Published online before print December 2, 2004, doi: 10.1161/01.RES.0000152262.22968.72
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© 2005 American Heart Association, Inc.
Cellular Biology |
cGMP Catabolism by Phosphodiesterase 5A Regulates Cardiac Adrenergic Stimulation by NOS3-Dependent Mechanism
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-Universität Bochum, Medizinische Fakultät, Bochum, Germany; Department of Pharmacology (D.C.M., K.A., C.J.S.), New York Medical College, Valhalla; and Department of Pharmacology (W.R.D.), University of Vermont, Burlington.
Correspondence to David A. Kass, Ross Research Bldg 835, Johns Hopkins University Hospital, 720 Rutland Ave, Baltimore, MD 21205. E-mail dkass{at}jhmi.edu
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Abstract |
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ß-Adrenergic agonists stimulate cardiac contractility and simultaneously blunt this response by coactivating NO synthase (NOS3) to enhance cGMP synthesis and activate protein kinase G (PKG-1). cGMP is also catabolically regulated by phosphodiesterase 5A (PDE5A). PDE5A inhibition by sildenafil (Viagra) increases cGMP and is used widely to treat erectile dysfunction; however, its role in the heart and its interaction with ß-adrenergic and NOS3/cGMP stimulation is largely unknown. In nontransgenic (control) murine in vivo hearts and isolated myocytes, PDE5A inhibition (sildenafil) minimally altered rest function. However, when the hearts or isolated myocytes were stimulated with isoproterenol, PDE5A inhibition was associated with a suppression of contractility that was coupled to elevated cGMP 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 ß-stimulated contractility or PKG-1 activation. This lack of response was not attributable to altered PDE5A gene or protein expression or in vitro PDE5A activity, but rather to an absence of sGC-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 and the antiadrenergic efficacy of PDE5A inhibition. These data support a novel regulatory role of PDE5A in hearts under adrenergic stimulation and highlight specific coupling of PDE5A catabolic regulation with NOS3-derived cGMP attributable to protein subcellular localization and targeted synthetic/catabolic coupling.
Key Words: PDE5 • phosphodiesterase • sildenafil • nitric oxide synthase • contractility • z-band
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Introduction |
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Beta-adrenergic regulation of cardiac contraction is coupled to elevations in adenosine (cAMP) and guanosine (cGMP) cyclic nucleotides. Increased cAMP enhances contractility1,2 by activating protein kinase A (PKA), whereas concomitant stimulation of cGMP opposes this in part by activating protein kinase G (PKG-1).3,4 The latter response is thought to be attributable to stimulation of soluble guanylate cyclase (sGC) by NO.3–9 Cyclic GMP is also synthesized by receptor GC (rGC) coupled to natriuretic peptide stimulation, and both sources can modulate cardiac function and structure, particularly in hearts stimulated by neurohormones or mechanical stress.3–5,10–13 cGMP is also regulated by catabolic phosphodiesterases such as phosphodiesterase 5A (PDE5A), and PDE5A inhibition by sildenafil (Viagra; SIL) and similar compounds augments cGMP in vascular tissue and is the primary therapy for erectile dysfunction.14,15 However, the role for PDE5A in regulating cardiac function has remained unclear.16–18 Such clarification has become increasingly important because PDE5A inhibitors are poised to become chronic treatments for diseases such as pulmonary hypertension.19,20
One intriguing feature of cGMP catabolic regulation by PDE5A is that in vascular tissues, it appears coupled to NO synthase (NOS3)-dependent synthetic pathways. For example, SIL counters hypoxia-induced pulmonary hypertension more effectively in nontransgenic (NTG) mice than in animals lacking the NOS3 isoform (NOS3–/–).21 Similarly, SIL enhances erectile tone less effectively in diabetic rats than healthy controls, yet this is improved by penile NOS3 gene transfer to augment NO synthesis.22 On the basis of such findings, we hypothesized that PDE5A regulates cardiac ß-adrenergic stimulation in a NOS3-dependent manner. Using mice with or lacking the NOS3 gene or with NOS pharmacologically inhibited, we found that ß-adrenergic contractile stimulation is suppressed by PDE5A inhibition, and this regulation requires NOS3 activity. This dependence on NOS3 is attributable to specific targeting of sGC/cGMP to PDE5A and to a role of NOS/NO in properly localizing PDE5A to z-band regions within cardiomyocytes.
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Materials and Methods |
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Male wild-type (WT) and NOS3–/– mice (C57BL6; 6 to 8 weeks old; The Jackson Laboratory) were studied. PDE5A was inhibited in vivo with SIL (100 µg/kg per minute; 37±5.2 nmol/L free plasma concentration); or EMD-360527/5 (160 to 300 µg/kg per minute; Merck). Both compounds have an IC50 of
10 nmol/L for purified PDE5A (versus 1 to 20 µmol/L for PDE1 or PDE3). In vitro studies used 0.1 to 1 µmol/L SIL, 0.05 µmol/L tadalafil (prepared in 1xPBS), or 0.1 µmol/L EMD-360527/5 in buffered 1% propanediol. In vivo and in vitro studies of vehicles alone confirmed no effects.
In Vivo Studies
Isoproterenol (ISO; 20 ng/kg per minute intravenous infusion for 5 minutes) with or without PDE5A inhibitor was given to anesthetized intact 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, PDE5A inhibition, and PDE5A inhibition+ISO. The ISO-only response was highly reproducible.
Isolated Myocyte Studies
Excised hearts were retroperfused by buffer containing 2,3-butanedione monoxime (1 mg/mL) and taurine (0.628 mg/mL) for 3 minutes, 0.9 mg/mL collagenase (type 2; 299 U/mg; Worthington), and 0.05 mg/mL protease (Sigma) for 6 to 7 minutes. Ventricles were gently chopped, filtered (150-micron mesh), centrifuged (500 rpmx1 minute), and rinsed in Tyrode’s solution with increasing calcium (final 1.8 mmol/L Ca2+). Cells were incubated with 5 µmol/L Indo-1 AM (Molecular Probes), rinsed, and studied at 27°C by field stimulation in an inverted fluorescence microscope (Diaphot 200; Nikon). Sarcomere length (IonOptix) and whole-cell calcium transient were measured. After baseline, cells were exposed to 10 nmol/L ISO, then ISO+SIL, or ISO+EMD-360527/5 at pH 7.45. SIL was diluted in 0.1% dimethyl sulfoxide and EMD in 0.001% propanediol; control solutions contained similar vehicle concentrations.
Gene and Protein Expression
PDE5A gene expression was assessed by quantitative real-time polymerase chain reaction (PCR). Residual genomic DNA was removed from mRNA by treatment with DNase I and cDNA synthesized with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Relative abundance of PDE5A mRNA was determined by SYBR Green I assay (QuantiTect SYBR Green PCR; Qiagen) using the following primers: PDE5A (GenBank No. NM_153422.1) upper-primer-1493 5'-TGAGCAGTTCCTGGAAGCCT-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 GeneAmp 5700 Sequence Detection System (Applied Biosystems). PCR samples were run in triplicate and GAPDH content used to normalize PDE5A content of different samples. Reactions (20 µL) were performed with 300 nmol/L of the specific primer pairs for 40 cycles of amplification (denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s). Amplification specificity of PCR products was confirmed by melting curve analysis.24 Subsequent to the final PCR cycle, reactions were heat denatured over a 35°C temperature gradient at 0.03°C/s from 60°C to 95°C.
Protein lysates from whole myocardium and isolated cardiac myocytes 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 x30 minutes, protein was quantified (No. 23235; Pierce), NuPAGE lithium dodecyl sulfate sample buffer added (No. 161 to 0737; Bio-Rad), and lysates electrophoresed on NuPAGE 4% to 12% Bis-Tris polyacrylamide gels (Invitrogen). Membranes were incubated with rabbit polyclonal antibodies raised against purified bovine lung PDE5A (1:5000; Cell Signaling), the amino terminal PDE5A domain (1:5000; gift from Mauro Giorgi, University of L’Aquila, L’Aquila, Italy), or recombinant PDE5A (1:10 000; gift from S.S. Visweswariah, Indian Institute of Science, Bangalore, India).
Fluorescence Resonance Energy Transfer Imaging
Ventricular myocytes from 1- to 2-day-old Sprague-Dawley rats (Charles River Breeding Laboratories) were prepared and transfected with the vector carrying the cGMP sensor cygnet-2,25 in which enhanced yellow fluorescent protein was substituted with the less pH-sensitive variant citrine,26 and imaged 18 to 24 hours after transfection as described.27 Images (50- to 80-ms exposure) were acquired every 10 seconds using custom software and processed by ImageJ (NIH). Fluorescence resonance energy transfer (FRET) was the change in 480 nm/545 nm emission intensities (
R) on 430-nm excitation28 expressed as percentage change more than the basal intensity (R0). Cells were bathed in HEPES-buffered Ringer’s modified saline (1mmol/L CaCl2) at room temperature (20°C to 22°C). For NOS inhibition studies, cells were preincubated with 1 mmol/L NG-nitro-L-arginine methyl ester (L-NAME) for 30 minutes at 37°C.
PDE5A and PKG-1 Activity Analysis
Total low Km cGMP phosphodiesterase activity was assayed at 1 µmol/L substrate by fluorescence polarization (Molecular Devices) under linear conditions or a 2-step radiolabeled method,18 with or without added SIL (0.1 to 1 µmol/L), tadalafil (50 nmol/L), or isobutyl-methylxanthine (IBMX; 50 µmol/L). PDE assays at 1 µmol/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 µmol/L), tadalafil (50 nmol/L), or sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (3 µmol/L; Sigma). After 10 minutes, cells were lysed and PKG-1 activity determined.
Immunofluorescent Histology
WT and NOS3–/– cardiomyocytes were fixed in 50% methanol/50% acetone and incubated overnight with sequence-specific PDE5A antibody (gift from K. Omori, Tanabe Seiyaku Co, Saitama, Japan) at 1:5000 dilution and either mouse monoclonal 
-actinin (1:500 dilution; Chemicon) or NOS3 (1:3000; Transduction Laboratories). Secondary incubation used anti-rabbit Alexa 488 and anti-mouse Alexa 546 (1 hour; 27°C; Molecular Probes). Cells were imaged on a Zeiss inverted epifluorescence microscope with argon-krypton laser confocal scanning system (UltraVIEW; PerkinElmer Life Sciences).
In Vivo Adenoviral Gene Transfer
In vivo intact heart adenovirus gene transfer of NOS3 to NOS3–/– mice was performed as described.29 Hearts were exposed by limited thoracotomy, animals cooled to 18°C to 20°C, the distal thoracic aorta clamped, and recombinant adenovirus vector (30 µL) containing a cytomegalovirus promoter coupled to either a marker gene (ß-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 were performed 3 to 5 days later (peak adenoviral gene expression).
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Results |
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Minimal Effect of SIL (PDE5A inhibition) on Basal Function
SIL had minimal impact on basal cardiovascular function in both and NOS3–/– animals (Table). A <10% decline in systolic pressure was observed in NTG animals (similar to humans30), but this did not occur in NOS3–/– mice. Treatment of myocytes with SIL alone slightly enhanced basal shortening (12±5%; P=0.04) but did not alter calcium transients.
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Blunting of ß-Adrenergic Cardiac Stimulation by PDE5A Inhibition Requires NOS3
In contrast to rest function, ISO-stimulated contractility was blunted by PDE5A inhibition in isolated myocytes and intact hearts, but this required the presence of active NOS3. In NTG myocytes, ISO increased sarcomere shortening >200%, and this declined by more than half with coexposure to SIL (Figure 1A). There was no accompanying change in the calcium transient supporting a myofilament Ca2+ densitization mechanism. In contrast, ISO-stimulated shortening in NOS3–/– myocytes was unaltered by SIL. Because NOS3-derived NO modifies cell function via sGC/cGMP signaling or direct interactions of NO with excitation-contraction coupling proteins,9 we next tested the effect of inhibiting sGC activity by ODQ31 (20 mmol/L) in NTG myocytes (Figure 1B). ODQ prevented the decline in ISO response from PDE5A inhibition.
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P<0.001 vs groups without ISO stimulation). SIL alone had no effect but blocked dP/dtmax rise by ISO in NTG but not NOS3–/–. The bottom panel shows percentage change in ISO response by SIL. SV indicates stroke volume; SW, stroke work; PMXI, maximal power index. *P<0.03 vs NTG response.Figure 1C and 1D shows results from in vivo heart studies. In NTG animals, ISO stimulation shifted the LV PV loop leftward, increasing its width and area consistent with greater contractility (Figure 1C, top left). Cotreatment with SIL suppressed this response (Figure 1C, top right). In contrast, NOS3–/– hearts showed a similar rise in contractility from ISO before and after SIL infusion (Figure 1C, bottom panels). Figure 1D provides summary data for LV maximal rate of pressure increase (dP/dtmax) and other ejection-phase measures of systolic function.
NOS3 Is Required for PDE5A Inhibition to Stimulate PKG-1 and cGMP
We next tested whether PDE5A inhibition stimulated PKG-1 in adult myocytes and whether this was lacking in NOS3–/– cells (Figure 2A). PDE5A inhibition slightly enhanced PKG-1 activity under basal conditions (10%; P<0.05). ISO also increased PKG-1 activity, but both combined raised this activity 70%. However, these interventions did not change PKG-1 activity in cells lacking NOS3. Coincubation of NTG cells with ODQ also prevented PKG-1 activation by ISO or ISO+SIL, further supporting a central requirement of cGMP derived by NOS-stimulated sGC for SIL modulation.
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To directly monitor intracellular cGMP production, FRET analysis was performed in normal neonatal rat myocytes (Figure 2B and 2C). ISO, SIL, and the NO donor (diethylamine NONOate/NO) all enhanced the FRET signal, providing the first direct demonstration that PDE5A inhibition enhances cGMP in myocytes. However, pretreatment of myocytes with the NOS inhibitor L-NAME blocked the cGMP rise by SIL, but not with the other stimuli.
Lack of NOS3 Does Not Alter PDE5A Expression or In Vitro Activity
PDE5A mRNA expression was 100-fold lower in whole heart than 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 that in lung. A second 70-kDa band was consistently observed in heart tissue that either reflected a splice variant or proteolytic fragment. Similar findings were obtained with alternative antibodies18,32 (data not shown). In vitro PDE5A activity (30% of total cGMP-esterase activity) was also similar between genotypes (Figure 3D). Coincubation with IBMX (positive control) lowered activity by 90%. Similar results for PDE5A-dependent cGMP-esterase activity were obtained by radio enzyme assay18 (32±7.3% NTG [n=9]; 37.9±8.2% NOS3–/– [n=8]; P=NS).
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6 hearts in each group) are displayed. Two bands were observed, one similar to that in lung and another near 70 kDa; expression was similar in both genotypes. C, PDE5A protein expression in isolated myocytes. A 92-kDa band (a) was present concordant with lung, and a lower kDa band (b) was also observed. There were no genotype differences. D, cGMP-esterase activity assays. Total activity (CON; normalized fluorescence polarization [FP] units) was largely blocked by the broad PDE inhibitor IBMX (50 µmol/L), whereas SIL (100 nmol/L) lowered activity by
Lack of NOS3 or Chronic NOS Activity Alters Myocyte Localization of PDE5A
Given similar gene/protein expression and in vitro activity, we next tested whether PDE5A cellular localization was altered by the lack of NOS3. In NTG cells, PDE5A was present throughout the cell but also localized to z-band striations (Figure 4A, green PDE5A; red -actinin). PDE5A Immunostaining was inhibited by specific blocking peptide (4B, left), whereas this same peptide did not block PDE1C staining (4C, left), supporting assay specificity. PDE5A displayed colocalization with NOS3 (4D through 4F) at z-band striations. However, in NOS3–/– cells, PDE5A distribution was diffuse (Figure 4G) with less localization to z-bands (Figure 4H, -actinin; Figure 4I, colocalization).
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To test whether altered PDE5A localization resulted from the absence of NOS3 protein or NOS activity, L-NAME (1 mg/mL in drinking water or 50 mg/kg IP for acute) was administered to NTG mice for varying periods and myocytes then isolated and stained for PDE5A and -actinin. With subacute NOS inhibition, PDE5A remained localized to z-bands, whereas it shifted away from these structures (diffuse) with sustained L-NAME exposure (Figure 5A). Z-bands remained present (Figure 5B). Thus, chronic NOS activity was the key requirement for maintaining normal myocyte PDE5A localization.
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Because 1 to 2 hours of L-NAME exposure did not alter PDE5A z-band localization but inhibited NOS activity, this protocol was used to define the role each played in modulating the efficacy of PDE5A inhibition (Figure 5C). As in NOS3–/– hearts, acute NOS inhibition eliminated the antiadrenergic effect of SIL. This was partially rescued by coadministration of an exogenous NO donor (DEA/NO; 4 µg/kg per minute) supporting NO-generated cGMP as central to PDE5A-catabolic regulation. However, DEA/NO did not restore SIL efficacy in NOS3–/– mice. Thus, both NOS activity and PDE5A z-band localization were required for adrenergic modulation by PDE5A inhibitors.
Re-Expression of NOS3 in NOS3-Null Heart Restores PDE5A Localization and Function
To further test the importance of chronic NOS3 expression and activity on PDE5A cardiac regulation, we re-expressed NOS3 in NOS3-null hearts by means of in vivo whole heart adenoviral gene transfer. NOS3 was primarily expressed in myocytes, shown by the lack of coimmunostaining between NOS3 and endothelial-specific caveolin I (Figure 6A through 6C). NOS3 protein and calcium-dependent activity were restored to near control levels (Figure 6D and 6E), and re-expressed NOS3 was coimmunoprecipitated with myocyte-specific caveolin-3. Myocytes from these hearts again demonstrated PDE5A localization at z-bands (Figure 6F through 6H) and recovery of the PDE5A inhibitor effect to blunt ISO-stimulated contractility (Figure 6I).
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Discussion |
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This 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 to provide the critical substrate targeted to catabolism by PDE5A. Second, chronic NOS activity is required for PDE5A to be properly localized to z-band regions near adrenergic receptor-complex signaling proteins. Loss of either component was sufficient to inhibit antiadrenergic effects of PDE5A inhibitors, and chronic lack of NOS activity combined both changes. These data provide the most comprehensive analysis of PDE5A physiology in cardiac myocytes and the intact heart to date and reveal a novel mechanism to explain variable responses to PDE5A inhibitors in patients with impaired NO synthesis.
NO3,4,9 and ß-adrenergic stimulation6,7 enhance cGMP, and thereby PKG-1 activity, and this was confirmed in the current study (Figure 2). However, these are not the sole stimuli because natriuretic peptides acting through rGC also increase cGMP to activate PKG-1. In the NOS3–/– heart, this pathway may even be somewhat upregulated to help maintain basal myocardial cGMP33 and PKG-1 activity (Figure 2A). Yet we found that lack of NOS3 was sufficient to prevent PKG-1 activation and antiadrenergic effects of PDE5A inhibitors. Similar findings with acute NOS or sGC inhibition further support a specific interaction between NOS-derived cGMP and PDE5A catabolism.
The antiadrenergic effect of PDE5A inhibition likely resulted from PKG-1 activation, which depresses myofilament calcium sensitivity by enhancing troponin I phosphorylation.3,4 This was indicated by the lack of Ca2+-transient change despite blunted sarcomere shortening when SIL was added to ISO. cGMP also activates dual-sensitivity PDEs that lower adrenergic stimulated cAMP,34 but one would expect a change in calcium transient by this mechanism. As has been shown for NOS-induced cGMP synthesis,8,9,33,35 effects of PDE5A inhibition on contraction were negligible at rest but increased during ß-adrenergic costimulation. This likely stems from increased cGMP synthesis and PKG-1 activation because of calcium-activated NOS,6 ß3-GI-coupled signaling,7 and cAMP inhibition of cGMP-PDEs.36 The FRET analysis found cGMP increased similarly with ISO with or without L-NAME, although PKG-1 activation with ISO (±SIL) was absent in myocytes lacking NOS3 and those with sGC inhibited by ODQ. The latter suggests coupling of NOS activity to adrenergic stimulation was important.
NOS3 activity and cardiomyocyte PDE5A localization were altered in NOS3–/– hearts and NTG hearts exposed to chronic NOS inhibition, and either mechanism was sufficient to explain loss of a SIL response. When NOS was acutely inhibited, PDE5A remained localized to z-bands, yet the antiadrenergic effect of SIL was lost. NO infusion partially restored a SIL response in NTG hearts yet had no impact in NOS3–/– hearts because the latter still had PDE5A shifted away from the z-bands. Figure 7 summarizes our proposed model. After activation of the ß-adrenergic receptor, cAMP and cGMP are stimulated, the latter principally because of activation of NOS3 and its target sGC. The effect of this cGMP on PKG-1 activation is contained within a strategic compartment by the localization of PDE5A to z-bands, enabling modulation of PKA/calcium-stimulated contractility by SIL. Acute NOS3 inhibition removes the critical substrate to the PDE5A complex and so eliminates the antiadrenergic effect of SIL. Chronic NOS3 inhibition or NOS3–/– further results in the loss of PDE5A from this strategic location, eliminating SIL effectiveness even if NO is provided exogenously.
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The exact mechanism by which NOS3 activity regulates myocyte localization of PDE5A remains unknown, but several possibilities deserve mention. NOS3-stimulated cGMP could target PDE5A directly by binding to its GAF domain37 or activating its regulatory domain via PKG-1.38 This would serve to increase PDE5A enzyme activity39 and thereby provide an environment wherein the effects of PDE5A inhibition were enhanced. The data also suggest that NOS3 or its product could play a direct role in PDE5A localization, helping PDE5A interact with a scaffold protein that anchors it to z-bands. A-Kinase anchoring proteins perform such a role by targeting PKA to specific kinases, PDEs, and other signaling proteins to localize signaling.40–42 Corresponding cGMP-dependent protein kinase anchoring proteins remain poorly defined, but such proteins might play an analogous role in localizing PDE5A activity. Intriguingly, a similar loss of PDE5A from z-band regions has been observed in canine hearts with dilated cardiomyopathy, suggesting that altered localization and accompanying physiologic regulation may contribute to cardiac disease.
Previous studies have reported low levels of PDE5A expression in the myocardium43,44 and minimal effects of PDE5A inhibition on resting heart function.16–18,30 This has led to the conclusion that PDE5A plays little role in the heart. The current results support this under basal conditions, but they reveal a very different situation during ß-stimulation. Importantly, having a low expression level does not imply a lack of physiologic impact, particularly if strategic intracellular placement can confer targeted signaling effects. The current results are supported by previous in vivo canine heart data,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 possibility that a similar benefit may be obtained by manipulating cGMP catabolic pathways. They further imply that therapeutic efficacy of a PDE5A inhibitor depends on NOS3 expression and activity, and this could underlie hyporesponsiveness in many patients receiving this treatment. As the use of PDE5A inhibitors expands to diseases such as pulmonary hypertension, these considerations will take on more clinical significance. Strategies to upregulate NOS3 activity may prove a valuable approach to augment the influence of PDE5A and its inhibitors.
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Acknowledgments |
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This work was supported by NIH grants P01-HL59408, RO1-HL-47511, and P50-HL52307 (to D.A.K.); a Uehara Memorial Foundation grant and American Heart Association Fellowship Grant (to E.T.); Telethon Italy (TCP00089; the European Union (QLK3-CT-2002-02149); the Italian Cystic Fibrosis Research Foundation; the Fondazione Compagnia di San Paolo (to M.Z.); NSF (MCB-9983097), and the Totman Medical Research Trust (to W.R.D.); and R29HL54081, RO1HL069061, and AHANY:GIA-0151273T (to C.J.S.). We thank Doris Koesling and Nazareno Paolocci for assistance, Dr Mauro Giorgi for generously providing PDE5A antibody, and Sejal Shah, Naziya Rahman, Jessy Mathews, Qizhi Guan, and Shilpa Challa (CJS Laboratory) for technical assistance.
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Footnotes |
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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. 
Original received September 1, 2004; resubmission received October 26, 2004; revised resubmission received November 16, 2004; accepted November 19, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1. Xiao RP, Zhu W, Zheng M, Chakir K, Bond R, Lakatta EG, Cheng H. Subtype-specific beta-adrenoceptor signaling pathways in the heart and their potential clinical implications. Trends Pharmacol Sci. 2004; 25: 358–365.[CrossRef][Medline] [Order article via Infotrieve]
2. Xiang Y, Kobilka BK. Myocyte adrenoceptor signaling pathways. Science. 2003; 300: 1530–1532.
3. Wegener JW, Nawrath H, Wolfsgruber W, Kuhbandner S, Werner C, Hofmann F, Feil R. cGMP-dependent protein kinase I mediates the negative inotropic effect of cGMP in the murine myocardium. Circ Res. 2002; 90: 18–20.
4. Layland J, Li JM, Shah AM. Role of cyclic GMP-dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes. J Physiol. 2002; 540: 457–467.
5. Massion PB, Feron O, Dessy C, Balligand JL. Nitric oxide and cardiac function: ten years after, and continuing. Circ Res. 2003; 93: 388–398.
6. Kanai AJ, Mesaros S, Finkel MS, Oddis CV, Birder LA, Malinski T. Beta-adrenergic regulation of constitutive nitric oxide synthase in cardiac myocytes. Am J Physiol. 1997; 273: C1371–C1377.[Medline] [Order article via Infotrieve]
7. Varghese P, Harrison RW, Lofthouse RA, Georgakopoulos D, Berkowitz DE, Hare JM. Beta(3)-adrenoceptor deficiency blocks nitric oxide-dependent inhibition of myocardial contractility. J Clin Invest. 2000; 106: 697–703.[Medline] [Order article via Infotrieve]
8. Barouch LA, Harrison RW, Skaf MW, Rosas GO, Cappola TP, Kobeissi ZA, Hobai IA, Lemmon CA, Burnett AL, O’Rourke B, Rodriguez ER, Huang PL, Lima JA, Berkowitz DE, Hare JM. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature. 2002; 416: 337–339.[Medline] [Order article via Infotrieve]
9. Massion PB, Balligand JL. Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice. J Physiol. 2003; 546: 63–75.
10. Pilz RB, Casteel DE. Regulation of gene expression by cyclic GMP. Circ Res. 2003; 93: 1034–1046.
11. Kishimoto I, Rossi K, Garbers DL. A genetic model provides evidence that the receptor for atrial natriuretic peptide (guanylyl cyclase-A) inhibits cardiac ventricular myocyte hypertrophy. Proc Natl Acad Sci U S A. 2001; 98: 2703–2706.
12. Holtwick R, van Eickels M, Skryabin BV, Baba HA, Bubikat A, Begrow F, Schneider MD, Garbers DL, Kuhn M. Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A. J Clin Invest. 2003; 111: 1399–1407.[CrossRef][Medline] [Order article via Infotrieve]
13. Wollert KC, Fiedler B, Gambaryan S, Smolenski A, Heineke J, Butt E, Trautwein C, Lohmann SM, Drexler H. Gene transfer of cGMP-dependent protein kinase I enhances the antihypertrophic effects of nitric oxide in cardiomyocytes. Hypertension. 2002; 39: 87–92.
14. Rybalkin SD, Yan C, Bornfeldt KE, Beavo JA. Cyclic GMP phosphodiesterases and regulation of smooth muscle function. Circ Res. 2003; 93: 280–291.
15. Reffelmann T, Kloner RA. Therapeutic potential of phosphodiesterase 5 inhibition for cardiovascular disease. Circulation. 2003; 108: 239–244.
16. Corbin J, Rannels S, Neal D, Chang P, Grimes K, Beasley A, Francis S. Sildenafil citrate does not affect cardiac contractility in human or dog heart. Curr Med Res Opin. 2003; 19: 747–752.[CrossRef][Medline] [Order article via Infotrieve]
17. Cremers B, Scheler M, Maack C, Wendler O, Schafers HJ, Sudkamp M, Bohm M. Effects of sildenafil (Viagra) on human myocardial contractility, in vitro arrhythmias, and tension of internal mammaria arteries and saphenous veins. J Cardiovasc Pharmacol. 2003; 41: 734–743.[CrossRef][Medline] [Order article via Infotrieve]
18. Senzaki H, Smith CJ, Juang GJ, Isoda T, Mayer SP, Ohler A, Paolocci N, Tomaselli GF, Hare JM, Kass DA. Cardiac phosphodiesterase 5 (cGMP-specific) modulates beta-adrenergic signaling in vivo and is down-regulated in heart failure. FASEB J. 2001; 15: 1718–1726.
19. Sastry BK, Narasimhan C, Reddy NK, Raju BS. Clinical efficacy of sildenafil in primary pulmonary hypertension: a randomized, placebo-controlled, double-blind, crossover study. J Am Coll Cardiol. 2004; 43: 1149–1153.
20. Zhao L, Mason NA, Morrell NW, Kojonazarov B, Sadykov A, Maripov A, Mirrakhimov MM, Aldashev A, Wilkins MR. Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation. 2001; 104: 424–428.
21. Zhao L, Mason NA, Strange JW, Walker H, Wilkins MR. Beneficial effects of phosphodiesterase 5 inhibition in pulmonary hypertension are influenced by natriuretic Peptide activity. Circulation. 2003; 107: 234–237.
22. Bivalacqua TJ, Usta MF, Champion HC, Leungwattanakij S, Dabisch PA, McNamara DB, Kadowitz PJ, Hellstrom WJ. Effect of combination endothelial nitric oxide synthase gene therapy and sildenafil on erectile function in diabetic rats. Int J Impot Res. 2004; 16: 21–29.[CrossRef][Medline] [Order article via Infotrieve]
23. Georgakopoulos D, Kass D. Minimal force-frequency modulation of inotropy and relaxation of in situ murine heart. J Physiol. 2001; 534: 535–545.
24. Mergia E, Russwurm M, Zoidl G, Koesling D. Major occurrence of the new alpha2beta1 isoform of NO-sensitive guanylyl cyclase in brain. Cell Signal. 2003; 15: 189–195.[CrossRef][Medline] [Order article via Infotrieve]
25. Honda A, Adams SR, Sawyer CL, Lev-Ram V, Tsien RY, Dostmann WR. Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci U S A. 2001; 98: 2437–2442.
26. Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem. 2001; 276: 29188–29194.
27. Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T. A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol. 2000; 2: 25–29.[CrossRef][Medline] [Order article via Infotrieve]
28. Zaccolo M, Pozzan T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science. 2002; 295: 1711–1715.
29. Champion HC, Georgakopoulos D, Haldar S, Wang L, Wang Y, Kass DA. Robust adenoviral and adeno-associated viral gene transfer to the in vivo murine heart: application to study of phospholamban physiology. Circulation. 2003; 108: 2790–2797.
30. Herrmann HC, Chang G, Klugherz BD, Mahoney PD. Hemodynamic effects of sildenafil in men with severe coronary artery disease. N Engl J Med. 2000; 342: 1622–1626.
31. Cellek S, Kasakov L, Moncada S. Inhibition of nitrergic relaxations by a selective inhibitor of the soluble guanylate cyclase. Br J Pharmacol. 1996; 118: 137–140.[CrossRef][Medline] [Order article via Infotrieve]
32. Giordano D, De Stefano ME, Citro G, Modica A, Giorgi M. Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain. Biochim Biophys Acta. 2001; 1539: 16–27.[Medline] [Order article via Infotrieve]
33. Gyurko R, Kuhlencordt P, Fishman MC, Huang PL. Modulation of mouse cardiac function in vivo by eNOS and ANP. Am J Physiol Heart Circ Physiol. 2000; 278: H971–H981.
34. Vandecasteele G, Verde I, Rucker-Martin C, Donzeau-Gouge P, Fischmeister R. Cyclic GMP regulation of the L-type Ca(2+) channel current in human atrial myocytes. J Physiol. 2001; 533: 329–340.
35. Hare JM, Givertz MM, Creager MA, Colucci WS. Increased sensitivity to nitric oxide synthase inhibition in patients with heart failure: potentiation of beta-adrenergic inotropic responsiveness. Circulation. 1998; 97: 161–166.
36. Kim NN, Huang Y, Moreland RB, Kwak SS, Goldstein I, Traish A. Cross-regulation of intracellular cGMP and cAMP in cultured human corpus cavernosum smooth muscle cells. Mol Cell Biol Res Commun. 2000; 4: 10–14.[CrossRef][Medline] [Order article via Infotrieve]
37. Rybalkin SD, Rybalkina IG, Shimizu-Albergine M, Tang XB, Beavo JA. PDE5 is converted to an activated state upon cGMP binding to the GAF A domain. EMBO J. 2003; 22: 469–478.[CrossRef][Medline] [Order article via Infotrieve]
38. Francis SH, Bessay EP, Kotera J, Grimes KA, Liu L, Thompson WJ, Corbin JD. Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity. J Biol Chem. 2002; 277: 47581–47587.
39. Rybalkin SD, Rybalkina IG, Feil R, Hofmann F, Beavo JA. Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. J Biol Chem. 2002; 277: 3310–3317.
40. Mongillo M, McSorley T, Evellin S, Sood A, Lissandron V, Terrin A, Huston E, Hannawacker A, Lohse MJ, Pozzan T, Houslay MD, Zaccolo M. Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ Res. 2004; 95: 67–75.
41. Bauman AL, Scott JD. Kinase- and phosphatase-anchoring proteins: harnessing the dynamic duo. Nat Cell Biol. 2002; 4: E203–E206.[CrossRef][Medline] [Order article via Infotrieve]
42. Fink MA, Zakhary DR, Mackey JA, Desnoyer RW, Apperson-Hansen C, Damron DS, Bond M. AKAP-mediated targeting of protein kinase A regulates contractility in cardiac myocytes. Circ Res. 2001; 88: 291–297.
43. Kotera J, Fujishige K, Omori K. Immunohistochemical localization of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in rat tissues. J Histochem Cytochem. 2000; 48: 685–693.
44. Loughney K, Hill TR, Florio VA, Uher L, Rosman GJ, Wolda SL, Jones BA, Howard ML, McAllister-Lucas LM, Sonnenburg WK, Francis SH, Corbin JD, Beavo JA, Ferguson K. Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase. Gene. 1998; 216: 139–147.[CrossRef][Medline] [Order article via Infotrieve]
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