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Phosphodiesterase 2 Inhibition Preferentially Promotes NO/Guanylyl Cyclase/Cgmp Signaling to Reverse the Development of Heart Failure

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Phosphodiesterase 2 Inhibition Preferentially Promotes NO/Guanylyl Cyclase/Cgmp Signaling to Reverse the Development of Heart Failure Phosphodiesterase 2 inhibition preferentially promotes NO/guanylyl cyclase/cGMP signaling to reverse the development of heart failure Reshma S. Baligaa,1, Michael E. J. Preedya,1, Matthew S. Dukinfielda, Sandy M. Chua, Aisah A. Aubdoola, Kristen J. Bubba, Amie J. Moyesa, Michael A. Tonesb, and Adrian J. Hobbsa,2 aWilliam Harvey Research Institute, Barts & The London School of Medicine & Dentistry, Queen Mary University of London, EC1M 6BQ London, United Kingdom; and bPfizer, Inc., St. Louis, MO 63198 Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 26, 2018 (received for review January 18, 2018) Heart failure (HF) is a shared manifestation of several cardiovas- innervation, whereas nNOS-generated NO regulates basal myo- cular pathologies, including hypertension and myocardial infarc- cardial inotropy and lusitropy via inhibition of the inward calcium tion, and a limited repertoire of treatment modalities entails that current (ICa), sympathovagal balance, and limitation of the activity the associated morbidity and mortality remain high. Impaired nitric of oxidases (6, 7). Indeed, in LVH, such protective NO-mediated oxide (NO)/guanylyl cyclase (GC)/cyclic guanosine-3′,5′-monophos- systems are depressed, in part, due to diminished NO bioavail- phate (cGMP) signaling, underpinned, in part, by up-regulation of ability and elevated GC-1/GC-2 heme oxidation (8–10), driven by cyclic nucleotide-hydrolyzing phosphodiesterase (PDE) isozymes, an increase in the production of reactive oxygen species, particu- contributes to the pathogenesis of HF, and interventions targeted larly by NADPH oxidase isoforms (11). Likewise, natriuretic pep- to enhancing cGMP have proven effective in preclinical models and tides maintain cardiac structure and function in both physiological patients. Numerous PDE isozymes coordinate the regulation of car- and pathological settings, as illustrated by the hypertrophic, fibrotic diac cGMP in the context of HF; PDE2 expression and activity are up- cardiac phenotype in transgenic animals lacking these mediators or regulated in experimental and human HF, but a well-defined role for cognate receptors, and the exacerbated response of such mice to this isoform in pathogenesis has yet to be established, certainly in cardiac stress (12–14). terms of cGMP signaling. Herein, using a selective pharmacological From a pharmacological standpoint, NO donors, GC-1/GC-2 PHARMACOLOGY inhibitor of PDE2, BAY 60-7550, and transgenic mice lacking either [soluble GC (sGC)] stimulators, and exogenously applied natri- NO-sensitive GC-1α (GC-1α−/−) or natriuretic peptide-responsive uretic peptides have been shown to decrease hypertrophy in GC-A (GC-A−/−), we demonstrate that the blockade of PDE2 promotes cardiomyocytes and offset the development of HF in animal cGMP signaling to offset the pathogenesis of experimental HF (in- models and patients (15, 16). The common generation of cGMP and G-kinase activation affects a plethora of maladaptive, hy- duced by pressure overload or sympathetic hyperactivation), revers- + pertrophic pathways, including functional inhibition of Ca2 ing the development of left ventricular hypertrophy, compromised 2+ contractility, and cardiac fibrosis. Moreover, we show that this ben- channels and Ca sequestration (17, 18), calcineurin/nuclear − − eficial pharmacodynamic profile is maintained in GC-A / mice but is factor of activated T cells (NFAT) signaling (19), blockade of absent in animals null for GC-1α or treated with a NO synthase regulators of G protein-signaling (RGS) proteins (20), transient inhibitor, revealing that PDE2 inhibition preferentially enhances NO/GC/cGMP signaling in the setting of HF to exert wide-ranging Significance protection to preserve cardiac structure and function. These data substantiate the targeting of PDE2 in HF as a tangible approach to The morbidity and mortality associated with heart failure (HF) maximize myocardial cGMP signaling and enhancing therapy. are unacceptably high. Cyclic guanosine-3′,5′-monophosphate (cGMP) plays a key role in preserving cardiac structure and nitric oxide | natriuretic peptide | cyclic GMP | phosphodiesterase | function, and therapeutically targeting cGMP in HF has shown heart failure promise in experimental models and patients. Phosphodiester- ases (PDEs) metabolize and curtail the actions of cGMP (and eft ventricular hypertrophy (LVH) and subsequent heart cAMP), and increased PDE activity is thought to contribute to HF Lfailure (HF) are common to many cardiovascular disorders, pathogenesis. Herein, we show that inhibition of one specific including hypertension and myocardial infarction (MI), and, after isoform, PDE2, enhances the salutary effects of cGMP in the age, represent the most significant independent risk factors for context of HF, and that this beneficial action facilitates a distinct cardiovascular morbidity and mortality (1). Current therapy focuses pathway, driven by nitric oxide, that is impaired in this disorder. on reducing excess fluid load (e.g., diuretics) and blocking neuro- These observations validate PDE2 inhibitors as a demonstrable hormonal pathways (e.g., β-blockers, angiotensin-converting enzyme means of boosting cardiac cGMP and advancing HF therapy. inhibitors) (2). Unfortunately, these interventions do not offer a Author contributions: R.S.B., M.E.J.P., M.S.D., S.M.C., A.A.A., K.J.B., A.J.M., M.A.T., and cure but only slow deterioration in LV function. Consequently, HF A.J.H. designed research; R.S.B., M.E.J.P., M.S.D., S.M.C., A.A.A., K.J.B., A.J.M., and M.A.T. is still associated with a 5-y survival rate of ∼50%; the disorder performed research; M.A.T. contributed new reagents/analytic tools; R.S.B., M.E.J.P., therefore represents a clear unmet medical need. M.S.D., S.M.C., A.A.A., K.J.B., A.J.M., M.A.T., and A.J.H. analyzed data; and R.S.B., Generation of the second-messenger cyclic guanosine-3′,5′- M.E.J.P., M.S.D., S.M.C., A.A.A., K.J.B., A.J.M., M.A.T., and A.J.H. wrote the paper. monophosphate (cGMP) by activation of NO-sensitive guanylyl The authors declare no conflict of interest. This article is a PNAS Direct Submission. cyclases [α1β1 (GC-1) and α2β1 (GC-2)] (3) and natriuretic peptide- sensitive GCs (GC-A and GC-B) (4) plays a key role in main- This open access article is distributed under Creative Commons Attribution-NonCommercial- taining physiological cardiac contractility and integrity, and in NoDerivatives License 4.0 (CC BY-NC-ND). offsetting the pathogenesis of LVH and HF (5). From a homeo- 1R.S.B. and M.E.J.P. contributed equally to this work. static perspective, endothelial nitric oxide synthase (eNOS) and 2To whom correspondence should be addressed. Email: [email protected]. neuronal NOS (nNOS) appear to function in a complementary This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. manner; eNOS-derived NO is thought to contribute to LV com- 1073/pnas.1800996115/-/DCSupplemental. pliance and β-adrenergic inotropy, and to amplify parasympathetic www.pnas.org/cgi/doi/10.1073/pnas.1800996115 PNAS Latest Articles | 1of10 Downloaded by guest on September 25, 2021 receptor potential cation channel (TRPC) (21), and myosin Genotyping. Genomic DNA was prepared from ear biopsies for analysis by PCR binding protein (MBP)-C (22). This cardioprotective profile of using standard cycling parameters utilizing the forward and reverse primers cGMP in HF is exemplified by positive clinical outcomes with the listed in SI Appendix, Table S1, as we have described previously (47). dual neutral endopeptidase (an enzyme that inactivates natri- −/− uretic peptides) inhibitor-angiotensin receptor blocker LCZ696 Generation of GC-1α Knockout Mice. This GC-1α strain was developed at (23) and the sGC stimulator vericiguat (15). Pfizer, Inc. Briefly, heterozygous mouse embryonic stem cells containing one α−/− An alternative therapeutic strategy to augment cGMP signaling GC-1 allele modified by homologous recombination were obtained from Deltagen. Long-distance PCR was used to generate a 5.7-kb 5′ homology arm for cardioprotection is inhibition of phosphodiesterases (PDEs). ′ Cardiac cGMP concentrations are dynamically regulated by GC- and a 3.6-kb 3 homology arm flanking a 5.3-kb deletion encompassing exons 6 and 7 of the mouse GC-1α gene (GenBank accession no. NM_021896). driven synthesis in cooperation with degradation mediated by Exons 6 and 7 were replaced with a Lac-Z/Neor selection cassette, disrupting PDEs, a family of enzymes that hydrolyze cGMP and its sibling ′ ′ ′ the 3 end of the cyclase domain (SI Appendix, Fig. S1). Mouse 129P2/OlaHsd cyclic adenosine-3 -5 -monophosphate (cAMP) (24). In the heart, (E14) cells were transformed by established techniques (48), and G418- PDE isozymes 1, 2, 5, and 9 are believed to exert the most influ- resistant colonies were selected. Expanded clones were screened via long- ential effects on cGMP signaling (25–28).Ofthese,PDE1isthe distance PCR and Southern blot analysis. One clone with the correctly major isoform expressed in the healthy myocardium (29), whereas modified GC-1α allele was used to generate the mouse line via standard PDE5 is only minimally expressed under physiological conditions blastocyst injection (48). but significantly up-regulated in the failing myocardium (30, 31). In Successful deletion of GC-1α was confirmed by immunoblot, assay of HF, inhibition of cGMP-metabolizing PDEs 1, 5, and 9 slows enzyme activity in lung homogenates, functional vascular pharmacology in pathogenesis (27, 28); indeed, up-regulation of expression and/or response to NO donor, and measurement of mean arterial blood pressure − − activity of PDEs is thought to underpin the diminution of cGMP (MABP; SI Appendix, Fig. S1). The body weight (BW) of WT and GC-1α / signaling characteristic of HF (32). Several small clinical stud- mice did not differ significantly at the time of experimentation (WT: 30.92 ± − − ies support a beneficial role for PDE5i in HF (33–36), but the 0.63 g, GC-1α / : 30.95 ± 0.65 g; P > 0.05; n = 10).
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