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(The FASEB Journal. 2003;17:1380-1391.)
© 2003 FASEB

Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8)

MARIE GEORGET, PHILIPPE MATEO, GRÉGOIRE VANDECASTEELE, LARISSA LIPSKAIA*, NICOLE DEFER*, JACQUES HANOUNE*, JACQUELINE HOERTER, CLAIRE LUGNIER{ddagger} and RODOLPHE FISCHMEISTER1

Laboratoire de Cardiologie Cellulaire et Moléculaire, INSERM U-446, Université Paris-Sud, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France;
* Laboratoire de Régulation des Gènes et Signalisation Cellulaire, INSERM U-99, Hôpital Henri-Mondor, F-94010 Créteil, France; and
{ddagger} Laboratoire de Pharmacologie et de Physico-Chimie des Interactions Cellulaires et Moléculaires, CNRS-UMR 7034, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, F-67401, Illkirch, France

1Correspondence: INSERM U-446, Université Paris-Sud, Faculté de Pharmacie, 5, Rue J.-B. Clément, F-92296 Châtenay-Malabry Cedex, France. E-mail: Fisch{at}vjf.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hearts from AC8TG mice develop a higher contractility (LVSP) and larger Ca2+ transients than NTG mice, with (surprisingly) no modification in L-type Ca2+ channel current (ICa,L) (1) . In this study, we examined the cardiac response of AC8TG mice to ß-adrenergic and muscarinic agonists and IBMX, a cyclic nucleotide phosphodiesterase (PDE) inhibitor. Stimulation of LVSP and ICa,L by isoprenaline (ISO, 100 nM) was twofold smaller in AC8TG vs. NTG mice. In contrast, IBMX (100 µM) produced a twofold higher stimulation of ICa,L in AC8TG vs. NTG mice. IBMX (10 µM) increased LVSP by 40% in both types of mice, but contraction and relaxation were hastened in AC8TG mice only. Carbachol (10 µM) had no effect on basal contractility in NTG hearts but decreased LVSP by 50% in AC8TG mice. PDE assays demonstrated an increase in cAMP-PDE activity in AC8TG hearts, mainly due to an increase in the hydrolytic activity of PDE4 and PDE1 toward cAMP and a decrease in the activity of PDE1 and PDE2 toward cGMP. We conclude that cardiac expression of AC8 is accompanied by a rearrangement of PDE isoforms, leading to a strong compartmentation of the cAMP signal that shields L-type Ca2+ channels and protects the cardiomyocytes from Ca2+ overload.—Georget, M., Mateo, P., Vandecasteele, G., Lipskaia, L., Defer, N., Hanoune, J., Hoerter, J., Lugnier, C., Fischmeister, R. Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8).


Key Words: transgenic mouse • cAMP • isolated heart • L-type Ca2+ current • ß-adrenoceptors • muscarinic receptors • phosphodiesterase • compartmentation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CYCLIC AMP is one of the major second messengers involved in the regulation of cardiac function. Its production is controlled by the activity of adenylyl cyclase (AC), which is enhanced via heterotrimeric Gs protein, such as during activation of ß-adrenoceptors by catecholamines. A rise in intracellular cAMP leads to activation of cAMP-dependent protein kinase (PKA), which phosphorylates a multitude of regulatory proteins controlling the force of contraction and its relaxation: these include the L-type Ca2+ channel, phospholamban, myofilament proteins, and ryanodine receptors (2) . In contrast to catecholamines, acetylcholine (ACh) released during activation of the parasympathetic system generates a negative chronotropic and inotropic effect on the heart. By acting on M2-muscarinic receptors, ACh opposes the action of the sympathetic system mainly through Gi protein inhibition of AC (3 , 4) .

Cyclic AMP production by AC is counterbalanced by its hydrolysis into 5'-AMP by cyclic nucleotide phosphodiesterases (PDE, 5–7). PDEs constitute a large multigenic family of which four members have been found and characterized in the heart: PDE1, which is activated by Ca2+/calmodulin (CaM); PDE2, which is stimulated by cGMP; PDE3, which is inhibited by cGMP; and PDE4, which is insensitive to cGMP. While PDE3 and PDE4 hydrolyze cAMP, PDE1 hydrolyzes equally cAMP and cGMP and PDE2 hydrolyzes both with a lower apparent Km for cGMP than for cAMP (6 7 8) .

It has become well established that an increase in intracellular cAMP level may lead to different effects on cardiac function, depending on the pathway used. For instance, activation of prostaglandin E1 (PGE1), glucagon-like peptide-1 (GLP-1), and ß-adrenergic receptors, all of which increase cAMP level, produce different effects on cardiac function (9 10 11 12) : PGE1 does not modify contractile activity, whereas GLP-1 and isoprenaline, a ß-adrenergic agonist, exert negative and positive effects on myocyte contraction, respectively (11 , 12) . Functional and/or physical compartmentation of cAMP may provide a means by which a cell discriminates among different external stimuli acting on a promiscuous second messenger cascade (13 , 14) . A typical example of such compartmentation is given by the distinct effects on rat ventricular myocytes of ß1- and ß2-adrenergic receptor (AR) agonists. Although both ß-AR agonists increase cAMP concentration, L-type Ca2+ channel current (ICa,L), and contraction, only ß1-AR agonists induce the phosphorylation of phospholamban and contractile proteins, leading to an additional lusitropic effect not observed with ß2-AR agonists (15 , 16) . The cAMP signal generated by the ß2-AR pathway has been shown to be highly localized to the sarcolemma (17 , 18) possibly through a Gi protein-dependent mechanism (17 , 18) .

In a recent study, we proposed that compartmentation of cAMP might also serve a protective role in preserving cardiac myocytes from a deleterious Ca2+ overload (1) . Using the transgenic mouse line AC8TG, in which the human neuronal Ca2+/calmodulin activated AC8 (19 20 21) protein is specifically expressed in cardiac myocytes by coupling the exogenous gene cDNA to the {alpha}MHC promoter (19 20 21) , we found that the L-type Ca2+ channel current (ICa,L) was not enhanced in AC8TG mice, although these mice had a twofold increased cardiac function (1) as a result of a seven- and fourfold increase in total AC and PKA activities, respectively (19 20 21) . Since these mice showed no sign of hypertrophy or cardiomyopathy but on the contrary displayed all the hallmarks of an improved sarcoplasmic reticulum (SR) function (faster whole-heart and single-cell contractile relaxation, larger and shorter Ca2+ transients), we speculated that the additional cAMP production due to AC8 expression served to specifically activate Ca2+ uptake into the SR but not Ca2+ influx at the sarcolemma (1) . In the present study, our aim was to provide some clues about what might be the mechanisms responsible for the cAMP compartmentation that shields Ca2+ channels. We first examined how cardiac expression of AC8 affected the ß-adrenergic and muscarinic control of cardiac function and ICa,L. Next, we examined whether PDEs might play a role in this compartmentation, first by testing the effect of PDE inhibition on contractile activity and ICa,L amplitude in non transgenic (NTG) and AC8TG mice and second by measuring the activity of each PDE isoform in these two animal models.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The investigation conforms to institutional guidelines defined by the European Community guiding principles in the care and use of animals and the French decree #87/848 of October 19, 1987. 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 (#7475, May 27, 1997).

Ex vivo physiology
Four- to 5-month-old mice were anesthetized by intraperitoneal injection of pentothal (150 mg/kg). The heart was quickly removed and placed in oxygenated Krebs-Henseleit solution (95% O2 and 5% CO2, pH 7.35) containing low Ca2+ concentration (0.4 mM) and heparin. The aorta was cannulated on a 20-gauge cannula and perfused by the Langendorff method with a Krebs-Henseleit solution at constant pressure (75 mmHg). A small home made latex balloon was inserted in the left ventricle (LV) chamber. The temperature was monitored on line and maintained at 37 ± 0.2°C throughout the experiments. All hearts were initially perfused with oxygenated Krebs-Henseleit solution at a perfusate Ca2+ concentration ([Ca2+]o) of 1.8 mM and the LV balloon was progressively inflated to isovolumic condition of work corresponding to an end diastolic pressure (EDP) of 5–8 mmHg. The water-filled balloon was connected to a pressure transducer (Statham gauge Ohmeda, Bilthoven, Holland) for continuous measurement of LV systolic pressure (LVSP) on a paper recorder (Astro-Med, West Warrick, UK). Contractile parameters were measured on-line by programming a PC-compatible 486/50 microcomputer in Assembly language (Borland) to determine heart rate (HR), LVSP, rate-pressure product (RPP =LVSPxHR, used as an index of cardiac work), EDP, and the following kinetic parameters: time to peak of contraction (ttp), time for half relaxation (tR1/2), and maximal values of the first derivatives of LVSP (+dLVSP/dt and -dLVSP/dt). To dissociate specific changes in the kinetics of contraction and relaxation from those resulting from a modification of LVSP, these first derivatives were also expressed relative to LVSP (+dLVSP/dt/LSVP and -dLVSP/dt/LSVP). Some experiments were done under pacing conditions at a reduced [Ca2+]o (1 mM). In this case, the hearts were stimulated at 680 bpm via platinum wires (stimulator HSE, Hugo Sachs Electronics, March-Hugstetten, Germany). Ten minutes of equilibration in isovolumic working conditions were imposed before beginning the experiment.

Preparation of mouse ventricular myocytes
Ventricular myocytes were isolated from the hearts of NTG and AC8TG mice as described previously (22) . Three- to 4-month-old animals were anesthetized by intraperitoneal injection of pentothal (150 mg/kg), and the heart was quickly removed and placed into a cold Ca2+-free Tyrode’s solution. The aorta was cannulated and the heart was perfused with an oxygenated Ca2+-free Tyrode’s solution during 3–5 min using retrograde Langendorff perfusion at 37°C. For enzymatic dissociation, the heart was perfused with Ca2+-free Tyrode’s solution containing collagenase D and protease XXIV for 10 min. Then the heart was removed and placed into a dish containing an oxygenated Tyrode’s solution supplemented with 0.2 mM Ca2+ at room temperature. The ventricles were separated from atria, cut in small pieces, and triturated with a pipette to disaggregate myocytes. Ventricular cells were filtered on gauze and allowed to sediment by gravity for 10 min. The supernatant was removed and cells were suspended in 0.5 mM Ca2+-Tyrode’s solution. The procedure was repeated once again and cells were suspended in 1 mM Ca2+-Tyrode’s solution. The cells were stored at room temperature until use.

L-type Ca2+ channel current measurement
The whole-cell configuration of the patch-clamp technique was used to record L-type Ca2+ channel current (ICa,L). For routine ICa,L measurements, the cells were depolarized every 8 s from a holding potential of -50 mV to 0 mV for 400 ms. At this holding potential, fast Na+ current and T-type Ca2+ current were inactivated. External and internal (pipette) solutions contained Cs+ instead of K+ to eliminate K+ currents. The electrical resistance of the patch pipette varied between 0.5 and 1.2 m{Omega}. All experiments were done at room temperature. A challenger VM (Kinetic, Atlanta, GA, USA) generated voltage clamp protocols. The currents were measured by a patch-clamp amplifier (Biologic RK 400, Claix, France), filtered at 3 kHz, and sampled at a frequency of 10 kHz. The maximal amplitude of ICa,L was measured as the difference between the peak inward current at 0 mV and the current at the end of 400 ms pulse. Currents were not compensated for capacitative and leak currents. Two mV and 10 ms depolarizations from the holding potential were used to elicit capacitative currents, which were filtered at 10 kHz and sampled at a frequency of 20 kHz. Exponential analysis of these currents led to an estimate of the membrane capacitance and series resistance.

Solutions and drugs
The composition of the initial Krebs-Henseleit solution used for ex vivo experiments was (in mM): 113 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.8 CaCl2, 11 glucose, 10 Na-pyruvate. Isoprenaline (ISO), 3-isobutyl-1-methyl xanthine (IBMX), and carbachol (CCh) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Aliquots of stock solution of ISO (1 mM) containing 1 mg mL-1 ascorbic acid to prevent oxidation were kept frozen. All solutions of ISO, IBMX and CCh were freshly prepared in control external solution before each experiment. To obtain adequate concentrations in ex vivo experiments, the drugs were infused via a push syringe at a 20x concentration and the infusion flow that was adjusted to 5% of the coronary flow (estimated by continuously monitoring the volume of fluid dripping from the heart). This 5% increase in coronary flow did not modify contractility. All drugs were applied for 5 min and washed for 5 to 10 min until recovery of a new steady state.

The ionic composition of Ca2+-free Tyrode’s solution used for cell isolation was (in mM): NaCl 140, KCl 5.4, NaH2PO4 1, MgCl2 1.1, HEPES 5, glucose 10. The pH was adjusted to 7.3 with NaOH and the solution was oxygenated. The enzymatic solution for the cardiomyocyte dissociation was composed of Ca2+-free Tyrode’s solution to which was added 4.05 units collagenase D (Roche, Meylan, France), 17.8 units protease XXIV (Sigma, St. Quentin, Fallavier, France), 1 mg/mL BSA (ICN Pharmaceuticals, Orsay, France), and 1.4% fetal calf serum (FCS, Life Technology, Eragny/Oise, France). The 0.2-, 0.5-, and 1 mM Ca2+-Tyrode’s solutions were composed of Ca2+-free Tyrode solution to which 1.4% FCS and 0.2, 0.5 and 1 mM Ca2+ were respectively added. The composition of the control external solution for patch-clamp experiment was (in mM): 107.1 NaCl, 20 CsCl, 10 HEPES, 5 glucose, 4 NaHCO3, 0.8 NaH2PO4, 1.8 MgCl2, 1.8 CaCl2. The pH was adjusted to 7.4 with NaOH. Cells were exposed to different drugs with a system of capillary tubings. Patch pipettes solution contained (in mM): 119.8 CsCl, 10 HEPES, 5 EGTA, 4 MgCl2, 5 Na2CP (creatine phosphate disodium salt), 0.0062 CaCl2, 3.1 Na2ATP, 0.4 Na2GTP. The pH was adjusted to 7.3 with CsOH.

PDE assays
Ten mice hearts were homogenized by pair (NTG) or individually (AC8TG) in 10 v/w mL buffer (20 mM Tris-HCl pH 7.5, 2.0 mM Mg acetate, 5.0 mM EGTA, 1.0 mM dithiothreitol, 10 µg/mL leupeptin, 10 µg/mL soya trypsin inhibitor, 2000 U/mL aprotinin, and 0.33 mM Pefabloc) by using an ultra-turrax (6 fold for 10 s) and a glass-glass potter homogenizer, then stored until use in small aliquots at -80C°.

PDE activities were measured by radioenzymatic assay as described previously (23) at a substrate concentration of 1 µM cAMP or cGMP in the presence of 15000 cpm [3H]-cAMP or [3H]-cGMP as a tracer, respectively. The buffer solution was of the following composition: 50 mM Tris-HCl pH 7.5, 2 mM Mg acetate, and 1 mM EGTA. Assays of total cyclic nucleotide hydrolytic activity and isoform-specific PDE activity were run in the same batch of experiments. Tissues were diluted in order to have ~15% of hydrolysis in the absence of specific inhibitors. The proportion of cAMP-hydrolyzing PDE isoforms was determined with 1 µM cAMP as substrate in the presence of 1 mM EGTA by using 10 µM rolipram for PDE4, 10 µM cilostamide for PDE3. IBMX-sensitive cAMP-PDE activity was determined by using 100 µM IBMX. The proportion of cGMP-hydrolyzing PDE isoforms was determined with 1 µM cGMP as substrate either in basal state (in presence of 1 mM EGTA) by using 20 µM EHNA for PDE2 or in Ca2+/calmodulin-activated state (with 10 µM CaCl2 and 18 nM calmodulin instead of 1 mM EGTA) by using 10 µM nimodipine for PDE1. PDE isoform-specific activities were determined as the difference between PDE activity in the absence of inhibitor and the residual hydrolytic activity observed in the presence of the selective inhibitor (5) . The results were expressed as pmol·min-1·mg prot-1. Proteins were determined according to Lowry et al. (24) using bovine serum albumin as standard.

Data analysis
The results are expressed as means ± SE. Student’s paired t test was used to test the effect of drugs on NTG and AC8TG hearts in ex vivo experiments. Otherwise Student’s t test for unpaired data was used to compare NTG and AC8TG mice. A value of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ß-Adrenergic regulation of cardiac function
Since AC is a key enzyme mediating cardiac response to ß-adrenergic stimulation, we evaluated the consequences of the cardiac expression of AC8 on the contractile response to a ß-adrenergic agonist, isoprenaline (ISO). The dose-response curves to ISO were first compared in NTG and AC8TG mice under spontaneous beating condition and in 1.8 mM [Ca2+]o (Fig. 1 A, B). As shown earlier (1) , basal LVSP and HR were much larger in AC8TG than in NTG mice (Fig. 1A, B in the absence of ISO). In NTG mice, ISO induced a classical dose-dependent stimulation of LVSP (Fig. 1A ) and HR (Fig. 1B ), leading to a strong increase in the cardiac work index RPP (not shown). By contrast, none of these parameters was significantly affected by ß-adrenergic stimulation in AC8TG mice (Fig. 1A, B ).



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  		Figure 1. ß-Adrenergic regulation of cardiac function in NTG and AC8TG mice. A, B) Hearts were spontaneously beating and perfused with 1.8 mM [Ca2+]o. Increasing concentrations of isoprenaline (ISO) were added to the perfusate while continuously measuring left ventricular systolic pressure (LVSP, A) and heart rate (HR, B) in NTG ({circ}) and AC8TG mice ({blacktriangleup}). The points are means ± SE. NTG, n = 6; AC8TG, n = 5. *P < 0.05 and **P < 0.01 vs. NTG. C–G) The hearts were stimulated at 680 bpm and perfused with 1 mM [Ca2+]o in the absence (empty bars) or presence (filled bars) of 100 nM isoprenaline (ISO). LVSP (C), the maximal rate of contraction (+dLVSP/dt/LVSP, D), the time to peak of contraction (ttp, E), the maximal rate of relaxation (–dLVSP/dt/LVSP, F), and the time for half relaxation (tR1/2, G) were measured in each condition in NTG and AC8TG mice, as indicated. The bars are means ± SE. NTG, n = 6; AC8TG, n = 6. *P < 0.05, **P < 0.01, and ***P < 0.001 ISO vs. control.

The lack of responsiveness to ISO in AC8TG mice at 1.8 mM [Ca2+]o was likely due to a saturation of the cardiac contractile machinery due to AC8 expression. Indeed, we had found earlier that increasing [Ca2+]o from 1.8 to 2.5 mM did not significantly change the contractility in AC8TG hearts while it strongly increased LVSP in NTG hearts (1) . Therefore, we reexamined the effect of ISO at a lower [Ca2+]o (1 mM), corresponding to half activation of contraction in AC8TG mice (1) . These experiments were also performed under pacing conditions (at 680 bpm) to avoid any interference between HR and contractility changes. Under these new conditions, ISO (100 nM) was found to increase LVSP in AC8TG (from 48±5 to 79±9 mmHg, n=6) and NTG mice (from 21±2 to 95±16 mmHg, n=6) (Fig. 1C ). However, when expressed in relative percent variation, the ISO stimulation of LVSP was fivefold lower in AC8TG than with NTG mice (64±9% vs. 340±33% increase over basal, P<0.001). As expected, ISO accelerated the onset of contraction in NTG mice and this effect was also found in AC8TG mice, with a similar {approx}15% change in +dLVSP/dt/LVSP (Fig. 1D ) and ttp (Fig. 1E ) in both mice. ISO also exerted a net lusitropic effect in both mice (Fig. 1F, G ), albeit to a larger extent in NTG (47±4% decrease of tR1/2) than in AC8TG mice (28±4% decrease of tR1/2, P<0.01, Fig. 1G ). Notice that the differences in parameters observed under basal conditions between NTG and AC8TG mice were abrogated in the presence of ISO, indicating that ß-adrenergic stimulation and AC8 expression affect cardiac contractility via a common pathway.

Muscarinic regulation of cardiac function
We then investigated whether cardiac expression of AC8 modified the muscarinic regulation of cardiac contractility by comparing the effect of carbachol (CCh), a muscarinic receptor agonist, in NTG and AC8TG mice. As in most other mammalian species, muscarinic receptor activation had no direct negative inotropic effect in mouse ventricle as CCh (10 µM) had no effect on basal LVSP in NTG mice (–1.5±7% variation, n=6, Fig. 2 A). However, as in other species, CCh produced a clear, indirect negative inotropic effect best demonstrated after ß-adrenergic receptor activation with ISO (19±5% decrease of the response to 100 nM ISO, n=6, P<0.05, Fig. 2B ). This indirect effect of CCh, called "accentuated antagonism," was also observed in AC8TG mice (Fig. 2B ) with a similar amplitude (27±2% decrease of ISO response, n=6). However, unlike in NTG mice, CCh produced a large and significant decrease in basal LVSP in AC8TG hearts (46±5% reduction, n=6). Notice that, as with ISO, CCh abrogated the difference in LVSP between NTG and AC8TG mice, indicating that muscarinic stimulation counteracts the effects of AC8 expression on cardiac contractility.



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  		Figure 2. Muscarinic regulation of cardiac contraction in NTG and AC8TG mice. The hearts were stimulated at 680 bpm and perfused with 1 mM [Ca2+]o. A) Mean of 6 NTG and 6 AC8TG hearts are represented in the absence (empty bars) or presence (filled bars) of 10 µM carbachol (CCh). B) Mean of 6 NTG and 6 AC8TG ISO (100 nM)-stimulated hearts are represented in the absence (empty bars) or presence (filled bars) of 10 µM CCh. The bars are means ± SE. *P < 0.05 and **P < 0.01 AC8TG vs. NTG (paired t test).

Effect of PDE inhibition on cardiac function
Basal AC activity and cAMP concentration are both enhanced in the hearts of AC8TG compared with NTG mice (21) . Since cyclic nucleotide PDE also play a role in the control of intracellular cAMP level, we examined whether the contribution of PDE activity to the control of heart function is modified in AC8TG mice. For this, we tested the effect of IBMX, a broad-spectrum PDE inhibitor, on the contractility of paced hearts. Figure 3 shows two representative experiments performed in NTG (Fig. 3A, B ) and AC8TG hearts (Fig. 3C, D ) demonstrating that IBMX (10 µM) produces a positive inotropic effect in both groups of mice. On average, IBMX increased LVSP similarly by ~40% in NTG and AC8TG hearts (Fig. 3E ). However, when comparing the effects of IBMX with those of ISO (Fig. 1) , it appears that IBMX had a much smaller effect than a saturating concentration of ISO on contractility and kinetics in NTG mice, whereas IBMX and ISO produced a similar effect on all parameters in AC8TG mice (compare Fig. 3E-I and Fig. 1C-G ). Thus, AC8TG mice have a higher sensitivity to PDE inhibition than NTG mice, which may indicate a more pronounced contribution of PDE activity to the control of heart function in this transgenic animal model.



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  		Figure 3. Cardiac response to IBMX in NTG and AC8TG mice. On the top, original recordings of left ventricular contraction of NTG (A, B) and AC8TG (C, D) hearts under basal conditions (A, C) and after application of 10 µM IBMX (B, D). The hearts were stimulated at 680 bpm and perfused at 1 mM [Ca2+]o. E–I) Mean results of 6 NTG and 6 AC8TG hearts are compared in the absence (empty bars) or presence (filled bars) of 10 µM IBMX. LVSP (E), the maximal rate of contraction (+dLVSP/dt/LVSP, F), time to peak of contraction (ttp, G), maximal rate of relaxation (–dLVSP/dt/LVSP, H), and the time for half relaxation (tR1/2, I) were measured in each condition in NTG and AC8TG mice. The bars are means ± SE. *P < 0.05 and **P < 0.01 IBMX vs. control.

ß-Adrenergic regulation of basal ICa,L
To complement the experiments in isolated perfused heart, subsequent experiments were performed in isolated ventricular myocytes to examine how cardiac AC8 expression modifies the regulation of the L-type Ca2+ channel current (ICa,L). In our previous study (1) , we found that the basal ICa,L density was similar in AC8TG and NTG mice, although the former have a several-fold increase in total AC and PKA activities (19 20 21) . In the representative experiments shown in Fig. 4 , we now find that both NTG (Fig. 4A ) and AC8TG myocytes (Fig. 4B ) responded to ISO (100 nM), although the effect of ISO was substantially smaller in AC8TG than in NTG mice. On average (Fig. 4C ), ISO (100 nM) increased ICa,L amplitude by 125 ± 11% (n=12) in NTG and 55 ± 8% (n=14) in AC8TG mice (P<0.05), indicating a twofold lower responsiveness to ß-adrenergic stimulation in the transgenic mouse. This difference was not due to a reduced potency of ISO in AC8TG mice, since the twofold difference between the two groups remained even when ISO concentration was increased 10-fold (117.5±15.1%, n=3, in NTG vs. 46.1±9.8%, n=5, in AC8TG with 1 µM ISO). Forskolin (10 µM), a direct activator of AC, increased ICa,L amplitude by a similar amount in NTG and AC8TG mice (103±21%, n=9, and 131±21%, n=7, in NTG and AC8TG cells, respectively), comparable to the level obtained with ISO in NTG mice. This indicates that the reduced efficacy of ISO to activate ICa,L in AC8TG myocytes was not due to an impairment in the PKA phosphorylation of L-type Ca2+ channels, but rather to a reduced coupling between ß-adrenergic receptors and PKA in transgenic mice.



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  		Figure 4. ß-Adrenergic and muscarinic regulation of ICa,L in NTG and AC8TG ventricular myocytes. The ventricular myocytes from NTG (A) and AC8TG mice (B) were first exposed to the control external solution; solid lines indicate the period of drug perfusion. Each symbol corresponds to a measure of ICa,L at 0 mV obtained every 8 s (holding potential of -50 mV). The individual current traces shown on top of the graphs were recorded at the times indicated by the corresponding letters in the bottom graphs. The mean effects of ISO or forskolin (10 µM) on ICa,L and of CCh on ISO-stimulated ICa,L in NTG (empty bars) and AC8TG mice (filled bars) are shown in panels C and D, respectively. The bars indicate the means and the lines the SE of the number of cells given. *P < 0.001 vs. NTG.

Muscarinic regulation of ICa,L
In the experiments shown in Fig. 4 , the accentuated antagonism of CCh was examined by testing the effect of the muscarinic agonist in the presence of ISO. In NTG (Fig. 4A ) and AC8TG mice (Fig. 4B ), CCh strongly antagonized the ISO response. As shown in Fig. 4D , the inhibitory effect of 1 µM CCh was similar in both groups of cells and averaged 69.2 ± 8.6% (n=5) and 77.1 ± 9.0% (n=6) inhibition of the ISO (100 nM) response in NTG and AC8TG myocytes, respectively.

In addition to the accentuated antagonism observed in NTG and AC8TG mice, CCh produced a "direct" negative inotropic effect in the isolated perfused heart of AC8TG mice only (Fig. 2) . To examine whether a similar difference exists at the level of ICa,L, we tested the effect of CCh on basal ICa,L in NTG and AC8TG mice. However, we found that CCh (1 µM) had no effect on basal ICa,L in either type of mouse (6±1% decrease in ICa,L in NTG, n=4, and 4±2% in AC8TG, n=4).

Effect of IBMX on basal ICa,L
As AC8TG and NTG hearts differed in their sensitivity to PDE inhibition (Fig. 3) , we examined whether isolated myocytes from NTG and AC8TG hearts also responded differently to IBMX. The rationale behind these experiments is that an increase in PDE activity might explain the attenuation of ICa,L response to ISO in AC8TG myocytes. As seen in the individual experiments of Fig. 5 , a saturating concentration of IBMX (100 µM) enhanced basal ICa,L in both NTG (Fig. 5A ) and AC8TG (Fig. 5B ) mice. However, the effect of IBMX was substantially larger in AC8TG mice. The summary data in Fig. 5C show that IBMX (100 µM) increased basal ICa,L by 58 ± 6% (n=15) in NTG and by 134 ± 7% (n=18) in AC8TG myocytes (P<0.05). Thus, whereas ISO exerts a twofold lower effect in AC8TG than NTG mice, the situation is the opposite with IBMX, which has a twofold stronger effect in AC8TG than NTG mice; forskolin acts equally well in both animals. Altogether, these results support the hypothesis that cardiac AC8 expression is counterbalanced by an increase in PDE activity, which prevents a basal activation of L-type Ca2+ channels and attenuates their response to ß-adrenergic receptor stimulation.



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  		Figure 5. Effect of IBMX on ICa,L in NTG and AC8TG ventricular myocytes. The ventricular myocytes from NTG (A) and AC8TG mice (B) were first exposed to the control external solution. During the periods indicated by the solid lines, the cells were superfused by 100 µM of IBMX. Each symbol corresponds to a measure of ICa,L at 0 mV obtained every 8 s (holding potential of -50 mV). The individual current traces shown on top of the graphs were recorded at the times indicated by the corresponding letters in the bottom graphs. C) Mean effects of IBMX on ICa,L in NTG (empty bar) and AC8TG mice (filled bar). The bars indicate the means and the lines the SE of the number of cells given. *P < 0.001 vs. NTG.

Measurement of PDE activity
To directly test this hypothesis, PDE activities were measured in NTG and AC8TG mice. First, the total cAMP (Table 1 ) and cGMP specific activities (Table 2 ) were determined in heart homogenates from NTG and AC8TG mice in the presence of EGTA. Basal cAMP hydrolyzing activity was increased significantly by 41% in AC8TG compared with NTG hearts (Fig. 6 A and Table 1 ), whereas cGMP hydrolyzing activity was decreased by 10% (Fig. 6B and Table 2 ), modifying markedly the ratio of cAMP- to cGMP-PDE from 1 to 1.57 (Fig. 6C ).


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  		Table 1. cAMP-PDE activitya


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  		Table 2. cGMP-PDE activitya



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  		Figure 6. Cardiac phosphodiesterase activity in NTG and AC8TG mice. The results are expressed as pmol·min-1·mg·prot-1 and represent the mean results from 10 hearts homogenized by pair (NTG, empty bars) or individually (AC8TG, filled bars). Bars indicate the means and the lines the SE. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NTG.

Next, we examined which PDE subtypes were involved in these alterations of cAMP and cGMP activities. To characterize the relative contribution of PDE3 and PDE4 in cAMP activity, we used cilostamide (10 µM) and rolipram (10 µM) as specific inhibitors of each subtype (5) . The major cAMP hydrolytic activity was due to PDE4, which represents 58% of the total cAMP-PDE in NTG mice (Table 1) . PDE4 activity increased by 28% in AC8TG hearts (P<0.05, Fig. 7 D and Table 1 ). The activity of PDE3, which represents only 23% of the total cAMP hydrolytic activity in NTG hearts, was similarly increased by 27% in AC8TG heart (P<0.05, Fig. 7C and Table 1 ). Furthermore, 100 µM IBMX inhibited by 77% and 80% the total cAMP hydrolytic activity in NTG and AC8TG heart homogenates, respectively, increasing by 47% (P<0.0001) the IBMX-sensitive cAMP-PDE activity (Table 1) . The residual, IBMX-resistant hydrolytic activity, possibly due to PDE8 and PDE9, which are also expressed in mouse heart but are resistant to inhibition by IBMX (25 , 26) , accounted for ~20% of the total PDE activity and was not different in NTG and AC8TG hearts (Table 1) .



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  		Figure 7. Cardiac PDE specific isoform activities in NTG and AC8TG mice. The results are expressed as pmol·min-1·mg prot-1 and represent the mean results from 10 hearts homogenized by pair (NTG, empty bars) or individually (AC8TG, filled bars). To identify the specific PDE isoform activities, selective PDE inhibitors were added: 10 µM nimodipine for PDE1 (A), 20 µM EHNA for PDE2 (B), 10 µM cilostamide for PDE3 (C), and 10 µM rolipram for PDE4 (D). The bars indicate the means and the lines the SE. *P < 0.05 and ***P < 0.001 vs. NTG.

Addition of 5 µM cGMP stimulated the cAMP hydrolytic activity by 81% in NTG mice but only by 27% in AC8TG mice (Table 1) . Since these changes reflect a balance between cGMP inhibition of PDE3, cGMP activation of PDE2, and competition between cGMP and cAMP on PDE1, the difference between NTG and AC8TG mice might indicate a lower PDE2- and/or a larger PDE3+PDE1 contribution in the transgenic model. The contribution of PDE2 was evaluated with 1 µM cGMP as activator and substrate and using 20 µM EHNA as a selective inhibitor (5 , 27) . As shown in Table 2 and Fig. 7B , cGMP-activated PDE2 represented 79% of the total cGMP hydrolytic activity in NTG hearts but only 69% in AC8TG hearts (P<0.001).

PDE1 activity is stimulated by Ca2+/calmodulin (CaM), which is also an important regulator of AC8. Therefore, we determined the hydrolytic activities toward cAMP and cGMP in the presence of Ca2+/CaM. Ca2+/CaM-activated cAMP-PDE activity was 71% larger in AC8TG vs. NTG mice (P<0.0001, Table 1 ). The contribution of PDE1 was evaluated with 10 µM nimodipine as inhibitor (5) . This increase was mainly due to a 124% increase in cAMP-PDE1 activity (P<0.0001, Table 1 ). In contrast, Ca2+/CaM-activated cGMP-PDE activity decreased by 28% in AC8TG compared with NTG hearts (P<0.0001, Table 2 ), this alteration being associated with a 60% decrease in cGMP-PDE1 activity (P<0.0001, Fig. 7A and Table 2 ). Table 2 also shows that other, yet unidentified, PDEs hydrolyze cGMP in mouse heart and that their contribution is substantially larger in AC8TG than NTG mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present report, we show that cardiac specific expression of AC8 strongly modifies the regulation of heart function by sympathetic and parasympathetic neuromediators. Our results can be summarized as follows: 1) the positive inotropic effect of a ß-adrenergic stimulation was strongly attenuated in AC8TG mice, both at the level of the isolated heart and cardiac myocyte; 2) although CCh had no "direct" effect on basal ICa,L in either NTG or AC8TG and on contractility in NTG mice, it strongly reduced basal contractility in AC8TG hearts; 3) the "indirect" negative inotropic effect of CCh was similar in both mice, both at the level of contractility and ICa,L; 4) PDE inhibition induced a more pronounced effect on contractility and ICa,L in AC8TG than NTG mice; 5) this latter effect was due to an enhancement of cAMP-PDE activity in AC8TG, resulting mainly from an increase in the hydrolytic activity toward cAMP of PDE4 and PDE1 and a decrease in the hydrolytic activity toward cGMP of PDE1 and PDE2. We conclude that cardiac expression of AC8 is accompanied by a rearrangement of PDE isoforms, leading to a strong compartmentation of the cAMP signal that shields L-type Ca2+ channels and protects the cardiomyocyte from deleterious Ca2+ overload.

Data obtained in vivo after bilateral vagotomy showed an increased basal cardiac contractility and HR in AC8TG compared with NTG mice whereas in intact anesthetized mice, using echocardiography, HR and cardiac function were similar in both animal models (21) . While the use of anesthetics may have depressed heart function alleviating the differences between the two animal models, it is also possible that an enhanced vagal tone could mask the enhanced intrinsic cardiac function in AC8TG mice. In the present study, the hearts were isolated from neural input and the circulation, allowing a more precise analysis of the intrinsic cardiac contractile properties in response to various stimuli. Such ex vivo experiments unveiled a large increase in basal cardiac contractility in AC8TG compared with NTG mice (1) . In such conditions, CCh decreased basal cardiac contraction in AC8TG mice. This direct negative inotropic effect of the muscarinic agonist, which was not observed in NTG hearts (28) , reduced the contractility in AC8TG mice to the level of NTG mice, alleviating the difference in cardiac contractility between the two animal models. However, the question arises as to what underlines the "direct" negative inotropic effect of CCh in AC8TG mice? Clearly, this effect is unlikely due to a direct inhibition of AC8 by G-protein {alpha}i subunit, since AC8 activity has been reported to be insensitive to Gi stimulation directly (29 , 30) . One possibility is that the direct effect of CCh in AC8TG is due to G{alpha}i inhibition of the basal activity of endogenous AC5 and/or AC6, whose contributions might be amplified by the presence of AC8. Alternatively, muscarinic regulation of basal contractility in AC8TG hearts may involve other mechanisms besides AC inhibition, such as activation of phosphatase, stimulation of cGMP-stimulated PDE2, or phosphorylation via cGMP-dependent protein kinase (see ref 31 for a review). The two latter processes may be amplified in AC8TG hearts due to the reduced cGMP hydrolytic activity observed in this study.

Our previous in vivo study showed that cardiac function was not affected by ß-adrenergic stimulation in AC8TG hearts (21) . We found in this ex vivo study that AC8TG hearts did not respond to ISO when tested at near-physiological (1.8 mM) Ca2+ concentration. However, ISO produced a clear positive inotropic effect in AC8TG mice when the external Ca2+ concentration was reduced to 1 mM, i.e., a concentration at which cardiac contraction is half-maximal (1) . This indicates that the ß-adrenergic signaling pathway was still functional in AC8TG mice but that the lack of ISO effect at higher Ca2+ concentrations was due to a saturation of the contractile machinery. Consistent with this hypothesis, we earlier found that increasing [Ca2+]o from 1.8 to 2.5 mM did not significantly change the contractility in AC8TG hearts but strongly increased LVSP in NTG hearts (1) . Cardiac function was no longer different in NTG and AC8TG hearts upon a maximal ß-adrenergic stimulation with ISO. This confirms our hypothesis that AC8 expression and ß-adrenergic stimulation share a common pathway to produce a positive inotropic effect. It should be noted that the positive inotropic effect of ISO in AC8TG hearts is unlikely to result from a stimulation of AC8 by G-protein {alpha}s subunits since AC8 activity has been reported to be insensitive to Gs stimulation (30) . The positive inotropic effect of ISO in AC8TG mice observed in ex vivo experiments more likely reflects the stimulation of endogenous cardiac AC5 and/or AC6 cyclase activity.

Since the quantity of AC5 and AC6 proteins and ß-adrenergic receptors was not altered in AC8TG (21) , one might expect, at first approximation, that G{alpha}s stimulation of these endogenous adenylyl cyclases would generate similar responses in AC8TG and NTG mice. Our electrophysiological experiments in isolated myocytes clearly showed that this was not the case. Indeed, the response of ICa,L to ISO was twofold lower in AC8TG vs. NTG mice, although the response to forskolin was identical, demonstrating a comparable sensitivity to PKA phosphorylation of L-type Ca2+ channels in the two type of mice. This suggests that a mechanism prevents Ca2+ channels in AC8TG mouse from an excessive stimulation by cAMP. Hyperactivation of Gi protein pathways was unlikely to account for such a mechanism, as the muscarinic regulation of ICa,L, both at basal and ISO-stimulated level, was similar in AC8TG and NTG mice. A possible mechanism could be an inhibition of endogenous AC activity in AC8TG mice as a result of the increase in PKA activity. Indeed, both AC5 and AC6 are directly phosphorylated and inhibited by PKA (5 , 6 , 29 , 32 , 33) . Phosphorylation by PKA inhibits AC5 activity by decreasing the maximal velocity of the enzyme (33) . Phosphorylation of AC6 would disrupt the functional G{alpha}s binding site, leading to an attenuation of its stimulation by G{alpha}s-coupled receptor (32) . Although attractive, such a PKA-mediated AC inhibition might not easily explain our data. If one assumes that in AC8TG mice the ISO effect on ICa,L is reduced due to a PKA inhibition of AC5/AC6 activity, then one would expect the same pool of PKA to phosphorylate Ca2+ channels and activate basal ICa,L. However, as shown in our previous study, basal ICa,L is not increased in AC8TG mice (1) , indicating that L-type Ca2+ channels are not in contact with the cAMP/PKA pathway initiated at AC8.

Since cAMP level is not only controlled by its production but also by its degradation (5 6 7 , 29) , we examined the possibility that the contribution of PDE activity might be modified in AC8TG mice. Several observations led us to conclude that PDE activity is up-regulated in transgenic mice. First, application of IBMX, a broad-spectrum PDE inhibitor, increased cardiac function in AC8TG as well as in NTG mice, but the kinetic parameters were modified in AC8TG hearts only. Moreover, the same concentration of IBMX increased LVSP maximally in AC8TG hearts, to the level reached by a maximal concentration of ISO, but not in NTG hearts. Thus, AC8TG mice appeared more sensitive to PDE inhibition than NTG mice. Second, at the cellular level, application of IBMX induced a twofold larger stimulation of ICa,L in AC8TG than NTG myocytes. This was as if PDE inhibition "revealed" the presence of AC8 to L-type Ca2+ channels. Finally, PDE assay confirmed the enhancement of cAMP hydrolytic activity of these enzymes in AC8TG and their increased sensitivity to IBMX. This increase in PDE activity was essentially due to an increase in PDE4 and Ca2+/CaM-activated PDE1 hydrolytic activity toward cAMP. As shown earlier (34) , the expression of PDE isoforms might be directly controlled by the level of cAMP. Surprisingly, though we found an increase in Ca2+/CaM-activated PDE1 hydrolytic activity toward cAMP, we found at the same time a decrease in Ca2+/CaM-activated PDE1 hydrolytic activity toward cGMP in AC8TG hearts. One possible explanation for this apparent discrepancy is that the overall PDE1 activity corresponds to the activity of different PDE1 isoforms, such as PDE1A and PDE1C, which differ in their kinetic constants as characterized in rat heart (35) . The expression of PDE splice variants might also differ between NTG and AC8TG mice and this may contribute to the overall increase in cAMP-PDE activity. We believe that this up-regulation of cAMP-PDE activity is a compensatory mechanism to AC8 expression that serves a protective role by shielding L-type Ca2+ channels, hence preventing an excessive and deleterious entry of Ca2+ into the cell.

Such a contribution of PDE to the compartmentation of cAMP signaling pathway has already been reported. In frog myocyte, during ISO stimulation of ICa,L, PDE activity was shown to limit the cAMP broadcast throughout the cell, resulting in a local ß-adrenergic regulation of this current (18 , 36 , 37) . It has been shown that PDE3 and PDE4 are differently associated with subcellular structures in cardiac tissues: PDE4 is associated with sarcolemma (38 , 39) and nuclear envelope (40) whereas PDE3 is mainly associated with sarcoplasmic reticulum (41) . Thus, PDE3 and PDE4 might locally regulate cAMP level in the vicinity of functional proteins regulated by cAMP-dependent protein kinase (42) . In AC8TG ventricular myocytes, an increase in PDE activity, and particularly in PDE4, may play a role in limiting the spatial spread of cAMP that dissociates ICa,L from AC8 protein and reduces ß-adrenergic stimulation of this current. Moreover, it was shown in cardiac ventricle that rolipram, a selective PDE4 inhibitor, although increasing cAMP level, was unable to affect the phosphorylation state of phospholamban and the inhibitory subunit of troponin, supporting the role of PDE4 in cAMP compartmentation (43) . Many proteins may be involved in this spatial cAMP signal localization. A kinase-anchoring proteins (AKAP) have been shown to localize PKA near its target proteins during ß-adrenergic regulation of cardiac contractility (44) . Similar proteins were also shown to anchor PDE4 isoforms at specific organelles (45 , 46) . Finally, recent studies have emphasized the role of caveolae in the spatial organization of various signaling complexes, which might facilitate the interaction between molecules and contribute to the localization of the cAMP signal in a restrictive place (14 , 47 , 48) .

In conclusion, our study demonstrates that PDEs play a dynamic role in shaping the spatial profile of intracellular cAMP concentration in cardiac myocytes. Although cAMP is a relatively small molecule that should readily diffuse throughout the cell, we propose that its concentration is different in different cellular compartments (36) . In the unique model of the AC8TG mouse, the overall intracellular cAMP concentration is much increased (21) , but not necessarily in all compartments. For instance, the degree of cAMP-dependent phosphorylation may be increased for proteins located at the sarcoplasmic reticulum membrane (e.g., phospholamban, ryanodine receptor) and/or in the contractile machinery (e.g., troponin I), accounting for the strong positive inotropic and lusitropic effect seen in AC8TG mice both at the level of the whole heart and single myocyte (1) . However, the degree of cAMP-dependent phosphorylation may remain normal, or even become lower during ß-adrenergic stimulation, for sarcolemmal L-type Ca2+ channels, which might explain why the AC8TG mice survive and show no phenotypic alteration or any sign of hypertrophy or cardiomyopathy (1 , 21) . Thus, activation of cAMP-PDE may serve a protective role during a long-term activation of the cAMP cascade.


   ACKNOWLEDGMENTS
 
We thank Patrick Lechêne for skilful programming of the automated data analysis, Florence Lefebvre and Hélène Basaran for excellent technical help.

Received for publication October 8, 2002. Accepted for publication March 31, 2003.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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