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 and are expressed as means±SEM. n=5 independent experiments. Kruskal-Wallis test with Dunn's correction for multiple comparisons. D, Cell size measured in NRVM expressing mCherry, HDAC-1S406A-S436A-Flag (S>A), or HDAC-1S406D-S436D-Flag (S>D) and treated with DMSO, Cilo (10 μmol/L), or NE (10 μmol/L) for 48 hours. Values are expressed as fold change relative to untransfected and dimethyl sulfoxide (DMSO) treated within the same transfection group and are presented as mean±SEM. n=6 independent experiments (6 different differentiations from 3 independent human induced pluripotent stem cell lines, at least 27 cells per condition). Anderson-Darling log normality test and hierarchical analysis of log-transformed data followed by Bonferroni correction. E, Schematic illustration of the PDE3A/SMAD4 (SMAD family member 4)/HDAC-1 nuclear ND. Top, With active PDE3A2 at the SMAD4/HDAC-1 nuclear complex cAMP levels are locally low and PKA (protein kinase A) is inactive. HDAC-1 deacetylates histones, repressing expression of prohypertrophic genes. Bottom, Inhibition of PDE3A or displacement of PDE3A2 results in a local increase in cAMP, activation of local PKA, and phosphorylation of HDAC-1, leading to inhibition of its deacetylase activity. As a result, transcription of prohypertrophic genes is enhanced, leading to cardiac myocyte hypertrophy. Some elements used in E have been generated using BioRender.com.