Sarah Karam

Jean Piero Margaria

Aurélia Bourcier

Delphine Mika

Audrey Varin

Ibrahim Bedioune

Marta Lindner

Kaouter Bouadjel

Matthieu Dessillons

Françoise F Gaudin

Margaria Jp

Bourcier

Varin

Dessillons

Lechène

Gomez

Samuel Jl

Michel Jb

Vandecasteele

PhD Grégoire Vandecasteele
email: gregoire.vandecasteele@universite-paris-saclay.fr

Cardiac overexpression of PDE4B blunts β-adrenergic response and maladaptive remodeling in heart failure

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INTRODUCTION

Heart failure (HF) is the final common stage of many cardiovascular disorders including myocardial infarction and hypertension as common causes. Regardless of the underlying cause for HF, patients show a hyper-active sympathetic nervous system with elevated plasma catecholamine levels that correlate with disease severity. 1 Although beneficial on the short term, prolonged sympathetic stimulation promotes cardiac hypertrophy, myocardial cell death, fibrosis, and arrhythmias. 2 Accordingly, β-blockers improve survival in patients with chronic low ejection fraction 3 and are a mainstay of current HF therapy. However, in most instances, β-blockers can only slow disease progression and may cause significant side effects in some patients. Thus, there is a need for improved therapeutics in HF, alternative or complementary to β-blockers.

The β-adrenergic stimulation of cardiac function involves the second messenger cAMP which activates the cAMP-dependent protein kinase (PKA) to modulate key proteins of the excitationcontraction coupling (ECC) process. These include sarcolemmal L-type Ca 2+ channels (LTCC) that underlie the L-type Ca 2+ current (ICa,L), ryanodine receptors (RyR2), phospholamban (PLB), troponin I and myosin-binding protein C. 4 The intracellular levels of cAMP are determined by the balance between synthesis by adenylyl cyclases and degradation by cyclic nucleotide phosphodiesterases (PDEs). PDEs represent a highly diverse superfamily of enzymes, regrouped into 11 families according to their primary structure, enzymatic properties, regulation and pharmacology. 5 Among these, PDE3 and PDE4 are the main PDEs degrading cAMP with a high affinity in the heart and are critical to control the termination of cardiac β-AR responses. 6 Inhibition of PDE4 results in a massive potentiation of cAMP during β-AR stimulation not only in rodent 6 but also in dog, 7 pig 8 and human cardiomyocytes. 9 As a consequence, PDE4 activity modulates β-AR stimulation of ICa,L and ECC in these species. 6,9,10 PDE4 inhibition also increases cellular hypertrophy [START_REF] Zoccarato | Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2[END_REF] and sarcoplasmic reticulum (SR) Ca 2+ leak, thereby favoring cardiac arrhythmias. 10 The PDE4 family is encoded by four genes (Pde4a-Pde4d), but only three (Pde4a, Pde4b, and Pde4d) are expressed in the human and rodent heart. [START_REF] Richter | Conserved expression and functions of PDE4 in rodent and human heart[END_REF] Whereas PDE4D was proposed to associate with RyR2 to control SR Ca 2+ release [START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF] and with PLB to control SR Ca 2+ reuptake [START_REF] Beca | Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca2+ release and cardiac contractility, independently of L-type Ca2+ current[END_REF] we have found that PDE4B is physically associated to the principal CaV1.2 subunit of LTCCs to modulate β-AR regulation of ICa,L. [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF] Pde4d knock-out mice have enhanced SR Ca 2+ leak and arrhythmias and develop HF with age, [START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF] whereas Pde4b knock-out mice have exaggerated β-AR stimulation of ICa,L and Ca 2+ transients, leading to increased spontaneous Ca 2+ waves and enhanced susceptibility to ventricular tachycardia. [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF] PDE4B expression and activity are decreased in a rat model of pathological hypertrophy induced by pressure overload, [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on beta-adrenergic cAMP signals[END_REF] suggesting that increasing PDE4B in the heart could be beneficial in HF. To test this hypothesis, we first measured PDE4B expression in human hearts and found that it was decreased in HF. We then generated transgenic mice with cardiac-specific overexpression of PDE4B, and found that constitutive cardiac PDE4B overexpression decreases cardiac contractile function, blunts β-AR responses, and protects against the detrimental effects of chronic isoproterenol (Iso) infusion. To further examine the therapeutic potential of PDE4B, the gene was delivered to the heart with adenoassociated virus 9 (AAV9). This approach resulted in a drastic improvement of cardiac function in the chronic Iso model, and significantly delayed contractile dysfunction and attenuated remodeling induced by transverse aortic constriction (TAC) without impacting normal cardiac function. These results suggest that increasing PDE4B with gene therapy could represent a novel therapeutic strategy to treat HF.

METHODS

An expanded methods section is available in the Data supplement.

The authors declare that all supporting data are available within the article and its online supplementary files. Human heart tissue was obtained from the cardiovascular biobank of Bichat Hospital (BB-0033-00029, coordinator Dr. JB Michel) and from a tissue collection from the Pitié-Salpétrière Hospital (coordinator Dr. JL Samuel), with approval by the Inserm Institutional Review Board and the Consultative Committee for the Protection of Human Subjects in Biomedical Research at the Pitié-Salpétrière Hospital, respectively. Patients or their relatives were informed that anonymized tissue will be used for research and given the right to refuse. All experiments involving animals were carried out according to the European Community guiding principles in the care and use of animals (2010/63/UE), the local Ethics Committee (CREEA Ile-de-France Sud) guidelines and the French decree n° 2013-118 on the protection of animals used for scientific purposes. Mice overexpressing the murine PDE4B3 (NCBI NM_019840.2) under the α-MHC promoter (PDE4B-TG mice) were generated by pronuclear injection and the genotype was confirmed by PCR. Male PDE4B-TG mice and age-matched WT littermates were used for experiments. AAV9 encoding the murine PDE4B3 (NCBI NM_019840.2) or luciferase under the control of a CMV promoter were produced in AAV-293T cells. PDE4B3 expression as well as hypertrophic and fibrosis markers were evaluated by qRT-PCR. PDE expression was assessed by western blot, PDE activity by radioimmunoassay, and cAMP by ELISA. Transthoracic two-dimensional-guided M-mode echocardiography was performed with a ML6 linear probe of 15 MHz under 2% isoflurane gas and 0.8 L/min oxygen anaesthesia. Sixlead surface electrocardiograms (ECG) were recorded during isoflurane inhalation and telemetric ECG transmitters were used to record ECG in freely roaming animals. Ex vivo cardiac function was evaluated in Langendorff heart preparations. Isolated cardiomyocyte studies: β-AR regulation of cAMP levels and PKA activity were assessed by optical FRET methods. ICa,L was recorded with the patch-clamp technique, sarcomere shortening and Ca 2+ transients with an Ionoptix ® system. Pathological cardiac remodeling was induced by chronic Iso infusion (60 mg/kg/day during 14 days) using osmotic minipumps or by TAC.

RESULTS

PDE4B expression is decreased in human heart failure

To determine whether PDE4B is decreased in human HF like in experimental pathological hypertrophy, [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on beta-adrenergic cAMP signals[END_REF] PDE4B protein was measured by western blot in human heart samples collected from two Parisian hospitals. As shown in Figure 1, PDE4B protein expression was significantly decreased in hearts with either ischemic (Figure 1A) or dilated cardiomyopathy compared to non-failing controls (Figure 1B).

Cardiac phenotype of PDE4B transgenic mice

To evaluate the role of PDE4B in controlling cardiac function, two transgenic mouse lines with cardiomyocyte-specific overexpression of PDE4B3, the main PDE4B isoform expressed in the mouse myocardium, [START_REF] Mika | PDE4B mediates local feedback regulation of beta(1)-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes[END_REF] were generated (PDE4B-TG). In the first line, PDE4B mRNA and protein were increased ~50-fold compared to wild-type (WT), resulting in a ~25-fold increase in PDE4 activity and ~15-fold increase in total cardiac cAMP-hydrolytic activity (Fig. 2A-D). Hence, in the following this line is referred to as TG15. Consistent with increased PDE activity, cAMP was significantly decreased by ~30% in hearts from these mice (Fig. 2E). The expression of other major PDEs hydrolyzing cAMP in the heart (PDE3A, PDE4A and PDE4D) was not modified in TG15 mice compared to WT (Supplemental Fig. 1).

To evaluate the consequences of PDE4B overexpression on cardiac structure and function, young WT and TG15 male mice (10-15 weeks) were subjected to echocardiography and anatomical analysis.

Cardiac contractility, quantified as fractional shortening (FS), was significantly decreased by ~30% in TG15 mice (Fig. 2F). This result is in line with the reduced cAMP content, and suggests that PDE4B overexpression counteracts the positive inotropic effect of the endogenous sympathetic stimulation. In contrast, heart rate was unchanged (Fig. 2F). Echocardiography also revealed a trend toward an increase in left ventricle weight (Fig. 2F), a result that was confirmed by morphometric analysis (Fig. 2G), which indicated a mild but significant cardiac hypertrophy in TG15 mice, whereas lung weight was unchanged (Fig. 2G and Supplemental Table 1). Along with decreased FS, the phosphorylation of phospholamban (PLB), troponin I (TnI) and myosin-binding protein C (MyBPC) at PKA sites was decreased in cardiac tissues from TG15 mice (Fig 2H -J). In addition, PLB phosphorylation at Threonine 17, a specific CaMKII site, was also decreased (Fig. 2H).

In the course of this study, we characterized a second line of PDE4B-TG mice with substantially higher levels of overexpression: PDE4B mRNA was increased ~900-fold and total cAMP-hydrolytic activity was increased ~50-fold (Supplemental Fig. 2A,B). Hence this line is referred to as TG50 in the rest of the study. FS was decreased by ~50% in TG50, and this was accompanied by increased left ventricle (LV) dimension and mass, although there was no sign of pulmonary congestion (Supplemental Fig. 2C,D). Heart rate (HR) measured by echocardiography tended to be decreased in TG50, although the difference was not statistically significant (Supplemental Fig. 2C). In order to exclude confounding effects of anesthesia, telemetric ECG recordings were performed in freely roaming WT, TG15 and TG50 mice across a circadian cycle (Supplemental Fig. 3A). These measurements confirmed that HR was unchanged in TG15, whereas in TG50, HR was decreased during daytime, both at rest and during exercise on a treadmill (Supplemental Fig. 3A and3B). To determine whether this bradycardia was related to a difference in transgene expression at the supraventricular level, PDE4B protein was measured in the left ventricle, left atria and dissected sinoatrial node (SAN) of WT, TG15 and TG50. As shown in Supplemental Fig. 3C, PDE4B was modestly increased in SAN from TG15 whereas it was strongly overexpressed in TG50. Intriguingly, exercise capacity, as determined by the maximal distance run on a treadmill until exhaustion, was not different between WT and PDE4B-TG mice, with even a trend for increased distance run in TG15 compared to WT mice (Supplemental Fig. 3D).

To assess the long term consequences of cardiac PDE4B overexpression, serial echocardiography was performed in both lines during aging. As shown in Fig. 3A and 3B, in TG50 there was a progressive deterioration of systolic function accompanied by a prominent cardiac hypertrophy and dilatation. This led to premature death, with 50% mortality at 72 weeks (Fig. 3C). In contrast, in TG15, although FS remained lower than in WT, cardiac hypertrophy actually normalized with aging, and mortality was similar to WT (Fig. 3A-C). Consistent with these results, the fetal marker genes Nppa, Nppb and Myh7 were strongly re-expressed in hearts from TG50, both in young (10-15 weeks) and old (58-60 weeks) mice, whereas in TG15 these genes tended to be increased in young but normalized in old (Supplemental Fig. 4A-C). This pattern was also observed for Rcan1, a specific target gene of the calcineurin-NFAT pathway. [START_REF] Frey | Calsarcin-2 deficiency increases exercise capacity in mice through calcineurin/NFAT activation[END_REF] These results show that while substantial PDE4B overexpression in TG15 is well tolerated, drastic cardiac PDE4B overexpression in TG50 is detrimental. The reversible hypertrophy in TG15 suggested a potential role of PDE4B in physiological hypertrophy. Physiological hypertrophy has been associated with activation of the RAC-alpha serine/threonine-protein kinase (AKT), which can directly phosphorylate and inhibit glycogen synthase kinase 3β (GSK3β), and with activation of ERK1/2. [START_REF] Nakamura | Mechanisms of physiological and pathological cardiac hypertrophy[END_REF] However, the phosphorylation of these kinases was similar between young WT and TG15 (Supplemental Figure 5).

In contrast, the phosphorylation AKT and GSK3β were decreased in TG50 compared to WT mice, whereas that of ERK1/2 was increased. While these results do not support the implication of these pathways in the reversible hypertrophy of TG15, they suggest their contribution to the development of maladaptive remodeling in TG50. In the following, we focused on the detailed phenotypic characterization of the TG15 mouse line.

PDE4B overexpression blunts β-AR stimulation of heart function

To determine the role of PDE4B under acute β-AR stimulation, we first evaluated cardiac function under Iso infusion in anesthetized mice by echocardiography and ECG. As shown in Fig. 4A and4B, Iso increased FS and heart rate, which were significantly lower in TG15 than in WT hearts. To better assess the intrinsic properties of TG15 mouse hearts, isolated Langendorff-perfused hearts were investigated (Fig 4C -F). The developed pressure was similar in WT and TG15 hearts, revealing preserved contractile capacities of TG hearts in basal conditions (Fig. 4C). Basal heart rate was also unaffected by PDE4B overexpression (Fig. 4D), in agreement with the in vivo data. To evaluate βadrenergic responsiveness, increasing concentrations of Iso were applied in hearts paced at 650 bpm(Fig. 4E). Hill fit of the data revealed a significantly increased EC50 value for Iso in TG15 hearts compared to WT (Fig. 4F). Similar results were observed on the maximal rates of contraction and relaxation (Supplemental Fig. 6).

PDE4B overexpression blunts β-AR responses in isolated cardiomyocytes

To investigate how PDE4B overexpression affects the β-AR signaling cascade, intracellular cAMP and PKA activity were monitored in isolated ventricular myocytes expressing the FRET biosensors EPAC-S H187 20 and AKAR3-NES [START_REF] Allen | Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters[END_REF] , respectively. Whereas a brief application of Iso (30 nmol/L, 15s) produced a robust cAMP elevation in WT cells (+177% increase of the FRET ratio), it increased the FRET ratio by only ~35% in TG15 cells (Supplemental Fig. 7A). Similarly, PKA activation was strongly blunted (~15% increase in FRET ratio) in TG15 versus WT cardiomyocytes (~53% increase in FRET ratio, Supplemental Fig. 7B). As a consequence, the β-AR stimulation of ICa,L was severely diminished in TG15 myocytes (Supplemental Fig. 7C), while basal ICa,L density remained unchanged (WT: 7.0±0.3 pA/pF, n=44; TG:7.4±0.2 pA/pF, n=84). Importantly, attenuated β-AR responses in TG15 myocytes were restored to WT levels in the presence of the selective PDE4 inhibitor, Ro 20-1724 (Supplemental Fig. 8A-C). Next, we measured Ca 2+ transient and sarcomere shortening amplitudes in isolated ventricular myocytes (Fig. 5A-E), which revealed an attenuation of the inotropic and lusitropic effects of β-AR stimulation, whereas basal values were similar between WT and TG15 mice. Again, these effects were normalized by PDE4 inhibition (Supplemental Fig. 8D,E). These results are in line with the preserved basal cardiac function and the diminished β-AR responsiveness observed ex vivo. Furthermore, whereas about 80% of isolated WT ventricular myocytes exposed to Iso showed frequent occurrences of spontaneous Ca 2+ release events (SCRs), SCRs were rare in cardiomyocytes isolated from TG15 mice, and occurred in less than 50% of the cells (Fig. 5G,H). Thus, PDE4B overexpression protects against proarrhythmogenic events triggered by β-AR stimulation.

PDE4B overexpression protects from maladaptive remodeling induced by chronic infusion of isoproterenol

To determine the potential protective effects of cardiac PDE4B overexpression under stress conditions, WT and TG15 mice were subjected to chronic Iso infusion. Cardiac function was evaluated by echocardiography on the day of osmotic minipump implantation, at the end of the 2 weeks treatment, and before sacrifice at 4 weeks (Fig. 6A). WT and TG15 mice implanted with Iso minipumps displayed an increase in heart rate at two weeks, attesting the adequate infusion of the drug (Supplemental Fig. 9A). Interestingly, chronic Iso infusion decreased FS at 2 and 4 weeks in WT mice, while it was first increased then preserved in TG15 (Fig. 6B). Chronic Iso also significantly increased LV mass in WT but not in TG15 mice (Fig. 6B), a finding that was corroborated by measurements of heart weight to tibia length ratio at the time of sacrifice (Fig. 6C). Moreover, Iso increased lung weight in WT but not in TG15 mice (Fig. 6C). Finally, whereas Iso induced cardiac fibrosis in the LV of WT mice, it was drastically reduced in TG15 mice (Fig. 6D). Thus, these results indicate that despite their reduced basal contractile function, TG15 mice were protected against the maladaptive remodeling induced by chronic catecholamine infusion.

To further examine the therapeutic potential of enhanced cardiac PDE4B activity, adeno-associated type 9 viruses (AAV9) were engineered to express this isoform, while a virus encoding luciferase (Luc) was used as control (Supplemental Fig. 10A). We first conducted a pilot study on a limited number of healthy mice to validate our procedure. In these preliminary experiments, tail vein injection of 10 12 AAV9-PDE4B viral particles led to a ~2-fold increase of PDE4B protein in the heart and a ~50% increase in total cardiac cAMP-hydrolytic activity 8 weeks after injection (Supplemental Fig. 10B and10C). Interestingly, in contrast to what we observed in PDE4B-TG mice, this mild overexpression of PDE4B did not decrease FS (Supplemental Fig. 10D) nor did it induce cardiac hypertrophy or lung congestion (Supplemental Fig. 10E,F). We then tested whether PDE4B gene therapy could prevent HF induced by chronic Iso stimulation. For this, mice were injected with 10 12 viral particles of either AAV9-Luc or AAV9-PDE4B two weeks before minipump implantation and were followed by serial echocardiography during 6 weeks (Fig. 7A). As shown in Fig. 7B, the decrease in FS induced by chronic Iso in control mice expressing Luc was virtually absent in animals expressing PDE4B. In addition, PDE4B overexpression partially prevented LV hypertrophy induced by chronic Iso (Fig. 7B). Although PDE4B gene therapy could not fully prevent the increase in heart weight induced by Iso (Fig. 7C), Iso-induced fibrosis and apoptosis were prevented by AAV9mediated PDE4B overexpression (Fig. 7D,E). Western blot analysis indicated that myocardial PDE4B protein was increased ~5-fold in AAV9-PDE4B injected mice (Supplemental Fig. 11A). In summary, AAV9-mediated cardiac PDE4B overexpression partially prevented hypertrophy, but importantly, preserved against the cardiac dysfunction and remodeling induced by chronic Iso stimulation.

Next, cardiac gene therapy with PDE4B was evaluated in mice with transverse aortic constriction (TAC). Two days after the surgery, mice with a pressure gradient across the aorta ≥60 mmHg were randomly injected with either AAV9-Luc or AAV9-PDE4B (10 12 viral particles/mouse) and followed by serial echocardiography during six weeks (Fig. 8A). Although the extent of depressed FS was ultimately similar in mice injected with PDE4B compared to Luc, this decrease was significantly less pronounced at day 14, indicating that PDE4B overexpression delayed cardiac contractile dysfunction induced by TAC (Fig. 8B). In addition, PDE4B overexpression attenuated cardiac hypertrophy and pulmonary congestion induced by TAC (Fig. 8B,C) and reduced both fibrosis and apoptosis (Fig. 8D,E). Similar to what we observed in mice treated with Iso, cardiac PDE4B protein was increased ~6-fold in TAC mice injected with AAV9-PDE4B (Supplemental Fig. 11B).

DISCUSSION

In the present study, we tested whether the cAMP-specific PDE4B is decreased in human HF as reported previously in pressure overload-induced hypertrophy in rats. [START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on beta-adrenergic cAMP signals[END_REF] We characterized the phenotype of mice with myocardial overexpression of PDE4B and we tested whether increasing cardiac PDE4B confers protection against catecholamine toxicity and HF induced by pressure overload. Our main findings can be summarized as follows. First, PDE4B is decreased in failing human hearts compared to non-failing controls. Second, constitutive cardiac PDE4B overexpression in mice counteracts β-adrenergic stimulation of contractile function, which is well tolerated along life span in mice with a ~15-fold increase in cardiac cAMP-PDE activity (TG15), but becomes detrimental at higher levels (~50-fold increase in cardiac cAMP-PDE activity, TG50). Third, TG15 mice are protected from the pathological remodeling elicited by chronic β-adrenergic stimulation, including cardiac hypertrophy, systolic dysfunction, and fibrosis development. Fourth, cardiac PDE4B gene transfer with AAV9, resulting in a significantly lower increase in PDE4B protein and cAMP-PDE activity, also protected against chronic catecholamine stimulation and trans-aortic constriction, without depressing basal cardiac function. These results suggest that decreased PDE4B is maladaptive in HF and that a moderate PDE4B activation in the heart achieved by AAV9-mediated gene transfer could constitute a new promising strategy to limit the systolic dysfunction and pathological remodeling in HF.

Although PDE4 is known to be conserved in rodent, pig and human myocardium 8,[START_REF] Richter | Conserved expression and functions of PDE4 in rodent and human heart[END_REF] there is little information about its expression in human HF and, to our knowledge, nothing concerning PDE4B.

Our present results show that this variant is expressed in non-failing human hearts, and is decreased in HF. This finding, together with studies reporting decreased PDE4A and PDE4D in human failing hearts [START_REF] Richter | Conserved expression and functions of PDE4 in rodent and human heart[END_REF][START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF] indicates that the three PDE4 isoforms expressed in heart are reduced in human HF, a result that may explain in part the reported lack of effect of PDE4 inhibitors on force in the failing human myocardium. [START_REF] Molenaar | PDE3, but not PDE4, reduces beta(1) -and beta(2)-adrenoceptor-mediated inotropic and lusitropic effects in failing ventricle from metoprololtreated patients[END_REF][START_REF] Molenaar | Carvedilol induces greater control of beta2-than beta 1-adrenoceptor-mediated inotropic and lusitropic effects by PDE3, while PDE4 has no effect in human failing myocardium[END_REF] TG15 mice had a decreased contractility but preserved heart rate, whereas in TG50 contractility was further depressed and this was associated with bradycardia. Our results in Supplemental Fig. 3 show that these differences are the consequence of a higher expression of PDE4B in TG50, both in the ventricles and in the SAN. Lower expression of the transgene in TG15 SAN versus ventricle may be explained by a position effect secondary to random insertion of the transgene. [START_REF] Ohtsuka | One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)[END_REF] The less obvious effect of PDE4B overexpression on heart rate versus contractility may also be related to the mouse model. Indeed, previous studies found little chronotropic response to PDE4 inhibition in mice [START_REF] Galindo-Tovar | Phosphodiesterase-4 blunts inotropism and arrhythmias but not sinoatrial tachycardia of (-)-adrenaline mediated through mouse cardiac beta(1)adrenoceptors[END_REF] compared to rats. [START_REF] Christ | Inotropy and L-type Ca current, activated by beta-and beta-adrenoceptors, are differently controlled by phosphodiesterases 3 and 4 in rat heart[END_REF] In TG15, PDE4B overexpression was associated with decreased cAMP content and depressed phosphorylation of the major proteins involved in ECC. In addition, β-AR stimulation of cAMP production, PKA, ICa,L, Ca 2+ transients and cell contraction, as well as of developed pressure in isolated hearts, were attenuated in TG15. The reason why the maximal effect of Iso on developed pressure was similar in isolated perfused hearts from TG15 and WT mice (Fig. 4C) whereas maximal sarcomere shortening was decreased in TG15 myocytes (Fig. 5B) remains unclear. Nevertheless, these results are consistent with the key role of PDE4 in modulating β-AR responses in the rodent heart. 6,[START_REF] Nikolaev | Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2adrenergic receptor-mediated signaling[END_REF] The decreased β-AR stimulation of ICa,L and of pro-arrhythmic Ca 2+ release events in TG15 mice confirms the importance of PDE4B for LTCC regulation, as previously inferred from studies in PDE4 knock-out mice [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF] , while the decreased PLB, TnI and MyBPC phosphorylation indicates that in TG15, the overexpressed PDE4B also impacts other critical proteins for ECC.

The phenotype of TG15 mice reported here is, in several aspects, similar to that of mice with a ~10fold overexpression of PDE3A in the heart. [START_REF] Oikawa | Cyclic nucleotide phosphodiesterase 3A1 protects the heart against ischemiareperfusion injury[END_REF] This may not be too surprising, given that PDE3A represents the other major cAMP-PDE regulating cardiac function. One notable difference however, was that PDE3A-TG mice showed bradycardia, whereas heart rate was unchanged in TG15 mice.

Although not determined in the study of Oikawa et al. [START_REF] Oikawa | Cyclic nucleotide phosphodiesterase 3A1 protects the heart against ischemiareperfusion injury[END_REF] , this difference might be due to a high expression of the transgene in the SAN in PDE3A-TG mice. Alternatively, it could be due to different enzymatic properties or localization of these two PDEs. However, upon Iso challenge, heart rate was significantly lower in TG15 mice (Fig. 4), suggesting that PDE4B may contribute to SAN function during sympathetic stimulation. This could be due to the propensity of long PDE4 isoformsincluding the PDE4B3 variant overexpressed here-to be phosphorylated and activated by PKA upon β-AR stimulation. 6,[START_REF] Ghigo | PI3Kgamma Protects against Catecholamine-Induced Ventricular Arrhythmia through PKA-mediated Regulation of Distinct Phosphodiesterases[END_REF] Whereas the phenotype of PDE4B-TG mice resembles that of PDE3A-TG mice, it is nearly opposite to that of PDE2-TG mice in which bradycardia is associated with an increase in contractile force presumably due to increased diastolic filling. [START_REF] Vettel | Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction[END_REF] Although the exact reason for such difference remains uncertain, the lower affinity of PDE2 for cAMP should make this PDE more efficient to degrade the high cAMP levels present in the SAN compared to the ventricles. [START_REF] Vinogradova | High basal protein kinase A-dependent phosphorylation drives rhythmic internal Ca2+ store oscillations and spontaneous beating of cardiac pacemaker cells[END_REF] The intracellular blockade of β-AR stimulation by PDE4B induced adaptive changes of the heart, but was devoid of overt pathological consequences in TG15. Indeed, basal contractile parameters were similar in isolated hearts from WT and TG15 mice, indicating that constitutive PDE4B overexpression is not detrimental per se. Moreover, PDE4B overexpression did not negatively impact on maximal exercise capacity and there was even a tendency for increased distance run in TG15 mice.

Although counterintuitive at first sight, it should be noted that Iso was still able to enhance cardiac performance in TG15 mice (Fig. 4), indicating the persistence of a significant cardiac reserve. A similar phenotype was observed in β1-AR and double β1-/β2-AR knock-out mice, despite a near total impediment of chronotropic and inotropic reserve, [START_REF] Rohrer | Alterations in dynamic heart rate control in the beta 1-adrenergic receptor knockout mouse[END_REF][START_REF] Rohrer | Cardiovascular and metabolic alterations in mice lacking both beta1-and beta2-adrenergic receptors[END_REF] suggesting the contribution of intrinsic preload and afterload mechanisms in the adaptive response to exercise. It should also be acknowledged that the maximum running distance until exhaustion depends on a number of other factors, among which skeletal muscle capacity is critical. [START_REF] Habouzit | Decreased muscle ACE activity enhances functional response to endurance training in rats, without change in muscle oxidative capacity or contractile phenotype[END_REF] This is certainly one reason for the difference observed with βblockers in healthy subjects, especially the non-selective ones, which affect endurance capacity not only by their direct effects on the heart, but also by impacting skeletal muscle adaptation and metabolism as well as vascular and lung functions. [START_REF] Ladage | Cardio-selective beta-blocker: pharmacological evidence and their influence on exercise capacity[END_REF] The mild cardiac hypertrophy observed in young TG15 mice was associated with transient induction of the brain natriuretic peptide gene Nppb and the calcineurin/NFAT target gene Rcan1. In contrast, AKT and ERK1/2, which have been associated with adaptive hypertrophy, [START_REF] Nakamura | Mechanisms of physiological and pathological cardiac hypertrophy[END_REF] were not activated in young TG15 mice. These results differ with those obtained in TG50 mice, in which persistent expression of fetal genes and Rcan1 was associated with progressive cardiac hypertrophy, dilatation, and HF. These results are consistent with the notion that transient calcineurin/NFAT activation can contribute to physiological hypertrophy induced by exercise [START_REF] Eto | Calcineurin is activated in rat hearts with physiological left ventricular hypertrophy induced by voluntary exercise training[END_REF] or pregnancy [START_REF] Chung | Calcineurin activity is required for cardiac remodelling in pregnancy[END_REF] whereas permanent and excessive activation promotes pathological hypertrophy and HF. [START_REF] Molkentin | A calcineurin-dependent transcriptional pathway for cardiac hypertrophy[END_REF] Unexpectedly, ERK1/2 was activated in young TG50, suggesting that it contributes to the hypertrophic response, as it does in early stages of pathological hypertrophy induced by TAC. [START_REF] Li | Downregulation of survival signalling pathways and increased apoptosis in the transition of pressure overload-induced cardiac hypertrophy to heart failure[END_REF] Importantly, AKT and GSK3β phosphorylation were decreased in TG50. Given the well described cardioprotective effects of AKT, [START_REF] Sussman | Myocardial AKT: the omnipresent nexus[END_REF] decreased activation of this kinase appears as an important contributor to maladaptive remodeling in TG50. Indeed, these results are consistent with increased maladaptive hypertrophy observed in Akt1-knockout mice [START_REF] Debosch | Akt1 is required for physiological cardiac growth[END_REF] and with studies showing that in the context of pressure overload, decreased phosphorylation of AKT and of GSK3β are associated with cardiac dilatation and the transition to HF. [START_REF] Brancaccio | Melusin, a muscle-specific integrin beta1-interacting protein, is required to prevent cardiac failure in response to chronic pressure overload[END_REF][START_REF] Benard | Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling[END_REF] Importantly, our results show that TG15 mice were protected against pathological cardiac remodeling induced by chronic Iso infusion, although these results were mitigated by the reduced contractile function. However, cardiac delivery of PDE4B using an AAV9 resulted in a significantly lower increase in myocardial PDE4B protein which did not modify cardiac function in healthy mice, but efficiently protected against the detrimental effects of chronic Iso treatment and TAC. Indeed, in both models AAV9-mediated PDE4B overexpression counteracted the systolic dysfunction, attenuated the hypertrophic response, and efficiently prevented fibrosis, a hallmark of pathological remodeling.

Replacement interstitial fibrosis as observed here is a known consequence of cardiomyocyte death which occurs in both chronic Iso infusion models [START_REF] Benjamin | Isoproterenol-induced myocardial fibrosis in relation to myocyte necrosis[END_REF] and TAC models. [START_REF] Zhao | Deletion of Interleukin-6 Attenuates Pressure Overload-Induced Left Ventricular Hypertrophy and Dysfunction[END_REF] Accordingly, our results show that apoptosis induced by chronic Iso and TAC was prevented in mice treated with AAV9-PDE4B.

Several studies have demonstrated that Ca 2+ overload triggered by PKA-mediated β-AR stimulation of ICa,L is a critical mechanism for cardiomyocyte death and HF development. [START_REF] Communal | Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the beta-adrenergic pathway[END_REF][START_REF] Nakayama | Ca2+-and mitochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart failure[END_REF] Thus, our present finding that PDE4B overexpression blunts β-AR stimulation of PKA activity and ICa,L, provides a likely explanation for the potent anti-fibrotic action of PDE4B overexpression. This does not exclude that PDE4B acts on other determinants of cardiomyocyte death. For instance, PDE4B decreased CaMKII-mediated PLB phosphorylation (Fig 2H ), which was proposed to be involved in CaMKIImediated apoptosis in vivo. [START_REF] Yang | Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo[END_REF] In conclusion, our study demonstrates the potential benefits of increasing PDE4B to limit the cardiotoxic effects of chronic sympathetic stimulation and HF development in mice. Our results also underline the critical importance of transgene dosage for the beneficial or detrimental outcome of cardiac gene manipulation, as previously noted in the case of the β2-ARs. [START_REF] Liggett | Early and delayed consequences of beta(2)-adrenergic receptor overexpression in mouse hearts: critical role for expression level[END_REF] In this regard, the moderate increase in PDE4B conferred by AAV9 injection appears sufficient to counteract the adverse remodeling induced by chronic Iso and TAC while minimally impacting on normal cardiac function. One limit of our study is the low clinical applicability of these models, hence future studies should be performed in more relevant models such as myocardial infarction. Interestingly, long PDE4 isoforms such as PDE4B3 possess a PKA consensus phosphorylation site in Upstream Conserved Region 1 (UCR1) which, when phosphorylated, activates the enzyme. [START_REF] Mika | PDE4B mediates local feedback regulation of beta(1)-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes[END_REF] Recently, a small allosteric modulator that mimics the effect of PKA phosphorylation on PDE4 long forms was reported. [START_REF] Omar | Small-molecule allosteric activators of PDE4 long form cyclic AMP phosphodiesterases[END_REF] This prototypical PDE4 activator was shown to reduce the chronically elevated cAMP levels which drive abnormal cyst formation in animal and human cell models of autosomal dominant polycystic kidney disease. [START_REF] Omar | Small-molecule allosteric activators of PDE4 long form cyclic AMP phosphodiesterases[END_REF] These results suggest that such compound could constitute an alternative approach to gene therapy in HF.

ECG and Telemetry

Six-lead surface electrocardiograms (ECG) were recorded in littermate pairs of 2 month-old mice during isoflurane inhalation (1-1.5 % plus oxygen). Recordings of 10 s were used for signal averaging. To record an ECG in freely roaming animals, telemetric ECG transmitters (model TA11PA-C10, Data Sciences International, St. Paul, MN, USA) were implanted under isoflurane anesthesia. The telemetric ECG was analyzed during normal activity over a 24-hour period one week after implantation. All recordings were digitized (EMKA, Falls Church, VA, USA) at 2 kHz and analyzed off-line.

Forced treadmill activity

Prior to the experiments, all mice were acclimatized to the rodent treadmill and running exercise by 6 min activity in the treadmill apparatus as follows: 3 Four days before the day of the experiment (D-4; running for 2 min at 10 m.min -1 , 4 min at 13 m.min -1 , 2 min at 15 m.min -1 , 2 min at 16.5 m.min -1 and 2 min at 18 m.min -1 at a 4% slope); 3 days before the day of the experiment (D-3; running for 2 min at 13 m.min -1 , 2 min at 16 m.min -1 , 2 min at 18 m.min -1 , 4 min at 20 m.min -1 and 2 min at 22 m.min -1 at a 4% slope). Compressed air, triggered by a photocell at the end of each lane, was used, if necessary, to maintain the running motivation during acclimatization and experiments. On the day of the experiment, to measure the maximal aerobic running performance, all animals were briefly warmed-up by running 10 m.min -1 for 6 min. Then, animals were exercised until they were no longer able to maintain the increasing speed of the treadmill. Treadmill speed was increased by 3.3 m.min -1 every 1.5 min to reach 30 m.min -1 at a 4% slope, and then treadmill speed was increased by 3.3 m.min -1 every min at a 4% slope. This allowed the maximal distance exercise ran capacity until exhaustion to be determined for each animal.

Perfused heart preparation

Male mice (PDE4B-TG and WT littermates) were anaesthetized with intraperitoneal injection and the first derivatives of LV pressure (LV +dP/dtmax and LV -dP/dtmax) were measured online using a dedicated software (Emka technologies data analyzer, Paris, France). Then, the hearts were paced at 650 bpm using platinum electrode placed on the surface of the right ventricle and increasing concentrations of isoprenaline (Iso) were infused from 0.1 to 100 nmol/L. Pacing was stopped in the presence of 100 nmol/L Iso to record spontaneous cardiac parameters.

Isoprenaline infusion model

Male mice (WT or PDE4B-TG) at 10-weeks of age were treated for 14 days with either isoprenaline (60 mg/kg/day, Iso, Sigma) or vehicle (0.9% NaCl) administered via osmotic minipumps (2002, Alzet, USA) as described 4 with slight modifications. Two weeks after the end of the treatment, animal were anesthetized by intraperitoneal injection of pentothal (150 mg/kg). Hearts were rapidly removed and remaining blood was washed out in cold Ca 2+ -free KREBS solution (120 mmol/L NaCl, 4.8 mmol/L KCl, 2.4 mmol/L MgSO4-7H2O, 1.2 mmol/L KH2PO4 and 24 mmol/L NaHCO3). Transversal slice of 3-4 mm of width was cut in the middle of the heart and rapidly fixed in 10% formalin for histology. The rest of ventricular tissue was frozen in liquid nitrogen and stored at -80°C until use.

TAC model

Male C57/Bl6 mice aged 8 weeks were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed in a supine position atop a heating pad at 37°C. A 5 mm upper partial sternotomy was performed and the sternum retracted using a chest retractor. The thymus and fat tissue were gently separated from the aortic arch. Following identification of the transverse aorta, a small piece of a 4.0 silk suture was placed between the innominate and left carotid arteries. Two loose knots were tied around the transverse aorta and a small piece of 0.38 mm pin gauge was placed parallel to the transverse aorta. The first knot was tied against the pin, followed by the second and the needle was promptly removed in order to yield a constriction of 0.38 mm in diameter. In sham control mice, the entire procedure is identical except for the ligation of the aorta. The sternum and the skin were closed using a 6.0 prolene suture with an interrupted suture pattern and the animal were then administered s.c. 0.03 μg/mg buprenorphine and allowed to recover on a heating pad until fully awake.

Histological and immunohistochemical analysis

Histological staining was performed as described previously. .Mouse hearts were fixed for 24 h in 10% formalin and then were paraffin embedded and transversely sectioned. Sections (3 μm) were deparaffinized and subjected to heat-induced epitope retrieval (HIER) in a 10 mmol/L citrate buffer. Cardiac fibrosis was assessed using Masson's trichrome stain kit (Microm France). To analyze apoptotic myocytes in the heart, autofluorescence was quenched by treating paraffin-embedded sections with PBS/BSA (5%) for 2 h before performing TUNEL staining (Roche) according to the manufacturer's protocol and using Proteinase K treatment. Sections were counterstained with Alexa Fluor 594-conjugated WGA (Invitrogen) at 10 μg/mL for 1 h at room temperature to visualize cell membranes. Slides were scanned by the digital slide scanner NanoZoomer 2.0-RS (Hamamatsu, Japan), which allowed an overall view of the samples. Images were digitally captured from the scan slides using the NDP.view2 software (Hamamatsu).

Preparation of mouse ventricular myocytes

Mice were anesthetized by intraperitoneal injection of pentothal (150 mg/kg), and the heart was AMVMs were left to adhere for 2 h in a 95% O2, 5% CO2 atmosphere at 37°C, before the medium was replaced with FBS-free MEM containing adenoviruses encoding the cAMP FRET sensor Epac-S H187 5, 6 or the cytoplasmic PKA sensor AKAR3-NES 7,8 at a multiplicity of infection of 1000 active viral particles per cell for 24 h.

Preparation of protein extracts

For PDE assay, frozen adult mouse hearts were homogenized in ice-cold buffer containing (in Tablets and PhosSTOP TM phosphatase inhibitor tablets (Roche Diagnostics). Tissue lysates were centrifuged at 15,000 g and 4°C for 20 minutes, and supernatants were used.

Real-time quantitative PCR

Total RNA was extracted from ventricular tissue using Trizol reagent (MRCgene). Reverse transcription of RNA samples was carried out by using iScript cDNA synthesis kit (Bio-Rad)

according to manufacturer's instructions. Real-time PCR reactions were prepared using SYBR Green Supermix (Bio-Rad) and performed in a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad). The relative amount of mRNA transcripts was quantified using the Ct method. The average Ct obtained in non-treated cells was used as a calibrator and RPLP2 housekeeping gene was used as the reference for normalization. Sequences of the primers used are provided in Supplemental Table 2 below.

cAMP Assay

Basal cAMP levels in mouse hearts were measured with a cyclic AMP EIA kit (NewEast Biosciences) following the instructions from the manufacturer. Mouse ventricular tissue were first frozen and reduced to powder in liquid nitrogen. The tissue powder was weighted, diluted in 0.1 mol/L HCl, and used to detect the amount of cAMP.

PDE activity assay

Cyclic AMP-PDE activity was measured according to the method of Thompson and Appleman 9 as described previously. 10 In brief, samples were assayed in a 200 μL reaction mixture containing 40 mmol/L Tris-HCl (pH 8.0), 1 mmol/L MgCl2, 1.4 mmol/L β-mercaptoethanol, 1 μmol/L cAMP, 0.75 mg/ml bovine serum albumin, and 0.1 μCi of [ 3 H]cAMP for 15 minutes at 33°C. The reaction was terminated by heat inactivation in a boiling water bath for 1 minute.

The PDE reaction product 5′-AMP was then hydrolyzed by incubation of the assay mixture with 50 μg Crotalus atrox snake venom for 20 minutes at 33°C, and the resulting adenosine was separated by anion exchange chromatography using 1 ml AG1-X8 resin (Bio-Rad) and quantified by scintillation counting.

Western blot analysis

Protein samples were separated in denaturating acrylamide gels and subsequently transferred onto PVDF membranes. After blocking the membranes with 5% milk buffer for 1 h, the incubation with primary antibodies was carried out over night at 4°C. After incubation with appropriate secondary antibodies for 1 h, proteins were visualized by enhanced chemoluminescence and quantified with Quantity One software. The primary antibodies used were: rabbit anti-PDE4B (113-4) raised against the C-terminus of PDE4B, rabbit anti-PDE4A transients were assessed by the percentage of variation of the Fura-2 ratio by dividing the twitch amplitude (difference of end-diastolic and peak systolic ratios) to end-diastolic ratio. The Tau was used as an index of relaxation and Ca 2+ transient decay kinetics. All parameters were calculated offline using a dedicated software (IonWizard 6x).

Supplemental

A B

Figure 1 :

 1 Figure 1: PDE4B is decreased in human heart failure. A, Western blot analysis of PDE4B

Figure 2 :

 2 Figure 2: Cardiac phenotype of transgenic mice with cardiac-specific PDE4B overexpression,

Figure 3 :

 3 Figure 3: Long term consequences of PDE4B cardiac overexpression depend on expression

Figure 4 :

 4 Figure 4: PDE4B-TG15 mice have attenuated responses to isoproterenol in vivo and in isolated

Figure 5 :

 5 Figure 5: Decreased inotropic, lusitropic and pro-arrhythmic effects of isoproterenol in isolated

Figure 6 :

 6 Figure 6: PDE4B-TG15 mice are protected against maladaptive remodeling induced by chronic

Figure 7 :

 7 Figure 7: Gene therapy with PDE4B protects against maladaptive remodeling induced by

Figure 8 :

 8 Figure 8: Gene therapy with PDE4B protects against maladaptive remodeling induced by

  Figure1

  of pentobarbital (150 mg/kg). The heart was quickly removed and placed into a solution for dissection containing (in mmol/L): NaCl 116, D-glucose 15, NaHCO3 25, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 0.4, at 4°C, oxygenated (95% O2-5% CO2). Then, the aorta was cannulated and perfused by the Langendorff method with Krebs-Henseleit solution containing (in mmol/L): NaCl 116, D-glucose 11, NaHCO3 25, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.2, Pyruvate 2, EDTA 0.1, at a constant pressure of 75 mm Hg and a temperature of 37.0±0.5°C. A latex balloon filled with water and ethanol (90/10) connected to a pressure transducer (Statham gauge Ohmeda, Bilthoven, The Netherlands) was introduced into the left ventricle after crossing the mitral valve. For each heart, the experiment started with a progressive increase of the latex balloon inserted inside the left ventricle to generate a ventricular volume-developed pressure relationship. When the maximal developed pressure was reached, ten minutes of equilibration in isovolumic working conditions were imposed before measuring cardiac parameters. Heart rate, left ventricular developed pressure (LVDP)

  quickly removed and placed into a cold Ca 2+ -free Tyrode's solution containing 113 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4-7H2O, 0.6 mmol/L KH2PO4, 0.6 mmol/L NaH2PO4, 1.6 mmol/L NaHCO3, 10 mmol/L HEPES, 30 mmol/L Taurine, and 20 mmol/L glucose, adjusted to pH 7.4. The ascending aorta was cannulated, and the heart was perfused with oxygenated Ca 2+ -free Tyrode's solution at 37°C for 4 minutes using retrograde Langendorff perfusion. For enzymatic dissociation, the heart was perfused with Ca 2+ -free Tyrode's solution containing LiberaseTM Research Grade (Roche Diagnostics) for 10 minutes at 37°C. Then the heart was removed and placed into a dish containing Tyrode's solution supplemented with 0.2 mmol/L CaCl2 and 5 mg/ml BSA (Sigma-Aldrich). The ventricles were separated from the atria, cut into small pieces, and triturated with a pipette to disperse the myocytes. Ventricular myocytes were filtered on gauze and allowed to sediment by gravity for 10 minutes. The supernatant was removed, and cells were suspended in Tyrode's solution supplemented with 0.5 mmol/L CaCl2 and 5 mg/ml BSA. Cells were suspended in Tyrode's solution with 1 mmol/L CaCl2. For ICa,L recording and Ionoptix experiments, freshly isolated ventricular myocytes were plated in 35-mm culture dishes coated with laminin (10 μg/ml) and stored at room temperature until use. For primary culture, Tyrode's solution was replaced by Minimum Essential Medium (MEM, 51200, Gibco) supplemented with 5% FBS, 2% penicillinstreptomycin, 0.1% BSA, 2 mmol/L L-glutamine, Insulin Transferin Selenium 1X and plated on 35 mm culture dishes coated with laminin (10 μg/ml) at a density of 10 4 cells per dish.

  AC55), mouse anti-PDE4D (ICOS) (the three being kind gifts from Dr. Marco Conti, UCSF, California, USA), anti-phospholamban (PLB) (sc-21923, Santa Cruz), anti-p-PLB (Thr17) (A010-13, Badrilla), anti-p-PLB (Ser16) (A010-12, Badrilla), anti-troponin I (TnI) (4002, Cell Signaling), anti-p-TnI (Ser22-23) (4004, Cell Signaling), anti-myosin-binding protein C (MyBPC3) (sc-50115, Santa Cruz), anti-p-MyBPC3 (Ser282) (ALX-215-057-R050, Alexis), rabbit anti-PDE3A (a kind gift from Dr C. Yan, University of Rochester, Rochester, NY, USA), rabbit anti-PDE3B (University of Torino, Torino, Italy), mouse anti-β-actin (sc-47778, Santa Cruz), anti-calsequestrin (CSQ) (PA1-193, Pierce), mouse anti-vinculin (Vinc) (V9131, Sigma), rabbit anti-GAPDH (cell signalling). ICa,L current measurements The whole-cell configuration of the patch-clamp technique was used to record ICa,L. Patch electrodes with 1-2 MΩ resistance when filled with internal solution contained 118 mmol/L CsCl, 5 mmol/L EGTA, 4 mmol/L MgCl2, 5 mmol/L sodium phosphocreatine, 3.1 mmol/L Na2ATP, 0.42 mmol/L Na2GTP, 0.062 mmol/L CaCl2 (pCa 8.5), and 10 mmol/L HEPES, adjusted to pH 7.3. External Cs + -Ringer solution contained 107.1 mmol/L NaCl, 20 mmol/L CsCl, 4 mmol/L NaHCO3, 0.8 mmol/L NaH2PO4, 5 mmol/L glucose, 5 mmol/L Na pyruvate, 10 mmol/L HEPES, 1.8 mmol/L MgCl2, and 1.8 mmol/L CaCl2, adjusted to pH 7.4. The cells were depolarized every 8 seconds from -50 mV to 0 mV for 400 ms. The use of -50 mV as holding potential allowed the inactivation of voltage-dependent sodium currents. Potassium currents were blocked by replacing all K + ions with external and internal Cs + . Measurements of Ca 2+ transients and cell shortening Isolated cardiomyocytes were loaded with 3 μM Fura-2 AM (Invitrogen) at room temperature for 15 minutes and then washed with external Ringer solution containing (in mmol/L): NaCl 121.6, KCl 5.4, NaHCO3 4.013, NaH2PO4 0.8, 10 mM HEPES, glucose 5, Na pyruvate 5, MgCl21.8, and CaCl2 1, pH 7.4. The loaded cells were field stimulated (5 V, 4 ms) at a frequency of 0.5 Hz. Sarcomere length (SL) and Fura-2 ratio (measured at 512 nm upon excitation at 340 nm and 380 nm) were simultaneously recorded using an IonOptix System (IonOptix ® ). Cell contractility was assessed by the percentage of sarcomere shortening, which is the ratio of twitch amplitude (difference of end-diastolic and peak systolic SL) to end-diastolic SL. Ca 2+

Supplemental Figure 2 : 20 Supplemental Figure 3 ASupplemental Figure 5 : 5 C o n t r a c t io n v e lo c it

 220355 Figure 1A

Table 1 Morphometric and echocardiographic parameters WT and PDE4B-TG mice (line A) at 10 weeks of age

 1

	Student t test. **p<0,01, ***p<0,001.
	HW, heart weight; LW, lung weight; BW, body weight, TL, tibia length; EF, ejection fraction;
	LVIDd, end-diastolic left ventricular internal diameter; IVSd, end-diastolic interventricular
	septum; PWd, end-diastolic posterior wall; LVIDs, end-systolic left ventricular internal
	diameter; IVSs, end-systolic interventricular septum; PWs, end-systolic posterior wall.

ACKNOWLEDGEMENTS

We thank Agnès Hivonnait (Institut du thorax) for help with ECG recordings and Dr. Kees Jalink (The Netherlands Cancer Institute) for the Epac-S H187 sensor.

SOURCES OF FUNDING

UMR

-S1180 is a member of the Laboratory of Excellence LERMIT supported by the French National Research Agency (ANR-10-LABX-33) under the program "Investissements d'Avenir" ANR-11-IDEX-0003-01. This work was also funded by grant ANR-19-CE14-0038-02 to GV, ANR-16-ECVD-0007-01 to RF, ANR-16-CE14-0014 to DM, a grant from the Fédération Française de Cardiologie to VA, and grants from Italian Ministry of Health (RF-2013-02354892) and Fondazione Cariplo (2015-0880) to AG. SK was supported by Fondation Lefoulon-Delalande, JPM by the VINCI program of Università Italo-Francese (UIF-UFI), MD, KB and ML by the French Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation, IB and DM by the CORDIMM program of Région Ile-de-France and Fondation pour la Recherche Médicale.

DISCLOSURES

None.

SUPPLEMENTAL MATERIAL

Cardiac overexpression of PDE4B blunts β-adrenergic response and maladaptive remodeling in heart failure

Karam S 1 *, Margaria JP 2 *, Bourcier A 1 *, Mika D 1 , Varin A 1 , Bedioune I 1 , Lindner M 1 , Bouadjel K 1 , Dessillons M 1 , Gaudin F 3 , Lefebvre F 1 , Mateo P 1 , Lechène P 1 , Gomez S 1 , Domergue V 3 , Robert P 3 , Coquard C 1 , Algalarrondo V 1 , Samuel JL 4 , Michel JB 5 , Charpentier F 6 , Ghigo A 2 , Hirsch E 2 , Fischmeister R 1 , Leroy J 1 , Vandecasteele, G 1$ 1 Université Paris-Saclay, Inserm, Signaling and Cardiovascular Pathophysiology, UMR-S 1180, 92296 Châtenay-Malabry, France. 2 Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University di Torino, 10126 Torino, Italy. 3 Université Paris-Saclay, Inserm, UMS-IPSIT, 92296 Châtenay-Malabry, France. 4 UMR-S 942, INSERM, Paris University, 75010 Paris, France. 5 UMR-S 1148, INSERM, Paris University, X. Bichat hospital, 75018 Paris, France. 6 Institut du thorax, INSERM, CNRS, UNIV Nantes, 8 quai Moncousu, 44007 Nantes cedex 1, France.

Supplemental Methods

Reagents

Isoprenaline (Iso) was purchased from Sigma and Ro 20-1724 (Ro) was from Calbiochem.

Human heart tissue

Human heart tissue was obtained from the cardiovascular biobank of Bichat-Claude Bernard Hospital (BB-0033-00029, authorization AC-2018-3171 of the French Ministry of Research and Innovation, and PFS17-002 of the French Biomedicine Agency, coordinator Dr. JB Michel) and from a tissue collection from the Pitié-Salpétrière Hospital (coordinator Dr. Jane-Lise Samuel), with approval by the Inserm Institutional Review Board and the Consultative Committee for the Protection of Human Subjects in Biomedical Research at the Pitié-Salpétrière Hospital, respectively. The study was performed with left ventricular myocardium (LV) of end-stage failing hearts from patients with ischemic cardiomyopathy and idiopathic dilated cardiomyopathy. Non-failing LV was obtained from brain-dead organ donors that were unsuitable for transplantation for technical reasons. Patients or their relatives were informed that anonymized tissue will be used for research and given the right to refuse.

Transgenic mouse generation

Mice overexpressing PDE4B specifically in the heart (PDE4B-TG, C57/Bl6) were generated at the Institut Clinique de la Souris (Strasbourg, France). A mouse cDNA encoding for PDE4B3 (NCBI NM_019840.2, corresponding to mouse PDE4B transcript variant 1 in the NCBI nomenclature), kindly provided by Dr. J. Cherry (Boston University, Boston, MA, USA), 1 was subcloned between the 5.5-kb murine α-MHC promoter and the human growth hormone polyadenylation sequence of a pBluescript-based vector (a kind gift from Dr J. Robbins, Children's Hospital Research Foundation, Cincinnati, OH, USA). 2 The purified transgene

Cyclic AMP level and PKA activity measurements by fluorescent resonance energy transfer (FRET) imaging

Freshly isolated ventricular myocytes from PDE4B-TG and WT mice were infected with adenoviruses to measure cAMP and PKA activity, respectively. The cells were then washed once and maintained in the Ringer solution described above containing 1.8 mmol/L CaCl2, at room temperature, Images were captured every 5 seconds using the 40× oil immersion objective of an inverted microscope (Nikon) connected to a software-controlled (Metafluor, Molecular Devices) cooled charge coupled (CCD) camera (Cool SNAP HQ2). CFP was excited during 300 ms by a Xenon lamp (100W, Nikon) using a 440/20BP filter and a 455LP dichroic mirror.

Dual emission imaging of CFP and YFP was performed using a Dual-View emission splitter equipped with a 510LP dichroic mirror and 480/30 nm, 535/25 nm BP filters. Average fluorescence intensity was measured in a region of interest comprising the entire cell.

Background was subtracted and CFP bleed through in the YFP channel was corrected before calculating the YFP/CFP ratio for the AKAR3-NES sensor or the CFP/YFP ratio for the Epac-S H187 sensor. Ratio images were obtained using Image J software.

Statistics

All results are expressed as mean±SEM and were analyzed using the GraphPad Prism software (GraphPad software, Inc., La Jolla, CA, USA). Normal distribution was tested by the Shapiro-Wilk normality test. For simple two-group comparison, we used an unpaired Student t-test or a Mann-Whitney test when the data did not follow a normal distribution. Differences between multiple groups were analyzed using an ordinary one-way ANOVA with Tukey's multiple comparisons post-hoc test, or a Kruskal Wallis with Dunn's multiple comparisons post-hoc test, when the data did not follow a normal distribution. Two-way ANOVA (ordinary or mixedeffects) with Sidak's or Tukey's post-hoc test was used when appropriate. P-value<0.05 was considered statistically significant.

Supplemental Table 2

Sequences of the primers used in qRT-PCR experiments


Delphine Mika

Jérôme Leroy

Rodolphe Fischmeister

Dr Grégoire Vandecasteele
email: gregoire.vandecasteele@u-psud.fr

Rôle des phosphodiestérases des nucléotides cycliques de type 3 et 4 dans le couplage excitation-contraction et les arythmies cardiaques Role of cyclic nucleotide phosphodiesterases type 3 and 4 in cardiac excitation-contraction coupling and arrhythmias

Keywords:

Les phosphodiestérases des nucléotides cycliques (PDEs) représentent une superfamille d'enzymes spécialisées dans la dégradation de l'AMPc et du GMPc. Dans le coeur, les PDEs de type 3 et 4 sont les deux familles majeures impliquées dans la régulation des taux d'AMPc et dans le contrôle de l'inotropisme. Ces deux familles sont codées par plusieurs gènes, et les études récentes du phénotype cardiaque de souris invalidées pour ces différents gènes ont permis de mieux comprendre leur rôle dans la régulation des acteurs du couplage excitationcontraction (CEC) cardiaque. En particulier, ces travaux soulignent le caractère local de la régulation des acteurs du CEC par les PDEs, ainsi que leur rôle dans le maintien de l'homéostasie calcique intracellulaire et la prévention des troubles du rythme.

Introduction

Le myocarde se contracte sous l'effet d'oscillations périodiques de la concentration de calcium intracellulaire ([Ca 2+ ]i). Dans les myocytes ventriculaires, ces augmentations transitoires de la [Ca 2+ ]i naissent au niveau de structures spécialisées, les dyades, formées par l'apposition de deux systèmes membranaires : les tubules transverses (tubules T) et le réticulum sarcoplasmique (RS). Lors du potentiel d'action cardiaque, l'ouverture des canaux calciques de type L (LTCC) localisés dans la membrane tubulaire au niveau des dyades génère un influx de Ca 2+ (le courant calcique de type L, ou ICa,L) qui provoque l'ouverture des récepteurs de la ryanodine (RyR2) [START_REF] Lacampagne | Ryanodine receptor and heart disease[END_REF] localisés dans la membrane du RS. Ceci induit une libération massive du Ca 2+ contenu dans ce compartiment, permettant d'atteindre une [Ca 2+ ]i de l'ordre de la micromolaire nécessaire au développement de la contraction. Lors de la relaxation, la [Ca 2+ ]i diminue rapidement sous l'action des pompes calciques ATPdépendantes du RS (SERCA2), qui permettent la réintégration du Ca 2+ dans ce compartiment, tandis que le reste du Ca 2+ est extrudé, principalement par l'échangeur Na + -Ca 2+ (NCX) et dans une moindre mesure, par les pompes calciques de la membrane plasmique (PMCA) [START_REF] Bers | Cardiac excitation-contraction coupling[END_REF] (Figure 1).

Ce processus appelé couplage excitation-contraction (CEC) est sous le contrôle de la voie de l'adénosine 3',5'-monophosphate cyclique (AMPc) qui constitue un relais essentiel de la régulation nerveuse du coeur. La libération de noradrénaline des terminaisons sympathiques ou la décharge d'adrénaline par les glandes surrénales activent les récepteurs β-adrénergiques (β-AR), entraînant une activation, via les protéines G stimulatrices (Gs), des adénylate cyclases (AC) responsables de la synthèse d'AMPc. Classiquement, l'effet inotrope positif de l'AMPc est attribué à l'activation de la protéine kinase dépendante de l'AMPc (PKA) qui phosphoryle les LTCC et les RyR2, conduisant à une augmentation de la [Ca 2+ ]i et donc de la contraction. La PKA phosphoryle aussi le phospholamban (PLB), et ceci lève l'inhibition tonique exercée par cette protéine sur la SERCA2, et accélère le repompage du Ca 2+ dans le RS. Enfin, la PKA phosphoryle la troponine I (TnI) au niveau des myofilaments, ce qui diminue leur affinité pour le Ca 2+ . La phosphorylation du PLB et de la TnI concourent à une accélération de la relaxation cardiaque (effet lusitrope positif) [START_REF] Bers | Cardiac excitation-contraction coupling[END_REF]. En plus de la PKA, l'AMPc peut activer les canaux HCN (hyperpolarisation-activated cyclic nucleotide-gated channels), impliqués dans la régulation de la fréquence cardiaque [START_REF] Mangoni | Genesis and regulation of the heart automaticity[END_REF][START_REF] Lerebours | Sinus rhythm: mechanisms and function[END_REF] et le facteur d'échange des petites protéines G Rap, Epac (Exchange protein activated by cAMP) impliqué dans l'hypertrophie cardiaque [START_REF] Morel | cAMP-binding protein Epac induces cardiomyocyte hypertrophy[END_REF][START_REF] Metrich | Epac mediates beta-adrenergic receptor-induced cardiomyocyte hypertrophy[END_REF]. Des travaux récents proposent qu'Epac participe aussi à la régulation du CEC en régulant la sortie de Ca 2+ des RyR2 [START_REF] Pereira | The cAMP binding protein Epac modulates Ca2+ sparks by a Ca2+/calmodulin kinase signalling pathway in rat cardiac myocytes[END_REF] et l'affinité des myofilaments pour le Ca 2+ [START_REF] Cazorla | The cAMP binding protein Epac regulates cardiac myofilament function[END_REF].

Compartimentation de la voie AMPc dans les myocytes cardiaques

En plus des récepteurs β-AR, les myocytes cardiaques expriment d'autres récepteurs couplés à Gs, qui, bien qu'activant la même voie de signalisation, exercent des effets différents sur le coeur [START_REF] Steinberg | Compartmentation of G protein-coupled signaling pathways in cardiac myocytes[END_REF]. Une possibilité permettant d'expliquer ces observations est que la voie de l'AMPc est compartimentée au niveau subcellulaire. On sait en effet que les composants de la voie AMPc ne sont pas localisés au hasard dans les myocytes cardiaques. Par exemple, la distribution des récepteurs couplés aux protéines Gs et des adénylates cyclases 5/6 (isoformes cardiaques) n'est pas la même selon qu'on considère la membrane plasmique périphérique, les tubules T ou encore les cavéoles [START_REF] Gao | Identification and subcellular localization of the subunits of L-type calcium channels and adenylyl cyclase in cardiac myocytes[END_REF][START_REF] Vo | Beta2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation[END_REF]. De plus, la PKA est localisée au voisinage immédiat de ses cibles par une famille de protéines d'ancrage, les AKAPs (A-kinase anchoring proteins), qui lient aussi une variété d'autres protéines de signalisation et jouent un rôle important de « plateformes » de signalisation [START_REF] Scott | A-kinase anchoring proteins: getting to the heart of the matter[END_REF]. A cette localisation subcellulaire spécifique des protéines, s'ajoute celle du second messager lui-même, et ce malgré le caractère hautement diffusible de cette petite molécule. Les phosphodiestérases des nucléotides cycliques (PDEs), qui sont responsables de l'hydrolyse de l'AMPc et du 3',5'guanosine monophosphate cyclique (GMPc) en 5'-AMP ou 5'-GMP inactifs, jouent un rôle majeur dans le confinement de ces seconds messagers [START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases[END_REF]. Dans le coeur, on considère principalement quatre familles de PDEs (PDE1-4) qui sont capables de dégrader l'AMPc.

Alors que la PDE1 et la PDE2 hydrolysent l'AMPc et le GMPc, la PDE3 hydrolyse préférentiellement l'AMPc et la PDE4 est spécifique de l'AMPc. La PDE1 est activée par le complexe Ca 2+ /calmoduline ; la PDE2 est stimulée par le GMPc ; et la PDE3 est inhibée par le GMPc. Une cinquième PDE, la PDE8A, hydrolyse spécifiquement l'AMPc et a récemment été impliquée dans le contrôle du CEC cardiaque chez la souris [START_REF] Patrucco | Phosphodiesterase 8A (PDE8A) regulates excitation-contraction coupling in ventricular myocytes[END_REF].

Ainsi, un myocyte cardiaque exprime de multiples PDEs dont les propriétés enzymatiques, le mode de régulation, et la localisation subcellulaire diffèrent. Cette variété d'isoformes permet de contrôler localement de multiples « pools » d'AMPc en réponse à l'activation de différents récepteurs couplés à Gs, et joue un rôle crucial dans la spécificité d'action du second messager [START_REF] Mika | 16 Osadchii OE Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease[END_REF]. Le niveau d'expression des 4 familles présente une grande variabilité inter espèce, et dépend également du stade de développement et du territoire cardiaque considérés.

Toutefois, chez l'homme et dans la plupart des mammifères, les PDE3 et PDE4 sont les deux familles majeures qui contrôlent le CEC cardiaque [16].

Régulation du CEC cardiaque par les PDE3 et PDE4

 Régulation du CEC par les PDE3 La PDE3 possède une affinité submicromolaire pour l'AMPc et le GMPc, mais ce dernier est hydrolysé dix fois plus lentement que l'AMPc et se comporte donc comme un inhibiteur compétitif de l'AMPc. La PDE3 est codée par deux gènes, PDE3A et PDE3B. Bien que la PDE3B soit exprimée dans le coeur de souris [START_REF] Patrucco | PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent andindependent effects[END_REF], la PDE3A constitue l'isoforme de PDE3 dominante au niveau cardiaque [START_REF] Shakur | Regulation and function of the cyclic nucleotide phosphodiesterase (PDE3) gene family[END_REF]. Dans de nombreuses espèces animales dont l'homme, les inhibiteurs de PDE3 exercent des effets inotrope et lusitrope positifs puissants, et sont aussi capables de potentialiser les effets d'une stimulation β-AR sur le CEC cardiaque [16]. De ce fait, certains inhibiteurs de la PDE3 comme la milrinone (Corotrope  ) ou l'enoximone (Perfane) sont utilisés pour le traitement de l'insuffisance cardiaque aiguë. Toutefois, l'ampleur des effets contractiles des inhibiteurs de PDE3 varie considérablement selon l'espèce considérée, et il existe une corrélation positive entre l'efficacité des inhibiteurs de PDE3 et la quantité d'enzyme dans la fraction membranaire [START_REF] Weishaar | Subclasses of cyclic AMP-specific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility[END_REF]. Chez l'homme et le chien, deux espèces dans lesquelles l'effet des inhibiteurs de PDE3 est particulièrement évident, des expériences de purification du RS ont caractérisé la présence majoritaire de PDE3 [START_REF] Lugnier | Characterization of indolidan-and rolipram-sensitive cyclic nucleotide phosphodiesterases in canine and human cardiac microsomal fractions[END_REF]. Deux mécanismes principaux expliquent les effets inotrope et lusitrope positifs des inhibiteurs de PDE3: la stimulation du courant calcique ICa,L et l'activation du repompage du Ca 2+ dans le RS. En effet, dans plusieurs espèces, l'inhibition de la PDE3 exerce un effet stimulateur sur ICa,L : c'est par exemple le cas dans les myocytes atriaux humains ou de lapin, et dans les myocytes ventriculaires de cobaye.

Par ailleurs, les inhibiteurs de PDE3 augmentent l'accumulation de Ca 2+ par des vésicules de RS purifiées chez le chien, et accélèrent la cinétique de retour des transitoires Ca 2+ chez le chat ou le furet. Ces effets sont liés à la phosphorylation PKA-dépendante du PLB (pour revue, voir [16]).

Dans des myocytes ventriculaires isolés de rat, l'inhibition sélective de la PDE3 n'augmente pas ICa,L en conditions basales, mais exerce un effet significatif sur les transitoires calciques et la contraction, associé à une augmentation de la phosphorylation du PLB par la PKA. Lors d'une stimulation β-AR non maximale, l'inhibition de la PDE3 potentialise l'amplitude de ICa,L, des transitoires calciques et de la contraction, et ceci s'accompagne d'une augmentation de la phosphorylation PKA-dépendante des LTCC, de PLB, et de RyR2 [START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes[END_REF] (Mika, Leroy, Fischmeister, Vandecasteele résultats non publiés). Une situation similaire est retrouvée chez la souris, espèce dans laquelle l'inhibition de la PDE3 augmente les transitoires calciques et la contraction cardiaque basales, ainsi que le taux de phosphorylation par la PKA du PLB et des RyR2, sans augmenter l'amplitude d'ICa,L [START_REF] Kerfant | PI3Kgamma is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes[END_REF][START_REF] Beca | PDE3A Regulates Basal Myocardial Contractility Through Interacting with SERCA2a-Signaling Complexes in Mouse Heart[END_REF]. L'utilisation de souris déficientes pour chacun des gènes PDE3A et PDE3B (PDE3A -/-et PDE3B -/-) a permis de démontrer que l'effet inotrope positif des inhibiteurs de PDE3 était du à l'inhibition de PDE3A et non à celle de PDE3B. [START_REF] Beca | PDE3A Regulates Basal Myocardial Contractility Through Interacting with SERCA2a-Signaling Complexes in Mouse Heart[END_REF][START_REF] Sun | Role of phosphodiesterase type 3A and 3B in regulating platelet and cardiac function using subtype-selective knockout mice[END_REF]. Ex vivo, les coeurs de souris PDE3A -/-présentent une contractilité basale augmentée, associée à une hyperphosphorylation du PLB et des RyR2. Des expériences de co-immunoprécipitation ont montré l'existence de complexes macromoléculaires autour de SERCA2, associant la PDE3A, le PLB, les sous-unités régulatrices de la PKA et une AKAP [START_REF] Beca | PDE3A Regulates Basal Myocardial Contractility Through Interacting with SERCA2a-Signaling Complexes in Mouse Heart[END_REF] (Figure 2). Cette proximité spatiale suggère que la PDE3A contrôle le repompage calcique dans le RS en régulant localement les taux d'AMPc et l'activité de la PKA au voisinage immédiat du PLB.

 Régulation du CEC par les PDE4

Les PDE4 hydrolysent spécifiquement l'AMPc mais avec une affinité plus basse que la PDE3, de l'ordre de la micromole. Cette famille de PDE est codée par 4 gènes différents (PDE4A-D). Selon certains auteurs, seules les PDE4A, PDE4B et PDE4D seraient exprimés dans le tissu cardiaque [START_REF] Kostic | Altered expression of PDE1 and PDE4 cyclic nucleotide phosphodiesterase isoforms in 7-oxo-prostacyclin-preconditioned rat heart[END_REF] tandis que d'autres rapportent la présence de transcrits pour la PDE4C dans le coeur [START_REF] Mokni | Concerted regulation of cGMP and cAMP phosphodiesterases in early cardiac hypertrophy induced by angiotensin II[END_REF]. Dans la plupart des espèces, l'inhibition des PDE4 n'exerce peu ou pas d'effet inotrope positif direct, mais devient importante lorsque les taux d'AMPc sont augmentés [16,[START_REF] Muller | Involvement of rolipram-sensitive cyclic AMP phosphodiesterase in the regulation of cardiac contraction[END_REF]. Par exemple, chez le rat l'inhibition des PDE4 n'a aucun effet sur l'AMPc, ICa,L et le CEC en conditions basales, mais devient prédominante pour contrôler ces paramètres lors d'une stimulation β-AR ou lors de l'inhibition concomitante de la PDE3 [START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes[END_REF][START_REF] Leroy | Spatiotemporal dynamics of betaadrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF] (Mika, Leroy, Fischmeister, Vandecasteele résultats non publiés). Chez la souris, l'inhibition des PDE4 n'a pas d'effet sur ICa,L en conditions basales, mais potentialise fortement les effets d'une stimulation β-AR sur ICa,L et sur la contraction [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF]. Des travaux récents suggèrent que dans cette espèce, l'inhibition de la PDE4 exerce un effet inotrope positif direct sur le coeur isolé perfusé et augmente les transitoires calciques et la contraction de myocytes ventriculaires isolés [START_REF] Kerfant | PI3Kgamma is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes[END_REF][START_REF] Beca | Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca2+ release and cardiac contractility, independently of L-type Ca2+ current[END_REF]. Ces effets sont associés à une phosphorylation accrue par la PKA du PLB et du RyR2, indépendamment de ICa,L [START_REF] Kerfant | PI3Kgamma is required for PDE4, not PDE3, activity in subcellular microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes[END_REF]. Comme pour la PDE3, l'étude de souris invalidées pour les gènes PDE4A, PDE4B et PDE4D a permis de mieux comprendre le rôle des trois différents sous-types de PDE4 dans le CEC. Ces travaux ont permis de montrer qu'une isoforme issue du gène PDE4B régule préférentiellement le courant calcique de type L [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF] tandis que les isoformes issues du gène PDE4D régulent préférentiellement les flux calciques du RS [START_REF] Beca | Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca2+ release and cardiac contractility, independently of L-type Ca2+ current[END_REF][START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF] (Figure 2). En effet, il a d'abord été montré que dans les souris PDE4D -/-, la perte d'un variant particulier (PDE4D3) de ce gène normalement associé au RyR2, provoque l'hyperphosphorylation de ces récepteurs par la PKA, ce qui augmente leur activité [START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF]. L'ablation du gène PDE4D entraine aussi la phosphorylation accrue du PLB, et une isoforme longue du gène PDE4D co-immunoprécipite avec la pompe SERCA2 [START_REF] Beca | Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca2+ release and cardiac contractility, independently of L-type Ca2+ current[END_REF]. D'autres expériences de co-immunoprécipitation ont montré que les isoformes PDE4B et PDE4D peuvent s'associer à la sous-unité principale CaV1.2 des LTCC (Figure 2). Toutefois, la régulation β-AR de ICa,L n'est augmentée que dans les souris PDE4B -/-, alors qu'elle demeure inchangée dans les souris PDE4D -/-et dans les souris PDE4A -/- [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF]. Cette augmentation d'ICa,L dans les myocytes de souris PDE4B -/-conduit à une augmentation des transitoires calciques et de la contraction cellulaire sous stimulation β-AR. De façon intéressante, ces deux paramètres sont également augmentés dans les myocytes de souris PDE4D -/-, et ceci est associé à une phosphorylation accrue du PLB sous stimulation β-AR [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF]. Collectivement, ces études suggèrent que les isoformes PDE4B et PDE4D contrôlent différentes composantes du CEC dans les myocytes ventriculaires de souris : la PDE4B semble être l'isoforme principale de la PDE4 impliquée dans la régulation de l'influx calcique transitant par les canaux LTCC, tandis que la PDE4D régule la recapture de calcium par le RS, et peut être aussi sa libération par les RyR2 (Figure 2). Chez l'homme, l'inhibition de la PDE4 n'exerce pas d'effet inotrope positif direct marqué au niveau ventriculaire. Toutefois, il a été montré que la PDE4 contrôle les taux d'AMPc et l'amplitude d'ICa,L dans les myocytes atriaux humains. De plus, l'inhibition de la PDE4 potentialise l'effet d'une stimulation β-AR ou d'une inhibition de PDE3 sur la force de contraction dans des fibres atriales humaines [START_REF] Molina | Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias[END_REF].

Implications physiopathologiques

Les PDE3 et PDE4 constituent un mécanisme important de prévention des arythmies cardiaques liées à l'activation de la voie β-adrénergique. Ainsi, malgré des effets hémodynamiques bénéfiques à court terme, de nombreux essais cliniques ont montré que l'utilisation prolongée des inhibiteurs de PDE3 chez des patients en insuffisance cardiaque (IC) augmente le taux de mortalité et qu'une partie importante de cette augmentation est due à une incidence accrue de morts subites par arythmies [START_REF] Amsallem | Phosphodiesterase III inhibitors for heart failure[END_REF]. Plusieurs mécanismes peuvent être à l'origine de ces arythmies. L'inhibition chronique de la PDE3 active le répresseur transcriptionnel ICER, qui induit l'apoptose des myocytes cardiaques [START_REF] Yan | Regulation of phosphodiesterase 3 and inducible cAMP early repressor in the heart[END_REF]. Ce phénomène pourrait contribuer au remodelage structural du coeur, fournissant ainsi un substrat favorable aux troubles du rythme. Une autre possibilité est que les effets pro arythmiques soient une conséquence directe des changements de Ca 2+ intracellulaire responsables de l'augmentation de contractilité. En particulier, l'inhibition de la PDE3 pourrait induire une surcharge calcique à l'origine d'arythmies déclenchées. A notre connaissance, il n'a pas été reporté d'arythmie ou de pathologie cardiaque dans les modèles de souris invalidées pour la PDE3A ou la PDE3B [START_REF] Beca | PDE3A Regulates Basal Myocardial Contractility Through Interacting with SERCA2a-Signaling Complexes in Mouse Heart[END_REF][START_REF] Sun | Role of phosphodiesterase type 3A and 3B in regulating platelet and cardiac function using subtype-selective knockout mice[END_REF]. Ceci pourrait être lié à la contribution importante de PDE4 dans cette espèce. De fait, in vivo, les souris PDE4B -/-et PDE4D -/-présentent une susceptibilité augmentée aux tachycardies ventriculaires sous catécholamines ou en condition de stress [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF][START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF]. Dans le cas des souris PDE4B -/-, ces arythmies sont déclenchées par un train de stimulation rapide mais pas par des extrasystoles, et ne sont pas associées à une modification de la période réfractaire, ce qui est compatible avec une altération de l'homéostasie calcique plutôt qu'avec un mécanisme de réentrée ou une altération de la conduction [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF]. A la différence des souris PDE4D -/-, qui développent une cardiomyopathie dilatée en vieillissant [START_REF] Lehnart | Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias[END_REF], les souris PDE4B -/-ne présentent pas de signes de pathologie cardiaque évidents jusqu'à l'âge de quinze mois (Leroy & Vandecasteele, résultats non publiés). Au niveau cellulaire, les myocytes déficients pour PDE4B -/-ou PDE4D -/-présentent une augmentation de la fréquence des transitoires calciques spontanées sous stimulation β-AR [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice[END_REF]. Comme mentionné plus haut, ceci semble avoir pour origine une augmentation de l'influx de Ca 2+ dans les souris PDE4B -/-, tandis que des anomalies du repompage et de la libération de Ca 2+ par le RS sont associées à la perte de PDE4D. Le lien entre PDE et arythmies est encore renforcé par l'analyse du phénotype de souris invalidées pour la phosphatidyl-inositol 3 kinase γ (PI3Kγ -/- ), une kinase multifonctionnelle qui est aussi une protéine d'ancrage de la PKA [START_REF] Perino | Integrating cardiac PIP3 and cAMP signaling through a PKA anchoring function of p110gamma[END_REF] et qui peut lier certaines PDEs. Les souris PI3Kγ -/-présentent une sensibilité accrue à une surcharge cardiaque de pression, initialement attribuée à une diminution de l'activité PDE3B consécutive à la perte de la PI3Kγ [START_REF] Patrucco | PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent andindependent effects[END_REF]. Un étude récente montre que ces souris présentent aussi une vulnérabilité accrue aux arythmies ventriculaires induites par stimulation des récepteurs β2-AR, et a permis de montrer qu'en plus de la PDE3B, l'activité des PDE3A, PDE4A et PDE4B est diminuée dans les souris PI3Kγ -/- [START_REF] Ghigo | PI3Kgamma Protects against Catecholamine-Induced Ventricular Arrhythmia through PKA-mediated Regulation of Distinct Phosphodiesterases[END_REF]. Ceci est du à un défaut de d'activation de ces PDE par la PKA, suite à la perte de la PI3Kγ. La diminution d'activité de ces PDEs conduit à une augmentation de l'accumulation d'AMPc sous stimulation β2-AR, à l'hyperphosphorylation des canaux LTCC et du PLB, et à une augmentation des libérations spontanées de Ca 2+ dans les myocytes déficients pour PI3Kγ [START_REF] Ghigo | PI3Kgamma Protects against Catecholamine-Induced Ventricular Arrhythmia through PKA-mediated Regulation of Distinct Phosphodiesterases[END_REF].

Dans les fibres atriales humaines, l'inhibition sélective de la PDE3, mais aussi celle de la PDE4, augmentent les arythmies provoquées par stimulation des récepteurs β-AR [START_REF] Molina | Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias[END_REF]. Au niveau cellulaire, l'inhibition de la PDE4 augmente les libérations spontanées de Ca 2+ du RS et active l'échangeur Na + /Ca 2+ , ce qui produit un courant entrant dépolarisant qui peut être à l'origine de post-dépolarisations tardives et d'activités déclenchées [START_REF] Molina | Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias[END_REF]. L'activité PDE4 mesurée dans les oreillettes droites de patients en fibrillation atriale permanente tend à être diminuée par rapport à celle de patients en rythme sinusal, suggérant que la PDE4 pourrait jouer un rôle dans le développement de la fibrillation atriale chez l'homme [START_REF] Molina | Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias[END_REF].

Les taux de catécholamines circulants sont chroniquement augmentés dans l'insuffisance cardiaque (IC) et ceci est associé à un remodelage de la voie de signalisation β-adrénergique, caractérisé notamment par une diminution des récepteurs β1-AR, un découplage des récepteurs β2-AR à Gs, une augmentation des kinases associées à ces récepteurs, et une augmentation des protéines Gαi inhibitrices des AC [START_REF] Lohse | What is the role of beta-adrenergic signaling in heart failure?[END_REF]. On observe aussi des modifications Le schéma illustre les éléments fondamentaux responsables des mouvements calciques et de la contraction à l'intérieur d'un myocyte cardiaque. Les tubules transverses (tubules T) sont des invaginations de la membrane plasmique externe qui permettent de porter la dépolarisation (le potentiel d'action) à l'intérieur de la cellule cardiaque et dans lesquels sont insérés des canaux calciques de type L (LTCC), dont l'ouverture est responsable de l'influx calcique initial. En face des LTCC, au niveau de structures nommées dyades, se trouvent les canaux calciques des citernes terminales du réticulum sarcoplasmique, ou récepteurs de la ryanodine de type 2 (RyR2). L'ouverture des RyR2 est déclenchée par l'influx calcique provenant des LTCC et conduit à la libération du Ca 2+ contenu dans le réticulum sarcoplasmique. L'augmentation de la [Ca 2+ ] cytosolique qui en résulte permet l'activation des myofilaments et la contraction cardiaque. Lors de la relaxation, le Ca 2+ se détache des myofilaments et est transporté activement du cytoplasme dans le réticulum sarcoplasmique par les pompes ATPases calciques du RS (SERCA2). Ces pompes sont régulées négativement par une protéine accessoire, le phospholamban. La fraction de Ca 2+ entré par les LTCC est extrudée par l'échangeur Na + /Ca 2+ (NCX) et dans une moindre mesure par les pompes ATPases Ca 2+ de la membrane plasmique (PMCA).

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  du profil d'expression et d'activité des PDEs dans l'IC, notamment une diminution de l'expression et de l'activité de la PDE3 (bien que les résultats soient moins clairs chez l'homme) [16]. Dans un modèle d'hypertrophie pathologique par surcharge de pression chez le rat, nous avons observé une diminution de l'activité PDE totale due à une baisse d'expression et d'activité des PDE3A, PDE4A et PDE4B [38], tandis que dans un modèle d'installation d'hypertrophie cardiaque induite par l'angiotensine II, une augmentation d'activité PDE4 accompagnée d'une augmentation de la 69kDa-PDE4A et d'une diminution d'expression des 52 et 76 kDa-PDE4D sont observées [26]. Ces résultats suggèrent que le niveau d'expression des isoformes de PDE3 et PDE4 est régulé de façon spécifique selon le type de stimulus utilisé pour induire l'hypertrophie cardiaque et le stade de la pathologie. Alors qu'une augmentation des PDEs peut participer au phénomène de désensibilisation de la voie β-AR, leur diminution pourrait représenter un mécanisme compensateur visant à restaurer les niveaux d'AMPc et l'inotropisme. Toutefois, une diminution des PDEs altère également le degré de confinement de l'AMPc ce qui pourrait conduire à l'activation illégitime ou excessive de certains pools de PKA ou des facteurs d'échanges Epac et favoriser ainsi le remodelage pathologique et les troubles du rythme cardiaque. Cette hypothèse est étayée par les résultats d'une étude récente montrant qu'une isoforme particulière de PDE4D, la PDE4D5, régule l'activation d'Epac1 et le programme hypertrophique lors d'une stimulation des récepteurs β2-AR [39]. Une meilleure compréhension des altérations spécifiques des différentes isoformes de PDE3 et PDE4 dans l'hypertrophie et l'insuffisance cardiaque et de leurs conséquences sur le CEC et le remodelage hypertrophique pourrait permettre l'élaboration de stratégies thérapeutiques nouvelles visant à améliorer la fonction cardiaque sans augmenter le risque d'arythmies.

Figure 1 :

 1 Figure 1: Le couplage excitation-contraction dans un myocyte ventriculaire.

Figure 2 :

 2 Figure 2 : Régulation du couplage excitation-contraction des myocytes cardiaques par la voie β-adrénergique et les phosphodiestérases des nucléotides cycliques de type 3 et 4.L'activation par les catécholamines des récepteurs β-adrénergiques couplés aux protéines Gs active la synthèse d'AMPc par les adénylates cyclases 5 et 6 (AC 5/6), isoformes majoritaires au niveau cardiaque. L'AMPc diffuse et active différents pools de PKA localisés aux sites clés du couplage excitation-contraction par des protéines d'ancrage de la PKA, les AKAPs. L'AMPc peut aussi activer le facteur d'échange des petite protéines G Rap1 et Rap2, Epac. La dégradation de l'AMPc est assurée par plusieurs isoformes de phosphodiestérases (PDEs) de type 3 et 4 localisées notamment au niveau des cibles du couplage excitation-contraction. Dans le cas de certaines isoformes de PDE4D, cette localisation est assurée par des AKAPs. Certaines isoformes de PDE3 possèdent un domaine hydrophobe N-terminal impliqué dans leur localisation particulaire. Les PDE3 et PDE4 peuvent être localisées par interaction avec d'autres protéines, comme par exemple la phosphatidyl-inositol 3 kinase γ (PI3Kγ).

  Figure 1

Remerciements

Ce travail est soutenu par l'Agence Nationale de la Recherche (BLAN 1139 01) et la région Ile-de-France.

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