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Multiple cyclic nucleotide phosphodiesterases (PDEs) degrade cAMP in cardiomyocytes but the role of PDEs in controlling cAMP signaling during pathological cardiac hypertrophy (CH) is poorly defined.
Evaluate the β-adrenergic regulation of cardiac contractility and characterize the changes in cardiomyocyte cAMP signals and cAMP-PDE expression and activity following CH.
CH was induced in rats by thoracic aortic banding over a time period of 5 weeks and was confirmed by anatomical measurements and echocardiography. Ex vivo myocardial function was evaluated in Langendorff perfused hearts. Engineered cyclic nucleotide-gated channels (CNG) were expressed in single cardiomyocytes to monitor subsarcolemmal cAMP using whole-cell patch-clamp recordings of the associated CNG current (ICNG). PDE variant activity and protein level were determined in purified cardiomyocytes. Aortic stenosis rats exhibited a 67% increase in heart weight compared to sham-operated animals. The inotropic response to maximal β-adrenergic stimulation was reduced by ~54% in isolated hypertrophied hearts, along with a ~32% decrease in subsarcolemmal cAMP levels in hypertrophied myocytes. Total cAMP hydrolytic activity as well as PDE3 and PDE4 activities were reduced in hypertrophied myocytes, due to a diminution of PDE3A, PDE4A and PDE4B whereas PDE4D was unchanged. Regulation of β-adrenergic cAMP signals by PDEs was blunted in hypertrophied myocytes, as demonstrated by the diminished effects of IBMX (100 μmol/L) and of both the PDE3 inhibitor cilostamide (1 μmol/L) and the PDE4 inhibitor Ro 201724 (10 μmol/L).
β-adrenergic desensitization is accompanied by a reduction in cAMP-PDE and an altered modulation of β-adrenergic cAMP signals in CH.
Stimulation of cardiomyocyte β-adrenergic receptors (β-ARs) by noradrenaline released from the sympathetic nervous system (SNS) is the most powerful mechanism to increase cardiac output in response to stress or exercise. β-ARs signal primarily through Gαs-proteins resulting in the activation of adenylyl cyclases and the elevation in the intracellular concentration of the second messenger cAMP. The positive chronotropic, inotropic, and lusitropic effects of cAMP on cardiomyocytes are primarily mediated by the cAMP-dependent protein kinase (PKA) which phosphorylates and regulates many of the key proteins involved in cardiac excitation-contraction coupling.1
β-AR signaling is rapidly terminated through several mechanisms that limit cAMP production, such as re-uptake and/or metabolism of noradrenaline, uncoupling and desensitization of β-ARs, and inactivation of Gαs signaling upon GTP-hydrolysis. Intracellular cAMP is degraded by cyclic nucleotide phosphodiesterases (PDEs), thus inactivating PKA. Finally, protein phosphatases reverse the PKA-mediated effects on myocyte contraction by dephosphorylating the PKA down-stream targets. PDEs comprise a large group of isoenzymes that are divided into 11 PDE families based on their substrate and inhibitor specificity and sequence homology.2,3 Of these, PDE3 and PDE4 contribute the majority of the cAMP-hydrolytic activity in cardiomyocytes.4,5 PDE3 is encoded by two genes (PDE3A and PDE3B), with PDE3A being the predominant form expressed in cardiomyocytes.6 The PDE4 family consists of four genes (PDE4A to D), but only PDE4A, PDE4B, and PDE4D appear to be expressed in rat heart.7 These multiple PDEs contribute to the generation of intracellular cAMP microdomains within cardiomyocytes that are thought to be critical for the specificity of cAMP signalling.3,5
Whereas acute stimulation of cardiac output by the SNS is essential for the adaptation of the organism to its environment, chronic activation of the SNS promotes a pathological remodelling of the heart which may ultimately lead to heart failure (HF).8,9 It is well established that chronically elevated catecholamine levels, which are a hallmark of HF, lead to the desensitization of cardiac β-AR signaling through several mechanisms which limit cAMP production. These include the down-regulation of β1-ARs, the uncoupling of β2-ARs from Gαs, an increased activity of β-AR kinases and an increase in Gαi subunits which promotes the signaling of β-ARs through Gαi to inhibit adenylyl cyclase. In canine models of HF, Gαs and adenylyl cyclases type V and VI are also decreased.10
In comparison, the signaling mechanisms acting downstream of cAMP synthesis have received much less attention. In particular, only a few studies have investigated the potentially critical role of PDEs in controlling cAMP signaling during pathological cardiac hypertrophy (CH). These studies focused on PDE3, reporting a decreased PDE3 expression and/or activity in the failing heart.11 Such a decrease in PDE3 is thought to have adverse consequences on the heart as it promotes cardiomyocyte apoptosis12 and exaggerates cardiac dysfunction induced by chronic pressure overload.13 Although largely ignored until recently, a role of PDE4 in HF is supported by the late onset cardiomyopathy developed in mice with deletion of the PDE4D gene.14
Intrigued by these findings, we determined the expression pattern of PDE3 and PDE4 enzymes and investigated the role of these PDEs in the control of β-AR cAMP signals in a rat model of compensated CH. Using CNG channels, we provide the first live cell recordings of cAMP in hypertrophied cardiomyocytes and demonstrate that PDE3 and PDE4 regulation of β-AR cAMP signals is blunted in hypertrophy. We report a decreased protein expression of PDE3A, PDE4A, and PDE4B but not of PDE4D in hypertrophied cardiomyocytes compared to sham controls that is paralleled by similar changes in cAMP-PDE activities for these PDE subtypes. These results document profound defects in the two main PDEs degrading cAMP in hypertrophied myocytes.
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 supplement material at http://circres.ahajournlas.org.
Briefly, CH was induced in rats by thoracic aortic banding over a time period of 5 weeks and was confirmed by anatomical measurements and echocardiography. Ex vivo myocardial function was evaluated in Langendorff perfused hearts. Engineered cyclic nucleotide-gated channels (CNG) were expressed in single isolated cardiomyocytes to monitor subsarcolemmal cAMP using whole-cell patch-clamp recordings of the associated CNG current (ICNG). PDE variant activity and protein level were determined in purified cardiomyocytes.
To generate a model of CH, male Wistar rats were subjected to aortic constriction over a time period of 5 weeks as described in Materials and Methods. The development of CH was then assessed through anatomical data and echocardiography (see Table 1). Heart weight (HW) of the CH rats was significantly higher (+67%) than that of the sham-operated rats whereas body weight (BW) and tibia length (TL) were similar in both groups. The weight of lung, liver, kidney, and right ventricle were also unchanged (data not shown). Telediastolic interventricular septum, telediastolic posterior wall thickness and left ventricular end diastolic diameter as determined by echocardiography were significantly larger in CH rats compared to sham (Table 1). However, the fractional shortening was similar between both groups indicating a preserved left ventricular (LV) function in CH rats. From these data, we can classify this model as compensated CH.15 Measurement of cell capacitance, perimeter, and surface confirmed that LV myocytes from CH rats exhibited substantial hypertrophy when compared to sham-operated animals (Table 1).
To further characterize our model, myocardial function was evaluated using an isolated Langendorff heart preparation. As shown in Table 2, spontaneous heart rate was similar between sham-operated and CH hearts and was increased to the same extent by a saturating concentration of the β-AR agonist isoprenaline (ISO, 1 μmol/L). Basal left ventricular developed pressure (LVDP) and maximal rate of contraction, measured as the maximal value of LV +dP/dtmax, were not different between the two groups, as was the maximal rate of cardiac relaxation, measured as the maximal value of LV -dP/dtmax (Table 2). ISO (1 μmol/L) increased these three parameters by 2 to 4-fold in normal hearts. In contrast, the response to maximal β-AR stimulation was reduced in hypertrophied hearts (Table 2). To further document this difference, concentration-response curves (CRC) to ISO were generated for both groups. Figure 1A shows the CRC obtained for LV +dP/dtmax by fitting the experimental data with the Hill function. The two resulting curves were statistically different (Fischer test, p<0.001) with a decreased maximal effect (Emax) of ISO in CH hearts (Emax was 8205.0±182.7 mmHg.s-1 in sham and 3758.7±24.4 mmHg.s-1 in CH) but identical apparent potency (EC50 was 3.4±0.3 nmol/L in sham versus 3.3±0.2 nmol/L in CH). Similar results were obtained when the hearts were electrically paced (at 400 bpm). Thus, in this model of compensated CH, basal cardiac function is preserved but the inotropic reserve in response to β-AR stimulation is reduced.
It is well established that β-adrenergic control of cardiac contractility is associated with changes in the particulate, but not the soluble pool of cellular cAMP.16,17 To determine whether the attenuated inotropic response to ISO in CH was related to a decrease in plasma membrane cAMP, engineered cyclic nucleotide-gated (CNG) channels were over-expressed in sham and CH cardiomyocytes and the associated CNG current (ICNG) was recorded as an index of subsarcolemmal cAMP.18 Figure 1B summarizes the results of initial experiments in which the effect of ISO at 100 nmol/L was tested on sham and CH cardiomyocytes expressing either the low-affinity E583M CNGA2 channel (K1/2cAMP=10.3 μmol/L) or the high affinity C460W/E583M CNGA2 channel (K1/2 cAMP=1.4 μmol/L). In order to correct for cell size, ICNG density was calculated by dividing the current amplitude by the cell capacitance for each experiment. As shown in Figure 1B, E583M CNGA2 detected ISO-induced cAMP signals in cardiomyocytes from sham-operated rats (ICNG density was increased to 4.2±1.5 pA/pF in the presence of 100 nmol/L ISO, n=19) but not in cardiomyocytes from CH rats (ICNG density was 0.7±0.2 pA/pF, n=12). In contrast, using the high affinity C460W/E583M CNGA2 allowed unambiguous detection of β-AR-dependent cAMP signals in both groups. On average, ISO increased ICNG density to 15.1±1.9 pA/pF (n=28) in sham cardiomyocytes and to 8.3±1.8 pA/pF (n=24) in CH myocytes. These results demonstrate a decrease in the subsarcolemmal concentration of cAMP upon β-AR stimulation in hypertrophy. To fully characterize the β-AR response, CRCs to ISO were generated in both groups using C460W/E583M CNGA2 (Figure 1C). Consistent with the measurements of contractility of whole hearts, ISO dose-dependently increased cAMP in sham and CH cardiomyocytes , but maximal stimulation was attenuated in the CH myocytes. Fit of the data to the Hill equation yielded two statistically different curves (Fischer test, p<0.001) with a decreased maximal effect in hypertrophy (Emax was 15.1±0.7 pA/pF in sham versus 10.3±0.2 pA/pF in CH cardiomyocytes) but similar apparent potency (EC50 was 9.9±2.3 nmol/L in sham versus 6.2 ± 0.7 nmol/L in CH cardiomyocytes). CNGA2 channel expression was the same in sham and CH myocytes as indicated by immunoblot analysis (see Supplement Material, online Figure 1). In contrast, the response to the forskolin analogue L-858051 (100 μmol/L) was identical in sham and CH cardiomyocytes (30.3±3.0 pA/pF in sham versus 29.4±5.7 pA/pF in CH cardiomyocytes, Figure 1D). Since at this concentration L-858051 was shown to saturate the probe,23 this result only indicates a similar density of functional CNG channels in sham and CH cardiomyocytes. Thus, the ~32% decrease in maximal ISO response in CH cells reflects a real decrease in the concentration of cAMP at the membrane.
As a first experiment to elucidate the role of PDEs on cAMP signals in sham-operated and CH rat hearts, the effect of the general PDE inhibitor IBMX was tested in cardiomyocytes using the low-affinity E583M CNGA2 channel. We showed previously that 100 μmol/L IBMX had no effect on basal ICNG in cardiomyocytes expressing the E583M CNGA2 channel.18 In Figure 2, the effects of IBMX in combination with 100 nmol/L ISO are compared. Although IBMX increased the response of ICNG to ISO in both sham (Figure 2A) and CH myocytes (Figure 2B), the response remained on average ~50% smaller in CH cells (Figure 2C). Because most of the cAMP hydrolytic activity is inhibited at the concentration of IBMX used,19 this 50% difference between sham and CH cardiomyocytes likely reflects a commensurate reduction in cAMP synthesis. Using the higher affinity C460W/E583M CNGA2 channel to elicit larger basal responses to ISO, we evaluated the contributions of PDE3 and PDE4 to the β-AR response. Preliminary experiments were performed to check the effect of global PDE inhibition on basal ICNG in rat ventricular myocytes expressing the C460W/E583M CNGA2 channel. In agreement with our previous reports,5,20 global PDE inhibition with IBMX (100 μmol/L) failed to significantly increase the current in sham (ICNG was 0.9±0.5 pA/pF in the presence of IBMX, and 17.3±4.4 pA/pF in the presence of 100 nmol/L ISO, n=3) and CH cardiomyocytes (ICNG was 1.1±0.45 pA/pF in the presence of IBMX, and 5.4±0.8 pA/pF the presence of 100 nmol/L ISO, n=6). As shown in Figure 3, the results were quite different upon β-AR stimulation. In sham cardiomyocytes, the effect of ISO on ICNG was potentiated ~2-fold upon PDE3 inhibition with cilostamide (CIL, 1 μmol/L) and 3 to 5-fold upon PDE4 inhibition with Ro 201724 (RO, 10 μmol/L) (Figure 3A, C & D). In CH cardiomyocytes, CIL potentiated the ISO response by only ~40% whereas RO increased the ISO response ~2-fold (Figure 3B, C & D). Consequently, the responses of ICNG to ISO in combination with either PDE inhibitor were significantly reduced in CH versus sham cells (p<0.01, Figure 3C & D).
The above results suggest that in addition to a decreased β-AR cAMP production, cAMP degradation by PDE3 and PDE4 is also decreased in CH cardiomyocytes. To test this hypothesis further, the expression of PDE3 and PDE4 proteins was measured in cardiomyocytes isolated from sham-operated (n=6) or CH rats (n=4). Equal amounts of proteins prepared from cardiomyocytes isolated from CH or sham-operated animals were separated on SDS/PAGE and PDE3A, PDE4A, PDE4B, and PDE4D proteins were subsequently detected by Western blotting using PDE subtype-selective antibodies. Detergent extracts prepared from brain and heart of PDE KO mice and wild type control mice were analyzed on the same blots to control for the specificity of the antibodies. As shown in Figure 4A, all immunoreactive bands detected with PDE4-subtype selective antibodies in tissues of wild type mice were not present in the respective PDE4 KO tissue. This finding confirms the specificity of the antibodies used, and indicates that the proteins detected in the rat heart, given their identical migration on SDS-PAGE, are authentic PDEs. Most PDEs are expressed as multiple variants through the use of different promoters and alternative splicing which explains the presence of multiple immunoreactive bands detected in Western blotting for several PDE subtypes. Three immunoreactive bands migrating at approximately 125 kDa, 106 kDa, and 97 kDa were detected for PDE3A in rat cardiomyocytes. All three appeared to be decreased in CH myocytes compared to sham cells. Probing cardiomyocyte extracts with PDE4A-selective antibodies also labeled three bands of approximately 105, 95 and 79 kDa. All PDE4A immunoreactive signals were attenuated in CH myocytes compared to sham. A single band migrating at approximately 92 kDa was detected in ventricular cardiomyocytes for PDE4B. Its expression was decreased in CH cardiomyocytes. A single band migrating at approximately 91 kDa was detected in cardiomyocytes extracts using PDE4D-selective antibodies. Migration of this band corresponds to the migration of PDE4D variants PDE4D3, PDE4D8, and PDE4D9.21 In contrast to the other PDE subtypes analyzed, the PDE4D immunoreactive signal intensity was not significantly different in CH compared to sham cells (Figure 4B).
To determine whether changes in PDE protein expression are reflected in similar changes of PDE activity, total cAMP-hydrolytic activity in isolated cardiomyocytes from CH and sham-operated rats was measured (Figure 5). Total cAMP-PDE as well as PDE3 and PDE4 activities were significantly decreased by 42%, 42% and 53%, respectively (Figure 5A). To further dissect the contributions of single PDE4 subtypes, total extracts from cardiomyocytes isolated from sham (n=5) and CH (n=4) rats were immunoprecipitated using subtype-specific antibodies against PDE4A, PDE4B, and PDE4D and the PDE activity recovered in the IP pellets was assayed (Figure 5B). The activity of both PDE4A and PDE4B was significantly reduced in CH cells compared to sham. Conversely, PDE4D activity was not significantly different between the two groups.
With this study, we provide evidence that cardiac hypertrophy induced by chronic pressure overload in the rat is associated with major changes in the cAMP signaling machinery that controls excitation-contraction coupling. Together with marked β-AR desensitization, we demonstrate a decrease in relative activities and protein densities for PDE isoforms belonging to the PDE3 and PDE4 families, the major cAMP hydrolyzing forms expressed in cardiomyocytes from different species.
In rat cardiomyocytes, PDE3 and PDE4 contribute between 70% and 90% of the total cAMP-hydrolyzing activity.4,18,20,22 Therefore, a molecular characterization of the various PDE3A and PDE4 isoforms expressed in cardiomyocytes was undertaken. The three immunoreactive bands of PDE3A detected here in the rat heart likely represent the counterparts of human PDE3A1, PDE3A2 and PDE3A3 because of their similar migration in SDS/PAGE.23 Of the three immunoreactive species detected for PDE4A, the long 95 kDa form was previously detected in rat heart but not identified.7 To our knowledge, the two others species migrating at 105 kDa and 79 kDa were not described in cardiomyocytes. The size of the first corresponds to a long form, such as PDE4A5, PDE4A8, PDE4A10 or PDE4A11.24,25 The short form detected likely corresponds to PDE4A1, which has a mobility similar to the form migrating at ~75 kDa in rat brain.24 Only one immunoreactive band at approximately 92 kDa was detected in cardiomyocytes for PDE4B. This species was previously reported in rat ventricle24 and likely represents long PDE4B form(s) such as PDE4B1 and/or PDE4B3, for which transcripts were detected in rat heart.7 In contrast to earlier studies, we could not detect expression of PDE4B2, which is expected to migrate at approximately 75 kDa.4,7 Finally, we detected one PDE4D immunoreactive band which exhibited a migration similar to splice variants PDE4D3, PDE4D8 and PDE4D9.21 These diverse PDE isoforms have been shown to be localized in specific compartments so as to exert a local control of cAMP signaling: for instance, PDE4D3 associates with the ryanodine receptor complex14 and with the IKs potassium channel complex,26 PDE4D5 with β-arrestin near the β2-AR,27 whereas PDE4D8 has been shown to bind to the β1-AR.28 In addition, a long isoform of PDE4D was shown recently to coimmunoprecipitate with SERCA2a.29
When comparing sham and hypertrophied cardiomyocytes, profound quantitative differences were observed: relative activity and protein expression of PDE3A, PDE4A and PDE4B were decreased in hypertrophy, whereas that of long PDE4D isoforms remained unchanged. While our experiments do not exclude the possibility that changes in other PDE families also occur in cardiac hypertrophy, the observed decrease in PDE levels is comparable to the reduction of β1-ARs30 and the sarcoplasmic reticulum Ca2+ ATPase31 previously reported in rat heart using a similar model of hypertrophy. Although the mechanisms were not determined, our findings suggest that the genes encoding PDE3A, PDE4A and PDE4B are not part of the hypertrophic program. A small number of previous studies have investigated the variations of PDE3 and PDE4 during CH with contradictory results.32 In dogs with CH due to aortic valve stenosis, cAMP-PDE activity was reported to be unchanged,33,34 whereas PDE3 and PDE4 expression and activities were found to be enhanced in Dahl salt-sensitive rats.35 Our results are consistent with a decreased expression of PDE3A in rodent models of CH induced by chronic infusion of isoprenaline or angiotensin II12 and with several studies showing a reduction in PDE3 expression and/or activity at the HF stage.36-39 However, some studies found no difference in PDE3 activity during HF.34,40 Concerning PDE4, a constant mRNA level for PDE4D was reported in dog with HF,37 which is consistent with what we observe in rats with CH. However, the PDE4D3 isoform associated to RyR2 was found decreased in human HF.19 This difference might be due to the different species or to the fact that we measured the total cellular PDE4D pool. Therefore, a decrease in PDE4D3 could be compensated by an increase in another splice variant such as PDE4D8 or PDE4D9 which are also expressed in heart.26 It should be noted that in this study protein expression and activity measurements were performed using highly purified cardiomyocytes, thus excluding possible variations due to other cell types present in the heart.
Despite an important decrease in the two main PDEs involved in cAMP hydrolysis, there was a marked impairment of contractile responsiveness to isoprenaline in hypertrophied hearts (Figure 1A) which correlated with reduced subsarcolemmal cAMP levels upon β-AR stimulation in hypertrophied cells (Figure 1B and 1C). These results indicate that the PDE diminution was unable to compensate for β-AR desensitization. They are in agreement with numerous studies showing a loss of the β-AR inotropic reserve,41-44 a decreased β-AR density,30,41,45 a decreased ISO-stimulated cAMP formation,46,47 and a decreased β-AR stimulation of the L-type Ca2+ current48 in rodent models of CH induced by pressure overload. In contrast, the response to the forskolin analog L-858051 at 100 μM was unchanged (Figure 1D). We showed previously that this concentration of L-858051 saturates the CNG channels.23 Thus, this result indicates that the same density of functional channels was expressed in normal and hypertrophied cells.
In conclusion, our study provides strong biochemical and functional evidence for a decreased cAMP-hydrolytic reserve during cardiac hypertrophy, and identifies PDE3A, PDE4A and PDE4B as being specifically altered. PDE down-regulation in CH might be regarded as an initial adaptive process because it partly compensates for the deficit in cAMP synthesis. However, such PDE remodeling may be maladaptive in the long term, because of a loss of cAMP compartmentation.3,18 This in turn may cause unrestricted diffusion of cAMP and chronic activation of cAMP effectors, such as PKA and the exchange factor for Rap1, Epac, both of which were shown to induce pathological cardiac hypertrophy.49-51
We are grateful to Valérie Domergue-Dupont and the animal core facility of IFR141 for efficient handling and preparation of the animals, to Paul Milliez for echocardiography and to Patrick Lechêne, Françoise Marotte and Camille Rodriguez for skillful technical assistance. We thank Dr. Bertrand Crozatier for expert assistance with Langendorff-perfused heart experiments and critical reading of the manuscript.
A.A.-G. was the recipient of doctoral grants from the French Ministry of Education and Research and from the Groupe de Reflexion en Recherche Cardiovasculaire. This work was supported by grants from the Fondation Leducq 06CVD02 cycAMP (to R.F. and M.C.), EU contract LSHM-CT-2005-018833/EUGeneHeart (to R.F.), NIH grant HL092788 (to M.C.), and the Fondation de France (to G.V.).
Conflict of interest
All authors declare no conflict of interest.