The rewiring of cAMP/cGMP and LDH signalling drives cardiac hypertrophy in Pde5a −/− mice

Figure 1.. Beating rates in hearts isolated from Pde5a+/+ and Pde5a−/− mice in basal condition and after beta-adrenergic stimulation. (A) Heart rate response to perfusion with different concentrations of isoproterenol was measured as beats per minute (bpm) by a Langendorff system (upper panel); Δ hearts rate: beats while perfusing with isoproterenol subtracted by resting cardiac rate (lower panel). (B) Heart rate taken at various times in isolated hearts receiving single pulse of 10−7 M isoproterenol (upper panel). Δ hearts rate: beats while perfusing with isoproterenol subtracted by resting cardiac rate (lower panel). For each experiment, eight mice for a group were analysed and one-way ANOVA was performed (*P ≤ 0.05).

Figure 2.. Functional, morphological, and molecular analyses of hearts of Pde5a+/+ and Pde5a−/− mice after moderate induced hypertrophy. (A) Representative images of echocardiography from Pde5a+/+ and Pde5a−/− mice under different experimental conditions. Yellow double arrows indicate left ventricular end-systolic diameter (LVESD), and green double arrows indicate left ventricular end-diastolic diameter (LVEDD). Scale bars (red) correspond to 0.1 s. (B) Histograms of cardiac performance for FS (%) and EF (%). (C) Representative pictures of freshly isolated hearts of Pde5a+/+ and Pde5a−/− mice. Scale bar = 1 cm. (D) Histogram of the ratios VW/BW. (E) Representative pictures of transversal heart sections of Pde5a+/+ and Pde5a−/− mice stained with wheat germ agglutinin (WGA). (F) Histogram of CSA of Pde5a+/+ and Pde5a−/−. (G) Heart mRNA expression of Bnp assessed by RT-qPCR. Fold change versus Pde5a+/+ SHAM is shown. (H) Representative picture of protein expression of Pde5a in heart samples from Pde5a+/+ and Pde5a−/− mice. MW, molecular weight. Densitometry is shown. For each experiment, at least four mice for a group were analysed and two-way ANOVA was performed (*P ≤ 0.05, **P ≤ 0.01, ***P ≤…

Figure 3.. Fibrosis analysis in Pde5a+/+ and Pde5a−/− heart sections under basal and moderate induced hypertrophy. (A) Representative picro sirius red (PSR) staining of transversal sections of Pde5a+/+ and Pde5a−/− for fibrosis detection. (B) Histograms of interstitial and perivascular fibrosis of Pde5a+/+ and Pde5a−/−. (C) Heart mRNA expression of Col1a and Ctgf assessed by RT-qPCR. Fold change versus Pde5a+/+ SHAM is shown. For each experiment, at least three mice for a group were analysed and two-way ANOVA was performed (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001).

Figure S1.. Echo and morphological parameters in hearts from Pde5a+/+ and Pde5a−/− mice in the presence or absence of Sildenafil. (A, B, C, D) FS, fractional shortening; (B) EF, ejection fraction; (C) VW/BW, ventricular weight/body weight; (D) CSA, cross-sectional area. Each column represents the mean value ± SD of at least three mice.

Figure 4.. Functional, morphological, and molecular analysis of hearts of Pde5a+/+ and Pde5a−/− mice after severe induced hypertrophy. (A) Representative images of echocardiography from Pde5a+/+ and Pde5a−/− mice under different experimental conditions. Yellow double arrows indicate left ventricular end-systolic diameter (LVESD) and green double arrows indicate left ventricular end-diastolic diameter (LVEDD). Scale bars (red) correspond to 0.1 s. (B) Histograms of cardiac performance for FS (%) and EF (%). (C) Representative pictures of freshly isolated hearts of Pde5a+/+ and Pde5a−/− mice. Scale bar = 1 cm. (D) Histogram of the ratios VW/BW. (E) Representative pictures of transversal heart sections of Pde5a+/+ and Pde5a−/− mice stained with wheat germ agglutinin (WGA). (F) Histogram of CSA of Pde5a+/+ and Pde5a−/−. (G) Heart mRNA expression of Bnp assessed by RT-qPCR. Fold change versus Pde5a+/+ SHAM is shown. For each experiment, at least four mice for a group were analysed and two-way ANOVA was performed (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

Figure S2.. Western blot analysis of PKG signalling cascade in the heart of Pde5a+/+ and Pde5a−/− mice. (A, B) Gel of protein extracts from different conditions were separated on SDS–PAGE and then probed with antibody against PKG1α, vinculin, pVASP, and VASP; (B) quantitation by densitometric analysis. One-way ANOVA was performed (*P ≤ 0.05 and **P ≤ 0.01).

Figure 5.. Fibrosis analysis in Pde5a+/+ and Pde5a−/− heart sections under basal and severe induced hypertrophy. (A) Representative picro sirius red (PSR) of staining transversal sections of Pde5a+/+ and Pde5a−/− for fibrosis detection. (B) Histograms of interstitial and perivascular fibrosis of Pde5a+/+ and Pde5a−/−. (C) Heart mRNA expression of Col1a and Ctgf assessed by RT-qPCR. Fold change versus Pde5a+/+ SHAM is shown. For each experiment, at least three mice for a group were analysed and two-way ANOVA was performed (*P ≤ 0.05 and **P ≤ 0.01).

Figure S3.. Expression of cardiac Pde isoforms in sham and TAC Pde5a+/+ and Pde5a−/− hearts. RT-qPCR of Pde isoforms that selectively hydrolyze cAMP or cGMP or both cyclic nucleotides. Each column represents the mean value ± SD of at least four mice.

Figure S4.. Enzymatic activity of Pde isoforms in hearts from Pde5a+/+ and Pde5a−/− hearts. (A) Protein extracts were treated with the following inhibitors: Bay 60-7550 and PF04449613, specific for Pde2a and Pde9a, respectively. (B) Protein extracts were treated with calcium–calmodulin to specifically activate Pde1c. Each column represents the mean value ± SD of at least four mice. One-way ANOVA was performed (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

Figure S5.. Cyclic nucleotide levels and signalling in heart samples from different experimental conditions. (A, B) Determination of cGMP (A) and cAMP (B) in Pde5a+/+ and Pde5a−/− mice under SHAM, TAC, and TAC and Sildenafil conditions. Each column represents the mean value ± SD of at least four mice (two-way ANOVA, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001). (C) Western blot of pERK1/2 and BCL-2 relative to ERK 2 and vinculin, respectively, in protein extracts from Pde5a−/− hearts. n = 2 mice.

Figure 6.. Cyclic nucleotide levels and signalling in heart samples from different experimental conditions. (A) Determination of cAMP/cGMP ratio in Pde5a+/+ and Pde5a−/− mice under SHAM, TAC, and TAC and Sildenafil conditions. Each column represents the mean value ± SD of at least 4 mice (two-way ANOVA; *P ≤ 0.05 and **P ≤ 0.01). (B) Bnp mRNA levels in H9C2 pre-treated with isoproterenol to mimic hypertrophy and then treated with different ratio of cyclic nucleotides. Each column represents the mean value ± SD of five experiments and two-way ANOVA was performed (*P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001). (C) Western blot and densitometry of pERK1/2 and Bcl2 relative to ERK2 and vinculin, respectively, in protein extracts from Pde5a+/+ hearts. Each column represents the mean value ± SD of three mice and two-way ANOVA was performed (*P ≤ 0.05).

Figure 7.. In-gel LDH-stained activities from heart protein extracts of Pde5a+/+ and Pde5a−/− mice. (A) In-gel LDH staining assays with relative quantification of whole protein extracts (30 μg) from Pde5a+/+ and Pde5a−/− (SHAM and TAC) hearts. Each column represents the mean value ± SD of at least three mice and one-way ANOVA was performed (***P ≤ 0.001 and ****P ≤ 0.0001). (B) Representative in-gel LDH-stained assays with relative quantification of extracts from Pde5a+/+ and Pde5a−/− hearts (TAC and TAC+SILD conditions). (C) In-gel LDH staining assays of protein extracts from 26G and 27G TAC Pde5a+/+ hearts after in vitro addition of 50 μM cGMP for different time points. (D) Western blot analysis of protein extract from 26G and 27G TAC Pde5a+/+ hearts treated in vitro with 50 μM cGMP and probed with an antibody against LDHA C- terminal (aa 173–332).