genes; B RT-PCR results of the selected three genes from the altered seven genes (n = 4). C Western blotting of phosphorylated PKA and total PKA (n = 4)

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  Fig. 4. Inhibition of PKA promoted EdU-DNA incorporation in cardiomyocytes. A, B EdU-DNA incorporation assay in primary cardiomyocytes treated with DMSO and the PKA activator (1μM, 5μM, 10μM bucladesine) or the PKA inhibitor (10nM, 100nM, 1 μM H-89). EdU staining (red fluorescence), DAPI staining (blue fluorescence), scale bar = 20 μm; C, D Statistical analysis of EdU results. 5μM and 10μM bucladesine can inhibit cardiomyocyte replication (from 13.03% to 7.13% p = 0.0023 and 7.78% p = 0.0102) and 1μM H-89 can promote cardiomyocyte replication (from 12.26% to 19.25%, p = 0.0004)

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  Fig. 5. Inhibition of PKA-promoted cardiomyocyte replication and upregulated cell cycle–related genes. A Ki67 results of PKA inhibitor and activator; 5μM bucladesine inhibited cardiomyocyte replication (from 15.3% to 9.5%, p = 0.0047) and 1μM H-89 promoted cardiomyocyte replication (from 15.3% to 24.4%, p = 0.0017). B Western blotting results of PKA α/β/γ pT197, PKA α/β/γ, CDK4, CylinD1, and CylinE1