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Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease

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Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease1,2. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation3. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP4,5, nitric oxide signalling is often depressed by heart disease6. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

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Figure 1: PDE9A expression in heart and myocytes increases with disease.
Figure 2: PDE9A inhibition suppresses cardiac hypertrophy via natriuretic-peptide–cGMP pathway.
Figure 3: Pressure-overload-induced cardiac pathobiology is suppressed in Pde9a−/− mice.
Figure 4: Chronic PDE9A inhibition reverses pre-established hypertrophy/dysfunction in a NOS-independent manner.

Change history

  • 25 March 2015

    The received date year was corrected.


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We thank students R. D. Wardlow and X. Hu for their assistance with some of the assays and studies. This research was supported by: the National Institutes of Health (NIH) (HL-119012, HL-089297, HL-07227), Fondation Leducq TransAtlantic Network of Excellence, The Peter Belfer Foundation, Abraham and Virginia Weiss Professorship (D.A.K.); HL-093432 (E.T.), American Heart Association (D.I.L.) and Max Kade Fellowship of the Austrian Academy of Sciences (P.P.R). Procurement of human heart tissue was enabled by grants from the National Institutes of Health (HL089847 and HL105993) to K.B.M. N.H. and W.J.P. were supported by the European Commission FP7 project 2010 Health (MEDIA; 261409). R.H. and J.E.V.E. were supported by The Johns Hopkins Innovation Proteomics Center in Heart Failure (NHLBI-HV-10-05 (2) and HHSN268201000032C). W.R.D. was supported by NIH grant HL68891 and the Totman Trust for Biomedical Research. We thank Pfizer and in particular C. Schmidt and R. Kleiman for providing the Pde9a−/− mouse and PF-04449613, and L. Jaffe at the University of Connecticut Health Center for providing the PDE9A antibody.

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Authors and Affiliations



D.I.L. and D.A.K. conceived and directed the project, designed experiments and prepared the manuscript. D.I.L. conducted most of the experiments and analysed the data. G.Z. helped with in vivo experiments. T.S., S.-H.J., T.D., M.Z. and P.P.R. conducted molecular biology experiments. G.-S.C. and C.K. carried out immunostaining and in situ hybridization analyses. N.H. and W.J.P. performed experiments for HFPEF and aortic stenosis human samples. M.J.R. and R.H. performed the experiments for phosphoproteomics and J.A.K. contributed to the data analysis. D.B. performed echocardiography and drug treatment. K.B.M. coordinated the human sample and data collection at the University of Pennsylvania, and W.R.D., J.E.V.E. and E.T. helped with data interpretation and presentation.

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Correspondence to David A. Kass.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Development of Pde9a knockout (Pde9a−/−) and specificity of Pde5a or Pde9a siRNA.

a, Pde9a knockout (Pde9a−/−) mice were developed by replacing the exon 12 region with lacZ-neomycin cassette in the catalytic domain of the carboxy terminal in the Pde9a gene. The genotyping was performed using specific primers designed between exons 11 and 13 including neomycin as following: Gs1 (5′-CACAGATGATGTACAGTATGGTCTGG-3′), Gs2 (5′-TGCAGTCATCAGGACCAAGATGTCC-3′) and Neo (5′-GACGAGTTCTTCTGAGGGGATCGATC-3′). b, The typical genotyping pattern of Pde9a−/− mice was shown on 2% agarose gel (250 bp for wild type and 500 bp for Pde9a−/− mice). c, Selective gene silencing using siRNAs targeting PDE5A or PDE9A. PCR confirms specificity and substantial gene knockdown achieved in cell culture (n = 6 per group).

Extended Data Figure 2 Expression of PDE9A protein in RNCM, mouse brain and human heart.

a, Immunoblot for PDE9A in neonatal cardiomyocytes transfected with either scrambled control siRNA (Sc) or Pde9a siRNA (siR), confirming the suppression of protein expression by the siRNA. A control gel for comparison was derived from brain tissue using Pde9a−/− mice and littermate controls. The band identified at 60 to 65 kDa was similar in both tissues. PDE9A bands are usually identified between 55–70 kDa depending on the splice variants expressed in a given tissue and species. b, Control immunohistochemistry showing that PDE9A detected by antibody can be largely quenched (inactivated) selectively by preincubation with recombinant ligand; scale bar, 50 μm. c, Immunostaining of PDE9A from all 8 control and DCM patients; scale bar, 200 μm. There was consistent enhanced staining in DCM patients versus controls. All images were obtained at an identical level of laser illumination and have not been altered.

Extended Data Figure 3 Selectivity of PDE9A inhibitors.

a, The dose responses of recombinant PDE9A or PDE5A to a selective PDE9A inhibitor (PF-9613) and sildenafil. Data performed in triplicate at each point. A dose of 5 μM PF-9613 inhibited PDE9A effectively, but had negligible impact on PDE5A. By contrast, the PDE5A inhibitor sildenafil inhibited PDE5A by 80% at a dose of 1 μM, commonly used for cells and tissue, but had no impact on PDE9A at this dose. These doses were therefore used in our cell-based studies. b, Confirmation that an alternative PDE9A inhibitor (PF-04447943), currently being tested in humans, shows similar anti-hypertrophic effects as PF-9613 in cardiac myocytes (n = 4 per group); P < 0.001 vs. baseline; #P < 0.01 vs. phenylephrine. c, Inhibition of PKG activity with DT3 reverses the suppression of phenylephrine-stimulated Nppb gene expression by PF-9613. This is a companion panel to Fig. 2b, bottom. n = 6 for basal (no drugs), n = 8 for other groups; P < 0.001 vs. baseline; #P < 0.01 vs. phenylephrine. Data are mean ±s.e.m.

Extended Data Figure 4 PKG activity or cGMP measurement of ANP with inhibitors in RNCM.

a, PF-9613 significantly increases PKG activity assessed by in vitro assay upon stimulation with ANP; n = 4 per group. P < 0.01 vs. other groups. b, PF-9613 augmentation of ANP-stimulated cGMP is not altered due to gene silencing of Pde5a; n = 5. This differs from the complete suppression of cGMP modulation by PF-9613 in myocytes with genetically silenced Pde9a (Fig. 1g). c, SIL does not enhance cGMP stimulated by ANP. This contrasts to its augmentation of nitric-oxide-donor-derived cGMP (Fig. 1h); n = 4. P < 0.01 vs. basal state, #P < 0.01 vs. ANP. Data are mean ±s.e.m.

Extended Data Figure 5 Confocal immunostaining of cardiomyocyte PDE9A and PDE5A.

a, PDE9A does not co-localize with α-actinin at the Z-band, whereas PDE5A does. b, PDE9A does not co-localize with α-actinin in rat neonatal myocytes. c, PDE9A co-localizes with T-tubular membranes as defined by antibody staining against the sarcoplasmic reticular ATPase-2 (SERCA2a). This differed from the localization of PDE5A. Scale bars, 20 μm.

Extended Data Figure 6 Myocardial cAMP levels in controls (sham wild type) and Pde9a−/− mice before and after TAC.

The cAMP levels were increased in TAC wild type, but they were not affected by modulation of PDE9A expression. Sham wild type, n = 4; TAC wild type, n = 5; sham Pde9a−/− n = 6; TAC Pde9a−/− n = 10. Data are mean ±s.e.m.

Extended Data Figure 7 Effect of PF-9613 on blood pressure and cardiac function in mouse.

a, Acute administration of PF-9613 by gavage was studied to assess effects on cardiac pressures, and contractility (end-systolic elastance; Ees). Over a 1-h observation period (peak plasma concentrations found after 30 min) there was no change in any of these parameters; n = 3. b, Chronic treatment of sham control mice with PF-9613 for 3 weeks (n = 3) revealed no effect on cardiac function, mass or volumes. EF, ejection fraction; FS, fractional shortening; LV-ESD, left ventricular end-systolic cross-sectional dimension; LV-EDD, left ventricular end-diastolic cross-sectional dimension. Data are mean ±s.e.m.

Extended Data Figure 8 Effect of chronic PDE9A inhibition on left ventricular mass, lung weight and alteration of TAC-responsive genes.

a, Post-mortem analysis of heart mass and lung weight (both normalized to tibia length) from mice subjected to 5 weeks of pressure overload (TAC) and co-treated with either a vehicle control, PDE9A inhibitor or PDE5A inhibitor. A sham-operation control group is also shown; sham, n = 6; TAC, n = 9; TAC + PF9613, n = 9; TAC + SIL, n = 5. b, Molecular analysis of TAC-responsive (increased expression) genes, including showing similar reductions from either PDE inhibitor in some (for example, Trpc6), a disparity between inhibitors with significant or borderline greater efficacy from PDE9A inhibition in others (for example, Ctgf, Nppa, P < 0.02 and P < 0.1, respectively between PDE5A and PDE9A inhibitor response), and substantial disparities in others (for example, Fn1, P < 0.001 between PDE5A and PDE9A inhibition). Sham, n = 5; TAC, n = 5; TAC + PF9613, n = 6; TAC + SIL, n = 5. P < 0.01 vs. sham; †P < 0.001 vs. sham; #P ≤ 0.05; ‡P < 0.01; §P < 0.001 vs. TAC. Data are mean ±s.e.m.

Extended Data Figure 9 Gene expression of cGMP-hydrolyzing PDEs in Pde9a−/− and littermate controls.

n = 10 per group. The mouse model deleted Pde9a gene expression (normalized to Gapdh), but did not impact the expression of the two other cGMP-regulating PDEs in mouse: Pde1a or Pde5a.

Extended Data Table 1 Clinical characteristics of dilated heart failure and donor control patients

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Lee, D., Zhu, G., Sasaki, T. et al. Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease. Nature 519, 472–476 (2015).

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