cGMP Catabolism by Phosphodiesterase 5A RegulatesCardiac Adrenergic Stimulation by

NOS3-Dependent MechanismEiki Takimoto,* Hunter C. Champion,* Diego Belardi,* Javid Moslehi, Marco Mongillo,Evanthia Mergia, David C. Montrose, Takayoshi Isoda, Kate Aufiero, Manuela Zaccolo,

Wolfgang R. Dostmann, Carolyn J. Smith, David A. Kass

Abstract—�-Adrenergic agonists stimulate cardiac contractility and simultaneously blunt this response by coactivating NOsynthase (NOS3) to enhance cGMP synthesis and activate protein kinase G (PKG-1). cGMP is also catabolicallyregulated by phosphodiesterase 5A (PDE5A). PDE5A inhibition by sildenafil (Viagra) increases cGMP and is usedwidely to treat erectile dysfunction; however, its role in the heart and its interaction with �-adrenergic and NOS3/cGMPstimulation is largely unknown. In nontransgenic (control) murine in vivo hearts and isolated myocytes, PDE5Ainhibition (sildenafil) minimally altered rest function. However, when the hearts or isolated myocytes were stimulatedwith isoproterenol, PDE5A inhibition was associated with a suppression of contractility that was coupled to elevatedcGMP and increased PKG-1 activity. In contrast, NOS3-null hearts or controls with NOS inhibited by NG-nitro-L-arginine methyl ester, or soluble guanylate cyclase (sGC) inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one,showed no effect of PDE5A inhibition on �-stimulated contractility or PKG-1 activation. This lack of response was notattributable to altered PDE5A gene or protein expression or in vitro PDE5A activity, but rather to an absence ofsGC-generated cGMP specifically targeted to PDE5A catabolism and to a loss of PDE5A localization to z-bands.Re-expression of active NOS3 in NOS3-null hearts by adenoviral gene transfer restored PDE5A z-band localization andthe antiadrenergic efficacy of PDE5A inhibition. These data support a novel regulatory role of PDE5A in hearts underadrenergic stimulation and highlight specific coupling of PDE5A catabolic regulation with NOS3-derived cGMPattributable to protein subcellular localization and targeted synthetic/catabolic coupling. (Circ Res. 2005;96:100-109.)

Key Words: PDE5 � phosphodiesterase � sildenafil � nitric oxide synthase � contractility � z-band

Beta-adrenergic regulation of cardiac contraction is cou-pled to elevations in adenosine (cAMP) and guanosine

(cGMP) cyclic nucleotides. Increased cAMP enhances con-tractility1,2 by activating protein kinase A (PKA), whereasconcomitant stimulation of cGMP opposes this in part byactivating protein kinase G (PKG-1).3,4 The latter response isthought to be attributable to stimulation of soluble guanylatecyclase (sGC) by NO.3–9 Cyclic GMP is also synthesized byreceptor GC (rGC) coupled to natriuretic peptide stimulation,and both sources can modulate cardiac function and structure,particularly in hearts stimulated by neurohormones or me-chanical stress.3–5,10–13 cGMP is also regulated by catabolicphosphodiesterases such as phosphodiesterase 5A (PDE5A),and PDE5A inhibition by sildenafil (Viagra; SIL) and similar

compounds augments cGMP in vascular tissue and is theprimary therapy for erectile dysfunction.14,15 However, therole for PDE5A in regulating cardiac function has remainedunclear.16–18 Such clarification has become increasingly im-portant because PDE5A inhibitors are poised to becomechronic treatments for diseases such as pulmonaryhypertension.19,20

One intriguing feature of cGMP catabolic regulation byPDE5A is that in vascular tissues, it appears coupled to NOsynthase (NOS3)–dependent synthetic pathways. For exam-ple, SIL counters hypoxia-induced pulmonary hypertensionmore effectively in nontransgenic (NTG) mice than in ani-mals lacking the NOS3 isoform (NOS3�/�).21 Similarly, SILenhances erectile tone less effectively in diabetic rats than

Original received September 1, 2004; resubmission received October 26, 2004; revised resubmission received November 16, 2004; accepted November19, 2004.

From the Division of Cardiology (E.T., H.C.C., D.B., J.M., T.I., D.A.K.), Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Md;Dulbecco Telethon Institute at the Venetian Institute of Molecular Medicine (M.M., M.Z.), Padova, Italy; Department of Pharmacology and Toxicology(E.M.), Ruhr-Universitat Bochum, Medizinische Fakultat, Bochum, Germany; Department of Pharmacology (D.C.M., K.A., C.J.S.), New York MedicalCollege, Valhalla; and Department of Pharmacology (W.R.D.), University of Vermont, Burlington.

This manuscript was sent to James Willerson, Consulting Editor, for review by expert referees, editorial decision, and final disposition.*These authors contributed equally to this work.Correspondence to David A. Kass, Ross Research Bldg 835, Johns Hopkins University Hospital, 720 Rutland Ave, Baltimore, MD 21205. E-mail

dkass@jhmi.edu© 2005 American Heart Association, Inc.

Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000152262.22968.72

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healthy controls, yet this is improved by penile NOS3 genetransfer to augment NO synthesis.22 On the basis of suchfindings, we hypothesized that PDE5A regulates cardiac�-adrenergic stimulation in a NOS3-dependent manner. Us-ing mice with or lacking the NOS3 gene or with NOSpharmacologically inhibited, we found that �-adrenergiccontractile stimulation is suppressed by PDE5A inhibition,and this regulation requires NOS3 activity. This dependenceon NOS3 is attributable to specific targeting of sGC/cGMP toPDE5A and to a role of NOS/NO in properly localizingPDE5A to z-band regions within cardiomyocytes.

Materials and MethodsMale wild-type (WT) and NOS3�/� mice (C57BL6; 6 to 8 weeks old;The Jackson Laboratory) were studied. PDE5A was inhibited in vivowith SIL (100 �g/kg per minute; 37�5.2 nmol/L free plasmaconcentration); or EMD-360527/5 (160 to 300 �g/kg per minute;Merck). Both compounds have an IC50 of �10 nmol/L for purifiedPDE5A (versus 1 to 20 �mol/L for PDE1 or PDE3). In vitro studiesused 0.1 to 1 �mol/L SIL, 0.05 �mol/L tadalafil (prepared in1�PBS), or 0.1 �mol/L EMD-360527/5 in buffered 1% propanediol.In vivo and in vitro studies of vehicles alone confirmed no effects.

In Vivo StudiesIsoproterenol (ISO; 20 ng/kg per minute intravenous infusion for 5minutes) with or without PDE5A inhibitor was given to anesthetizedintact mice and in vivo heart function assessed by pressure–volume(PV) relations23 at a fixed atrial pacing rate of 600 to 650 minutes�1.Data were measured at baseline, with ISO, rebaseline, PDE5Ainhibition, and PDE5A inhibition�ISO. The ISO-only response washighly reproducible.

Isolated Myocyte StudiesExcised hearts were retroperfused by buffer containing 2,3-butanedione monoxime (1 mg/mL) and taurine (0.628 mg/mL) for 3minutes, 0.9 mg/mL collagenase (type 2; 299 U/mg; Worthington),and 0.05 mg/mL protease (Sigma) for 6 to 7 minutes. Ventricles weregently chopped, filtered (150-micron mesh), centrifuged (500rpm�1 minute), and rinsed in Tyrode’s solution with increasingcalcium (final 1.8 mmol/L Ca2�). Cells were incubated with5 �mol/L Indo-1 AM (Molecular Probes), rinsed, and studied at27°C by field stimulation in an inverted fluorescence microscope(Diaphot 200; Nikon). Sarcomere length (IonOptix) and whole-cellcalcium transient were measured. After baseline, cells were exposedto 10 nmol/L ISO, then ISO�SIL, or ISO�EMD-360527/5 at pH7.45. SIL was diluted in 0.1% dimethyl sulfoxide and EMD in

0.001% propanediol; control solutions contained similar vehicleconcentrations.

Gene and Protein ExpressionPDE5A gene expression was assessed by quantitative real-timepolymerase chain reaction (PCR). Residual genomic DNA wasremoved from mRNA by treatment with DNase I and cDNAsynthesized with the SuperScript First-Strand Synthesis System forRT-PCR (Invitrogen). Relative abundance of PDE5A mRNA wasdetermined by SYBR Green I assay (QuantiTect SYBR Green PCR;Qiagen) using the following primers: PDE5A (GenBank No.NM_153422.1) upper-primer-1493 5�-TGAGCAGTTCCTGGAAG-CCT-3�, lower-primer-1596 5�-ATGTCACCATCTGCTTGGCC-3�,product 104 bp; GAPDH (NM_008084.1) upper-primer-263 5�-ACCATCTTCCAGGAGCGAGAC-3�, lower-primer-363 5�-GCCTTCTCCATGGTGGTGAA-3�, product 101 bp; with a Gene-Amp 5700 Sequence Detection System (Applied Biosystems). PCRsamples were run in triplicate and GAPDH content used to normalizePDE5A content of different samples. Reactions (20 �L) wereperformed with 300 nmol/L of the specific primer pairs for 40 cyclesof amplification (denaturation at 95°C for 15 s, annealing at 60°C for30 s, and extension at 72°C for 30 s). Amplification specificity ofPCR products was confirmed by melting curve analysis.24 Subse-quent to the final PCR cycle, reactions were heat denatured over a35°C temperature gradient at 0.03°C/s from 60°C to 95°C.

Protein lysates from whole myocardium and isolated cardiacmyocytes were extracted in lysis buffer (No. 9803; Cell Signaling)with miniprotease inhibitor (No. 1 to 836-153; Roche) and 5% Triton(Sigma). After12 000g centrifugation �30 minutes, protein wasquantified (No. 23235; Pierce), NuPAGE lithium dodecyl sulfatesample buffer added (No. 161 to 0737; Bio-Rad), and lysateselectrophoresed on NuPAGE 4% to 12% Bis-Tris polyacrylamidegels (Invitrogen). Membranes were incubated with rabbit polyclonalantibodies raised against purified bovine lung PDE5A (1:5000; CellSignaling), the amino terminal PDE5A domain (1:5000; gift fromMauro Giorgi, University of L’Aquila, L’Aquila, Italy), or recom-binant PDE5A (1:10 000; gift from S.S. Visweswariah, IndianInstitute of Science, Bangalore, India).

Fluorescence Resonance Energy Transfer ImagingVentricular myocytes from 1- to 2-day-old Sprague-Dawley rats(Charles River Breeding Laboratories) were prepared and transfectedwith the vector carrying the cGMP sensor cygnet-2,25 in whichenhanced yellow fluorescent protein was substituted with the lesspH-sensitive variant citrine,26 and imaged 18 to 24 hours aftertransfection as described.27 Images (50- to 80-ms exposure) wereacquired every 10 seconds using custom software and processed byImageJ (NIH). Fluorescence resonance energy transfer (FRET) wasthe change in 480 nm/545 nm emission intensities (�R) on 430-nmexcitation28 expressed as percentage change more than the basal

Baseline Hemodynamic Effects of PDE5A Inhibition by SIL in NTG Mice and NOS3�/�

NTG

P

NOS3�/�

PBaseline SIL Baseline SIL

SBP (mm Hg) 94�5 86�6 �0.005 120�2 111.8�3 NS

CO (ml/min) 10.6�3 11.1�2.3 NS 9.8�1.2 10.4�1.4 NS

EF (%) 62�12 64�7 NS 54�10 59�12 NS

dP/dtmax (mm Hg/s) 11 885�2758 11 604�2387 NS 10 107�1338 11 211�1498 NS

dP/dtmax/IP (sec�1) 201�53 227�56 NS 125�21 143�23 NS

dP/dtmin (mm Hg/s) �9623�946 �8405�209 NS �11 134�1298 �11 618�1418 NS

PMX/EDV (mm Hg/s) 35.3�6.1 33.6�8.3 NS 29.2�4.1 31.8�7.0 NS

Tau (ms) 3.9�0.5 3.4�0.8 NS 5.0�0.8 4.6�0.7 NS

SBP indicates systolic blood pressure; CO, cardiac output; EF, ejection fraction; IP, instantaneous pressure at timeof dP/dtmx; PMX/EDV, maximal ventricular power normalized to end-diastolic volume; Tau, time constant of pressurerelaxation.

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intensity (R0). Cells were bathed in HEPES-buffered Ringer’smodified saline (1mmol/L CaCl2) at room temperature (20°C to22°C). For NOS inhibition studies, cells were preincubated with1 mmol/L NG-nitro-L-arginine methyl ester (L-NAME) for 30 min-utes at 37°C.

PDE5A and PKG-1 Activity AnalysisTotal low Km cGMP phosphodiesterase activity was assayed at1 �mol/L substrate by fluorescence polarization (Molecular Devices)under linear conditions or a 2-step radiolabeled method,18 with orwithout added SIL (0.1 to 1 �mol/L), tadalafil (50 nmol/L), orisobutyl-methylxanthine (IBMX; 50 �mol/L). PDE assays at1 �mol/L cGMP detected several high-affinity cGMP-PDEs(PDE5A and PDE9A) and dual-specificity PDEs (eg, PDE1C,PDE3A, PDE10A, and PDE11A).

PKG-1 activity was assayed by colorimetric analysis (CycLex)performed in whole myocytes incubated with or without added ISO(10 nmol/L), SIL (1 �mol/L), tadalafil (50 nmol/L), or sGC inhibitor1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (3 �mol/L; Sigma).After 10 minutes, cells were lysed and PKG-1 activity determined.

Immunofluorescent HistologyWT and NOS3�/� cardiomyocytes were fixed in 50% methanol/50%acetone and incubated overnight with sequence-specific PDE5A anti-

body (gift from K. Omori, Tanabe Seiyaku Co, Saitama, Japan) at1:5000 dilution and either mouse monoclonal �-actinin (1:500 dilution;Chemicon) or NOS3 (1:3000; Transduction Laboratories). Secondaryincubation used anti-rabbit Alexa 488 and anti-mouse Alexa 546 (1hour; 27°C; Molecular Probes). Cells were imaged on a Zeiss invertedepifluorescence microscope with argon-krypton laser confocal scanningsystem (UltraVIEW; PerkinElmer Life Sciences).

In Vivo Adenoviral Gene TransferIn vivo intact heart adenovirus gene transfer of NOS3 to NOS3�/� micewas performed as described.29 Hearts were exposed by limited thora-cotomy, animals cooled to 18°C to 20°C, the distal thoracic aortaclamped, and recombinant adenovirus vector (30 �L) containing acytomegalovirus promoter coupled to either a marker gene (�-galactosidase) or NOS3 (109 particles) injected into the left ventricular(LV) cavity. The aorta was clamped for 9 to 10 minutes, then released,the chest closed, and animals rewarmed. Hemodynamic studies wereperformed 3 to 5 days later (peak adenoviral gene expression).

ResultsMinimal Effect of SIL (PDE5A inhibition) onBasal FunctionSIL had minimal impact on basal cardiovascular functionin both and NOS3�/� animals (Table). A �10% decline in

Figure 1. A, PDE5A inhibition (SIL) blunts ISO-enhanced sarcomere shortening without changing the calcium transient in NTG myo-cytes but has no effect in NOS3�/� cells (example tracings on left, summary data on right; n12 from 4 hearts; *P�0.01). B, Pretreat-ment of myocytes with ODQ prevents antiadrenergic effect of PDE5A inhibition (EMD-360527/5) on ISO response (n10; exampleshown). C, LV PV loops for baseline and ISO stimulation with or without concurrent PDE5A inhibition (SIL). ISO-enhanced contractilitywas blocked by SIL in NTG but not NOS3�/� hearts. D, Summary data for maximal rate of pressure rise (dP/dtmax; n4 all groups;*P�0.001 vs all other groups; †P�0.001 vs groups without ISO stimulation). SIL alone had no effect but blocked dP/dtmax rise by ISO inNTG but not NOS3�/�. The bottom panel shows percentage change in ISO response by SIL. SV indicates stroke volume; SW, strokework; PMXI, maximal power index. *P�0.03 vs NTG response.

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systolic pressure was observed in NTG animals (similar tohumans30), but this did not occur in NOS3�/� mice.Treatment of myocytes with SIL alone slightly enhancedbasal shortening (12�5%; P0.04) but did not altercalcium transients.

Blunting of �-Adrenergic Cardiac Stimulation byPDE5A Inhibition Requires NOS3In contrast to rest function, ISO-stimulated contractility wasblunted by PDE5A inhibition in isolated myocytes and intacthearts, but this required the presence of active NOS3. In NTGmyocytes, ISO increased sarcomere shortening 200%, andthis declined by more than half with coexposure to SIL(Figure 1A). There was no accompanying change in thecalcium transient supporting a myofilament Ca2� densitiza-tion mechanism. In contrast, ISO-stimulated shortening inNOS3�/� myocytes was unaltered by SIL. Because NOS3-derived NO modifies cell function via sGC/cGMP signalingor direct interactions of NO with excitation–contractioncoupling proteins,9 we next tested the effect of inhibiting sGCactivity by ODQ31 (20 mmol/L) in NTG myocytes (Figure1B). ODQ prevented the decline in ISO response fromPDE5A inhibition.

Figure 1C and 1D shows results from in vivo heart studies.In NTG animals, ISO stimulation shifted the LV PV loopleftward, increasing its width and area consistent with greatercontractility (Figure 1C, top left). Cotreatment with SIL

suppressed this response (Figure 1C, top right). In contrast,NOS3�/� hearts showed a similar rise in contractility fromISO before and after SIL infusion (Figure 1C, bottom panels).Figure 1D provides summary data for LV maximal rate ofpressure increase (dP/dtmax) and other ejection-phase mea-sures of systolic function.

NOS3 Is Required for PDE5A Inhibition toStimulate PKG-1 and cGMPWe next tested whether PDE5A inhibition stimulated PKG-1in adult myocytes and whether this was lacking in NOS3�/�

cells (Figure 2A). PDE5A inhibition slightly enhancedPKG-1 activity under basal conditions (�10%; P�0.05). ISOalso increased PKG-1 activity, but both combined raised thisactivity �70%. However, these interventions did not changePKG-1 activity in cells lacking NOS3. Coincubation of NTGcells with ODQ also prevented PKG-1 activation by ISO orISO�SIL, further supporting a central requirement of cGMPderived by NOS-stimulated sGC for SIL modulation.

To directly monitor intracellular cGMP production, FRETanalysis was performed in normal neonatal rat myocytes(Figure 2B and 2C). ISO, SIL, and the NO donor (diethyl-amine NONOate/NO) all enhanced the FRET signal, provid-ing the first direct demonstration that PDE5A inhibitionenhances cGMP in myocytes. However, pretreatment ofmyocytes with the NOS inhibitor L-NAME blocked thecGMP rise by SIL, but not with the other stimuli.

Figure 2. A, PKG-1 activity in myocytesfrom NTG or NOS3�/� hearts. SIL andtadalafil (TAD) alone slightly raisedPKG-1 activity (P�0.05) but increased itby 70% (P�0.001) when combined withISO (30% over ISO alone; P�0.001).NOS3�/� cells showed no changes inPKG-1 activity with these interventions.ODQ blocked SIL�ISO activation ofPKG-1 in NTG myocytes. B, cGMP pro-duction measured by FRET in control ratneonatal myocytes. SIL (500 nmol/L) andISO (100 nmol/L) raised cellular cGMP(P�0.01), with a greater change by theircombination. Addition of the NO donorDEA/NO (5 �mol/L) increased this fur-ther. NOS inhibition (5 �mol/L L-NAME)blocked cGMP increase by SIL with orwithout ISO (P�0.05 vs without L-NAME)but did not alter DEA/NO or ISO-onlyresponses. C, Summary data for relativeFRET change (*P�0.05 vs untreatedcells).

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Lack of NOS3 Does Not Alter PDE5A Expressionor In Vitro ActivityPDE5A mRNA expression was 100-fold lower in whole heartthan in lung and even lower in isolated myocytes. However,expression levels were similar in NTG and NOS3�/� tissues(Figure 3A). Protein expression was also similar between

genotypes in whole heart (Figure 3B) and myocytes (Figure 3C),with a prominent band observed at �95 kDa that matched thatin lung. A second �70-kDa band was consistently observed inheart tissue that either reflected a splice variant or proteolyticfragment. Similar findings were obtained with alternative anti-bodies18,32 (data not shown). In vitro PDE5A activity (�30% of

Figure 4. Confocal immunostaining ofcardiomyocyte PDE5A distribution. A,PDE5A in NTG myocytes was present incytosol but more prominent at z-bands.Corresponding red staining is for z-bandprotein �-actinin. B, PDE5A staining isprevented by coincubation with blockingpeptide (5:1 BP/Ab; red �-actinin; CellSignaling); whereas PDE1C staining isunaffected by this blocking peptide (C,left; right panel �-actinin). D, PDE5Astaining in another NTG cell, costainedfor NOS3 (E, red) with colocalization atz-bands (F, yellow). In contrast, NOS3�/�

myocytes displayed diffuse PDE5A distri-bution with less z-band localization (Gand I). Z-bands were preserved asshown by �-actinin staining in the samecell (H).

Figure 3. A, PDE5A gene expression byRT-PCR under basal conditions in NTGand NOS3�/� hearts (HRT) and isolatedmyocytes (MYO). Lung from NTG animalis shown for comparison (n4 eachgroup; *P�0.01 vs myocyte). B, PDE5Aprotein expression in whole myocardium.Example Western blot (protein loadingshown) and summary data from four tosix separate blots (n�6 hearts in eachgroup) are displayed. Two bands wereobserved, one similar to that in lung andanother near 70 kDa; expression wassimilar in both genotypes. C, PDE5A pro-tein expression in isolated myocytes. A92-kDa band (a) was present concordantwith lung, and a lower kDa band (b) wasalso observed. There were no genotypedifferences. D, cGMP–esterase activityassays. Total activity (CON; normalizedfluorescence polarization [FP] units) waslargely blocked by the broad PDE inhibi-tor IBMX (50 �mol/L), whereas SIL (100nmol/L) lowered activity by �30%(P�0.001). There were no significant ge-notype differences in enzyme activity forwhole myocardium or myocytes.

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total cGMP–esterase activity) was also similar between geno-types (Figure 3D). Coincubation with IBMX (positive control)lowered activity by �90%. Similar results for PDE5A-dependent cGMP–esterase activity were obtained by radioenzyme assay18 (32�7.3% NTG [n9]; 37.9�8.2% NOS3�/�

[n8]; PNS).

Lack of NOS3 or Chronic NOS Activity AltersMyocyte Localization of PDE5AGiven similar gene/protein expression and in vitro activity,we next tested whether PDE5A cellular localization wasaltered by the lack of NOS3. In NTG cells, PDE5A waspresent throughout the cell but also localized to z-bandstriations (Figure 4A, green PDE5A; red �-actinin). PDE5AImmunostaining was inhibited by specific blocking peptide(4B, left), whereas this same peptide did not block PDE1Cstaining (4C, left), supporting assay specificity. PDE5Adisplayed colocalization with NOS3 (4D through 4F) atz-band striations. However, in NOS3�/� cells, PDE5A distri-bution was diffuse (Figure 4G) with less localization toz-bands (Figure 4H, �-actinin; Figure 4I, colocalization).

To test whether altered PDE5A localization resulted fromthe absence of NOS3 protein or NOS activity, L-NAME (1mg/mL in drinking water or 50 mg/kg IP for acute) was

administered to NTG mice for varying periods and myocytesthen isolated and stained for PDE5A and �-actinin. Withsubacute NOS inhibition, PDE5A remained localized toz-bands, whereas it shifted away from these structures (dif-fuse) with sustained L-NAME exposure (Figure 5A). Z-bandsremained present (Figure 5B). Thus, chronic NOS activitywas the key requirement for maintaining normal myocytePDE5A localization.

Because 1 to 2 hours of L-NAME exposure did not alterPDE5A z-band localization but inhibited NOS activity, thisprotocol was used to define the role each played in modulat-ing the efficacy of PDE5A inhibition (Figure 5C). As inNOS3�/� hearts, acute NOS inhibition eliminated the antiad-renergic effect of SIL. This was partially rescued by coad-ministration of an exogenous NO donor (DEA/NO; 4 �g/kgper minute) supporting NO-generated cGMP as central toPDE5A-catabolic regulation. However, DEA/NO did notrestore SIL efficacy in NOS3�/� mice. Thus, both NOSactivity and PDE5A z-band localization were required foradrenergic modulation by PDE5A inhibitors.

Re-Expression of NOS3 in NOS3-Null HeartRestores PDE5A Localization and FunctionTo further test the importance of chronic NOS3 expressionand activity on PDE5A cardiac regulation, we re-expressed

Figure 5. A, Chronic NOS inhibition by L-NAME alters PDE5A myocyte distribution. With 1- to 2-hour L-NAME exposure, PDE5A (green)remained localized to z-band structures; however, sustained inhibition reduced such localization. Z-bands (B, �-actinin) were pre-served. C, Impact of SIL on ISO-mediated inotropy is modified by NOS3 activity or an NO donor. Data show percentage change inISO-enhanced dP/dtmax attributable to SIL in NTG and NOS3�/�, NTG cotreated with 1 to 2 hours of L-NAME; the same condition withDEA/NO added as an NO donor, and NOS3�/� cotreated with DEA/NO. Inhibiting NOS with L-NAME prevented SIL from impeding anISO-inotropic response, but this was partially restored by DEA/NO. The latter did not restore SIL effects in NOS3�/� hearts. *P�0.01 vsother conditions.

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NOS3 in NOS3-null hearts by means of in vivo whole heartadenoviral gene transfer. NOS3 was primarily expressed inmyocytes, shown by the lack of coimmunostaining betweenNOS3 and endothelial-specific caveolin I (Figure 6A through6C). NOS3 protein and calcium-dependent activity wererestored to near control levels (Figure 6D and 6E), andre-expressed NOS3 was coimmunoprecipitated withmyocyte-specific caveolin-3. Myocytes from these heartsagain demonstrated PDE5A localization at z-bands (Figure6F through 6H) and recovery of the PDE5A inhibitor effect toblunt ISO-stimulated contractility (Figure 6I).

DiscussionThis study reveals a strong interdependence between PDE5A-mediated cGMP catabolism and cGMP synthesis by NOS3 in

regulating adrenergic-stimulated cardiac contractility. First,NOS3 and consequent NO/sGC activity would appear toprovide the critical substrate targeted to catabolism byPDE5A. Second, chronic NOS activity is required for PDE5Ato be properly localized to z-band regions near adrenergicreceptor-complex signaling proteins. Loss of either compo-nent was sufficient to inhibit antiadrenergic effects of PDE5Ainhibitors, and chronic lack of NOS activity combined bothchanges. These data provide the most comprehensive analysisof PDE5A physiology in cardiac myocytes and the intactheart to date and reveal a novel mechanism to explainvariable responses to PDE5A inhibitors in patients withimpaired NO synthesis.

NO3,4,9 and �-adrenergic stimulation6,7 enhance cGMP,and thereby PKG-1 activity, and this was confirmed in the

Figure 6. Adenoviral in vivo gene transfer of NOS3 to NOS3�/� heart. A through C, Confocal images show vascular caveolin-1 staining(A, green), NOS3 (B, red), and combined images (C) in NOS3�/� hearts transfected with AdNOS3. NOS3 was primarily restored to myo-cytes by gene transfer. D, Western blot for NOS3 in control (WT), NOS3�/� transfected with �-galactosidase (Ad�gal), and NOS3�/�

transfected with NOS3 (AdNOS3). Bottom panel shows immunoprecipitate to NOS3 probed for caveolin-3 (cav3). E, NOS activityassessed by arginine–citrulline conversion was restored to NTG levels in NOS3�/��AdNOS3. F through H, Confocal images of PDE5A(F, green), NOS3 (G, red), and colocalization of both proteins (H, yellow) in myocyte from NOS3�/� heart transfected with AdNOS3.NOS3 restoration led to PDE5A being relocalized to z-bands and colocalizing with NOS3. I, Restored effectiveness of PDE5A-I (SIL) forblunting the contractile (dP/dtmax) response to ISO.

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current study (Figure 2). However, these are not the solestimuli because natriuretic peptides acting through rGC alsoincrease cGMP to activate PKG-1. In the NOS3�/� heart, thispathway may even be somewhat upregulated to help maintainbasal myocardial cGMP33 and PKG-1 activity (Figure 2A).Yet we found that lack of NOS3 was sufficient to preventPKG-1 activation and antiadrenergic effects of PDE5A in-hibitors. Similar findings with acute NOS or sGC inhibitionfurther support a specific interaction between NOS-derivedcGMP and PDE5A catabolism.

The antiadrenergic effect of PDE5A inhibition likely re-sulted from PKG-1 activation, which depresses myofilamentcalcium sensitivity by enhancing troponin I phosphoryla-tion.3,4 This was indicated by the lack of Ca2�-transientchange despite blunted sarcomere shortening when SIL wasadded to ISO. cGMP also activates dual-sensitivity PDEs thatlower adrenergic stimulated cAMP,34 but one would expect achange in calcium transient by this mechanism. As has beenshown for NOS-induced cGMP synthesis,8,9,33,35 effects ofPDE5A inhibition on contraction were negligible at rest butincreased during �-adrenergic costimulation. This likelystems from increased cGMP synthesis and PKG-1 activationbecause of calcium-activated NOS,6 �3-GI–coupled signal-ing,7 and cAMP inhibition of cGMP-PDEs.36 The FRETanalysis found cGMP increased similarly with ISO with orwithout L-NAME, although PKG-1 activation with ISO(�SIL) was absent in myocytes lacking NOS3 and those withsGC inhibited by ODQ. The latter suggests coupling of NOSactivity to adrenergic stimulation was important.

NOS3 activity and cardiomyocyte PDE5A localizationwere altered in NOS3�/� hearts and NTG hearts exposed tochronic NOS inhibition, and either mechanism was sufficientto explain loss of a SIL response. When NOS was acutelyinhibited, PDE5A remained localized to z-bands, yet theantiadrenergic effect of SIL was lost. NO infusion partiallyrestored a SIL response in NTG hearts yet had no impact inNOS3�/� hearts because the latter still had PDE5A shiftedaway from the z-bands. Figure 7 summarizes our proposedmodel. After activation of the �-adrenergic receptor, cAMPand cGMP are stimulated, the latter principally because ofactivation of NOS3 and its target sGC. The effect of thiscGMP on PKG-1 activation is contained within a strategiccompartment by the localization of PDE5A to z-bands,enabling modulation of PKA/calcium–stimulated contractili-ty by SIL. Acute NOS3 inhibition removes the criticalsubstrate to the PDE5A complex and so eliminates theantiadrenergic effect of SIL. Chronic NOS3 inhibition orNOS3�/� further results in the loss of PDE5A from thisstrategic location, eliminating SIL effectiveness even if NO isprovided exogenously.

The exact mechanism by which NOS3 activity regulatesmyocyte localization of PDE5A remains unknown, but sev-eral possibilities deserve mention. NOS3-stimulated cGMPcould target PDE5A directly by binding to its GAF domain37

or activating its regulatory domain via PKG-1.38 This wouldserve to increase PDE5A enzyme activity39 and therebyprovide an environment wherein the effects of PDE5A

Figure 7. Model of NOS3-PDE5A coregulation of �-adrenergic stimulated contractility in the heart. In a wild-type (WT) normal myocyte(left), �-adrenergic stimulation triggers adenylate–cyclase (AC)-coupled activation of cAMP and NOS3/sGC generation of cGMP. cAMPenhances intracellular calcium and activates PKA to enhance contractility. cGMP signaling is compartmentalized by regional placementof PDE5A, which targets the cGMP–PKG activity to a region strategically linked to adrenergic regulation. Acute PDE5A inhibitionenhances localized cGMP and PKG activity to blunt adrenergic stimulation. Acute NOS3 blockade by L-NAME removes the requiredcGMP substrate and thus efficacy of PDE5A inhibition, but exogenous NO can still restore efficacy. With genetic lack of NOS3 or withchronic NOS inhibition (right), PDE5A moves away from z-bands (left). This removes its physiologic regulation of acute adrenergic sig-naling, and exogenous NO can no longer restore the efficacy of PDE5A inhibition.

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inhibition were enhanced. The data also suggest that NOS3 orits product could play a direct role in PDE5A localization,helping PDE5A interact with a scaffold protein that anchorsit to z-bands. A-Kinase anchoring proteins perform such arole by targeting PKA to specific kinases, PDEs, and othersignaling proteins to localize signaling.40–42 CorrespondingcGMP-dependent protein kinase anchoring proteins remainpoorly defined, but such proteins might play an analogousrole in localizing PDE5A activity. Intriguingly, a similar lossof PDE5A from z-band regions has been observed in caninehearts with dilated cardiomyopathy, suggesting that alteredlocalization and accompanying physiologic regulation maycontribute to cardiac disease.

Previous studies have reported low levels of PDE5Aexpression in the myocardium43,44 and minimal effects ofPDE5A inhibition on resting heart function.16–18,30 This hasled to the conclusion that PDE5A plays little role in the heart.The current results support this under basal conditions, butthey reveal a very different situation during �-stimulation.Importantly, having a low expression level does not imply alack of physiologic impact, particularly if strategic intracel-lular placement can confer targeted signaling effects. Thecurrent results are supported by previous in vivo canine heartdata,18 and testing in humans would seem warranted.

Increasing cGMP in the heart by natriuretic peptide–coupled synthesis can counter stress-response remodeling/cardiac hypertrophy.11,12 The current data raise the possibilitythat a similar benefit may be obtained by manipulating cGMPcatabolic pathways. They further imply that therapeuticefficacy of a PDE5A inhibitor depends on NOS3 expressionand activity, and this could underlie hyporesponsiveness inmany patients receiving this treatment. As the use of PDE5Ainhibitors expands to diseases such as pulmonary hyperten-sion, these considerations will take on more clinical signifi-cance. Strategies to upregulate NOS3 activity may prove avaluable approach to augment the influence of PDE5A and itsinhibitors.

AcknowledgmentsThis work was supported by NIH grants P01-HL59408, RO1-HL-47511, and P50-HL52307 (to D.A.K.); a Uehara Memorial Founda-tion grant and American Heart Association Fellowship Grant (toE.T.); Telethon Italy (TCP00089); the European Union (QLK3-CT-2002-02149); the Italian Cystic Fibrosis Research Foundation; theFondazione Compagnia di San Paolo (to M.Z.); NSF (MCB-9983097), and the Totman Medical Research Trust (to W.R.D.); andR29HL54081, RO1HL069061, and AHANY:GIA-0151273T (toC.J.S.). We thank Doris Koesling and Nazareno Paolocci forassistance, Dr Mauro Giorgi for generously providing PDE5Aantibody, and Sejal Shah, Naziya Rahman, Jessy Mathews, QizhiGuan, and Shilpa Challa (CJS Laboratory) for technical assistance.

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Dostmann, Carolyn J. Smith and David A. KassMergia, David C. Montrose, Takayoshi Isoda, Kate Aufiero, Manuela Zaccolo, Wolfgang R.

Eiki Takimoto, Hunter C. Champion, Diego Belardi, Javid Moslehi, Marco Mongillo, EvanthiaNOS3-Dependent Mechanism

cGMP Catabolism by Phosphodiesterase 5A Regulates Cardiac Adrenergic Stimulation by

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