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the Dulbecco Telethon Institute (M.M., A.T., V.L., M.Z.) and Venetian Institute of Molecular Medicine (M.M., A.T., V.L., T.P., M.Z.), Padova, Italy
Department of Cardiology (C.G.T., D.A.K., N.P.), Johns Hopkins Medical Institutions, Baltimore, Md
Division of Biochemistry and Molecular Biology (Y.-F.C., M.D.H.), Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, United Kingdom
Department of Pharmacology (W.R.D.), University of Vermont, Burlington
Department of Biomedical Sciences (T.P.), University of Padova, Italy.
Abstract
-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving 3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by -adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the -adrenergic pathway. In this, activation of 3-adrenergic receptors counteracts cAMP generation obtained via stimulation of 1/2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.
Key Words: PDE2 cAMP cardiomyocytes fluorescence resonance energy transfer compartmentalization
Introduction
The activation of the -adrenergic system is the primary neurohormonal mechanism controlling force and frequency of cardiac contraction. The majority of -adrenergic effects are driven by the elevation of intracellular cAMP levels, followed by activation of a cAMP-dependent protein kinase A (PKA). Given the multiple PKA targets within the myocyte, a spatially confined PKA activity is essential to warrant response specificity.1 It is becoming increasingly evident that the compartmentalization of proteins within specific regions of cardiac myocytes is pivotal to the appropriate functioning of the cAMP/PKA signaling pathway, although the machinery that underpins compartmentalized cAMP signaling is only now becoming fully appreciated.2 Thus receptors, Gs proteins and adenylyl cyclase (AC) are confined in discrete parts of the plasma membrane,3 and PKA is anchored via AKAPs (A kinase anchoring proteins)4 close to its specific targets.5 cAMP can be compartmentalized in discrete cell regions,6eC8 leading to activation of selected pools of PKA.8
Fundamental to the generation and shaping of cAMP gradients is the activity of phosphodiesterases (PDEs),9 a superfamily of enzymes grouped in 9 families able to hydrolyze cAMP with more than 40 isoenzymes.9 Instrumental to the generation and shaping of cAMP gradients in cells is the association of PDEs to specific subcellular structures or signaling scaffold complexes via targeting domains.10 PDE isoenzymes are thus poised to perform unique functional roles that depend on the particular combination of regulatory mechanism, kinetics, and localization.
Members of at least 4 cAMP-PDE families coexist in cardiac myocytes, namely (1) PDE1, a Ca2+/calmodulin activated PDE; (2) PDE2, a cGMP-activated PDE; (3) PDE3, a cGMP-inhibited PDE; and (4) PDE4, a cGMP-independent, cAMP-specific PDE. Of these, PDE3 and PDE4 provide the major PDE activity in the heart,11 and most studies performed to date have focused on these 2 enzyme families. However, several pieces of evidence suggest that PDE2 may also control important biological functions in heart cells. PDE2 is able to hydrolyze both cAMP and cGMP but is unique in that its N-terminal, paired GAF domains are able to bind cGMP, leading to a concomitant increase in hydrolytic activity.10 Regarding cardiac function in particular, a cGMP-mediated, PDE2-dependent decrease of cAMP has been reported to be responsible for cholinergic attenuation of -adrenergic signaling in rabbit atrioventricular nodal cells.12 Similarly, in pacemaker cells, the effect of NO on heart rate appears to be mediated, at least in part, by inhibition of L-type Ca2+ current (ICa) via a signaling pathway involving guanylyl cyclase activation, synthesis of cGMP, and stimulation of a cGMP-activated PDE.13 The role of PDE2 in the contractile myocardium is less clear. Although, in amphibian ventricular myocytes14 and human atrial cells,15 cGMP has been reported to induce a strong inhibition of cAMP-enhanced ICa via activation of a cGMP-stimulated PDE, the same effect was not found in other species.16
Here we test the novel hypothesis that in cardiac myocytes compartmentalized PDE2 activity blunts -agonisteCinduced cardiac contractility via a 3-receptoreCcoupled NO-dependent pathway. By using real-time, fluorescence resonance energy transfer (FRET)-based imaging of i in living cells,17 we report, for the first time, that consequent to activation of 3-receptors, there follows a rise in intracellular NO-cGMP levels, which serve to activate membrane-associated PDE2 activity, thereby selectively attenuating spatially confined pools of intracellular cAMP that, ultimately, affect contractile function.
Materials and Methods
Primary cultures of cardiac ventricular myocytes were imaged for FRET changes as described.8 Confocal immunostaining of PDE2A was performed with a polyclonal antibody (FabGennix). Cell lysis, fractionation method, and PDE assay are described in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org. Cai was assessed by Indo-1 fluorescence, and sarcomere length was measured by real-time Fourier transform (IonOptix MYO100 MyoCam) and whole calcium transient. All methods used in this work are described in detail in the online data supplement.
Results
PDE2 Exerts Tight Control Over cAMP Generated by -Adrenoceptor Stimulation
We have previously shown that PDE4 and PDE3 families provide the major cAMP PDE activity in rat neonatal ventriculocytes, whereas PDE2 activity is only a minor fraction of the total activity.11 However, we found that the spatial confinement of specific PDEs can enhance the importance of particular PDE isoforms in determining cAMP signaling.11 With this in mind, we set out to evaluate the role of PDE2 in the control of cAMP signaling using the specific PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and a genetically encoded, FRET-based sensor for cAMP.18
We treated rat neonatal ventriculocytes expressing the genetically encoded cAMP sensor with 10 eol/L EHNA and found a small increase in i, with a /R0=0.9±0.2% (mean±SEM; n=10) (Figure 1A). Inhibition of all PDE activity with the nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) (100 eol/L), however, generated a rise in i that was at least ten times larger, with a /R0 of 10.3±1.5% (n=8) (Figure 1A).
Addition of 10 eol/L EHNA to ventriculocytes that had previously been stimulated with a submaximal concentration of norepinephrine (NE) (5 nmol/L) resulted in a very large, saturating increase in i (Figure 1B). The same concentration of NE applied to control cells caused a marginal i rise. Comparable results were obtained in ventriculocytes from neonatal mice and stimulated with either NE or isoproterenol alone or in combination with EHNA (not shown). To quantify more clearly the effect of PDE2 inhibition in the presence of catecholamines, we used a variant of the cAMP sensor (RII-R230K) showing &100-fold higher EC50 value in a cAMP-dependent dissociation assay in vitro11 and found that the response to 1 eol/L NE showed a 32 R/R0 of 1.2±1.0% and the subsequent addition of 10 eol/L EHNA generated a 7-fold increase in i (32 R/R0=6.9±1.7%) (Figure 1C and 1D). Such a response was slightly higher than that recorded with the same sensor after inhibition of total PDE activity with IBMX (100 eol/L) (Figure 1D).
The low-affinity cAMP probe RII-R230K allows for the investigation of the high range of cAMP concentrations invariably achieved after PDE inhibition. We thus used such a probe in all subsequent experiments, unless otherwise specified. To exclude the possibility that the effect of EHNA on i was attributable to its known ability to inhibit adenosine deaminase, we challenged cells with NE and EHNA in the presence of the A2a receptor antagonist SCH5826119 (1 eol/L). However, this made no difference to the effect of EHNA either in basal or in stimulated conditions (Figure 1E), indicating that EHNA functioned to alter cAMP levels in these cells by inhibiting PDE2 activity.
PDE2 Selectively Controls cAMP Generated by -Adrenoreceptor Agonists
Although PDE2 represents only a small fraction of total PDE activity in rat neonatal ventriculocytes, the experiments presented in Figure 1 demonstrate that PDE2 inhibition leads to a dramatic augmentation of the -adrenoreceptor (AR)eCinduced i. This suggests that PDE2 may be tightly coupled to a pool of AC activated by -AR. To test this hypothesis, we measured the cAMP response induced by PDE2 inhibition in cells treated with the pharmacological AC activator forskolin. Cells treated with 1 eol/L forskolin show a rise in i comparable to that determined by challenge with 1 eol/L NE (R/R0=1.9±0.1%; compare Figures 1B and 2A). Inhibition of PDE2 by EHNA induced an almost undetectable amplification of the cAMP rise in forskolin-treated cells (R/R0 of 2.2±0.2%) (Figure 2). Thus, the effect of EHNA is more than 30 times larger in the presence of NE than after stimulation with forskolin.
Localization of PDE2
To gain insight into the possibility that PDE2 underpins a compartmentalized effect on cAMP signaling in ventriculocytes, we performed biochemical fractionations of neonatal rat myocytes to determine whether or not PDE2 is membrane associated. In doing this, we determined the activity of PDE2, PDE3, and PDE4 in the low-speed (P1) and high-speed (P2) membrane fractions from ventriculocytes as well as in the "soluble" high-speed supernatant fraction (S2). As shown in supplemental Table I, all of the PDE2 activity was recovered in the membrane fraction and no PDE2 activity was detectable in the supernatant. This finding suggests that the PDE2 activity in these cells is entirely attributable to the PDE2A2 isoform, which is membrane associated because of its unique N-terminal extension.20
We further investigated the subcellular localization of PDE2 by immunocytochemistry and confocal microscopy. As shown in Figure 3, PDE2 was found to localize at the plasma membrane and in particular in correspondence of the cell-to-cell junctions. In addition, localization in correspondence of the sarcomeric Z line was clearly detected (Figure 3). A similar localization at the Z line has been shown for PDE4D,11 an enzyme involved in the selective control of cAMP generated after -AR stimulation.
Determination of PDE2 Activity in Lysates From Unstimulated and NE-Treated Myocytes
Unlike other PDEs expressed in heart cells (eg, PDE321 and PDE422), no stable activation of PDE2 activity has been reported to date. To establish whether our experimental conditions could lead to a stable activation of PDE2 enzymes, we tested PDE2 activity in lysates prepared from both unstimulated and NE-treated cells. The total PDE activity corresponded to 72±6 pmol/min per milligram of protein (mean±SD, n=5), and the relative PDE2 activity in the same preparations was 2.2±0.8 pmol/min per milligram of protein (n=5). The PDE2 activity in lysates of cells treated, for various lengths of time, with either NE or NE plus EHNA was then determined. The in vitro activity of PDE2 in lysates treated with 5 nmol/L NE for 1 to 10 minutes and expressed as percent of PDE2 activity at time 0, was 100±7% at 1 minute, 118±11% at 2 minutes, 82±15% at 5 minutes, 122±12% at 8 minutes, and 97±18% at 10 minutes (n=4). Similarly, the effect on PDE2 activity after treatment with 5 nmol/L NE plus 10 eol/L EHNA was determined as 88±12% at 8 minutes and 91±14% at 14 minutes (n=4). We also tested PDE2 activity on membrane preparations found that the addition of NE had no discernible effect on PDE2 activity (see supplemental Table I). Collectively, these data indicate that no stable increase in PDE2 activity ensued as a result of treating cells with either NE alone or NE together with EHNA.
Indeed, the only known way of markedly potentiating the ability of PDE2 to hydrolyze cAMP is through an increase in cGMP levels.23 In fact, a 4-fold increase in cAMP-hydrolyzing PDE2 activity has been reported to result from exposure to low micromolar concentrations of cGMP.24
NO/cGMP Pathway Modulates the cAMP Response to -AR Stimulation via PDE2 Activation
To evaluate whether a rise in i could be responsible for potentiating PDE2 activity after -AR stimulation, we exploited the fact that the synthesis of cGMP by soluble guanylate cyclases (sGC) is potently activated by NO.
As shown in Figure 4A and 4B, treatment of myocytes with the NO donor sodium nitroprusside (SNP) (10 eol/L), in the presence of 1 eol/L NE, reduced the i rise by &50%. SNP, however, had no effect on i when added to resting cells (not shown). A similar result (reduction of i of &30%) was observed with another NO donor, diethylamine/NO complex (10 eol/L) (not shown). The reduction of i induced by SNP was completely reversed and, indeed, was even slightly potentiated by the addition of the sGC inhibitor 1H-[1,2,4]oxadiazolol[4,3,2]quinoxalin-1-one (ODQ) (10 eol/L25) (Figure 4A and 4B). These data indicate that the effect of SNP is mediated by cGMP synthesized by a NO-activated GC. As might be predicted, preincubation of myocytes with ODQ completely prevented the effect of SNP on i (not shown).
The role of sGC activation in the modulation of NE-generated i was confirmed by direct activation of sGC using the NO-independent activator BAY41eC2272 (10 eol/L) (not shown).
Interestingly, the effect of NO donors on i was dependent on the mechanism of the cAMP rise. Indeed, in cells treated with 1 eol/L forskolin, addition of neither SNP (10 eol/L) nor ODQ (10 eol/L) resulted in significant changes in i concentration (Figure 4C).
The data presented above are compatible with the hypothesis that cGMP modulates -AReCgenerated cAMP by increasing PDE2 activity. To confirm this, we measured the effect of SNP and ODQ on NE-generated i under conditions of PDE2 inhibition. In the presence of 25 eol/L EHNA, neither SNP nor ODQ elicited any significant change in the i generated by 1 eol/L NE (Figure 4D). When the selective PDE3 inhibitor cilostamide (10 eol/L) was used in place of EHNA, the effect of SNP and ODQ on NE-generated i was unchanged (not shown).
NE, via 3-AR, Counteracts 1/2 Signaling by Increasing Intracellular cGMP
NE activates 1-, 2-, and 3-ARs. Whereas 1 and 2 receptors are known to couple to AC to produce cAMP, stimulation of the 3 receptor isoform leads to the production of NO, which, in turn, stimulates sGC to produce cGMP.26 To assess whether NE generates a rise of cGMP in cardiac myocytes, we used a genetically encoded, FRET-based probe for cGMP, namely cygnet 2.1', which allows real-time imaging of cGMP levels in living cells.27 Transfected ventriculocytes, as expected, responded to SNP with a rapid and dose-dependent increase in i, as detected by a raise in the ratio of 480 nm/535 nm emission after excitation at 430 nm (/R0) (not shown).
Perfusion with NE (1 eol/L) of cells transfected with cygnet 2.1 induced a rise in cGMP with a /R0 of 4.22±0.8% (n=10) (Figure 5A). This corresponds with &20% of the maximal effect obtained with 10 eol/L SNP. An identical effect was observed with a combination of 1 eol/L NE and the 1 and 2 specific antagonists, CGP20712A (300 nmol/L) and ICI118,551 (100 nmol/L), respectively (Figure 5A). Conversely, blockade of the 3 receptor with the selective antagonist SR59230A (10 eol/L) dramatically reduced the NE effect on i; (Figure 5A). Blockade of cGMP response to NE was also obtained by preincubation of the myocytes with ODQ (10 eol/L) (Figure 5A).
These experiments indicate the involvement of 3-AR in the synthesis of cGMP induced by NE and lead to the prediction that 3-AR stimulation should negatively modulate the cAMP increase triggered by activation of 1- and 2-AR. As shown in Figure 5B and 5C, when the 3-AR was selectively antagonized, NE generated a rise in i that was &1.5 times higher than that in the absence of the 3-AR antagonist.
3-ARs are known to be expressed at very low levels in cardiac myocytes. By performing real-time RT-PCR on neonatal myocytes from rat hearts, we estimated that 3-ARs represent approximately 0.02% of the total -ARs (see the online data supplement for details). The very low abundance of 3-AR in these cells points to a tight coupling between the 3-AR and its downstream signaling machinery, PDE2, and the pool of 1/2-AReCactivated ACs (see also Figure 8).
PDE2 Potently Modulates the Effect of Catecholamine on Intracellular Ca2+ Transients and Myocyte Contractility
To evaluate the impact of PDE2 inhibition on ventricular myocyte function, we measured Ca2+ transients in spontaneously beating rat neonatal myocytes expressing the genetically encoded Ca2+ sensor aequorin.28 As shown in Figure 6A through 6E, addition of 5 nmol/L NE generated a 1.5-fold increase in the amplitude of Ca2+ transients over basal (amplitude of individual peaks averaged over 1 minute interval, P<0.05, n=5). A concentration of 10 eol/L EHNA generated a further significant increase (&35%, P<0.05) of such amplitude, resulting in a 2-fold increase over basal (n=5). We next examined the effect of PDE2 inhibition on myocyte contractility. To this end, we measured sarcomere shortening (SS) in isolated myocytes from adult mouse hearts. In these cells, a submaximal concentration of isoproterenol (ISO) (2.5 nmol/L) increased SS by 34.5±10% (P<0.005 versus basal, n=20) and ISO coinfusion with EHNA (10 eol/L showed a >3-fold increase over ISO [SS: 117±18%, P<0.0005 versus ISO alone and P<0.00005 versus basal, n=20]; Figure 6F and 6G). Incubation of the cells with ODQ (10 eol/L) blunted ISO+EHNAeCinduced SS by more than 50% (41±9% with ISO, P<0.01 versus 76±15% with ISO+EHNA, P<0.01; all n=16), with a relative increase of ISO+EHNA over ISO of only 84% (P<0.01 versus cells not treated with ODQ). Notably, the effect of PDE2 inhibition on contractility was dramatically reduced in myocytes obtained from mice in which the expression of endothelial NO synthase (eNOS) had been abolished by targeted gene ablation (eNOSeC/eC).29 In eNOSeC/eC myocytes, we titrated ISO dose (2 nmol/L) to achieve a similar contractility increase as with WT cells. This was done to avoid any possible "saturation" effect, taking into account that eNOSeC/eC adult murine myocytes show increased cardiac contractile force in response to -adrenergic stimulation when compared with wild-type (WT) littermates.30 In presence of 2 nmol/L ISO, SS increase was 32±10% (P<0.001 versus basal; P=NS versus ISO in WT cells). In contrast, combining ISO with EHNA led to an additional increase in fractional shortening of only 23% over ISO alone, from 32±10% to 55±17% (P=0.015 versus ISO alone, n=20), that was significantly smaller when compared with the same conditions in WT cells (P<0.01) (Figure 6F and 6G). The residual effect on fractional shortening after PDE2 inhibition observed in eNOSeC/eC mice may result from ISO-induced generation of cGMP through an eNOS-independent mechanism. In fact, as shown in Figure 5A, the 3-AR antagonist SR59230A does not completely abolish NE-induced cGMP, compatible with the existence of a parallel pathway for cGMP generation. In further support of this hypothesis is the observation that in cardiac myocytes cGMP is synthesized in response to ISO even in the presence eNOS inhibitors.31 The nature of this eNOS-independent pathway to cGMP synthesis remains to be investigated.
The contractility trend was paralleled by consistent changes in Ca2+ transients. Basal Ca2+ transients were similar between WT and eNOSeC/eC myocyte groups. ISO stimulation produced an increase in Ca2+ transient amplitude of +10.6±3% in WT (P<0.005 versus basal) and +5.2±3.3% in eNOSeC/eC mice (P=0.05 versus basal; P=NS versus ISO WT) (Figure 6H and 6I). However, in WT coinfusion of ISO with EHNA increased Ca2+ transients by 42.3±8.8% (P<0.005 versus ISO alone; P<0.0005 versus basal), whereas in eNOSeC/eC myocytes, this increment was significantly less: +12.4±3% (P<0.05 versus ISO/EHNA in WT; P<0.005 versus basal; P<0.05 versus ISO) (Figure 6H and 6I).
Interestingly, the potentiation effect on SS and Ca2+ transient amplitude obtained after PDE2 inhibition in adult myocytes was solely generated in the presence of -AR stimulation and was not detectable when all AC were stimulated with forskolin (Figure 7), confirming our findings in neonatal myocytes and supporting the tight coupling between PDE2 and the pool of cyclases activated by -ARs.
Discussion
A large body of evidence indicates that PDEs contribute to the compartmentalization of cAMP signaling.7,8,10,32 In particular, in rat neonatal ventriculocytes, PDE3 and PDE4 show distinct localization11 and PDE4 appears to be primarily involved in regulating cAMP in a domain harboring the -AReCactivated AC, whereas PDE3 acts in a distinct compartment.11 In addition, specific targeting of a PDE5A to Z-bands has been reported to selectively enable modulation of ISO-induced contractility in cardiac myocytes.31 Thus the view is emerging that the functional coupling of distinct PDEs to individual receptors provides an efficient way to control local levels of cAMP in a stimulus-specific manner.11,17,33
The present study makes a substantial advancement in our understanding of the role of PDEs in compartmentalized cAMP signaling in that provides evidence that PDE2 plays a key role in the selective control of cAMP generated after -AR stimulation. Although PDE2 activity provides only &3% of total PDE activity in rat neonatal ventriculocytes and PDE2 inhibition showed only a minimal ability to increase i in unstimulated cells, the contribution of PDE2 was strikingly potentiated in the presence of NE. Such a robust effect of PDE2 on the cAMP response to catecholamines suggests a functional coupling between PDE2 and a pool of AC activated by -AR stimulation. The finding that PDE2 inhibition has no effect on i generated through the nonselective activation of all AC with forskolin confirms such a hypothesis.
Targeting of PDE2 to lipid rafts has been reported34 and, interestingly, -AR, AC,3 and NOS35 are known to reside in the cardiac subtype of lipid rafts known as caveolae. In the present study we found that PDE2 is entirely confined to a membrane compartment, in agreement with the notion that localization of the enzyme to a restricted domain potently enhances its effects on catecholamine-induced cAMP generation. 3-AR can activate distinct intracellular pathways by coupling to Gs,36 Gi,37 and NOS.26 Although the presence of a 3-AR/NO/cGMP pathway exerting a feedback control over cAMP has been reported in cardiac myocytes,26,38 the functional relevance of 3-AR signaling in the heart is still controversial.30,39,40 Our data show that 3-ARs are involved in NE-induced NO generation, leading to sGC activation, synthesis of cGMP, and activation of PDE2. This defines a key role for PDE2 in the attenuation of -adrenergic signaling via a 3AR-activated feedback loop (Figure 8).
The functional compartmentalization of PDE2 and -AR may have important clinical implications. The downregulation of 1/2-AR found in failing cardiac myocytes41 is accompanied by a significant increase in 3-AR expression.42 In addition, 3-ARs show a relative resistance to homologous desensitization as compared with 1/2-AR.43 As a consequence, in conditions of high-adrenergic tone, typical of heart failure, 3-AReCmediated signaling may assume a prominent role and may underlie the progressive nature of functional impairment in heart failure. Interestingly, an increase in PDE2 activity has been recently found in pressure-overloaded ventricles of aortic-banded rats.44 Thus, in the context of a failing heart, activation of PDE2 via a 3-AR/NOS/cGMP pathway may further reduce the inotropic reserve and aggravate systolic dysfunction. Selective inhibition of PDE2 may attenuate the negative inotropic effect of 3-AR signaling and positively influence cardiac performance.
Acknowledgments
This work was supported by Telethon Italy (TCP00089), the Italian Cystic Fibrosis Research Foundation, and the Fondazione Compagnia di San Paolo (to M.Z.); The European Union (QLK3-CT-2002-02149) (to M.Z. and M.D.H.); the Medical Research Council (G8604010) and British Heart Foundation (FS/1999043) (to M.D.H.); Telethon, the Italian Association for Cancer Research, the Italian Ministry of Education, and the Italian Ministry of Health (to T.P.); the National Science Foundation (MCB-9983097) and the Totman Medical Research Trust (to W.R.D.); and the American Heart Association (SDG, 0430144N) and National Institutes of Health (R01 HL075265) (to N.P.).
This manuscript was sent to H. Michael Piper, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
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