Published online before print March 27, 2008, doi: 10.1161/CIRCRESAHA.107.167817
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© 2008 American Heart Association, Inc.
Cellular Biology |
Spatiotemporal Dynamics of β-Adrenergic cAMP Signals and L-Type Ca2+ Channel Regulation in Adult Rat Ventricular Myocytes
Role of Phosphodiesterases
From INSERM U769 (J.L., A.A.-G., P.L., J.-L.M., R.F., G.V.), Châtenay-Malabry, France; Univ Paris-Sud 11 (J.L., A.A.-G., P.L., J.-L.M., R.F., G.V.), Faculté de Pharmacie, Châtenay-Malabry, France; Rudolf-Virchow-Center (V.O.N.), Deutsche Forschungsgemeinschaft-Research Center for Experimental Biomedicine, University of Würzburg, Germany; and Division of Reproductive Biology (W.R., M.C.), Department of Obstetrics and Gynecology, Stanford University School of Medicine, Palo Alto, Calif.
Correspondence to Dr Rodolphe Fischmeister, INSERM U-769, Université Paris-Sud 11, Faculté de Pharmacie, 5, Rue J.-B. Clément, F-92296 Châtenay-Malabry Cedex, France. E-mail fisch{at}vjf.inserm.fr
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Abstract |
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Steady-state activation of cardiac β-adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer–based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca2+ channel current (ICa,L) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. ICa,L amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for
5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 µmol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 µmol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-l-methylxanthine (300 µmol/L), cAMP signals and ICa,L declined with a time constant >10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to 50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct β-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4.
Key Words: cAMP • L-type calcium current • 5'-3' cyclic nucleotide phosphodiesterases • β-adrenergic receptors • compartmentation
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Introduction |
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CAMP is an ubiquitous second messenger regulating a myriad of cellular functions. In the heart, cAMP mediates the positive inotropic and lusitropic effects of β-adrenergic receptor (β-AR) stimulation by activating the cAMP-dependent protein kinase (PKA), thereby promoting the phosphorylation and activation of key components of the excitation–contraction coupling process. These include the sarcolemmal L-type Ca2+ channels (CaV1.2), which are responsible for the initial Ca2+ influx; the ryanodine receptors, which allow Ca2+ release from the sarcoplasmic reticulum (SR); troponin I, which controls myofilament sensitivity to Ca2+; and phospholamban, which regulates Ca2+ withdrawal from the cytosol and reuptake into the SR.1
These phosphorylation events are thought to be tightly regulated by multiprotein signaling complexes organized around A kinase anchoring proteins, thus providing a molecular basis for subcellular compartmentation of the cAMP pathway.2,3 In support of this view, PKA-mediated phosphorylation gradients were recently reported.4 However, differential regulation of localized PKA also requires a nonuniform distribution of the PKA activator, cAMP. Indeed, the presence of subcellular compartments with different cAMP concentrations, also called cAMP microdomains, are inferred from L-type Ca2+ channel current (ICa,L) measurements in response to local β-AR stimulation in cardiomyocytes5 and by direct monitoring of cAMP using fluorescence resonance energy transfer (FRET)-based sensors6,7 or cyclic nucleotide-gated (CNG) channels.8 These and other studies9,10 also underlined the importance of cAMP phosphodiesterases (PDEs) for the spatiotemporal control of cAMP signals.11
In the present study, we used a unique approach, combining live-cell imaging and electrophysiology to investigate the spatiotemporal dynamics of β-AR responses in adult rat ventricular myocytes (ARVMs). We performed direct measurements of the cyclic nucleotide level in the subsarcolemmal and cytosolic compartments using a recombinant CNG channel12 and the FRET-based Epac2-camps sensor,13 respectively. Changes in the cAMP level in these 2 compartments were compared in real time with the degree of PKA phosphorylation of CaV1.2 channels by monitoring the amplitude of ICa,L. Unlike most previous studies performed under steady-state conditions, here, we only used brief (15-second) β-AR stimulations, similar to those elicited during a startle response. This approach allowed us to follow in detail the onset and recovery phases for the 3 parameters and to delineate the intricate spatiotemporal contribution of PDE isoforms 3 and 4 to the β-AR response of cardiac myocytes.
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Materials and Methods |
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All experiments performed conform to the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J no. L358, 18 December 1986), the local ethics committee (CREEA Ile-de-France Sud) guidelines, and the French decree no. 87-848 of October 19, 1987 (J Off République Française, 20 October 1987, pp 12245–12248). Authorizations to perform animal experiments according to this decree were obtained from the French Ministère de l’Agriculture, de la Pêche et de l’Alimentation (no. 92-283, June 27, 2007).
Detailed methods are included in the online data supplement at http://circres.ahajournlas.org.
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Results |
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Compartmentalized cAMP and ICa,L Responses to β-AR Stimulation
To compare β-adrenergic cAMP signals in the subsarcolemmal and cytosolic compartments, E583M/C460W CNGA214 and Epac2-camps13 were expressed in ARVMs using recombinant adenoviruses. These sensors can detect rapid changes in cAMP with similar sensitivity (EC50
1 µmol/L).13,14 Application of isoprenaline (Iso) (100 nmol/L) during a 15-second pulse produced a rapid and transient increase in cAMP at the plasma membrane, as monitored by the CNGA2 current density (dICNG; Figure 1A). As shown on the images in Figure 1B, cyan (CFP) and yellow (YFP) fluorescent protein fluorescence were uniform throughout cells expressing Epac2-camps, indicating cytosolic localization of the probe. The same 15-second Iso pulse elicited a transient rise of [cAMP]i, as monitored by FRET changes between CFP and YFP (Figure 1B) and the consequent changes in CFP/YFP ratio (Figure 1C). With both sensors, cAMP detection occurred immediately after Iso application, confirming that β-AR and Gs activation occurred in less than 5 seconds.4,15 As shown by the CFP/YFP ratio images in Figure 1C (also see the Movie in the online data supplement), Iso application produced a global increase of the cAMP signal, with no obvious striation pattern. Once produced, cAMP binds to tetrameric PKA, which dissociates into catalytic and regulatory subunits. Free catalytic subunits then phosphorylate various substrates, including the L-type Ca2+ channels. Figure 1D shows an example of ICa,L increase elicited by a 15-second pulse of Iso, thus allowing to further compare cAMP kinetics with that of the phosphorylation of 1 of its final effectors. The average of several similar experiments measuring cAMP responses with the different indicators and ICa,L potentiation are presented in Figure 2. Iso-induced subsarcolemmal cAMP transients led to dICNG that peaked at 10.7±1.6 pA/pF in tmax=38.3±3.1 seconds (Figure 2A and 2D) before returning rapidly to baseline (
decay=33.9±2.6 seconds, n=26; Figure 2E). In the cytosol, Iso elicited a maximal FRET variation of 9.1±0.5%, with a significantly slower time course than that measured at the plasma membrane by ICNG (tmax=57.6±1.6 seconds and decay=73.4±2.0 seconds, n=49; P<0.001 versus dICNG values; Figure 2B, 2D, and 2E). Thus, cAMP transients rise and decrease faster at the plasma membrane than in the cytosol. The responses of [cAMP]i to Iso in both compartments preceded that of ICa,L (Figure 2C). The maximal increase in ICa,L (+133.5±10.4%, n=18) occurred at tmax=82.2±5.4 seconds (n=18; P<0.001 versus tmax for ICNG), and ICa,L was still strongly enhanced when [cAMP]i had already returned to basal levels (decay=259.2±34.8 seconds, P<0.001 versus ICNG). This suggests that molecular events occurring downstream of cAMP are rate-limiting for both the rise and decay of ICa,L.
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PDE Modulation of cAMP and ICa,L Responses to β-AR Stimulation
[cAMP]i is tightly controlled by PDEs, with PDE3 and PDE4 contributing the majority of cAMP-PDE activity in rat cardiac myocytes.8–10 Therefore, we examined the role of these 2 PDEs in regulating cAMP and ICa,L signals in response to β-AR stimulation using the selective inhibitors cilostamide (Cil) (PDE3) and Ro 20-1724 (Ro) (PDE4), respectively. We showed previously that global PDE inhibition with 3-isobutyl-l-methylxanthine (IBMX) barely affected basal cAMP levels measured with CNG channels but strongly increased basal ICa,L.8,16 This effect on ICa,L could be reproduced by concomitant blockade of PDE3 and PDE4, whereas selective inhibition of a single PDE family was without effect.16 We confirmed this finding here and characterized the effect of both inhibitors on basal cAMP monitored by Epac2-camps (see Figure I in the online data supplement). Application of Cil (1 µmol/L) or Ro (10 µmol/L) alone generally induced a slight (<0.5%) but significant increase of basal FRET ratio. Concomitant inhibition of PDE3 and PDE4 resulted in either a substantial cAMP elevation or, in some cells, had no effect. On average, Cil+Ro increased the FRET ratio by 4.3±1.86% (n=9). These results suggest a low and variable basal cAMP synthesis in ARVMs counterbalanced by PDE3 and PDE4.
We next determined the role of PDE4 in terminating β-AR stimulation (Figure 3). Incubation of ARVMs with Ro (10 µmol/L) before the Iso pulse increased the amplitude of the cAMP response to Iso both at the membrane (by 88.9±3.3%, n=20; P<0.05 versus Iso alone; Figure 3A) and in the cytosol (by 58.0±7.9%, n=19; P<0.001 versus Iso alone; Figure 3B), along with an increase in the amplitude of the ICa,L response (by 55.2±8.5%, n=12; P<0.001 versus Iso alone; Figure 3C). Ro also greatly prolonged the effect of Iso on cAMP in both the subsarcolemmal and cytosolic compartments by increasing the duration of the rising phases (tmax=84.2±9 seconds, n=20 for ICNG and tmax=128.9±13.6 seconds, n=19 for the FRET signal; P<0.001 versus Iso alone for each; Figure 3D) and the duration of the decay phases (decay=106.6±12.2 seconds for ICNG and decay= 367.6±54.2 seconds for the FRET signal; P<0.001 versus Iso alone for each; Figure 3E). Ro had similar effects on ICa,L: time to peak and decay were significantly increased on PDE4 inhibition (tmax=129.3±10.2 seconds, P<0.001 and decay=380.8±55.4 seconds, P<0.05 versus Iso alone, n=12). These results identify PDE4 as an essential regulator of β-AR cAMP signals and L-type Ca2+ channel in ARVMs.
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In contrast, when PDE3 activity was inhibited using 1 µmol/L Cil, we detected only a slight, but significant, prolongation of the cAMP response at the plasma membrane (Figure 4A). Time to maximal ICNG density was delayed (tmax=54.1±6.4 seconds, n=25, P<0.01) in the presence of the inhibitor (Figure 4A and 4D), but the maximal amplitude (13.5±1.8 pA/pF) and the rate of decay (decay=37.8±2.7 seconds, Figure 4E) were not significantly different from the control values obtained with Iso alone. Moreover, Cil did not affect Iso-induced cytosolic cAMP signals or ICa,L (Figure 4B through 4E). In the presence of Cil, the FRET ratio increased by 9.7±0.9% (n=18) on an Iso challenge, with similar kinetics as in control (tmax=59.2±2.0 seconds and decay=63.6±2.5 seconds). Similarly, Cil neither modified the amplitude (164.0±14.5% increase over control, n=13) nor the time course (tmax=75.0±6.5 seconds and decay=272.2±42.5 seconds) of the Iso increase of ICa,L.
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These results suggest that PDE3 plays a minor role in the regulation of cAMP and ICa,L responses to a brief β-AR stimulation in ARVMs. Yet, other PDE(s) besides PDE4 must be involved in degrading β-AR-dependent cAMP because ICNG density and FRET ratio still declined in the presence of Ro (Figure 3). To determine whether the remaining PDE activity after PDE4 blockade was attributable to PDE3, the cells were preincubated with Ro (10 µmol/L) during 2 minutes, then Cil (1 µmol/L) was added together with Iso. After 15 seconds, Iso was washed out in the continuous presence of Cil and Ro. As shown in Figure 5A through 5C, simultaneous inhibition of PDE3 and PDE4 did not further increase the amplitude of Iso-dependent responses compared with Ro alone (dICNG was increased to 15.3±2.0 pA/pF, n=13; the FRET ratio was increased by 12.3±0.6%, n=10, and ICa,L was potentiated by 191.4±21.2%, n=10 in the presence of Cil+Ro). The values for tmax obtained in these conditions were similar to those obtained when only PDE4 was blocked (Figure 3) most likely because, unlike in Figure 4, PDE3 was not fully inhibited during the rising phase of the Iso response. However, we observed a considerable prolongation of Iso-triggered responses, so that the rates of decay could not be accurately estimated by exponential fitting over a period of 500 seconds. Thus, even though PDE3 inhibition alone had almost no effect on any parameters, PDE3 activity was sufficient to terminate β-AR–dependent cAMP and ICa,L responses when PDE4 was inhibited. We tested the participation of additional PDEs by including the wide spectrum PDE inhibitor IBMX (300 µmol/L) during the Iso stimulation and washout. The effect of IBMX was similar to that of Cil+Ro on membrane cAMP (dICNG) and ICa,L (Figure 5A and 5C). In contrast, IBMX suppressed the residual cAMP decrease in the cytosol (Figure 5B), suggesting that other PDEs, such as PDE1 and PDE2, are active in this compartment. The IBMX-resistant, slow decrease in ICNG density and ICa,L likely reflects cAMP diffusion from the membrane compartment to the cytosol dialyzed by the internal patch pipette solution.12 However, the rate of dICNG decrease in the presence of IBMX was approximately 1 order of magnitude smaller than the rate of dICNG decrease without IBMX, making cAMP leak through the patch pipette a negligible contributor of dICNG kinetics in the previous experimental conditions.
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PKA-Mediated PDE4 Activation Regulates β-AR cAMP Signals in ARVMs
Using CNG channels, we have shown previously that subsarcolemmal cAMP signals are controlled by a negative feedback involving PKA activation of PDE3 and PDE4.8 Coexpression of Epac2-camps with rabbit muscle cAMP-dependent protein kinase inhibitor (PKI)17 allowed us to examine the role of PKA in cytosolic cAMP degradation. As shown in Figure 6A through 6C, PKI overexpression increased the amplitude (by 58.9±9.6%, n=26; P<0.001 versus Iso alone) and duration (tmax=91.0±6.3 seconds and decay=123.9±3.4 seconds, n=26; P<0.001 versus Iso alone) of the FRET response to Iso. Additional PDE4 inhibition further slowed the cAMP signal detected by the FRET sensor, but we obtained a similar time course as with Ro alone (see Figure 3B, 3D, and 3E). Thus, PKI had no additional effect over PDE4 inhibition, suggesting that PKA regulation of β-AR cAMP levels in the cytosol of ARVMs is mainly attributable to PDE4 activation. We tested whether this mechanism could mask the participation of PDE3. In the presence of PKI, Cil had a small but significant effect on the decay phase of Iso-induced cAMP (Figure 6A through 6C). The fact that Cil had a much larger effect in the presence of Ro (Figure 5B), which fully blocks PDE4 activity, than with PKI, which only prevents its activation by PKA,18 suggests that basal PDE4 activity is sufficient to hydrolyze most of the cAMP produced by β-AR stimulation even when PDE3 is inhibited. This hypothesis was confirmed in subsequent biochemical experiments (Figure 6D and 6E). Total cAMP hydrolytic activity was measured from homogenates of isolated cardiomyocytes, and specific PDE3 and PDE4 activities were determined using selective inhibitors. As shown in Figure 6D, total basal PDE activity was 99.4±8.29 pmol/min per milligram of protein, of which >60% was attributable to PDE4 (62.1±5.5 pmol/min per milligram of protein) and 30% to PDE3 (33.2±2.8 pmol/min per milligram of protein, n=10). Application of Iso (100 nmol/L, 15 seconds) increased total cAMP-hydrolytic activity by 47.0±4.4% (n=8, P<0.01 versus basal), and this was mainly attributable to PDE4 activation (by 69.4±7.3%, n=8; P<0.01), although there was also a small increase in PDE3 activity (by 35.6±6.4%, n=8; P<0.05). Furthermore, this activation was dependent on the level of β-AR stimulation (Figure 6E), suggesting that the level of PDE activation follows cAMP elevation induced by β-AR agonists.
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Discussion |
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In the present study, we determined the kinetics of β-AR–induced cAMP signals in different subcellular compartments of ARVMs using real-time cAMP sensors and compared them with the activation of an endogenous downstream target of cAMP/PKA signaling, ICa,L. We also evaluated the contribution of PDEs to the regulation of these signaling events. The main findings were as follows: (1) submembrane and cytosolic cAMP signals display different kinetics, indicating a restricted cAMP diffusion between these compartments; (2) ICa,L stimulation develops >2-fold slower, and its return to basal levels develops >7-fold slower compared with membrane cAMP responses, suggesting that Cav1.2 phosphorylation and dephosphorylation are rate-limiting in the β-AR cascade; (3) PDE4 is the main PDE subtype modulating β-AR cAMP transients and, as a consequence, ICa,L recovery after β-AR stimulation; (4) PKA shapes β-AR cAMP transients by activating PDE4; and (5) β-AR cAMP signals are differentially regulated by PDEs at the membrane and in the cytosol.
Although several recent studies investigated cAMP signaling and compartmentation of cyclic nucleotides using live-cell imaging in cardiac cells, most of these were performed using neonatal cells.4,6,9 Because subcellular structures may play an important role in cAMP compartmentation, we used highly differentiated adult cardiac myocytes in the present study. Despite the presence of developed T-tubular network in ARVMs, fluorescence images of Epac2-camps probe showed no obvious striated pattern on the ratio images in either quiescent cells or following β-AR stimulation. This contrasts with the result of a previous report where a cAMP sensor developed on the basis of the R and C subunits of PKA did show striated cAMP patterns6 but corroborates subsequent studies using other FRET-based cAMP sensors7 and A-kinase activity reporter (AKAR2).4
Following a 15-second Iso pulse, cAMP transients in both subsarcolemmal and cytosolic compartments started almost immediately after Iso application, in agreement with earlier estimates of the kinetics of the β-AR/Gs/adenylyl cyclase cascade.4,15 However, comparing the CNG current with the Epac2-camps signal showed that cAMP kinetics (rise and decay) were faster at the membrane than in the cytosol. These kinetics were too fast to be notably influenced by cAMP diffusion in the patch pipette (as estimated from measurements with IBMX, see also Rich et al12). Also, ICNG and Epac2-camps have a similar sensitivity to cAMP (KD1 µmol/L),13,14 and the binding and dissociation kinetics of cAMP on the 2 sensors are much faster than the measured kinetics during Iso response (see the online data supplement for details). Thus, β-AR stimulation of ARVMs leads to cAMP signals with different dynamics in distinct compartments, as previously reported in HEK-293 cells.19–21 The cytosolic versus membrane cAMP signals described here were delayed to the same extent as recently reported for cytosolic versus membrane PKA activity using AKAR2 sensors in neonatal cardiomyocytes.4 Such synchronization indicates that a cAMP gradient between membrane and cytosol creates a phosphorylation gradient mediated by PKA in cardiac cells. As noted previously,4,21 the delay between membrane and cytosolic cAMP-mediated events is not compatible with cAMP diffusion rates measured in the cytosol.7 Thus, cAMP diffusion from the membrane to the bulk cytosol must be restricted either by an enzymatic or a physical barrier.12,22
The rate of ICa,L stimulation reported here is similar to those reported earlier for ICa,L in frog ventricular myocytes,23 for neuronal potassium channels,24 and for the membrane-associated PKA sensor AKAR2.4 Activation of Cav1.2 developed >2-times slower than the rise of cAMP at the membrane, suggesting that the rate-limiting step in the β-AR stimulation of ICa,L occurs after cAMP accumulation. This is likely attributable to PKA activation, as suggested by the slower response of the PKA-based cAMP probe, FlCRhR, to β-AR stimulation in frog ventricular myocytes.23 The recovery of ICa,L from β-AR stimulation was also comparable to previous results in frog cells23 and to dephosphorylation kinetics of AKAR2.4 The considerable delay between ICa,L recovery and cAMP decay indicates that mechanisms other than cAMP degradation by PDEs are rate-limiting in the recovery of ICa,L: these include the deactivation of PKA, dephosphorylation of the L-type Ca2+ channel by phosphatases, or recovery of basal Ca2+ channel gating.23
The rapid decay of ICNG compared to Epac2-camps signal could be attributable to diffusion of cAMP to the cytosol and degradation by PDEs. PDEs have been shown to be localized at the plasma membrane through membrane-association domains (such as PDE325) or protein–protein interactions26,27 and are more concentrated at the plasma membrane than in the cytosol.28 Differential cAMP degradation might lead to faster phosphorylation/dephosphorylation of membrane proteins (such as the calcium channels) as compared with cytosolic proteins on a given stimulus, as previously suggested in neurons.24
A number of previous studies demonstrated that basal cAMP levels are controlled by PDEs in resting cardiac cells.6,9,10,16,29 Basal cAMP turnover is mainly regulated by PDE4 in neonatal cardiomyocytes,9 whereas both PDE3 and PDE4 regulate resting cAMP levels and ICa,L in adult cells.16 We previously reported that IBMX had no effect on subsarcolemmal cAMP levels measured with CNG channels in ARVMs.8 Here, using Epac2-camps, we detected a variable but significant increase in basal cAMP on PDE3 and PDE4 inhibition (supplemental Figure I). Because both sensors present similar affinities for cAMP13,14 but are located in distinct subcellular compartments, this result suggests that basal cAMP levels are different in distinct parts of the cell. This could be related to the presence of IBMX insensitive PDE830 or cAMP transporters such as multidrug resistance protein 5,31 both of which are expressed in heart and might lower cAMP selectively at the membrane. Following β-AR stimulation, we confirm here that PDE4 is the main PDE degrading cAMP in ARVMs7–9 and is crucial for Ca2+ channel regulation.16 Interestingly, PDE4 inhibition had a more drastic effect on cytosolic cAMP than subsarcolemmal cAMP, in agreement with a major fraction of PDE4 activity being cytosolic in the rat myocardium.32 Furthermore, inhibition of all PDE activities with IBMX had no additional effect at the membrane compared to simultaneous inhibition of PDE3 and PDE4, whereas it further delayed the decay of cytosolic cAMP signals (Figure 5B), suggesting that other PDEs, such as PDE1 or PDE2, are contributing to cAMP degradation in the cytosol.10 In contrast, inhibition of PDE3 had only a minor effect on ICNG and no effect on the FRET signal or ICa,L. These results are in agreement with previous studies in rodent cardiac myocytes7,9 and may indicate a preferential location of PDE3 at the membrane.25 However, when PDE4 was inhibited, PDE3 became predominant to hydrolyze membrane and cytosolic cAMP, as well as regulating ICa,L recovery. Thus, it appears that PDE4 is able to compensate for PDE3 inhibition, at least in the cytosol and in the vicinity of L-type Ca2+ channels. This could be related to integration of PDE4 into A kinase anchoring protein–organized signaling complexes,3,33 hence facilitating activation of PDE4 by PKA.3,8,18 This mechanism is supported by the fact that PKA inhibition mimics, to some extent, the effect of Ro on cytosolic cAMP and that PDE4 activity is enhanced on β-AR stimulation.8
More generally, these results raise the question of the respective roles of PDE3 and PDE4 in regulating cardiac contractility in larger mammals, where PDE3 is thought to be predominant. This idea is based mainly on the fact that PDE3 inhibitors, contrary to PDE4 inhibitors, have a direct inotropic effect on basal contractility. However, PDE4 inhibitors exert strong inotropic responses in the presence of β-AR tone.32 Thus, it is tempting to speculate that PDE3 regulates a "constitutive" cAMP pool linked to contractility, whereas PDE4 regulates the cAMP microdomains mobilized by β-AR stimulation. Further experiments in large mammals and humans are necessary to verify this hypothesis.
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Acknowledgments |
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We are indebted to Florence Lefebvre for superb technical assistance. We thank Valérie Domergue-Dupont and the animal core facility of IFR141 for efficient handling and preparation of the animals. We are also grateful to Dr Hazel Lum (University of Illinois, Chicago) for providing us the adenovirus encoding PKI, Dr Dermot Cooper (University of Cambridge, UK) for the adenovirus encoding CNGA2 mutants, and Drs Stefan Engelhardt and Martin Lohse (University of Würzburg, Germany) for the adenovirus encoding Epac2-camps.
Sources of Funding
This work was supported by grants from the Fondation Leducq 06CVD02 cycAMP (to R.F. and M.C.), European Union contract LSHM-CT-2005-018833/EUGeneHeart (to R.F.), the Fondation de France (to G.V.), and NIH grant HD20788 (to M.C.). A.A.-G. was a recipient of doctoral grants from the French Ministry of Education and Research and the Groupe de Réflexion sur la Recherche Cardiovasculaire. J.L. was recipient of a post doctoral grant from Fondation Lefoulon Delalande and Fondation pour la Recherche Médicale.
Disclosures
None.
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Footnotes |
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Original received November 13, 2007; revision received March 6, 2008; accepted March 13, 2008.
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References |
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