Background:Noonan syndrome (NS) is a relatively common developmental disorder caused by dysregulation of the RAS/mitogen-activated protein kinase (MAPK) signalling pathway, leading to multisystem involvement including short stature, skeletal abnormalities, cardiac defects, and neurocognitive impairment. Preclinical studies suggest that statins attenuate RAS/MAPK pathway hyperactivation and improve neurocognitive deficits, growth impairment, and survival in experimental models. However, no randomised controlled trial has evaluated the efficacy and safety of statin therapy in children with NS. We aimed to evaluate the efficacy and safety of simvastatin in children with NS. Methods:In this national, multicentre, double-blind, placebo-controlled phase 3 trial conducted across 13 French centres, children aged 6-15 years with genetically confirmed NS and growth impairment were randomly assigned (1:1) to receive oral simvastatin (10 mg/day during month 1, 20 mg/day during month 2, then 20 or 40 mg/day according to age) or matching placebo once daily for 12 months. The primary outcome was the change in serum insulin-like growth factor 1 (IGF-1) levels. Secondary outcomes included growth, bone, cardiac, metabolic, and behavioural parameters. Analyses followed the intention-to-treat principle using mixed-effects and adjusted regression models. The trial was prospectively registered at ClinicalTrials.gov (NCT02713945). Findings:Fifty-three children were randomised (26 simvastatin, 27 placebo), and 49/53 (92.5%) completed the study. After 12 months, simvastatin did not significantly improve IGF-1 levels compared with placebo (between-group difference -0.08 log ng/mL, 95% CI -0.18 to 0.01; p = 0.08). No differences were observed between groups for height, growth velocity, bone mineral density, metabolic measures, or behavioural outcomes. Simvastatin was associated with lower circulating Dickkopf-related protein 1 (DKK1) concentrations (-0.27, 95% CI -0.52 to -0.01; p = 0.04), a biomarker of bone metabolism, and a higher ratio of early-to-late diastolic mitral annular velocity (Ea/Aa ratio) (0.27, 95% CI -0.004 to 0.55; p = 0.05), an echocardiographic parameter related to left ventricular diastolic function. However, these isolated findings were of borderline statistical significance, and were not accompanied by corresponding clinical improvements. Treatment was generally well tolerated. One serious adverse event occurred in the simvastatin group and was considered doubtfully related to study treatment. Interpretation:In children with NS, 12 months of simvastatin did not result in significant improvements in the primary endpoint or in major outcomes of clinical relevance under the conditions tested. Changes in DKK1 concentrations and the Ea/Aa ratio should be interpreted cautiously as exploratory secondary findings requiring confirmation in adequately powered studies. Further research is needed to explore alternative treatment strategies and more direct approaches to RAS/MAPK pathway modulation. Funding:French Ministry of Health.

CONTEXT:Insulin-like growth factor-1 (IGF-1) and C-type natriuretic peptide (CNP) promote endochondral bone growth. We previously reported that vosoritide, a CNP analogue, increases annualized growth velocity (AGV) and height SD in prepubertal children with hypochondroplasia (HCH). OBJECTIVE:We hypothesized that IGF-1 and CNP will be inversely associated at baseline and will respond differentially during a single-arm, unblinded, standardized 12-month phase 2 clinical trial of vosoritide in prepubertal patients aged 3 to 11 years with HCH and height less than -2.25 SD. Participants were followed for a baseline 6-month observation period and then received a dose of 15 μg/kg/day of vosoritide subcutaneous daily injections for 12 months. Anthropometrics, IGF-1, and NTproCNP were measured every 6 months. Longitudinal changes in IGF-1 and NTproCNP as well as the correlations between these 2 analytes and changes in height SD and AGV were analyzed. RESULTS:Baseline IGF-1 values are reduced in children with HCH and increased throughout the study, but IGF-1 SD did not change significantly. NTproCNP was raised at baseline and declined throughout the study, with NTproCNP SD significantly lower at months 6 and 12 on treatment. Change in height SD or AGV were not correlated to IGF-1 or NTproCNP SD at 12 months. The change in IGF-1 concentrations at 12 months was positively correlated with the change in NTproCNP plasma concentrations (ρ = 0.57; P = .024). CONCLUSION:In children with HCH, IGF-1 levels are reduced and NTproCNP levels are elevated at baseline. A correlation between changes in these levels during treatment suggests a possible interaction between IGF-1 and CNP signaling.

Skeletal dysplasias (SD) are rare genetic disorders affecting skeletal development and bone growth. Whereas specific gene mutations have been identified in many, however, the molecular signaling pathways contributing to the phenotype are poorly understood. The C-type natriuretic peptide (CNP) signaling pathway is a driver of normal endochondral bone growth and underlies the impact of several genetic disorders of bone growth, including achondroplasia, the most common form of SD. In this cross-sectional study of 73 children with SD, comprising 7 distinct forms, we have examined the association of plasma concentrations of bioactive CNP and bio-inactive NTproCNP (recognized bio markers driving growth) and of collagen X marker (CXM) (an established biomarker of the growth plate response) with age and with annualized height velocity (HV). Although significant associations were identified with age in several disorders, the association of NTproCNP and of CXM with HV was aberrant except in type II collagen disorders and MOPD II. In OI, CNP and NTproCNP were reduced in proportion to severity of OI phenotype. Reduction in CNP exceeded NTproCNP, suggesting that higher rate of clearance/degradation of bioactive CNP occurs in OI. Across a wide range of HV in subjects with OI, biomarkers were dissociated and unrelated to HV. Similar changes were observed in 3 other forms of SD (multiple epiphyseal dysplasia, microcephalic osteodysplastic primordial dwarfism type II, and Morquio A syndrome). Although limited numbers of affected individuals within each group were studied employing a single sample at one time point, the results indicate aberrant responses both within biomarkers and when related to HV. Importantly, we identify enhanced rates of CNP clearance in OI and other forms of SD, which suggests CNP agonists could have therapeutic benefits.

Background Natriuretic peptides (NP) have important roles in regulating fat balance and metabolic health. Reduced concentrations of ANP and BNP in plasma are associated with increased insulin resistance in obesity. Whether this is due to increased clearance or reduced bioactivity of immunoreactive NP forms is unclear.Design and Method: These questions were addressed in a community study of mildly obese subjects at middle age. The ratio of amino-terminal (NT) pro-NP to bioactive C-terminal NP was used as a putative index of the clearance of bioactive forms. Results Lower ratios of amino-terminal pro-NP to bioactive C-terminal NP were associated with increased insulin resistance. In linear regression models, NT-proANP and NT-proBNP outperformed ANP and BNP in predicting insulin resistance. Pro-NP glycosylation, which can impair NP and NT-proNP production in obesity, does not account for the diminished impact of ANP or BNP. Plasma concentrations of osteocrin, which competes for the NP clearance receptor (NPR-C) and potentially enhances NP bioactivity, was not associated with NPs, but did positively predict insulin resistance in females. Conclusions We find no evidence that increased clearance/degradation of NPs contributes to insulin resistance. Among the nine NP variants assessed, only NT-proANP and NT-proBNP independently predicted insulin resistance in both sexes. The impact of CNP on fat mass or insulin resistance was minor but significant in females. Lower concentrations of immunoreactive plasma ANP and BNP remains unexplained and requires closer study.

Natriuretic peptides (NP) have multiple actions benefitting cardiovascular and metabolic health. Although many of these are mediated by Guanylyl Cyclase (GC) receptors NPR1 and NPR2, their role and relative importance in vivo is unclear. The intracellular mediator of NPR1 and NPR2, cGMP, circulates in plasma and can be used to examine relationships between receptor activity and tissue responses targeted by NPs. Plasma cGMP was measured in 348 participants previously recruited in a multidisciplinary community study (CHALICE) at age 50 years at a single centre. Associations between bio-active NPs and bio-inactive aminoterminal products with cGMP, and of cGMP with tissue response, were analysed using linear regression. Mediation of associations by NPs was assessed by Causal Mediation Analysis (CMA). ANP’s contribution to cGMP far exceed those of other NPs. Modelling across three components (demographics, NPs and cardiovascular function) shows that ANP and CNP are independent and positive predictors of cGMP. Counter intuitively, findings from CMA imply that in specific tissues, NPR1 responds more to BNP stimulation than ANP. Collectively these findings align with longer tissue half-life of BNP, and direct further therapeutic interventions towards extending tissue activity of ANP and CNP.

AbstractOsteocrin (OSTN) is an endogenous protein sharing structural similarities with the natriuretic peptides [NPs; atrial (ANP), B‐type (BNP) and C‐type (CNP) NP], which are hormones known for their crucial role in maintaining pressure/volume homeostasis. Osteocrin competes with the NPs for binding to the receptor involved in their clearance (NPR‐C). In the present study, having identified, for the first time, the major circulating form of OSTN in human and ovine plasma, we examined the integrated haemodynamic, endocrine and renal effects of vehicle‐controlled incremental infusions of ovine proOSTN (83–133) and its metabolism in eight conscious normal sheep. Incremental i.v. doses of OSTN produced stepwise increases in circulating concentrations of the peptide, and its metabolic clearance rate was inversely proportional to the dose. Osteocrin increased plasma levels of ANP, BNP and CNP in a dose‐dependent manner, together with concentrations of their intracellular second messenger, cGMP. Increases in plasma cGMP were associated with progressive reductions in arterial pressure and central venous pressure. Plasma cAMP, renin and aldosterone were unchanged. Despite significant increases in urinary cGMP levels, OSTN administration was not associated with natriuresis or diuresis in normal sheep. These results support OSTN as an endogenous ligand for NPR‐C in regulating plasma concentrations of NPs and associated cGMP‐mediated bioactivity. Collectively, our findings support a role for OSTN in maintaining cardiovascular homeostasis.

ABSTRACT C‐type natriuretic peptide (CNP) is a paracrine growth factor essential in driving endochondral bone growth in mammals including humans. Despite evidence from animal experiments and tissues that CNP signaling stimulates osteoblast proliferation and osteoclast activity, whether CNP participates in bone remodeling in the mature skeleton is unknown. Using stored plasma samples from a previous randomized controlled clinical trial (RESHAW) of resveratrol supplementation in postmenopausal women exhibiting mild osteopenia, we have studied changes in plasma aminoterminal proCNP (NTproCNP) and concurrent change in bone turnover markers of formation (osteocalcin [OC] and alkaline phosphatase [ALP]) and resorption (C‐terminal telopeptide type 1 collagen [CTX]) with bone mineral density (BMD) over a 2‐year period of study in 125 subjects. In year one, subjects received placebo or resveratrol, switching to resveratrol or placebo, respectively, in year two. Across all time points, there were no significant associations of NTproCNP with CTX, ALP, or OC. During year one, plasma NTproCNP declined significantly in both groups. In the crossover comparison, analysis of change within individuals showed that, compared with placebo, NTproCNP declined after resveratrol ( p = 0.011) and ALP increased ( p = 0.008), whereas CTX and OC were unchanged. Inverse association of NTproCNP ( r = −0.31; p = 0.025) and positive association of OC ( r = 0.32, p = 0.022) with BMD at the lumbar spine were identified after resveratrol but not found after placebo. Decline in NTproCNP was independently associated with resveratrol treatment. This is the first evidence that CNP is modulated during a period of increasing BMD in postmenopausal women. Further study of NTproCNP and associations with drivers of bone formation or resorption can be expected to clarify CNP's role during other interventions directed to bone health in adults. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

BACKGROUND:Heart disease and stroke are major and often unheralded causes of serious morbidity and premature death in middle age. Early detection of those most at risk is an urgent unmet need for instituting preventative measures. In an earlier community study (Canterbury Health, Ageing and Life Course [CHALICE]) of healthy people aged 50 years, contrary to previous reports, low levels of the heart hormone B-type natriuretic peptide (BNP) were associated with reduced measures of heart function and higher markers of vascular risk. A specific gene variant (rs198358) was found to be an independent contributor to higher BNP levels. A closely related vascular hormone (C-type natriuretic peptide [CNP]) showed opposite associations-higher levels were correlated with higher vascular risk and reduced cardiac function. To determine whether these novel findings predict serious heart or vascular disease in later life, this proposal re-examines the same CHALICE participants 15 years later.OBJECTIVE:The primary objective is to determine the predictive value of (1) low plasma concentrations of the circulating cardiac hormones (atrial natriuretic peptide [ANP] and BNP) and (2) high levels of the vascular hormone CNP at age 50 years in detecting impaired cardiac and vascular function 15 years later. Secondary objectives are to determine specific associations of individual analytes (ANP, BNP, CNP, cyclic guanosine monophosphate [cGMP]) with echo-derived changes in cardiac performance at ages 50 years and 65 years.METHODS:All of the 348 participants (205/348, 58.9% female; 53/348, 15.2% Māori or Pacifica ethnicity) participating in the original CHALICE study-free of history of heart or renal disease at age 50 years and who consented to further study-will be contacted, recruited, and restudied as previously described. Data will include intervening health history, physical examination, heart function (speckle-tracking echocardiography), vascular status (carotid intimal thickness), and genetic status (genome-wide genotyping). Laboratory measures will include fasting blood sampling and routine biochemistry, ANP, BNP, CNP, their downstream effector (cGMP), and their bio-inactive products. Humoral metabolic-cardiovascular risk factors will be measured after an overnight fast. Primary outcomes will be analyzed using multiple linear regression.RESULTS:The study will commence in 2022 and be completed in 2024.CONCLUSIONS:Proving our hypothesis-that low BNP and high CNP at any age in healthy people predict premature aging of heart and blood vessels, respectively-opens the way to early detection and improved outcomes for those most at risk. Confirmation of our hypotheses would improve current methods of screening and, in appropriate cases, enable interventions aimed at increasing natriuretic hormones and reducing risk of serious cardiovascular complications using drugs already available. Such advances in detection, and from interventional corrections, have the potential to not only improve health in the community but also reduce the high costs inevitably associated with heart failure.INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID):PRR1-10.2196/37011.

C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) catalyzes the synthesis of cGMP in chondrocytes and osteoblasts. Elevated cGMP stimulates long bone growth, and inactivating mutations in CNP or GC-B reduce cGMP, which causes dwarfism. GC-B 7E/7E mice that express a GC-B mutant that cannot be inactivated by dephosphorylation exhibit increased CNP-dependent GC-B activity, which increases bone length, as well as bone mass and strength. Importantly, how GC-B increases bone mass is not known. Here, we injected 12-week-old, wild type mice once daily for 28 days with or without BMN-111 (Vosoritide), a proteolytically resistant CNP analog. We found that BMN-111 treated mice had elevated levels of osteocalcin and collagen 1 C-terminal telopeptide (CTX) as well as increased osteoblasts and osteoclasts. In BMN-111 injected mice, tibial mRNAs for Rank ligand and osteoprotegrin were increased and decreased, respectively, whereas sclerostin mRNA was elevated 400-fold, consistent with increased osteoclast activity and decreased osteoblast activity. Mineral apposition rates and trabecular bone mass were not elevated in response to BMN-111. Because 9-week-old male GC-B 7E/7E mice have increased bone mass but do not exhibit increased mineral apposition rates, we examined 4-week-old male GC-B 7E/7E mice and found that these animals had increased serum osteocalcin, but not CTX. Importantly, tibias from these mice had 37% more osteoblasts, 26% fewer osteoclasts as well as 36% and 40% higher mineral apposition and bone formation rates, respectively. We conclude that GC-B-dependent bone formation is coupled to an early juvenile process that requires both increased osteoblasts and decreased osteoclasts.

Collagen X marker (CXM) is a degradation fragment of collagen type X. It is a real-time biomarker of height velocity with established norms. Plasma C-type natriuretic peptide (CNP) and NTproCNP levels have also been found to correlate with growth velocity in the general population and are elevated in individuals with achondroplasia compared with age- and sex-matched controls. Collagen X marker levels in people with fibroblast growth factor receptor 3 (FGFR3)-opathies have never been systematically measured. The objective of this study was to measure CXM in a population of dwarfism caused by FGFR3-opathies. Using the same cohort in which CNP and NTproCNP levels were previously measured, archived serum aliquots from 63 children with achondroplasia, six with hypochondroplasia, and two with thanatophoric dysplasia had CXM concentrations measured. Results were plotted against age- and sex-specific norms, and standard deviation scores were plotted for comparison between clinical diagnoses. CXM levels were significantly decreased ( p < 0.0001) in children with achondroplasia compared with age- and sex-matched controls. Temporal patterns of change in CXM levels were sex-dependent. As the FGFR3 pathway was more constitutively active, CXM levels decreased. New tools are emerging to study impact of skeletal dysplasia on growth plate regulation and function.

Context: Natriuretic peptide receptor-C (NPR-C, encoded by NPR3) belongs to a family of cell membrane-integral proteins implicated in various physiological processes, including longitudinal bone growth. NPR-C acts as a clearance receptor of natriuretic peptides, including C-type natriuretic peptide (CNP), that stimulate the cGMP-forming guanylyl cyclase-coupled receptors NPR-A and NPR-B. Pathogenic variants in CNP, NPR2, and NPR3 may cause a tall stature phenotype associated with macrodactyly of the halluces and epiphyseal dysplasia. Objective: Here we report on a boy with 2 novel biallelic inactivating variants of NPR3. Methods: History and clinical characteristics were collected. Biochemical indices of natriuretic peptide clearance and in vitro cellular localization of NPR-C were studied to investigate causality of the identified variants. Results: We identified 2 novel compound heterozygous NPR3 variants c.943G>A p.(Ala315Thr) and c.1294A>T p.(Ile432Phe) in a boy with tall stature and macrodactyly of the halluces. In silico analysis indicated decreased stability of NPR-C, presumably resulting in increased degradation or trafficking defects. Compared to other patients with NPR-C loss-of-function, the phenotype seemed to be milder: pseudo-epiphyses in hands and feet were absent, biochemical features were less severe, and there was some co-localization of p.(Ile432Phe) NPR-C with the cell membrane, as opposed to complete cytoplasmic retention. Conclusion: With this report on a boy with tall stature and macrodactyly of the halluces we further broaden the genotypic and phenotypic spectrum of NPR-C-related tall stature.

Evidence from genetic disorders of CNP signalling suggests that plasma concentrations of CNP are subject to feedback regulation. In subjects with Achondroplasia (Ach), CNP intracellular activity is suppressed and plasma concentrations are raised but the therapeutic impact of exogenous CNP agonists on endogenous CNP is unknown. In this exploratory dose finding and extension study of 28 Ach children receiving Vosoritide over a 5 year period of treatment, endogenous CNP production was assessed using measurements of plasma aminoterminal proCNP (NTproCNP) adjusted for age and sex and normalised as standard deviation score (SDS), and then related to skeletal growth. Before treatment NTproCNP SDS was raised. Within the first 3 months of accelerating growth, levels were significantly reduced. Across the 5 years of sustained growth, levels varied widely and were markedly increased in some subjects during adolescence. Plasma NTproCNP was suppressed at 4 h post-injection in proportion to the prevailing level of hormone resistance as reflected by SDS before injection. We conclude CNP remains subject to regulation during growth promoting doses of Vosoritide. Fall in CNP during accelerating growth is consistent with an indirect feedback whereas the fall at 4 h is likely to be a direct effect from removal of intra cellular CNP resistance.

C-type Natriuretic Peptide (CNP) is expressed in the vascular endothelium where it has anti-inflammatory, anti-proliferative and vasodilator actions—reducing intimal injury. Statin therapy is widely employed to prevent coronary artery disease and stroke. Statins reduce serum cholesterol and have contributed to reduced morbidity and mortality of cardiovascular disease. In vitro studies have suggested that statins may have additional beneficial effects on important regulators of vascular structure and function. CNP is acutely modulated by statins in vitro. Whether these actions of statins in vitro are reflected in vivo is unknown. In a prospective study of 21 males without vascular risk factors or history of cardiovascular disease, and 22 older age matched subjects recently diagnosed as acute coronary syndrome (Group 2), plasma concentrations of bioactive CNP and bio-inactive aminoterminal proCNP (NTproCNP) were measured together with lipids before and at intervals of 1, 2 and 7 days after commencing atorvastatin 40 mg/day. Plasma lipids fell significantly in both group by day 7. In both groups, plasma CNP and NTproCNP were unchanged by atorvastatin. In Group 2, baseline CNP but not NTproCNP were significantly raised compared to values in age-matched controls. The ratio of NTproCNP to CNP was significantly lower in Group 2 at baseline and throughout the study but unaffected by statin. Conventional doses of atorvastatin do not affect plasma CNP products either in healthy males or those with symptomatic coronary artery disease. Elevated CNP after cardiac injury likely Results from regulated changes in clearance, not enhanced production.

C-type Natriuretic Peptide (CNP) and Endothelin-1 (ET-1) have reciprocal roles in maintaining vascular homeostasis and are acutely modulated by statins in human cultured endothelial cells. Whether these actions of statins in vitro are reflected in studies in vivo is unknown. In a prospective study of 66 subjects with or without post- acute coronary syndrome (ACS), plasma concentrations of bioactive CNP and bio-inactive aminoterminal proCNP (NTproCNP), ET-1, B-type Natriuretic Peptide (BNP) and high sensitivity C Reactive Protein (hsCRP) were measured together with lipids before and at intervals of 1, 2 and 7 days after commencing atorvastatin 40 mg/day - and for a further period of 6months in those with ACS. Plasma lipids fell significantly in all subjects but plasma CNP, NTproCNP and ET-1 were unchanged by atorvastatin. In ACS, baseline hsCRP, BNP and CNP but not NTproCNP or ET-1 were significantly raised compared to values in age-matched controls. The ratio of NTproCNP to CNP was significantly lower in ACS throughout the study and was unaffected by statin therapy. We conclude that conventional doses of atorvastatin do not affect plasma CNP products or ET-1. Elevated CNP after cardiac injury likely results from regulated changes in clearance, not enhanced production.

Paracrine actions of CNP and rapid degradation at source severely limit study of CNP's many roles in vivo. However provided sensitive and validated assays are used, there is increasing evidence that low concentrations of bioactive CNP in plasma, and the readily detectable concentrations of the bio-inactive processed product of proCNP (aminoterminal proCNP), can be used to advance understanding of the hormone's role in pathophysiology. Provided renal function is normal, concordant changes in both CNP and NTproCNP reflect change in tissue production of proCNP whereas change in CNP alone results from altered rates of bioactive CNP degradation and are reflected in the ratio of NTproCNP to CNP. As already shown in juveniles, where plasma concentration of CNP products are higher and are associated with concurrent endochondral bone growth, measurements of plasma CNP products in mature adults have potential to clarify organ response to stress and injury. Excepting the role of CNP in fetal-maternal welfare, this review examines evidence linking plasma CNP products with function of a wide range of tissues in adults, including the impact of extraneous factors such as nutrients, hormone therapy and exercise.

Although studies in experimental animals show that blood levels of C-type natriuretic peptide (CNP) and its bioinactive aminoterminal propeptide (NTproCNP) are potential biomarkers of long bone growth, a lack of suitable assays and appropriate reference ranges has limited the application of CNP measurements in clinical practice. Plasma concentrations of the processed product of proCNP, NTproCNP - and to a lesser extent CNP itself - correlate with concurrent height velocity throughout all phases of normal skeletal growth, as well as during interventions known to affect skeletal growth in children. Since a change in levels precedes a measurable change in height velocity during interventions, measuring NTproCNP may have predictive value in clinical practice. Findings from a variety of genetic disorders affecting CNP signaling suggest that plasma concentrations of both peptides may be helpful in diagnosis, provided factors such as concurrent height velocity, feedback regulation of CNP, and differential changes in peptide clearance are considered when interpreting values. An improved understanding of factors affecting plasma levels, and the availability of commercial kits enabling accurate measurement using small volumes of plasma, can be expected to facilitate potential applications in growth disorders including genetic causes (C) affecting the CNP signaling pathway. (c) 2019 S. Karger AG, Basel

BACKGROUND Phosphodiesterase-9 (PDE9) reduces natriuretic peptide (NP) signaling and may be involved in the pathophysiology of heart failure (HF). OBJECTIVES This study investigated for the first time the integrated hemodynamic, endocrine, and renal effects of phosphodiesterase-9 inhibition (PDE9-I). METHODS A total of 8 normal sheep and 8 sheep with pacing-induced HF received incremental intravenous boluses of PDE9-I (30, 100, and 300 mg PF-04749982 at 1-h intervals). RESULTS PDE9-I dose-dependently increased plasma cyclic guanosine monophosphate (cGMP) in normal sheep (p < 0.05) while concurrently reducing circulating atrial natriuretic peptide levels (p < 0.01). Similar trends were evident in HF, resulting in significant elevations in the cGMP/NP ratio in both states (p < 0.001 and p < 0.05, respectively). PDE9-I also produced progressive falls in arterial pressure (HF: p < 0.001), atrial pressure (Normal: p < 0.001; HF: p < 0.001), and peripheral resistance (HF: p < 0.001), and transiently increased cardiac output at the top dose (Normal: p < 0.05; HF: p < 0.001). Inhibition of PDE9 had a negligible effect on circulating hormones at the lower doses, but post-high dose, acutely increased renin activity (Normal: p < 0.001; HF: p < 0.05), vasopressin (Normal: p < 0.001; HF: p < 0.01), and cyclic adenosine monophosphate (HF: p < 0.001). Plasma aldosterone increased briefly after high-dose PDE9-I in normal sheep, and fell following the top dose in HF. PDE9-I dose-dependently increased urinary cGMP in both states (both p < 0.001). In HF, this was associated with increases in urine volume (p < 0.01), sodium excretion (p < 0.01), and creatinine clearance (p < 0.001). CONCLUSIONS PDE9-I improves NP efficacy in conjunction with beneficial hemodynamic and renal effects in experimental HF. These results support a role for PDE9 in HF pathophysiology and suggest its inhibition may constitute a novel therapeutic approach to this disease. (C) 2019 by the American College of Cardiology Foundation.