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

Lee DI, Zhu G, Sasaki T, Cho GS, Hamdani N, Holewinski R, Jo SH, Danner T, Zhang M, Rainer PP, Bedja D, Kirk JA, Ranek MJ, Dostmann WR, Kwon C, Margulies KB, Van Eyk JE, Paulus WJ, Takimoto E, Kass DA - Nature (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA.

ABSTRACT
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 disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. 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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PDE9A inhibition suppresses cardiac hypertrophy via NP-cGMP pathwaya, Effect of PF-9613 or gene-silencing on hypertrophic gene-activation in RNCMs (n=12/group for all but Nppa-siRNA-PDE9A−/− n=8) and adult myocytes (n=6); #-p<0.05;$-p<0.01;§-p<0.01;*-p<0.001;. b, Upper: PDE9A-inhibition reduces agonist-stimulated protein synthesis; *-p<0.001-vs-baseline; #-p<0.01 vs PE/ET-1. Lower: PKG-inhibitor DT3 prevents anti-hypertrophic effect of PF-9613;*-p<0.001;#-p<0.05. c, d, PF-9613 or PDE9A-siRNA augments cGMP from ANP but not DEANO simulation in neonatal and adult myocytes; all groups p<0.01 vs baseline; *-p<0.01 vs ANP. e, FlincG-cGMP fluorescence in RNCM before and after ANP stimulation (color-coded for sampled intracellular location); right: time course normalized to baseline. f, Myocyte cGMP stimulated by ANP rises with PF-9613 in RNCMs but not cells with Pde9a silenced. g, PF-9613 does not alter DEANO stimulated cGMP whereas PDE5A inhibitor (SIL) does. h, ANP±PF-9613 effects are unchanged by ODQ, whereas DEANO±SIL are inhibited; *-p<0.01-versus-baseline; #-p<0.01-vs-ANP; $-p<0.05 vs DEANO. i, RNMCs exposed to L-NAME and PE±siRNA to PDE5A, PDE9A, or both; *p<0.05 vs baseline; #p<0.05-vs-PE-scrambled siRNA.
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Figure 2: PDE9A inhibition suppresses cardiac hypertrophy via NP-cGMP pathwaya, Effect of PF-9613 or gene-silencing on hypertrophic gene-activation in RNCMs (n=12/group for all but Nppa-siRNA-PDE9A−/− n=8) and adult myocytes (n=6); #-p<0.05;$-p<0.01;§-p<0.01;*-p<0.001;. b, Upper: PDE9A-inhibition reduces agonist-stimulated protein synthesis; *-p<0.001-vs-baseline; #-p<0.01 vs PE/ET-1. Lower: PKG-inhibitor DT3 prevents anti-hypertrophic effect of PF-9613;*-p<0.001;#-p<0.05. c, d, PF-9613 or PDE9A-siRNA augments cGMP from ANP but not DEANO simulation in neonatal and adult myocytes; all groups p<0.01 vs baseline; *-p<0.01 vs ANP. e, FlincG-cGMP fluorescence in RNCM before and after ANP stimulation (color-coded for sampled intracellular location); right: time course normalized to baseline. f, Myocyte cGMP stimulated by ANP rises with PF-9613 in RNCMs but not cells with Pde9a silenced. g, PF-9613 does not alter DEANO stimulated cGMP whereas PDE5A inhibitor (SIL) does. h, ANP±PF-9613 effects are unchanged by ODQ, whereas DEANO±SIL are inhibited; *-p<0.01-versus-baseline; #-p<0.01-vs-ANP; $-p<0.05 vs DEANO. i, RNMCs exposed to L-NAME and PE±siRNA to PDE5A, PDE9A, or both; *p<0.05 vs baseline; #p<0.05-vs-PE-scrambled siRNA.

Mentions: PDE9A upregulation by heart disease suggested its inhibition might blunt pathological stress responses. To test this, RNCMs and adult myocytes were stimulated with phenylephrine or endothelin-1 (ET-1), increasing protein synthesis and hypertrophic fetal gene (Nppa, Nppb) expression. Co-incubation with a selective PDE9A antagonist (PF-04449613 [PF-9613], 5μM, Extended Data Fig. 3a for selectivity of PDE9A vs PDE5A), or Pde9a gene silencing/deletion (Fig. 2a, b; upper) reversed these changes. Cells lacking Pde9a were unaffected by PF-9613, confirming the drug’s selectivity (Fig. 2a). Similar results were obtained with PF-04447943, another PDE9A-inhibitor now used in human trials (NCT00930059, Extended Data Fig. 3b). Anti-hypertrophic effects of PDE9A inhibition required activation of PKG, as they were blocked DT3 (Fig. 2b; lower and Extended Data Fig. 3c).


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

Lee DI, Zhu G, Sasaki T, Cho GS, Hamdani N, Holewinski R, Jo SH, Danner T, Zhang M, Rainer PP, Bedja D, Kirk JA, Ranek MJ, Dostmann WR, Kwon C, Margulies KB, Van Eyk JE, Paulus WJ, Takimoto E, Kass DA - Nature (2015)

PDE9A inhibition suppresses cardiac hypertrophy via NP-cGMP pathwaya, Effect of PF-9613 or gene-silencing on hypertrophic gene-activation in RNCMs (n=12/group for all but Nppa-siRNA-PDE9A−/− n=8) and adult myocytes (n=6); #-p<0.05;$-p<0.01;§-p<0.01;*-p<0.001;. b, Upper: PDE9A-inhibition reduces agonist-stimulated protein synthesis; *-p<0.001-vs-baseline; #-p<0.01 vs PE/ET-1. Lower: PKG-inhibitor DT3 prevents anti-hypertrophic effect of PF-9613;*-p<0.001;#-p<0.05. c, d, PF-9613 or PDE9A-siRNA augments cGMP from ANP but not DEANO simulation in neonatal and adult myocytes; all groups p<0.01 vs baseline; *-p<0.01 vs ANP. e, FlincG-cGMP fluorescence in RNCM before and after ANP stimulation (color-coded for sampled intracellular location); right: time course normalized to baseline. f, Myocyte cGMP stimulated by ANP rises with PF-9613 in RNCMs but not cells with Pde9a silenced. g, PF-9613 does not alter DEANO stimulated cGMP whereas PDE5A inhibitor (SIL) does. h, ANP±PF-9613 effects are unchanged by ODQ, whereas DEANO±SIL are inhibited; *-p<0.01-versus-baseline; #-p<0.01-vs-ANP; $-p<0.05 vs DEANO. i, RNMCs exposed to L-NAME and PE±siRNA to PDE5A, PDE9A, or both; *p<0.05 vs baseline; #p<0.05-vs-PE-scrambled siRNA.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4376609&req=5

Figure 2: PDE9A inhibition suppresses cardiac hypertrophy via NP-cGMP pathwaya, Effect of PF-9613 or gene-silencing on hypertrophic gene-activation in RNCMs (n=12/group for all but Nppa-siRNA-PDE9A−/− n=8) and adult myocytes (n=6); #-p<0.05;$-p<0.01;§-p<0.01;*-p<0.001;. b, Upper: PDE9A-inhibition reduces agonist-stimulated protein synthesis; *-p<0.001-vs-baseline; #-p<0.01 vs PE/ET-1. Lower: PKG-inhibitor DT3 prevents anti-hypertrophic effect of PF-9613;*-p<0.001;#-p<0.05. c, d, PF-9613 or PDE9A-siRNA augments cGMP from ANP but not DEANO simulation in neonatal and adult myocytes; all groups p<0.01 vs baseline; *-p<0.01 vs ANP. e, FlincG-cGMP fluorescence in RNCM before and after ANP stimulation (color-coded for sampled intracellular location); right: time course normalized to baseline. f, Myocyte cGMP stimulated by ANP rises with PF-9613 in RNCMs but not cells with Pde9a silenced. g, PF-9613 does not alter DEANO stimulated cGMP whereas PDE5A inhibitor (SIL) does. h, ANP±PF-9613 effects are unchanged by ODQ, whereas DEANO±SIL are inhibited; *-p<0.01-versus-baseline; #-p<0.01-vs-ANP; $-p<0.05 vs DEANO. i, RNMCs exposed to L-NAME and PE±siRNA to PDE5A, PDE9A, or both; *p<0.05 vs baseline; #p<0.05-vs-PE-scrambled siRNA.
Mentions: PDE9A upregulation by heart disease suggested its inhibition might blunt pathological stress responses. To test this, RNCMs and adult myocytes were stimulated with phenylephrine or endothelin-1 (ET-1), increasing protein synthesis and hypertrophic fetal gene (Nppa, Nppb) expression. Co-incubation with a selective PDE9A antagonist (PF-04449613 [PF-9613], 5μM, Extended Data Fig. 3a for selectivity of PDE9A vs PDE5A), or Pde9a gene silencing/deletion (Fig. 2a, b; upper) reversed these changes. Cells lacking Pde9a were unaffected by PF-9613, confirming the drug’s selectivity (Fig. 2a). Similar results were obtained with PF-04447943, another PDE9A-inhibitor now used in human trials (NCT00930059, Extended Data Fig. 3b). Anti-hypertrophic effects of PDE9A inhibition required activation of PKG, as they were blocked DT3 (Fig. 2b; lower and Extended Data Fig. 3c).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA.

ABSTRACT
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 disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. 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.

Show MeSH
Related in: MedlinePlus