Published online before print April 9, 2007, doi: 10.1161/CIRCULATIONAHA.106.643536
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(Circulation. 2007;115:2159-2167.)
© 2007 American Heart Association, Inc.
Molecular Cardiology |
Compartmentalization of Cardiac ß-Adrenergic Inotropy Modulation by Phosphodiesterase Type 5
From the Division of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Md.
Correspondence to Dr David A. Kass, Ross Research Building 835, Johns Hopkins University Hospital, 720 Rutland Avenue, Baltimore, MD 21205. E-mail dkass{at}jhmi.edu
Received June 2, 2006; accepted February 16, 2007.
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Abstract |
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Background— Recent cell-based studies have found that cGMP synthesis and hydrolysis by phosphodiesterase (PDE) appear compartmentalized, with nitric oxide synthase–derived and/or PDE type 5 (PDE-5)–hydrolyzable cGMP undetected at the sarcolemmal membrane in contrast to cGMP stimulated by natriuretic peptide. In the present study, we determine the functional significance of such compartments with a comparison of ß-adrenergic modulation by PDE-5 inhibition to that of natriuretic peptide stimulation in both cardiomyocytes and intact hearts. The potential role of differential cGMP and protein kinase G stimulation by these 2 modulators was also studied.
Methods and Results— Intact C57/BL6 mouse hearts were studied with pressure-volume analysis, and adult isolated myocytes were studied with fluorescence microscopy. PDE-5 inhibition with 0.1 to 1 µmol/L sildenafil (SIL) suppressed isoproterenol (ISO)-stimulated contractility, whereas 10 µmol/L atrial natriuretic peptide (ANP) had no effect. ISO suppression by SIL was prevented in cells pretreated with a protein kinase G inhibitor. Surprisingly, myocardial cGMP changed little with SIL+ISO yet rose nearly 5-fold with ANP, whereas protein kinase G activation (vasodilator-stimulated protein phosphorylation; ELISA assay) displayed the opposite: increased with SIL+ISO but unaltered by ANP+ISO. PDE-5 and ANP compartments were functionally separated, as inhibition of nitric oxide synthase by Nw-nitro-L-arginine methyl ester eliminated antiadrenergic effects of SIL, yet this was not restorable by co-stimulation with ANP.
Conclusions— Regulation of cardiac ß-adrenergic response by cGMP is specifically linked to a nitric oxide–synthesis/PDE-5–hydrolyzed pool signaling via protein kinase G. Natriuretic peptide stimulation achieves greater detectable increases in cGMP but not protein kinase G activity and does not modulate ß-adrenergic response. Such disparities likely contribute to differential cardiac regulation by drugs that modulate cGMP synthesis and hydrolysis.
Key Words: catecholamines • contractility • myocytes • natriuretic peptides • nitric oxide synthase • cyclic GMP • phosphodiesterases, type 5
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Introduction |
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Cyclic guanosine monophosphate (cGMP) is a central second-messenger regulator of cardiovascular function.1–3 In the heart, cGMP dampens acute and chronic stress responses such as ß-adrenergic stimulation4–6 and pressure-load hypertrophy.7–10 Two enzymes are responsible for cGMP synthesis: a soluble guanylate cyclase (sGC) activated by nitric oxide (NO) and membrane-bound particulate GC coupled to natriuretic peptide (NP) receptors. Although these pathways have traditionally been depicted as providing a common cGMP pool, recent cell-based studies that employed a subsarcolemmal cGMP reporter found that these pathways are compartmentalized, with NP-stimulated cGMP detected at the outer membrane in contrast to NO-stimulated cGMP.11,12 The implications of such findings on differential myocardial cGMP and protein kinase G (PKG) activation and on cardiac function remain unknown. In addition, these data were obtained under rest conditions, where myocardial cGMP exerts modest effects, rather than in stimulated states (eg, ß-adrenergic agonists), where effects are enhanced.4–6,13
Clinical Perspective p 2167
A central mechanism for cyclic nucleotide compartmentation is its targeted hydrolysis by selective phosphodiesterases (PDEs). Among potential cardiac myocyte cGMP-PDEs (PDE types 1, 2, 5, and potentially 9), only type 5 (PDE-5) has been shown thus far to modify heart function. Although PDE-5 was previously thought unimportant for cardiac regulation,14 recent studies have shown that its inhibition by drugs such as sildenafil potently suppresses cardiac ß-adrenergic stimulation in dogs,15 mice,13 and humans16; enhances cardiac protection to ischemia-reperfusion injury17; and blunts pressure-overload hypertrophy.10 PDE-2, a dual-substrate esterase, appears to hydrolyze cGMP under rest conditions11 but targets cAMP (coupled to cGMP binding) in the presence of ß-adrenergic stimulation.18 PDE-5 regulation appears compartmentalized in cardiac myocytes, where it interacted with NO- but not NP-stimulated cGMP in the recent study of Castro et al11; however, because cGMP was only detected at sarcolemmal membrane in this study, internal pools that regulate ß-adrenergic reserve could still exist. This may be important as studies show that PDE-5 localizes to z-bands within myocytes,13,15 and this localization is required for its regulation of ß-stimulation.13 Accordingly, the present study tested the hypothesis that PDE-5 and NP modulation of ß-adrenergic responses are functionally compartmentalized in intact myocytes and hearts and further assessed whether this is associated with differential changes in myocardial cGMP and/or PKG.
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Methods |
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In Vivo Studies
Adult mice (male C57/Bl6, 6 to 8 weeks; The Jackson Laboratory, Bar Harbor, Me) were anesthetized, underwent thoracotomy, and instrumented with a miniature pressure-volume catheter (SPR-839 PV; Millar Instruments, Inc,Houston, Tex) via the left ventricular apex, as described13,19 (details in online-only Data Supplement). Isoproterenol (ISO; 20 ng/kg per min IV over 5 minutes) with or without a concomitant PDE-5 inhibitor (sildenafil [SIL] 100 µg/kg per min, 37±5.2 nM free plasma concentration, or EMD-360527/5 160 to 300 µg/kg per min; gift of Merck KgA, Darmstadt, Germany) was infused into a central jugular vein, and heart function was assessed with pressure-volume loops at a fixed atrial pacing rate of 600 minutes–1 with a transesophageal lead.19 Both PDE-5 inhibitors have an IC50 of
10 nmol/L for purified PDE-5 (versus 1 to 20 µmol/L for PDE-1 or PDE-3). Hemodynamic data were obtained at baseline, after ISO stimulation, after re-baseline, after infusion of a PDE-5 inhibitor, A-type NP (ANP; 10 µg/3 minutes IV; Sigma-Aldrich, St Louis, Mo), or both combined, and then with a second exposure to ISO in the presence of these agents. Previous studies have confirmed high reproducibility of repeated ISO infusion studies alone.13 In separate studies, animals first received pretreatment with the NO synthase (NOS) inhibitor Nw-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg IP) and 10 to 15 minutes later underwent the same pharmacological protocol.
Isolated Myocyte Studies
Adult mouse cardiomyocytes were freshly isolated and sarcomere length (Myocam, IonOptix, Milton, Mass) and whole-cell calcium transient (Indo-1am AM 1 mol/L [5 µmol/L]; Invitrogen–Molecular Probes, Carlsbad, Calif) measured at 27°C as described13 (online-only Data Supplement). After a 10-minute period for baseline stabilization, myocytes were exposed to 1 to 10 nmol/L ISO, then ISO+PDE-5 inhibitor (SIL 0.1 to 1 µmol/L, or EMD-360527/5 0.1 µmol/L buffered in 1% propanediol), ISO+ANP (10 µmol/L), or ISO+ANP+PDE-5 inhibitor. In other studies, myocytes were preincubated with the NOS inhibitor L-NAME (5 µmol/L over 30 minutes), and then subjected to similar ISO±(PDE-5 inhibitor±ANP) protocol.
cGMP Measurement and PKG Activity
cGMP was determined by enzyme immunoassay (Amersham, Buckinghamshire, UK) according to manufacturer instructions. Hearts were washed in ice-cold PBS, homogenized in 6% trichloroacetic acid, centrifuged, and extracted with water-saturated ether. The aqueous layer was vacuum dried and then resuspended in sodium acetate buffer for cGMP assay.
Myocardial PKG-1 activity was assayed in intact myocardium by assessment of PKG-phosphorylated vasodilator-stimulated protein (VASP) with a monoclonal antibody to phosphorylated VASP20 (pSer239) (Alexis Corp, Lausen, Switzerland) at a final dilution of 1:1000. Myocyte PKG activity was determined by colorimetric immunoassay (CycLex, Nagano, Japan) according to manufacturer instructions. Isolated myocytes were incubated with ISO (10 nmol/L) and then with either SIL (1 µmol/L), ANP (10 µmol/L), or L-NAME (10 µmol/L) alone or in combination. After 10 minutes of incubation, cells were lysed, and PKG-1 activity was determined.
Statistical Analysis
Results were analyzed by either ANOVA, repeated measures 1-way ANOVA, or a paired t test. Bonferroni correction was used for multiple comparisons.
All authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Basal Effect of PDE-5 Inhibition (SIL) and ANP on In Vivo and In Vitro Cardiac Function
Acute infusion of both SIL and ANP lowered systolic pressure associated with a decline in ventricular afterload from arterial vasodilation (Table 1). However, neither agent significantly altered basal systolic function (maximal rate of pressure increase, ejection fraction, CO) or chamber relaxation (
). The lack of direct cardiac effects from SIL or ANP under basal conditions was further confirmed by studies in isolated myocytes. Exposure to 1 µmol/L SIL or 10 µmol/L ANP did not change basal sarcomere shortening (3.8±0.4 versus 4.2±0.4, and 4.3±1.5 versus 4.6±1.8, respectively, P=NS for paired t test) or the resting calcium transient (peak change of 1% to 3%, P=NS for both).
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Differential Regulation of ISO Response by PDE-5 Inhibition Versus ANP
In contrast to their similar basal effects, SIL and ANP had very different effects on the response to ISO in both intact hearts and myocytes. ISO increased systolic function as shown by the pressure-volume loop example (Figure 1A), with a left shift of the end-systolic pressure-volume relation, and by the summary data for maximal rate of pressure increase and maximal power index (Figure 1B). As previously reported,13 SIL markedly blunted this response. However, ANP had essentially no effect. This disparity was further revealed in isolated adult myocytes (Figure 1C). ISO stimulation increased sarcomere shortening by 
200% with a corresponding rise in the calcium transient. The addition of SIL blunted the shortening response to ISO by nearly half without altering the calcium transient. In contrast, ANP (10 µmol/L) had negligible impact on both shortening and the calcium transient. Similar results were observed with 1 µmol/L ANP (data not shown). Because studies typically have used 0.01 to 1 µmol/L ANP, we do not believe the result was caused by insufficient dosing. One possibility for a lack of ANP myocyte effect was that the cGMP generated was extruded via ATP-binding cassette transporters.21 This typically requires 10 minutes or more22; yet examination of sarcomere shortening time tracings (Figure 1D) did not reveal even transient ANP depressant effects.
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Differential Change in Myocardial cGMP by ANP Versus PDE-5 Inhibition
Functional disparities between ANP and PDE-5 inhibition effects on ß-adrenergic stimulation could be caused by corresponding differences in total cGMP levels. To test this, we administered ANP or PDE-5 inhibitor+ISO to intact mice according to the same infusion protocol used to assess in vivo function, and then rapidly removed the heart and measured ventricular myocardial cGMP (Figure 2A). ANP markedly increased cGMP whereas PDE-5 inhibitor+ISO had only a small borderline effect (P=0.058 versus control). This supports different cGMP pools and indicates that what one measures does not necessarily predict physiological modulation. Furthermore, when ANP and a PDE-5 inhibitor were combined, the net rise in cGMP was similar to ANP alone, which supports the lack of an interaction between these pools.
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P=0.058) in cGMP, whereas ANP and ANP+PDE5-I resulted in 5- to 6-fold increases (*P<0.001). B, Disparity in VASP s239 phosphorylation as a marker of PKG activity from similar left ventricular myocardium. Hearts were exposed to infusion of ISO, ISO+ANP, and ISO+PDE-5-I (sildenafil). pVASP rose only with PDE-5 inhibition, opposite to changes observed in total cGMP. C, VASP phosphorylation is similarly increased by ISO+PDE-5-I as with ISO+PDE-5-I+ANP, suggesting minimal interaction between these regulators on PKG activation. Levels for control and ISO+ANP are again shown to be minimal (*P<0.001 versus control). D, PKG modulates suppression of ß-adrenergic stimulation by sildenafil. Myocytes treated with the PKG inhibitor Rp-8br-PET-cGMPs responded to ISO, but SIL no longer blunted this response.
Role of PKG Activation and Differential Changes With ANP Versus PDE-5 Inhibition
To further explore potential mechanisms for disparate functional responses, in vivo PKG activation was assessed by S239-VASP phosphorylation (pVASP) (Figure 2B). The results were consistent with modulation of ß-adrenergic stimulation yet strikingly discordant with measured changes in cGMP. ISO alone had little effect on pVASP, consistent with selectivity of the S239 site for PKG stimulation. ISO+ANP did not significantly alter pVASP, whereas ISO+SIL increased it nearly 200%. Lack of increased PKG activity with ANP was further confirmed in isolated myocytes by ELISA assay (142.9±33.4 versus 165.2±21.8 activity units, P=NS). In contrast, we previously reported modest increases with SIL alone and 40% rise in activity with SIL+ISO13 by the same methodology. We further tested whether combination of ANP+SIL increased pVASP more than SIL alone, but we found it did not (Figure 2C). Finally, we directly tested the role of PKG activation on the ability of SIL to blunt ISO stimulation by preexposing myocytes to the PKG inhibitor Rp-8Br-PET-cGMPs (Figure 2D). These cells responded to ISO in a manner similar to controls, but now SIL did not suppress this response.
Influence of NOS Inhibition With or Without ANP Infusion
We previously reported that the antiadrenergic effect of PDE-5 inhibition is absent after short-term inhibition of NOS.13 Because myocardial cGMP rose substantially with ANP stimulation, we tested whether ANP infusion might restore anti–ß-adrenergic effects of PDE-5 inhibitor despite inhibition of NOS activity. Intact mice were studied with or without pretreatment with the NOS inhibitor L-NAME (50 mg/kg IP). They were first stimulated with ISO and allowed to return to baseline, and then they received combined ANP+SIL, followed by a second ISO challenge. ANP+SIL itself minimally altered basal function (Table 2). In mice with functional NOS, ISO-induced inotropy declined with combined ANP+SIL to the same extent as observed with SIL alone (Figure 3A). However, the combination did not alter the ISO response in mice pre-treated with L-NAME (Figure 3B, upper panels). Similar findings were observed in isolated myocytes (Figure 3, A and B, lower panels). Figure 4 summarizes the in vivo results of these studies with a display of data for systolic function parameters.
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We next tested whether PKG activation associated with SIL+ISO was prevented by NOS inhibition and if this remained unchanged despite the addition of ANP. Adult myocytes were exposed to ANP+ISO with or without addition of SIL. Addition of SIL significantly increased PKG activity (Figure 5) but this was blocked when cells were pretreated with L-NAME. Furthermore, in cells treated with L-NAME, SIL+ISO elicited the same level of PKG activation as SIL+ISO+ANP, which indicated that ANP could not substitute for the loss of NOS activity.
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Discussion |
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The present study shows essentially full functional compartmentalization of NP-cGMP synthetic versus PDE-5 hydrolytic regulation of cardiac ß-adrenergic stimulation in intact hearts and isolated myocytes. Further, this disparity is accompanied by directionally opposite changes in measured total myocardial cGMP but concordant differences in PKG activation assessed by myocardial pVASP. Moreover, inhibition of NOS-derived cGMP blocks both PKG activation and antiadrenergic effects of SIL, and this is not compensated by NP. These data support differential stimulation of PKG pools and specific cGMP regulatory partnering between sGC and PDE-5.
Two recent studies directly revealed compartmentalization of cGMP generation in isolated myocytes,11 human embryonic kidney–NP receptor A cells,12 and vascular smooth muscle cells.12 In both studies, NP but not NO stimulation induced detectable cGMP at the outer cell membrane where a cyclic nucleotide-sensitive ion channel was expressed. Neither study examined whether this resulted in different activation of PKG and/or cellular functional response, but the present findings suggest that both occur. Furthermore, the cell studies examined basal responses, where cGMP modulation is usually very modest,13 rather than in ß-adrenergic–stimulated cells or cells stimulated by some other pathway. This is particularly true for effects mediated by PDE-5. There also may be differences between myocyte and vascular smooth muscle cGMP signaling, as the relative increase in cGMP with NO-sGC stimulation exceeded that with NP–particulate GC in vascular smooth muscle,12 opposite to what we observed in the myocardium.
Modulation of cardiac contractility by cGMP displays both dose- and adrenergic state–dependent features. Low concentrations enhance myocyte contractility that has been attributed to inhibition of PDE-3,23,24 a cAMP phosphodiesterase, whereas increased doses blunt contractility.4,25 The latter appears to be linked to activation of PKG and subsequent phosphorylation of troponin I to desensitize the myofilaments to calcium.25,26 Such modulation has been revealed with cGMP analogs such as 8-br-cGMP, or NO donors and NOS inhibitors, the latter studied extensively in intact hearts and in humans.5,27 However, basal effects remain modest and are truly negligible when triggered by PDE-5 inhibition, whereas with ß-AR costimulation cGMP can blunt contractility more markedly.4–6 The myocyte NOS3 isoform is particularly important in this regard as revealed by NOS3-gene transfer6 and myocyte-targeted overexpression models.28
In contrast, studies of NP peptide effects on basal or ß-adrenergic–stimulated function have yielded mixed results. Given the plasma membrane localization of the NP receptor and particulate GC, modification of calcium currents might be predicted and reduction of Ca2+ efflux has been reported in ECV304 cells,29 whereas in ventricular myocytes Ca2+ transients have been found to be reduced when stimulated by C-type NP.30 In frog ventricular myocytes, ANP has little impact on ICa under basal conditions, but inhibits it 33% in cells stimulated by ISO,31 an effect thought to be caused by activation of cAMP esterase. Opposite findings have also been reported, as CNP enhanced contractility in isolated hearts and myocyte shortening and Ca2+ transients via PKG-dependent phospholamban phosphorylation.32 Finally, dobutamine-stimulated contractility in intact conscious dogs was found unaltered by ANP inhibition or infusion.33 Although ANP infusion clearly had physiological effects (ie, vasodilation) in the present study, it had no impact on rest or ß-adrenergic–stimulated function, or on Ca2+ transients, results that are most consistent with the previous intact canine study.33
A primary mechanism to target cyclic nucleotide signaling within the cell is the strategic placement of downstream effectors (ie, protein kinase A, PKG), and regulatory PDEs. This paradigm has been extensively studied for cAMP modulation,34,35 but far less so for cGMP. Individual cAMP-PDEs functionally couple to specific targets in cardiac myocytes. For example, selective inhibition of PDE-3 and PDE-4 potentiates ß1 adrenergic receptor–stimulated cAMP, whereas glucagon receptor–stimulated cAMP is augmented only by PDE-4 inhibition.36 PDE-4b has been reported to locally regulate ryanodine receptor protein kinase A phosphorylation.37 The current data supports functional cGMP compartmentalization and reveals that measured levels of both the cyclic nucleotide and associated PKG activity appear regionally controlled. The finding of little PKG activation with ANP stimulation is a bit surprising, although consistent with the functional data. It remains possible that a subsarcolemmal compartment with NP-stimulated PKG activation exists,11 but this would seem to be smaller in magnitude and not detected from total cell lysates. The measurable activation, in contrast, may relate more to that involved with antagonizing ß-adrenergic stimulation.
The precise mechanism for localized cGMP-hydrolytic control by PDE-5, PDE-2, or other PDEs remains unresolved. The notion that a local cGMP pool can be isolated from outside sources by the placement of a high-gain esterase is an attractive model. Yet when both NOS and PDE-5 were inhibited, exogenous ANP stimulation still did not modify adrenergic reserve. PDE-2 is largely localized to the outer membrane at sites of t-tubules18 and closely couples with receptor/ligand signaling. Although PDE-2 can hydrolyze cGMP stimulated by ANP,11 in the presence of ß-adrenergic stimulation (ISO), it primarily hydrolyzes cAMP, and PDE-2 inhibition now augments myocyte shortening and Ca2+ transients.18 The latter has been attributed to ß3-adrenergic receptor–coupled NOS3 stimulation. It remains unclear whether particulate GC–generated cGMP also activates PDE-2 through its cGMP, adenylyl cyclase, FhlA (GAF) binding domain, and if so, whether cAMP or cGMP is preferentially hydrolyzed particularly when cAMP is costimulated by ISO. Our data suggest that NP-stimulated cGMP did not effectively diffuse beyond this local region. Direct assessment by means of fluorescent bioprobes for cGMP38,39 is under way to try to better identify these compartments and localized signaling pools.
Recent evidence that PDE-5 inhibition can blunt acute and chronic cardiac stress responses has refocused attention on potential cardiac applications of drugs previously used to treat erectile dysfunction and pulmonary hypertension.40 Interactions with therapies that enhance NO signaling or stimulate NP receptors may become more prevalent, which increases the importance of understanding targeting between cGMP synthesis and catabolism. Although our results support acute compartmentalization, it remains unknown whether chronic elevation of NP stimulation, as occurs in various heart disease conditions, interacts with cardiac PDE-5 catabolism, or if this separation is organ-specific. The selective interaction of PDE-5 with sGC-generated cGMP may have additional ramifications in that chronic depression (or genetic loss) of NOS3 activity leads to altered cellular localization of PDE-5 (from z-banding pattern to a more diffuse pattern) and with it an additional mechanism for loss of stress modulation by the PDE-5.13 Understanding of the interplay between sGC and PDE-5 will be central to the therapeutic use of agents that modulate them.
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Acknowledgments |
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Sources of Funding
The present study was supported by the National Institutes of Health (NIH) grants P01-HL59408, RO1-HL-47511, P50-HL52307, the Peter Belfer Laboratory, and the Abraham and Virginia Weiss Professorship (to Dr Kass); the Uehara Memorial Foundation Grant and the American Heart Association Fellowship and Scientist Development Grant (to Dr Takimoto); and NIH T32-HL07227 (to Drs Belardi and Vahebi).
Disclosures
None.
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Footnotes |
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*Drs Belardi, Tocchetti, and Vahebi contributed equally to this work.
The online-only Data Supplement, consisting of expanded Methods, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.106.643536.
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