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Pro–Atrial Natriuretic PeptideA Novel Guanylyl Cyclase-A Receptor Activator That GoesBeyond Atrial and B-Type Natriuretic Peptides

Tomoko Ichiki, MD, PHD, Brenda K. Huntley, BS, S. Jeson Sangaralingham, PHD, John C. Burnett, JR, MD

ABSTRACT

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OBJECTIVES The aim of this study was to determine if the atrial natriuretic peptide (ANP) precursor proANP is

biologically active compared with ANP and B-type natriuretic peptide (BNP).

BACKGROUND ProANP is produced in the atria and processed to ANP and activates the guanylyl cyclase receptor–A

(GC-A) and its second messenger, cyclic guanosine monophosphate (cGMP). ProANP is found in the human circulation,

but its bioavailability is undefined.

METHODS The in vivo actions of proANP compared with ANP, BNP, and placebo were investigated in normal

canines (667 pmol/kg, n ¼ 5/group). cGMP activation in human embryonic kidney 293 cells expressing GC-A or guanylyl

cyclase receptor–B was also determined. ProANP processing and degradation were observed in serum from normal

subjects (n ¼ 13) and patients with heart failure (n ¼ 14) ex vivo.

RESULTS ProANP had greater diuretic and natriuretic properties, with more sustained renal tubular actions,

compared with ANP and BNP in vivo in normal canines, including marked renal vasodilation not observed with ANP or BNP.

ProANP also resulted in greater and more prolonged cardiac unloading than ANP but much less hypotensive effects than

BNP. ProANP stimulated cGMP generation by GC-A as much as ANP. ProANP was processed to ANP in serum from normal

control subjects and patients with heart failure ex vivo.

CONCLUSIONS ProANP represents a novel activator of GC-A with enhanced diuretic, natriuretic, and renal vasodilating

properties, and it may represent a key circulating natriuretic peptide in cardiorenal and blood pressure homeostasis.

These results support the concepts that proANP may be a potential innovative therapeutic beyond ANP or BNP for

cardiorenal diseases, including heart failure. (J Am Coll Cardiol HF 2015;3:715–23) © 2015 by the American College of

Cardiology Foundation.

T he prevalence of acute decompensated heartfailure (HF) continues to grow, and the prog-nosis remains poor despite the availability of

many drugs to treat HF (1). Conventional diureticagents and vasodilators remain mainstays of therapy(1), but currently no new drugs have improved theprognosis of patients with acute decompensated HF(2–5). In addition, myocardial remodeling is drivenby decreased cyclic guanosine monophosphate(cGMP) activity in cardiac myocytes in both HF withpreserved ejection fraction (EF) and advanced HFwith reduced EF (6), suggesting that cGMP replace-ment therapy may be useful in patients with HF.

m the Cardiorenal Research Laboratory, Division of Cardiovascular Disease

ded by grants R01 HL36634 and P01 HL76611 from the National Institutes

sociationPostdoctoral Fellowship (10POST3600045) andaScientistDevelopm

Mayo Foundation. The authors have reported that they have no relationsh

nuscript received December 31, 2014; revised manuscript received March

The cardiac guanylyl cyclase receptor–A (GC-A)activators atrial natriuretic peptide (ANP) and B-typenatriuretic peptide (BNP) play roles in circulatingvolume and blood pressure homeostasis (7,8). Car-peritide (recombinant human ANP) and nesiritide(recombinant human BNP) have been used as thera-peutic agents for patients with HF. The use of car-peritide continues in Japan, with favorable resultsfrom clinical trials (9,10), but the use of nesiritide inpatients with HF has decreased in the United Statessince the ASCEND-HF (Acute Study of Clinical Effec-tiveness of Nesiritide and Decompensated HeartFailure) trial because there was no evidence of

s, Mayo Clinic, Rochester, Minnesota. This studywas

of Health awarded to Dr. Burnett, an American Heart

entGrant (12SDG11460017) awarded toDr. Ichiki, and

ips relevant to the contents of this paper to disclose.

13, 2015, accepted March 31, 2015.

ABBR EV I A T I ON S

AND ACRONYMS

ANP = atrial natriuretic peptide

BNP = B-type natriuretic

peptide

cGMP = cyclic guanosine

monophosphate

CNP = C-type natriuretic

peptide

DFRNa = distal fractional

reabsorption of sodium

EF = ejection fraction

GC-A = guanylyl cyclase

receptor–A

GC-B = guanylyl cyclase

receptor–B

HEK293 = human embryonic

kidney 293

HF = heart failure

His = 6-histidine

HR = heart rate

PCWP = pulmonary capillary

wedge pressure

PFRNa = proximal fractional

reabsorption of sodium

UV = urine volume

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ProANP Bioavailability Compared With ANP or BNP S E P T E M B E R 2 0 1 5 : 7 1 5 – 2 3

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improved prognosis beyond conventionaltherapies (4).

The molecular precursor of ANP, proANP,is formed after removal of the signal peptidefrom pre-proANP, which is produced fromthe NPPA gene in the heart (11,12). ProANP isthen processed into amino-terminal proANPand the biologically active ANP by corin(13,14) (Online Figure 1). ProANP is stored insecretory granules in atrial cardiomyocytesand cleaved to ANP upon secretion, inresponse to stretch (15). ANP is thendegraded into smaller molecular fragmentsby neprilysin and insulin-degrading enzyme(16–18). Hunter et al. (19) reported thatproANP circulates in canines and humans.Although initially circulating pro–natriureticpeptides were not thought to be biologicallyactive, we and others have recently estab-lished proBNP is active, has a longer half-lifethan BNP in rats (20), and can be processed toBNP in human serum (21), but it is unclear ifthis is true for proANP.

SEE PAGE 724

As with proBNP (20), we hypothesized that

proANP would be biologically active in vivo, with alonger half-life and longer lasting cardiorenal actionsthan ANP or BNP. Furthermore, we hypothesizedthat proANP can stimulate cGMP production viaGC-A receptors and be processed to ANP in the circu-lation. These studies advance our understanding ofproANP, ANP, GC-A, and cGMP signaling in the circu-lation, with potential physiological and therapeuticimplications.

METHODS

All human and animal experimental protocols used inthe present study were approved by the InstitutionalReview Board and the Animal Care and Use Commit-tee at the Mayo Clinic. Detailed methods are providedin the Online Appendix.ProANP, ANP, BNP, AND C-TYPE NATRIURETIC PEPTIDE

SYNTHESIS AND REAGENTS. Recombinant human andcanine proANP were synthesized by ProMab Bio-technologies (Richmond, California). ANP, canineBNP, and C-type natriuretic peptide (CNP) were fromPhoenix Laboratories (Burlingame, California). Hu-man proANP was tagged with Trx on the N-terminusand with 6-histidine (His) on the C-terminus forpeptide isolation, but canine proANP was untagged.

IN VIVO STUDIES IN NORMAL CANINES. We per-formed procedures (22–24) to assess the actions by

intravenous bolus injection of equimolar doses (667pmol/kg) (25) of canine proANP, ANP, canine BNP, andplacebo (0.9% saline) in normal canines (21 to 30 kg, n¼5 of each group). Short-term procedures permitted thecharacterization of pharmacokinetics and cardiorenalfunction up to 3 h after peptide or placebo injection. Inbrief, lithium carbonate tablets were given the nightbefore the study to assess renal tubular function. He-modynamic parameters were assessed with an arterialline and a Swan-Ganz catheter. Blood and urine werecollected from an arterial line and a ureter catheter,respectively. Renal blood flow was monitored with anelectromagnetic flow probe placed on the renal artery.Inulin was continuously infused for measurement ofglomerular filtration rate, which was calculated byinulin clearance. Plasma and urinary sodium, potas-sium, and lithium levels were determined. Urinaryexcretion of each parameter (X), such as cGMP,sodium, or potassium, was determined as X � urinevolume (UV) (milliliters per minute). Using the lithiumclearance technique, we calculated renal tubularfunction, proximal fractional reabsorption of sodium(PFRNa), and distal fractional reabsorption of sodium(DFRNa) (23,24). Levels of cGMPwere assayed using theRIA cGMP kit (Perkin-Elmer, Waltham,Massachusetts)(22–24). Pharmacokinetic profiles were calculated bynoncompartmental analysis using Phoenix WinNon-line 6.3 software (Certara, Princeton, New Jersey) (26).

IN VITRO STUDY IN HUMAN EMBRYONIC

KIDNEY 293 CELLS

Human embryonic kidney 293 (HEK293) cells werestably transfected with either human GC-A or gua-nylyl cyclase receptor–B (GC-B) using Lipofectamine(Invitrogen, Grand Island, New York) (27). Cells weretreated with or without 10�10 to 10�8 mol/l proANP,ANP, BNP, or CNP for 10 min, and intracellular cGMPlevels were measured.

EX VIVO HUMAN STUDIES. Fresh serum was obtainedfrom healthy volunteers (normal control subjects)and patients with HF (New York Heart Associationfunctional class II to IV), with informed consent.Fresh serum (100 ml) with or without 500 ng Trx- andHis-tagged proANP (1.9 � 10�7 mol/l) added wasincubated for varying times from 5 to 180 min at 37�C,then immunoprecipitated and Western-blotted usingHis-tag antibody (21,28).

STATISTICAL ANALYSIS. Data are reported asmean�SEM. Unpaired Student t tests were performed forcomparison between groups. Comparisons within agroup in time-course ex vivo and in vivo studieswere made by 1-way analysis of variance followedby Bonferroni multiple-comparison post-test analyses

FIGURE 1 In Vivo Urinary Changes

(A,B) Time course of urine volume (UV) (A) and urinary sodium excretion (UNaV) (B) after peptide injections. Data are calculated from the

difference from baseline. *p < 0.05 versus baseline (0 min); †p < 0.05 versus placebo; ‡p < 0.05 versus atrial natriuretic peptide (ANP), and

§p < 0.05 versus B-type natriuretic peptide (BNP). (C,D) Total UV (C) and sodium excretion (D) in the kidneys for 180 min after drug injection.

FIGURE 2 In Vivo RBF and Hct

(A) Renal blood flow (RBF) and (B) hematocrit (Hct). Data are calculated from the difference from baseline. *p < 0.05 versus baseline (0 min);

†p < 0.05 versus placebo; ‡p < 0.05 versus atrial natriuretic peptide (ANP); and §p < 0.05 vs B-type natriuretic peptide (BNP).

J A C C : H E A R T F A I L U R E V O L . 3 , N O . 9 , 2 0 1 5 Ichiki et al.S E P T E M B E R 2 0 1 5 : 7 1 5 – 2 3 ProANP Bioavailability Compared With ANP or BNP

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FIGURE 3 In Vivo Hemodynamic Changes

(A) Mean arterial pressure (MAP), (B) pulmonary capillary wedge pressure (PCWP), (C) systemic vascular resistance (SVR), and (D) heart rate

(HR). Data are calculated from the difference from baseline. *p < 0.05 versus baseline (0 min); †p < 0.05 versus placebo; ‡p < 0.05 versus

atrial natriuretic peptide (ANP); and §p < 0.05 versus B-type natriuretic peptide (BNP).

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when the global test was significant. Two-way analysisof variance was used to compare between groups intime-course in vivo studies, followed by Bonferronipost-tests. GraphPad Prism 5 (GraphPad Software, LaJolla, California) and JMP 10 (SAS Institute, Cary,North Carolina) were used for these calculations. Sta-tistical significance was accepted at p < 0.05.

RESULTS

IN VIVO RENAL ACTIONS. Online Table 1 illustratesbaseline characteristics in each group. There wasno statistical difference among the groups. Wecalculated the value of the difference from baseline(0 min) before drug injection for each parameter tocompare among the groups.

ProANP resulted in biphasic increases in UV andurinary sodium excretion with the first peak at 10 minand the second peak between 60 and 90 min, whereas

ANP and BNP showed a monophasic increase peak at10 min (Figures 1A and 1B). ANP also had a lower peakthan proANP and BNP. Urinary potassium excretionfollowed the same pattern with proANP, whereas ANPandBNPdecreased sharply in thefirst 45min,withBNPremaining low while ANP slowly increased over thenext 150min (Online Table 2). ProANPhad significantlygreater total UV for 3 h, but neither ANP nor BNPsignificantly changed from placebo (Figure 1C). ProANPand BNP had significantly greater total sodium excre-tion than placebo, but ANP did not (Figure 1D). All 3peptides had no significant differences compared withplacebo in total potassium excretion (data not shown).

ProANP significantly increased renal bloodflow from45 to 150 min compared with ANP and BNP (Figure 2A),and proANP had a greater increase in glomerularfiltration rate at 10 min compared with ANP or BNP(Online Table 2). ProANP showed the greatest increasein hematocrit from 30 to 180 min, possibly because of

FIGURE 4 In Vitro cGMP Response of Synthetic Peptides

Guanylyl cyclase receptor–A (GC-A) (A) or guanylyl cyclase receptor–B (GC-B) (B) expressing human embryonic kidney 293 cells were treated

with or without 10�8 to 10�6 mol/l of pro–atrial natriuretic peptide (proANP), ANP, B-type natriuretic peptide (BNP), or C-type natriuretic

peptide (CNP) for 10 min. *p < 0.05 versus no treatment; †p < .05 versus equimolar dose of BNP; and ‡p < 0.05 versus equimolar dose of ANP.

J A C C : H E A R T F A I L U R E V O L . 3 , N O . 9 , 2 0 1 5 Ichiki et al.S E P T E M B E R 2 0 1 5 : 7 1 5 – 2 3 ProANP Bioavailability Compared With ANP or BNP

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volume loss caused by diuresis (Figure 2B). To deter-mine which nephron segment was involved in thegreater natriuretic response to proANP, we analyzedPFRNa and DFRNa, as shown in Online Table 2. ProANPsignificantly decreased PFRNa at 10 min, similar to ANPandBNPbut showed a biphasic effect likeUV (Figure 1A)and urinary sodium excretion (Figure 1B), with asignificantly prolonged decrease of PFRNa over 120 mincompared with the other 3 groups. ProANP also signif-icantly decreased DFRNa, with a peak at 10 min, andshowed a significantly prolonged decrease of DFRNa for60 min compared with its baseline as well as placebo.ANP and BNP lacked the prolonged effects.

IN VIVO HEMODYNAMIC ACTIONS. Figure 3 andOnline Table 2 report hemodynamic responses beforeand after natriuretic peptide injection compared withplacebo. All 3 natriuretic peptides showed significantdecreases in mean arterial pressure, but only pro-ANP and BNP decreased pulmonary capillary wedgepressure (PCWP) and systemic vascular resistancecompared with placebo (Figures 3A to 3C). BNP hadthe most potent sustained decreases of mean arterialpressure, PCWP, and systemic vascular resistance.ProANP decreased mean arterial pressure similar toANP but had a prolonged decrease in PCWP for 2 h,whereas ANP did not. BNP increased heart rate (HR)for the first 20 min (Figure 3D), but after 30 min,proANP resulted in the greatest HR increase, togetherwith increased hematocrit (Figure 2B).

IN VIVO PLASMA AND URINARY cGMP IMMUNO-

REACTIVITY. Online Figures 2A and 2B illustrate

plasma and urinary cGMP levels. BNP had the greatestand most persistent increases of plasma and urinarycGMP compared with proANP or ANP, whereas proANPhad longer effects on both plasma and urinary cGMPlevels than ANP. In pharmacokinetic analyses (Table 1),BNP showed the highest plasma and urinary cGMPmaximal concentration and area under the curve.ProANP had significant increases on both parameterswith a longer half-life comparedwith either ANP or BNP.

IN VITRO cGMP ACTIVATION IN HEK293 CELLS. Weinvestigated the ability of proANP to activate cGMPgeneration compared with ANP and BNP in HEK293cells expressing GC-A and GC-B (Figures 4A and 4B).CNP was used as a positive GC-B control. BNP, ANP,and proANP all significantly induced GC-A-stimulatedcGMP production in a dose-dependent manner(Figure 4A). ProANP had significantly more effectsthan BNP and a similar effect to ANP at the highestdose (10�8 mol/l). BNP, ANP, and proANP showedlittle cGMP stimulation in GC-B cells (Figure 4B).

EX VIVO ProANP PROCESSING IN HUMAN SERUM.

We assessed whether proANP is processed inthe normal or HF human circulation ex vivo. Charac-teristics of normal control subjects and patients withHF and individual characteristics of patients with HFare shown in Online Tables 3 and 4. All patients werein the hospital and had moderate to severe HF.

As shown in Figure 5A, proANP was produced withan N-terminal Trx-tag to aid in peptide purificationand a C-terminal His-tag to aid in the isolation ofANP forms after processing. His-tag antibodies willdetect unprocessed proANP, proANP without the

FIGURE 5 Ex Vivo Analysis of proANP Processing

(A) Exogenous pro–atrial natriuretic peptide (proANP) was incubated in fresh human serum for indicated times at 37�C. Unprocessed or processed proANP was isolated by

immunoprecipitation and detected by Western blot. (B,C) Representative Western blot for normal serum (B) and heart failure (HF) serum (C). (D) Densitometric analysis

of ANP. *p < 0.05 versus 0 min.

TABLE 1 cGMP Pharmacokinetics

PcGMPCmax

(pmol/ml)

PcGMPTmax(min)

PcGMPAUClast

(pmol$min/ml)

PcGMPT1/2(min)

UcGMPVCmax

(pmol/ml)

UcGMPVTmax(min)

UcGMPVAUClast

(pmol$min/ml)

UcGMPVT1/2(min)

ANP 57.7 � 11.6 6.0 � 0.6 1,205.6 � 176.9 13.3 � 1.8 5,255.4 � 816.7 14.0 � 2.4 191,179.3 � 38,770.7 25.9 � 3.6

BNP 108.3 � 14.2* 9.0 � 1.8 3,498.6 � 488.1* 17.7 � 1.6 10,415.2 � 1,190.6* 18.0 � 3.7 498,780.3 � 51,977.7* 23.6 � 2.2

ProANP 51.5 � 4.7† 7.6 � 0.7 2,720.0 � 342.9* 40.3 � 6.7*† 5,984.9 � 417.9† 20.0 � 3.2 383,668.1 � 28,010.2* 44.8 � 3.2*†

*p < 0.05 versus ANP. †p < 0.05 versus BNP.

ANP ¼ atrial natriuretic peptide; AUClast¼ area under the concentration-time curve from time 0 to the last measurable concentration (180 min); BNP¼ B-type natriuretic peptide; cGMP¼ cyclic guanosinemonophosphate; Cmax ¼ maximum concentration; PcGMP ¼ plasma cyclic guanosine monophosphate level; Tmax ¼ time to maximum concentration; T1/2 ¼ half-life; UcGMPV ¼ urinary cyclic guanosinemonophosphate excretion.

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Trx-tag, and ANP but not processed aminoterminalproANP (Figure 5A). ProANP (w23 kDa) was processedinto a smaller molecular weight form (w4 kDa)(Figures 5B and 5C) in serum from normal controlsubjects and patients with HF, which was confirmedto be ANP by sequencing. In some subjects, a faintband around 13 kDa was determined to be proANPwithout the Trx-tag (Figure 5B).

To more precisely assess proANP processing in thecirculation, we examined proANP processing inserum from normal control subjects versus patientswith HF at 5 time points: 0, 5, 15, 60, and 180 min. Asa control, we pre-treated samples for 0 min withethylenediaminetetraacetic acid, which inhibitsproANP processing. In both normal control subjectsand patients with HF, ANP appeared after 5 min in-cubation and persisted for 15 min, then began todecrease at 60 min through 180 min (Figures 5Band 5C). The density of the ANP band from Westernblots in each group was significantly higher at eachtime point compared with 0 min, and there was nosignificant difference between normal control sub-jects and patients with HF (Figure 5D).

DISCUSSION

This is the first study to investigate proANP as a GC-Aactivator, with more sustained natriuretic, diuretic,and renal vasodilating actions than ANP or BNP andwith greater and more sustained cardiac unloadingproperties than ANP. Our results suggest that proANPis a biologically active natriuretic peptide with bothphysiological and therapeutic implications (OnlineFigure 3).

To date, the in vivo physiological actions ofproANP have not been defined, although de Bold (29)previously reported that extracts from the atrialmyocardium, which contained atrial natriuretic fac-tor, but most likely proANP as well, had potentdiuretic, natriuretic, and blood pressure–loweringproperties. In our study, we observed that proANPhad greater and more prolonged cGMP activationthan ANP, while the area under the curve of BNP forboth plasma cGMP and urinary cGMP excretion(Online Figure 2) was the highest of all 3 peptides.

Unexpectedly, proANP had significantly greaternatriuretic and diuretic actions than ANP or BNP(Figures 1A and 1B). These greater renal actions were aresult of greater tubular actions and greater increasesin renal blood flow and glomerular filtration rate. Thecardiorenal differences between proANP and matureANP may be due to: 1) the longer half-life of proANP;2) “dual” activating properties of proANP via proANPand “processed to ANP” forms; and/or 3) conversion of

proANP to active ANP forms; such as vessel dilator(ANP31–67), which works in the kidney through a non-cGMP pathway (30). Indeed, we speculate that thevessel dilator form may explain the second peak ofproANP urinary actions, which seems to occur via non-cGMP activation, as urinary cGMP was not biphasic.

Beyond the kidney, we report that proANP reducedcardiac filling pressures, including reductions inpulmonary artery pressure, PCWP, right atrial pres-sure, and systemic vascular resistance, with increasesin HR and hematocrit compared with baseline. Incontrast, these hemodynamic effects were notobserved with ANP. Notably, proANP markedlyincreased HR over 120 min (Figure 3D). We speculatethat the increased HR might be in response to volumeloss associated with decreased hematocrit (Figure 2B),but this increase in HR may lead to adverse clinicalevents due to increased myocardial oxygen demand.Further studies are required to observe HR in an HFmodel and to investigate the modulating actions ofproANP on sympathetic activity.

Both proANP and ANP reduced blood pressure, mostlikely because of actions on both the kidney and thesystemic circulation, but with one-half the hypoten-sive effects of BNP. Surprisingly, BNP showed strongerand more sustained systemic vasodilation togetherwith greater cardiac unloading effects, but with muchless natriuresis, diuresis, and renal vasodilationcompared with proANP. We cannot explain why BNPplays a different role fromANP or proANP, as all are GC-A activators. Recognizing that plasma and urinarycGMP reflects guanylyl cyclase activation,we observedthat proANP had greater and more prolonged cGMPactivation than ANP, but the area under the curve ofBNP for both plasma cGMP and urinary cGMP excretionwas greatest for all 3 peptides (Online Figure 2, Table 1).Possible explanations include BNP signaling throughan unknown receptor, differences in processing anddegradation, or resistance to degrading enzymes (31).

We demonstrate that proANP activates cGMPgeneration in GC-A- but not GC-B-overexpressingHEK293 cells. Although less than ANP, proANPclearly increased cGMP production, consistent withthe properties of a GC-A activator. This may be a directaction of proANP on GC-A, as it has been reported thatHEK293 cells lack corin, which processes proANP intoANP (13). GC-A-activating actions by proANP supportour concept that proANP may represent a biologicallyactive peptide itself, complementing mature pro-cessed ANP actions.

Although our studies support proANP as a directactivator of GC-A, in fact a dual mechanism for GC-Aactivation may be at play, as we demonstrate thatproANP can be processed to biologically active ANP in

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE:

ProANP may be a unique “dual” activator of GC-A by

either direct activation or through processing to ANP,

contributing to sodium and volume regulation.

ProANP’s in vivo cardiorenal actions continued for up

to 3 h, and processing of proANP remained intact in

patients with HF, so HF may be a target disease for

proANP treatment. ProANP may serve as a long-

lasting novel diuretic drug that can be administered

by bolus injection and be more useful in the clinical

setting compared with carperitide (ANP) or nesiritide

(BNP). For widespread clinical application, proANP

should be produced as a recombinant peptide on the

basis of its extended amino acid length.

TRANSLATIONAL OUTLOOK: Our studies advance

our understanding of the heart as an endocrine organ

and the proANP, ANP, GC-A, and cGMP system with

biological, diagnostic, and therapeutic implications.

Because proANP circulates, we must now consider it

as a biologically active hormone, and it may be useful

to develop assays that explore its biomarker potential.

Finally, its unique renal and cardiac unloading actions

may also lay the foundation for its development as a

new therapy for cardiorenal disease.

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human serum, similar to our previous studies onproBNP processing in the circulation (21). It has beenreported that corin is present in the circulation andkidneys (32), supporting potential proANP cleavage toANP in these locations (13,14). Similar to normal hu-man serum, proANP was rapidly processed to ANP inpatients with HF. Thus, our human proANP couldactivate GC-A either directly or indirectly throughprocessing to ANP in either normal control subjects orpatients with HF. These results are in contrast tothose from our studies of proBNP processing anddegradation, in which the cGMP activity of proBNPwas much less than BNP, proBNP processing to BNPwas rapid with degradation complete within 60 minin normal control subjects, and proBNP processingwas delayed (28). These differences in metabolismand bioactivity between proANP and proBNP may bekey factors in cardiovascular homeostasis.

The concept of a cGMP-deficient state in HF raisesa therapeutic opportunity, especially in HF withpreserved EF and advanced HF with reduced EF(6,33), despite results from the ASCEND-HF trial,which were negative, and supported by encouragingphase 2 clinical trials for BNP in HF, which werepositive. The failure of ASCEND-HF may be related todose, duration, and clinical endpoints (4,34). Clinicaltrials for long-term augmentation of the endogenousnatriuretic peptide/cGMP system by angiotensin-neprilysin inhibition (LCZ696) showed positive re-sults compared with angiotensin receptor blockade orangiotensin-converting enzyme inhibition. Specif-ically, LCZ696, which inhibits the degradation ofendogenous natriuretic peptides, decreased levels ofaminoterminal proBNP in patients with HF with pre-served EF compared with valsartan (PARAMOUNT[Prospective Comparison of ARNI with ARB on Man-agement of Heart Failure With Preserved EjectionFraction]) (35), and reduced risk for mortality andrehospitalization in patients with HF with reduced EFcompared with enalapril (PARADIGM-HF [Efficacyand Safety of LCZ696 Compared to Enalapril onMorbidity and Mortality of Patients With ChronicHeart Failure]) (36) and increased cGMP (37). Thesetrials suggest that enhancement of the cGMP pathwaymay be useful treatment for patients with HF.STUDY LIMITATIONS. A limitation of our study wasthe relatively small number of canines in each group.However, large normal canines compared with smallanimal studies lack hemodynamic variability and arewell established in our laboratory, thus providingstatistical significance using a smaller number of ca-nines. A second limitation was that proANP effects inanimals may not translate to humans, so clinicalstudies are needed to confirm these results.

CONCLUSIONS

ProANP represents a novel GC-A activatorwith a longerhalf-life and beneficial cardiorenal actions beyondANP and BNP. The bioactivity of exogenous proANPmay be through direct stimulation of GC-A, as well asthrough processing into ANP, as proANP is processed inthe circulation of both normal control subjects andpatients with HF. Our findings lay the rationale forfurther investigations of proANP with a focus on notonly physiological mechanisms of action, but thera-peutic implications in HF.

ACKNOWLEDGMENTS The authors deeply appreciatethe participation of the healthy volunteers andpatients with HF from our institution. The authorsacknowledge the contributions of Dr. Ingrid A.Andersen, Gail J. Harty, Sharon M. Sandberg, andDenise M. Heublein to the data collection.

REPRINT REQUESTS AND CORRESPONDENCE: Dr.Tomoko Ichiki, Mayo Clinic, Division of Cardiovas-cular Diseases, Cardiorenal Research Laboratory, 2001st Street SW, Rochester, Minnesota 55905. E-mail:ichiki.tomoko@mayo.edu.

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KEY WORDS atrial natriuretic peptides,cGMP, kidney

APPENDIX For an expanded Methods sectionand supplemental tables and figures, please seethe online version of this article.