Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy

Figures

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  Figure 1.. Analysis of the β-adrenergic phosphoproteome selectively elicited by inhibition of PDE3 (phosphodiesterase 3) or PDE2A. A, Frequency distributions of log2 intensity ratios to illustrate thresholding of PDE3-dependent and PDE2A-dependent (B) phosphoproteomics data for pathway analysis. The top 16% quantified phosphopeptides (highlighted in green and purple for PDE3A and PDE2A, respectively) were carried forward for further analysis. C, Venn diagram comparing the 16% upregulated phosphorylation sites in samples treated with the PDE3 inhibitor (green) or the PDE2A inhibitor (purple). The CI (68%) derives from the selected peptides lying outside 1 SD of the mean distribution of peptides, as shown in A and B, respectively. BAY indicates BAY 60-7550; Cilo, cilostamide; and Iso, isoproterenol.

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  Figure 2.. Analysis of the PDE3A1 (phosphodiesterase 3A1), PDE3A2, and PDE2A2 interactomes. A, Volcano plot of the PDE3A1-mCherry interactome (false discovery rate [FDR], 0.03; S-zero is the fold-change threshold [S0], 1.3). B, PDE3A2-mCherry interactome (FDR, 0.05; S0, 0.8). C, PDE2A2-mCherry interactome (FDR, 0.03; S0, 1.3). D, Area-proportional Venn diagram to compare significant interactors in PDE3A1, PDE3A2, and PDE2A2 affinity purifications.

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  Figure 3.. Nuclear protein interaction network shared by PDE3A1 (phosphodiesterase 3A1) and PDE3A2. Functional and physical interaction network of PDE3A1/A2 interactors and phosphorylated downstream proteins generated with the STRING software. All proteins shown have been identified experimentally either in the cilostamide-dependent phosphoproteome or in the interactome shared by PDE3A1 and PDE3A2. *Proteins found in the cilostamide-dependent PDE3A phosphoproteome. **Proteins that are both in the cilostamide-dependent phosphoproteome and in the PDE3A isoform interactomes.

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  Figure 4.. PDE3A (phosphodiesterase 3A) isoforms are in a nuclear complex involving SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). A, Western blot analysis showing PDE3A2 and HDAC-1 in the pull down of endogenous SMAD4 obtained from neonatal rat ventricular myocyte (NRVM) cell lysates. Representative of n=3 independent cultures. B, Detection of endogenous SMAD4 and HDAC-1 in the immunoprecipitate obtained by pulling down endogenous PDE3A from NRVM lysates. n=3. C, Immunostaining of endogenous PDE3A and SMAD4 in NRVM cells treated either with DMSO (control) or with leptomycin B (LMB; 100 nmol/L) for 3 hours. Quantification of fluorescence intensity is shown on the right. n=4 independent experiments (at least 15 cells per experiment). Mann-Whitney U test. D, Western blot analysis showing endogenous PDE3A1 and PDE3A2 in the nuclear (N) and cytoplasmic (C) fractions obtained from NRVM treated with 100 nmol/L LMB or dimethyl sulfoxide (DMSO) (control) for 3 hours. Densitometric quantification is shown on the right. Values are shown as relative to the cytosolic content and are presented as mean±SEM. n=6 biological replicates. Wilcoxon matched pairs signed-rank test. WCL…

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  Figure 5.. Inhibition of PDE3A (phosphodiesterase 3A) leads to HDAC-1 (histone deacetylase 1) phosphorylation and inhibition of HDAC-1 deacetylase activity. A, Representative images showing the distribution of SMAD4 (SMAD family member 4)–cAMP universal tag for imaging experiments (CUTie) and CUTie reporters in neonatal rat ventricular myocytes (NRVMs) treated with 100 nmol/L leptomycin B (LMB) for 3 hours. Summaries of the Förster resonance energy transfer (FRET) change detected with the 2 sensors in NRVM on application of 0.5 nmol/L isoproterenol (ISO) followed by 10 μmol/L cilostamide (Cilo) or 100 nmol/L BAY 60-7550 (BAY) are shown on the right and are expressed as percent increase over baseline. n=5 independent NRVM preparations. Wilcoxon matched pairs signed-rank test. B, Representative Western blot showing PKA (protein kinase A)-dependent phosphorylation of HDAC-1 (left) and densitometric analysis of 5 independent experiments (right). HDAC-1-Flag was immunoprecipitated from lysates obtained from NRVM treated with 0.5 nmol/L ISO, 10 μmol/L PDE3 inhibitor Cilo, a saturating cAMP stimulus (Sat, 100 μmol/L 3-isobutyl-1-methylxanthine [IBMX] and 25 μmol/L forskolin), or with 30 μmol/L PKA…

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  Figure 6.. Inhibition of PDE3A (phosphodiesterase 3A) regulates GATA4 (GATA4 binding protein 4) expression. A, Quantitative real-time polymerase chain reaction analysis of GATA4 mRNA transcripts in neonatal rat ventricular myocyte (NRVM) cells treated with 10 μM cilostamide (Cilo), saturating treatment (Sat, 100 μmol/L 3-isobutyl-1-methylxanthine [IBMX] and 25 μmol/L forskolin) or 30 μmol/L H89 for 2.5 hours. Bars show GATA4 mRNA values normalized to GAPDH and dimethyl sulfoxide (DMSO) treatment and are presented as mean±SEM. n=4 biological replicates. Kruskal-Wallis test with Dunn correction for multiple comparisons. B, Western blot analysis and quantification of GATA4 protein expression in NRVM cells treated with Cilo (10 μmol/L), H89 (30 μmol/L), or saturating treatment. GAPDH served as a loading control. Values are normalized to GAPDH and are presented as mean±SEM. n=5 biological replicates. Kruskal-Wallis test with Dunn correction for multiple comparisons. C, Western blot analysis and quantification of endogenous GATA4 expression in cardiac tissue lysates obtained from PDE3A knockout or wild-type control rats. Hsp90 served as a loading control and for normalization in the…

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  Figure 7.. Inhibition of PDE3A (phosphodiesterase 3A) affects cardiac myocyte hypertrophic growth. A, Western blot analysis and densitometric quantification of GATA4 (GATA binding protein 4) expression in cells overexpressing HDAC-1 (histone deacetylase 1)-Flag. Values are normalized to GAPDH and presented as mean±SEM. n=5 biological replicates. Mann-Whitney U test. B, Western blot analysis and quantification showing expression of GATA4 in neonatal rat ventricular myocyte (NRVM) cells overexpressing HDAC-1-Flag and treated with cilostamide (Cilo; 10 μmol/L), saturating cAMP (Sat, 100 μmol/L 3-isobutyl-1-methylxanthine [IBMX] and 25 μmol/L forskolin) of PKA inhibitor (PKI; 20 μmol/L). n=6 biological replicates. Values are normalized to GAPDH and to Flag signal and are presented as mean±SEM. Friedman test and Dunn correction for multiple comparisons. C, Western blot analysis and quantification showing expression of GATA4 in NRVM cells overexpressing HDAC-1-Flag (wild type [WT]), the phospho-null mutant HDAC-1S406A-S436A-Flag (Ala), or the phospho-mimic mutant HDAC-1S406D-S436D-Flag (Asp). GAPDH was used as a loading control. For quantification, values were normalized to GAPDH and Flag signal…