Jérôme Leroy

Rodolphe Fischmeister

CaV1.2 and β-Adrenergic Regulation of Cardiac Function

Overview of the β-adrenergic regulation of L-type calcium channels

In cardiac cells, the L-type calcium channel (LTCC) current or ICa,L underlies the plateau phase of the action potential. Upon depolarisation, ICa,L reflects calcium influx via the CaV1.2 channels. This current initiates cardiac contraction by gating the ryanodine receptor, therefore triggering the calcium release from the sarcoplasmic reticulum. [START_REF] Bers | Cardiac excitation-contraction coupling[END_REF] Among several regulatory pathways of this current, the best described is the β-adrenergic stimulation which contributes to the positive inotropic effects of catecholamines. To date, three β-adrenergic receptors (β-AR), respectively β1-AR, β-AR and β3-AR, have been cloned [START_REF] Van Der Heyden | Molecular aspects of adrenergic modulation of cardiac L-type Ca 2+ channels[END_REF] and this major achievement has led to the 2012 Nobel prize award to Robert Lefkowitz and Brian Kobilka who paved the road to our current understanding of their structures and functions. The classical pathway for β-AR receptor signaling is activation of adenylyl cyclases via Gαs, resulting in increased intracellular cAMP levels. The primary target of cAMP is the cAMP-dependent protein kinase (PKA) that in turn phosphorylates the CaV1.2 channels among other key proteins of the excitation-contraction coupling (figure 1). While direct modulation by G proteins of LTCCs was first suspected to partially mediate the upregulation of ICa,L upon β-AR activation, it has been clearly established that a cAMP phosphorylation mediated by PKA is responsible for this increase. [START_REF] Hartzell | Sympathetic regulation of cardiac calcium current is due exclusively to cAMP-dependent phosphorylation[END_REF][START_REF] Catterall | Voltage-gated calcium channels[END_REF] This chapter reviews the literature on the β-AR regulation of LTCC, with emphasis on recent informations on the molecular mechanisms of PKA regulation of CaV1.2 channels and the local compartmentation of β-AR/cAMP/PKA signaling around these channels. We conclude with an overview of such modulation in a pathological context. Additional details concerning LTCCs can be found in Chapter 2.

Molecular mechanisms of PKA regulation of LTCCs

LTCCs display three distinct gating modes upon depolarisation: mode 1 corresponding to brief openings, mode 2 to long lasting openings and mode 0 to a silent mode because of unavailability. [START_REF] Hess | Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists[END_REF] PKA phosphorylation of CaV1.2 channels results in a shift of the channel from the gating mode 1 to mode 2. [START_REF] Yue | ß-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes[END_REF] As a result, ICa,L density is increased ~2-3 fold and its voltage dependence slightly shifted towards hyperpolarized potentials (figure 2A). Voltage steadystate activation and inactivation in adult mouse ventricular myocytes are presented in figure 2.

Activation for ICa,L starts at -40 mV and is maximal around 5 mV while inactivation begins at -45 mV and is maximal at about 0 mV. The overlap of the two curves defines a "window current" between ~-40 mV and 0 mV, i.e. near the action potential "plateau" phase. The βadrenergic stimulation leads to an increased "window current" because of the effect of PKA phosphorylation on channel activation. As presented in figure 2B, isoproterenol application at a maximal concentration of 100 nM shifts the activation by 5 mV towards negative potentials while a minor shift of availability of 2.5 mV is observed. During maintained depolarisations, ICa,L decreases with time, a phenomenon named "inactivation", which depends on time, voltage and intracellular calcium. [START_REF] Cens | Voltage-and calcium-dependent inactivation in high voltage-gated Ca 2+ channels[END_REF] Because β-AR activation enhances calcium entry, it also accelerates ICa,L inactivation, [START_REF] Argibay | Inactivation, reactivation and pacing dependence of calcium current in frog cardiocytes: correlation with current density[END_REF] and calcium-dependent inactivation becomes the main mechanism by which the channel inactivates. [START_REF] Findlay | Physiological modulation of inactivation in L-type Ca 2+ channels: one switch[END_REF] Overall, β-AR stimulation promotes ICa,L thus the calcium entry during action potential that is partially responsible for its positive inotropic effects.

The LTCC current ICa,L in the working myocardium is mediated by the predominantly expressed CaV1.2 channels. These channels are multimeric proteins composed of a central subunit, α1C, the pore forming subunit which determines the main biophysical and pharmacological properties of the channel. This subunit is a protein of about 240 kDa with 24 transmembrane segments according to its hydropathy profile, organized into four repeated domains (I-IV) of six transmembrane segments each (1-6), with intracellular N-and a large C-termini. [START_REF] Catterall | Voltage-gated calcium channels[END_REF] Like other high voltage gated calcium channels, CaV1.2 associates with a largely extracellular disulfide-linked α2-δsubunit of 170 kDa. [START_REF] Davies | Functional biology of the alpha(2)delta subunits of voltage-gated calcium channels[END_REF] It also binds CaVβ subunits to its α interaction domain (AID) present in its intracellular I-II loop via their guanylate kinase (GK)like domain. [START_REF] Dolphin | Beta subunits of voltage-gated calcium channels[END_REF] Four genes encode four CaVβ subunits (β1-4) but CaVβ2 is thought to be the main isoform expressed in the heart. [START_REF] Colecraft | Novel functional properties of Ca 2+ channel ß subunits revealed by their expression in adult rat heart cells[END_REF] Both α2-δ [START_REF] Fuller-Bicer | Targeted disruption of the voltage-dependent calcium channel alpha2/delta-1-subunit[END_REF] , and CaVβ 14 auxiliary subunits influence the biophysical properties and increase the trafficking of the channel at the plasma membrane. γ (4,6-8) subunits are also expressed in ventricular cells and, like γ1 for the skeletal muscle calcium channel CaV1.1, can interact with the CaV1.2 channel to modulate its function when co-expressed in HEK-293 cells; but the exact role for these accessory proteins on CaV1.2 in native cardiac tissues remains to be determined. [START_REF] Yang | Cardiac L-type calcium channel (Cav1.2) associates with gamma subunits[END_REF]

Alpha 1 Subunit

If the main effects of β-AR stimulation on voltage dependence and amplitude of the ICa,L and their consequences for the "fight or flight" response are well documented in the literature, the molecular events that mediate the increase of CaV1.2 activity during a sympathetic stimulation remain elusive. This is due to the difficulty of reconstituting such modulation in heterologuous overexpression systems. The α1C subunit of CaV1.2 channels exhibits multiple potential PKA phosphorylation sites in the N-and the C-terminal regions (figure 3). [START_REF] Van Der Heyden | Molecular aspects of adrenergic modulation of cardiac L-type Ca 2+ channels[END_REF] Despite the fact that α1C is a substrate for phosphorylation by PKA in vitro, [START_REF] Puri | Differential effects of subunit interactions on protein kinase A-and C-mediated phosphorylation of L-type calcium channels[END_REF][START_REF] Jongh | Specific phosphorylation of a site in the full-length form of the 1 subunit of the cardiac Ltype calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase[END_REF] several attempts to mimic the adrenergic stimulation of CaV1.2 channels in expression systems have failed. [START_REF] Perez-Reyes | Regulation of the cloned L-type cardiac calcium channel by cyclic-AMP-dependent protein kinase[END_REF][START_REF] Zong | On the regulation of the expressed L-type calcium channel by cAMPdependent phosphorylation[END_REF][START_REF] Mikala | cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels[END_REF] This led to the hypothesis that at least one missing link in heterologuous systems would preclude the reconstitution of such modulation of over expressed CaV1.2 channels. One of these could be an A-kinase-anchoring protein (AKAP) which allows the cAMP modulation and the PKA phosphorylation of serine 1928 in the C-terminus of CaV1.2 channels in HEK293 cells. [START_REF] Gao | cAMP-dependent regulation of cardiac L-type Ca 2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits[END_REF] A conserved leucine zipper motif in the C-terminus of CaV1.2 identified in native cardiac cells directly anchors a low molecular weight AKAP15-PKA complex to ensure a fast and efficient β-adrenergic modulation of ICa,L (figure 3) [START_REF] Hulme | ß-adrenergic regulation requires direct anchoring of PKA to cardiac Cav1.2 channels via a leucine zipper interaction with A kinase-anchoring protein 15[END_REF] allowing its phosphorylation at serine 1928 when β-ARs are stimulated. [START_REF] Hulme | Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Cav1.2 channels during ß1-adrenergic regulation[END_REF] Surprisingly, the C-terminal part of rabbit CaV1.2 undergoes proteolytic processing by calpain at residue 1821 leaving two size forms of the α1C subunit of these channels expressed in cardiac cells, [START_REF] Jongh | Specific phosphorylation of a site in the full-length form of the 1 subunit of the cardiac Ltype calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase[END_REF] whereas this distal 37-50 kDa peptide contains the serine 1928 phosphorylated by PKA and the binding site for AKAP15. In fact, this fragment constitutes a potent autoinhibitory domain that covalently associates with the proximal C-terminal part of α1C reducing its open probability and shifting the voltage dependence of activation to depolarized potentials. [START_REF] Hulme | Autoinhibitory control of the Cav1.2 channel by its proteolytically processed distal C-terminal domain[END_REF] Later on, the role of serine 1928 in ICa,L upregulation by β-AR has been challenged. A first study showed that a DHPresistant CaV1.2 channels mutated at position 1928 (S1928A) displays a similar response to the β-AR agonist isoproterenol than endogenous channels when overexpressed in isolated ventricular myocytes. [START_REF] Ganesan | ß-Adrenergic stimulation of L-type Ca 2+ channels in cardiac myocytes requires the distal carboxyl terminus of alpha1C but not serine 1928[END_REF] Moreover, the generation of a S1928A knock-in mouse model confirmed that the phosphorylation of this residue by PKA is not required for the functional effects of β-AR stimulation on CaV1.2 in cardiac cells, since these mice exhibit an essentially conserved response of ICa,L to isoproterenol and typical chronotropic and inotropic responses to β-AR stimulation. [START_REF] Lemke | Unchanged ß-adrenergic stimulation of cardiac L-type calcium channels in Cav1.2 phosphorylation site S1928A mutant mice[END_REF] Therefore, while phosphorylation of serine 1928 within the C-terminus of CaV1.2 definitely occurs when β-AR are stimulated [START_REF] Jongh | Specific phosphorylation of a site in the full-length form of the 1 subunit of the cardiac Ltype calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase[END_REF][START_REF] Gao | cAMP-dependent regulation of cardiac L-type Ca 2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits[END_REF][START_REF] Hulme | Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Cav1.2 channels during ß1-adrenergic regulation[END_REF] it does not correlate with the functional effects observed on ICa,L. Hence, PKA would phosphorylate other PKA sites within the channel or another binding partner to mediate the increase of its activity.

Beta Subunit

Three serines (S459, -478 and -479) of the cardiac the CaVβ2a subunit were identified as putative PKA phosphorylation sites. [START_REF] Gerhardstein | Identification of the sites phosphorylated by cyclic AMP-dependent protein kinase on the ß2 subunit of L-type voltage-dependent calcium channels[END_REF] Co-expression of this subunit with a CaV1.2 lacking the C-terminal part including the serine 1928 in TsA-201 cells allowed an up-regulation of the current by cAMP/PKA pathway while expression of mutated CaVβ2a at serine 478 and 479, but not at position 459, was unable to do so. [START_REF] Bunemann | Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the ß2 subunit[END_REF] Thus, the authors concluded that the CaVβ2a ancillary subunit is the main target for PKA to mediate the βAR stimulation of ICa,L. But this conclusion has also been questioned by experiments realized in a more physiological context.

Overexpression of a CaVβ2a construct mutated for its PKA phosphorylation sites in ventricular cardiac cells did not prevent the cAMP/PKA modulation of ICa,L. [START_REF] Miriyala | Role of Cavß subunits, and lack of functional reserve, in protein kinase A modulation of cardiac Cav1.2 channels[END_REF] Nonetheless, the CaVβ2 subunit can associate with the giant cytoskeletal protein of 700 kDa named ahnak, which emerged as an important player in the β-AR regulation of cardiac ICa,L (figure 3). Through its interaction with CaVβ2, ahnak would serve as a brake on ICa,L that would be relieved by PKA phosphorylation of the ancillary subunit and the cytoskeletal protein. [START_REF] Haase | Ahnak is critical for cardiac CaV1.2 calcium channel function and its ß-adrenergic regulation[END_REF] Interestingly, ahnak polymorphism occurs, and the genetic variant generated interferes with the βAR stimulation of ICa,L by reducing the CaVβ2 interaction with ahnak. [START_REF] Haase | Ahnak is critical for cardiac CaV1.2 calcium channel function and its ß-adrenergic regulation[END_REF] However, if the involvement of the CaVβ or other binding partners such as ahnak can not be totally ruled out, recent studies reaffirmed the importance of the proteolytically cleaved distal C-terminus of CaV1.2 for its upregulation upon β-AR stimulation and a new model for the molecular basis of β-AR stimulation of CaV1.2 has been proposed. [START_REF] Fuller | Molecular mechanism of calcium channel regulation in the fight-or-flight response[END_REF] Overexpression in TsA-201 cells of a truncated CaV1.2 channel at position A1800 (the site of in vivo proteolytic cleavage previously determined for skeletal muscle CaV1.1 channel [START_REF] Hulme | Sites of proteolytic processing and noncovalent association of the distal Cterminal domain of Cav1.1 channels in skeletal muscle[END_REF] ) with α2δ1 and CaVβ1b and the distal C-terminal part of the channel (peptide from 1821-2171) produced a functional channel which is auto-inhibited. Two presumed sites for PKA phosphorylation were identified upstream the proteolytic site, a serine at position 1700 and a threonine 1704 at the interface of the distal and the proximal C-terminal parts. While a mutation of S1928 confirmed that its phosphorylation is not required for the increase of the current, substitution of T1704 and S1700 for alanines revealed that phosphorylation of T1704 is required for basal CaV1.2 channel activity whereas S1700 is crucial for its cAMP/PKA induced upregulation. In this scheme, the AKAP15 is still required to coordinate the PKA phosphorylation of S1700 that disrupts the interaction of the non-covalently distal C-terminus, thus relieving the inhibition of the channel. [START_REF] Fuller | Molecular mechanism of calcium channel regulation in the fight-or-flight response[END_REF] This assumption is reinforced by the fact that mice expressing a CaV1.2 channel deleted for the distal C-terminus display altered cAMP/PKA regulation accompanied with reduced expression of AKAP15. [START_REF] Fu | Deletion of the distal C terminus of Cav1.2 channels leads to loss of ß-adrenergic regulation and heart failure in vivo[END_REF]

Compartmentation of cAMP/PKA regulation of LTCCs

In the light of the knowledge accumulated over the years, it is evident that intracellular cAMP is not uniformly distributed nor is PKA uniformly activated within cardiomyocytes upon β-AR stimulation. [START_REF] Xiang | Compartmentalization of beta-adrenergic signals in cardiomyocytes[END_REF] On the contrary, a tight cAMP/PKA compartmentation is required for adequate processing and targeting of the information generated at the cell surface, conferring the specificity of the response to various hormones linked to Gαs-coupled receptors. [START_REF] Rochais | A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes[END_REF][START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: The role of cyclic nucleotide phosphodiesterases[END_REF] In the case of β-ARs, several processes contribute to a localized cAMP/PKA response: catecholamines activate different β-AR subtypes located at different places of the cell surface (caveolae, t-tubules…); Gαs-activation of different AC isoforms may lead to cAMP synthesis at different locations; cAMP diffusion may be restricted due to localized phosphodiesterase (PDE) activity; anchoring of PKA to AKAPs position PKA at different subcellular compartments to selectively phosphorylate a local pool of proteins for specific cellular processes. [START_REF] Perino | Anchoring proteins as regulators of signaling pathways[END_REF][START_REF] Diviani | A-kinase anchoring proteins: Molecular regulators of the cardiac stress response[END_REF] In addition, AKAPs also ensure that PKA is coupled to its upstream activators, including membrane β-ARs and ACs, and to signal termination enzymes, such as PDEs and phosphatases. [START_REF] Perino | Anchoring proteins as regulators of signaling pathways[END_REF]

Receptor Specificity

Although to date 3 types of β-adrenergic receptors (β-ARs) have been cloned, respectively β1-, β2-and β3-ARs, the effect of catecholamines in human heart is generally attributed to β1and β2-ARs. β1-and β2-ARs are highly homologous receptors and are both positively coupled to AC/cAMP/PKA cascade, ICa,L and cardiac performance. [START_REF] Xiang | Compartmentalization of beta-adrenergic signals in cardiomyocytes[END_REF] However, they exert opposite effects on hypertrophy [START_REF] Morisco | ß-Adrenergic cardiac hypertrophy is mediated primarily by the ß1-subtype in the rat heart[END_REF][START_REF] Ahmet | Beneficial effects of chronic pharmacological manipulation of ß-adrenoreceptor subtype signaling in rodent dilated ischemic cardiomyopathy[END_REF] and apoptosis, [START_REF] Communal | Opposing effects of ß1-and ß2adrenergic receptors on cardiac myocyte apoptosis -Role of a pertussis toxin-sensitive G proteins[END_REF][START_REF] Zaugg | ß-Adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes[END_REF] and their respective contribution varies significantly depending on the cardiac tissue, the pathophysiological state, the age or the developmental stage. [START_REF] Brodde | Cardiac adrenoceptors: Physiological and pathophysiological relevance[END_REF] Part of the difference is due to the fact that β2-ARs not only couple to Gαs but also to Gαi proteins and this confines the cAMP-dependent signal to the membrane compartment and to activation of the LTCCs. [START_REF] Chen-Izu | Gidependent localization of 2-adrenergic receptor signaling to L-type Ca 2+ channels[END_REF] Another difference between the two β-AR subtypes is their location at the cell surface, with β2-ARs present in the caveolae/lipid rafts [START_REF] Xiang | Caveolar localization dictates physiologic signaling of β2-adrenoceptors in neonatal cardiac myocytes[END_REF][START_REF] Calaghan | Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte[END_REF] of the transverse-tubular structure [START_REF] Nikolaev | ß2-Adrenergic receptor redistribution in heart failure changes cAMP compartmentation[END_REF] and β1-ARs distributed throughout both caveolae/lipid rafts and nonlipid raft membrane domains [START_REF] Xiang | Caveolar localization dictates physiologic signaling of β2-adrenoceptors in neonatal cardiac myocytes[END_REF] and in both plasma and t-tubular membranes. [START_REF] Nikolaev | ß2-Adrenergic receptor redistribution in heart failure changes cAMP compartmentation[END_REF] Accordingly, the β2-AR downstream activation of ICa,L is sensitive to disruption of caveolae by cholesterol depletion, whereas the β1-AR stimulatory effect is not. [START_REF] Balijepalli | Localization of cardiac L-type Ca 2+ channels to a caveolar macromolecular signaling complex is required for ß2adrenergic regulation[END_REF] Thus, one can conclude that β2-AR stimulation exerts a local activation of LTCCs whereas β1-AR stimulation leads to activation of LTCCs in the distance. [START_REF] Chen-Izu | Gidependent localization of 2-adrenergic receptor signaling to L-type Ca 2+ channels[END_REF][START_REF] Xiao | ß1-Adrenoceptor stimulation and ß2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca 2+ , and Ca 2+ current in single rat ventricular cells[END_REF][START_REF] Nikolaev | Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching ß1-adrenergic but locally confined ß2-adrenergic receptor-mediated signaling[END_REF] The β3-AR differs from β1-and β2-AR subtypes in its molecular structure and pharmacological functions. [START_REF] Rozec | ß3-Adrenoceptors in the cardiovascular system: Putative roles in human pathologies[END_REF] Expression of β3-AR was demonstrated in human myocardium both at the mRNA [START_REF] Gauthier | Functional β3adrenoceptor in the human heart[END_REF][START_REF] Moniotte | Real-time RT-PCR for the detection of beta-adrenoceptor messenger RNAs in small human endomyocardial biopsies[END_REF] and protein level. [START_REF] Moniotte | Real-time RT-PCR for the detection of beta-adrenoceptor messenger RNAs in small human endomyocardial biopsies[END_REF][START_REF] Chamberlain | The tissue distribution of the human ß3-adrenoceptor studied using a monoclonal antibody: direct evidence of the ß3-adrenoceptor in human adipose tissue, atrium and skeletal muscle[END_REF][START_REF] Moniotte | Upregulation of β3-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium[END_REF][START_REF] De Matteis | Immunohistochemical identification of the β3-adrenoceptor in intact human adipocytes and ventricular myocardium: effect of obesity and treatment with ephedrine and caffeine[END_REF] Interestingly, β3-AR activation produces a negative inotropic effect in human endomyocardial biopsies from transplanted hearts [START_REF] Rozec | ß3-Adrenoceptors in the cardiovascular system: Putative roles in human pathologies[END_REF][START_REF] Gauthier | Functional β3adrenoceptor in the human heart[END_REF] and in left ventricular samples from failing and non-failing explanted hearts [START_REF] Rozec | ß3-Adrenoceptors in the cardiovascular system: Putative roles in human pathologies[END_REF][START_REF] Moniotte | Upregulation of β3-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium[END_REF] that is due to activation of the NO/cGMP pathway, but increases ICa,L and contractility in human atrial tissue via the cAMP/PKA pathway (figure 4). [START_REF] Skeberdis | ß3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca 2+ current[END_REF] This is reminiscent of the contractile effects of the serotonin 5-HT4 receptors [START_REF] Schoemaker | 5-Hydroxytryptamine stimulates human isolated atrium but not ventricle[END_REF] , which are also coupled to increases in force of contraction [START_REF] Jahnel | Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle[END_REF] or ICa,L 59 in atria but not in healthy ventricles.

Role of AKAPs

Cyclic AMP signaling components are organized into multiprotein complexes, an arrangement that increases both efficiency and specificity of the transduction cascade. AKAPs play an essential role in these arrangements. AKAPs form a large family of proteins comprising >50 members whose primary function is to anchor PKA in the vicinity of its substrates, thus ensuring the preferential phosphorylation of a limited number of targets. [START_REF] Scott | A-kinase anchoring proteins: getting to the heart of the matter[END_REF][START_REF] Kritzer | AKAPs: The architectural underpinnings of local cAMP signaling[END_REF] As discussed above, AKAP15 (also called AKAP 7 or AKAP15/18) is the main AKAP controlling the PKA phosphorylation of LTCCs. However, in a very recent study, CaV1.2 phosphorylation and -AR stimulation of ICa,L was found to be unchanged in mice in which the gene encoding this protein was inactivated, suggesting that PKA is anchored by a different protein to the channel. [START_REF] Jones | Cardiomyocytes from AKAP7 knockout mice respond normally to adrenergic stimulation[END_REF] Furthermore, another AKAP, AKAP5, was found to target adenylyl cyclases (AC), PKA and phosphatases within caveolae to allow specific PKA phosphorylation of the sub-population of channels present in this compartment upon -AR stimulation. [START_REF] Nichols | Sympathetic stimulation of adult cardiomyocytes requires association of AKAP5 with a subpopulation of L-type calcium channels[END_REF] While AKAPs share in common their ability to bind PKA, they are remarkably diverse scaffold proteins. Within each signalosome, AKAPs couple PKA to different substrates, enhancing the rate and fidelity of their phosphorylation by the kinase. Importantly, AKAPs not only bind PKA but act as scaffold proteins for other signaling components including phosphatases 1 and 2, [START_REF] Marx | PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (Ryanodine receptor): Defective regulation in failing hearts[END_REF][START_REF] Marx | Requirement of a macromolecular signaling complex for β adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel[END_REF] Epac, [START_REF] Dodge-Kafka | The protein kinase A anchoring protein mAKAP co-ordinates two integrated cAMP effector pathways[END_REF] adenylyl cyclases [START_REF] Efendiev | AKAP79 interacts with multiple adenylyl cyclase (AC) isoforms and scaffolds AC 5 and 6 to AMPA receptors[END_REF][START_REF] Bauman | Dynamic regulation of cAMP synthesis through anchored PKA-adenylyl cyclase V/VI complexes[END_REF] and PDEs. [START_REF] Dodge | mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module[END_REF] Recently, it has been demonstrated that the phosphoinositide 3-kinase p110, that was shown recently tether PKA, [START_REF] Perino | Integrating cardiac PIP3 and cAMP signaling through a PKA anchoring function of p110gamma[END_REF] orchestrates multi-protein complexes including different PDEs to control cardiac CaV1.2 phosphorylation during -AR stimulation. [START_REF] Ghigo | PI3Kγ Protects against catecholamine-induced ventricular arrhythmia through PKA-mediated regulation of distinct phosphodiesterases[END_REF] The combination of PDEs and phosphatases present in individual AKAP complexes will affect the duration, amplitude, and spatial extent of cAMP/PKA signaling. Thus, by bringing together different combinations of upstream and downstream signaling molecules, AKAPs provide the architectural infrastructure for specialization of the cAMP signaling network. [START_REF] Kritzer | AKAPs: The architectural underpinnings of local cAMP signaling[END_REF][START_REF] Dodge-Kafka | The protein kinase A anchoring protein mAKAP co-ordinates two integrated cAMP effector pathways[END_REF][START_REF] Terrenoire | The cardiac IKs potassium channel macromolecular complex includes the phosphodiesterase PDE4D3[END_REF]

Role of Phosphodiesterases

Localized cAMP signals may be generated by the interplay between discrete cAMP production sites and restricted diffusion within the cytoplasm. Restricted diffusion of cAMP may be achieved by several means. A first possibility is that physical barriers are created by specialized membrane structures within the cytoplasm. This was initially proposed to explain the differences in cAMP concentration elicited by PGE1 at the plasma membrane and in the bulk cytosol of HEK293 cells, although an experimental proof that this actually occurs is still lacking. [START_REF] Rich | Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion[END_REF] However, another important mechanism that limits cAMP diffusion is cAMP degradation by PDEs, which appears critical for the formation of dynamic microdomains that confer specificity of the response. [START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: The role of cyclic nucleotide phosphodiesterases[END_REF][START_REF] Scott | A-kinase anchoring proteins: getting to the heart of the matter[END_REF] Cardiac cAMP PDEs degrading belong to 5 families (PDE1-4 and PDE8) which can be distinguished by distinct enzymatic properties and pharmacology. [START_REF] Mika | PDEs create local domains of cAMP signaling[END_REF] Among these, various enzymes were shown to degrade cAMP to allow a fine tuning of ICa,L regulation by PKA in heart. Although PDE2 is not highly expressed in cardiomyocytes, it controls LTCCs activity in various species, including human atrial myocytes. [START_REF] Méry | Erythro-9-(2-hydroxy-3nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterase in isolated cardiac myocytes[END_REF][START_REF] Rivet-Bastide | cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes[END_REF][START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF][START_REF] Vandecasteele | Cyclic GMP regulation of the L-type Ca 2+ channel current in human atrial myocytes[END_REF] This enzyme is activated by cGMP, and stimulation of guanylyl cyclase strongly decreases local cAMP levels controlling ICa,L with only modest effects on its global concentration, suggesting existence of a cAMP microdomain including -AR and LTCC under tight control of PDE2. [START_REF] Dittrich | Local response of L-type Ca 2+ current to nitric oxide in frog ventricular myocytes[END_REF] On the contrary to PDE2, PDE3 is inhibited by cGMP. This explains in part why cGMP at low concentration can also increase basal ICa,L as shown in human atrial myocytes. [START_REF] Vandecasteele | Cyclic GMP regulation of the L-type Ca 2+ channel current in human atrial myocytes[END_REF][START_REF] Kirstein | Nitric oxide regulates the calcium current in isolated human atrial myocytes[END_REF] In rodents, PDE3 and PDE4 are the major contributors to the total cAMP-hydrolytic activity [START_REF] Rochais | A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes[END_REF][START_REF] Richter | Conserved expression and functions of PDE4 in rodent and human heart[END_REF] and PDE4 is dominant to modulate β-AR regulation of cAMP levels. [START_REF] Nikolaev | Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching ß1-adrenergic but locally confined ß2-adrenergic receptor-mediated signaling[END_REF][START_REF] Mongillo | Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases[END_REF][START_REF] Rochais | Negative feedback exerted by PKA and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes. An in vivo study using adenovirus-mediated expression of CNG channels[END_REF][START_REF] Leroy | Spatiotemporal dynamics of ß-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: Role of phosphodiesterases[END_REF] Multiple PDE4 variants associate with β-ARs, [START_REF] Baillie | β-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates βadrenoceptor switching from Gs to G i[END_REF][START_REF] Richter | Signaling from ß1and ß2-adrenergic receptors is defined by differential interactions with PDE4[END_REF][START_REF] De Arcangelis | Differential association of phosphodiesterase 4D isoforms with ß2-adrenoceptor in cardiac myocytes[END_REF] RyR2, 88 SERCA2, [START_REF] Kerfant | PI3KY 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 Ca 2+ release and cardiac contractility, independently of L-type Ca 2+ current[END_REF] ICa,L, [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias[END_REF] and IKs [START_REF] Terrenoire | The cardiac IKs potassium channel macromolecular complex includes the phosphodiesterase PDE4D3[END_REF] to exert local control of ECC. In larger mammals, PDE3 activity is dominant in microsomal fractions [START_REF] Weishaar | Subclasses of cyclic AMP-specific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility[END_REF][START_REF] Lugnier | Characterization of indolidansensitive and rolipram-sensitive cyclic nucleotide phosphodiesterases in canine and human cardiac microsomal fractions[END_REF][START_REF] Smith | Development of decompensated dilated cardiomyopathy is associated with decreased gene expression and activity of the milrinone-sensitive cAMP phosphodiesterase PDE3A[END_REF] and PDE3 inhibitors exert a potent positive inotropic effect. [START_REF] Osadchii | Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease[END_REF] Selective inhibition of PDE3 with milrinone has been shown to improve cardiac contractility in patients with congestive heart failure. [START_REF] Monrad | Assessment of long-term therapy with milrinone and the effects of milrinone withdrawal[END_REF] The role of PDE4 is less well defined but evidence is emerging that PDE4 may also play an important role in these species. In the canine heart, a large PDE4 activity is found in the cytoplasm [START_REF] Weishaar | Subclasses of cyclic AMP-specific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility[END_REF] but PDE4 is also present in microsomal fractions, where it accounts for ~20% of the activity. [START_REF] Lugnier | Characterization of indolidansensitive and rolipram-sensitive cyclic nucleotide phosphodiesterases in canine and human cardiac microsomal fractions[END_REF] Recent studies have indicated that PDE4 is expressed in human ventricle where, similar to rodents, it associates with β-ARs, RyR2 and phospholamban. [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] Moreover, PDE4 is the main PDE modulating LTCC activity in rodent cardiomyocytes [START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF][START_REF] Leroy | Spatiotemporal dynamics of ß-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: Role of phosphodiesterases[END_REF] and PDE4 was recently shown to control ICa,L, ECC and arrhythmias in human atrium. [START_REF] Molina | Cyclic AMP phosphodiesterase type 4 protects against atrial arrhythmias[END_REF] An early evidence of the contribution of PDEs to intracellular cyclic nucleotide compartmentation was obtained by comparing the effects of the non selective β-AR agonist Iso, or the nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or the PDE3 inhibitor, milrinone, in guinea pig perfused hearts. Whereas each of these treatments increased intracellular cAMP and produced positive inotropic and lusitropic effects, differences in the phosphorylation pattern of PLB, TnI and MyBP-C by PKA were observed. [START_REF] Rapundalo | Inotropic responses to isoproterenol and phosphodiesterase inhibitors in intact guinea pig hearts: comparison of cyclic AMP levels and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins[END_REF] These results were attributed to a functional cellular compartmentation of cAMP and PKA substrates due to a different expression of PDEs at the membrane and in the cytosol. [START_REF] Weishaar | Subclasses of cyclic AMP-specific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility[END_REF] In canine ventricular myocytes, increase in particulate but not total cAMP correlated with increase in Ca 2+ transient amplitude and decay kinetics. [START_REF] Hohl | cAMP compartmentation is responsible for a local activation of cardiac Ca 2+ channels by ß-adrenergic agonists[END_REF] In response to β-AR stimulation, about 45% of the total cAMP was found in the particulate fraction but this fraction declined to <20% when IBMX was added to Iso, although cAMP production was up to 3-4 fold greater. These results show that cAMP-PDEs reside predominantly in the cytoplasm, where they prevent excessive cAMP accumulation upon β-AR activation. Thus PDEs appear important to maintain the specificity of the β-AR response by limiting the amount of cAMP diffusing from membrane to cytoplasm. Similar results were obtained when studying the impact of PDE inhibition on ICa,L regulation by local application of Iso in frog ventricular myocytes. 100 In the absence of IBMX, application of Iso to half of the cell increased ICa,L half maximally, corresponding to activation of the channels located in the same part of the cell as the β-AR agonist. When IBMX was added with Iso on one half of the cell, the effect of Iso was greatly potentiated because in this condition, LTCCs in the remote part of the cells could be recruited. Thus, these results suggest that PDE activity is important for the definition of local cAMP pools involved in the β-AR stimulation of LTCCs. Subsequent studies using ratiometric FRET biosensors to directly monitor cAMP have shown that the second messenger increases preferentially in discrete microdomains corresponding to the dyad region under β-AR stimulation, and that cAMP diffusion is limited by PDE activity. 101,102 Other studies using recombinant cyclic nucleotide gated channels to measure cAMP generated at the plasma membrane identified specific functional coupling of individual PDE families, mainly PDE3 and PDE4, to β -AR, β2-AR, PGE1-R and Glu-R as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones. [START_REF] Leroy | Spatiotemporal dynamics of ß-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: Role of phosphodiesterases[END_REF] While PDE4 regulates ICa,L in cardiomyocytes, [START_REF] Verde | Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca 2+ current in rat ventricular myocytes[END_REF][START_REF] Leroy | Spatiotemporal dynamics of ß-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: Role of phosphodiesterases[END_REF] the molecular identity of the PDE4 regulating the LTCC was only recently unveiled. [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias[END_REF] In mouse cardiomyocytes, PDE4B and PDE4D, but not PDE4A, were found to be part of a CaV1.2 signaling complex. However, in mice deficient for the Pde4d gene (Pde4d -/-), basal or β-AR stimulated ICa,L were not different from wild-type mice, while in Pde4b -/-mice the β-AR response of ICa,L was increased, together with an increase in cell contraction and Ca 2+ transients. [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias[END_REF] In vivo, upon β-AR stimulation, catheter-mediated burst pacing triggers ventricular tachycardia in Pde4b -/-mice but not in wild-type. [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias[END_REF] Thus, PDE4B is the main PDE4 isoform regulating cardiac LTCC activity and plays a key role during β-AR stimulation of cardiac function. PDE4B, by limiting the amount of Ca 2+ that enters the cell via the LTCCs, prevents Ca 2+ overload and arrhythmias.

Role of Phosphatases

Formation of inside-out patches from rabbit ventricular myocytes causes run down of LTCC activity that is blocked by okadaic acid, a serine/threonine phosphatase inhibitor. 103 This observation provided early functional evidence that a phosphatase is anchored in close proximity to the channel that counteracts upregulation of CaV1.2 by phosphorylation. Later on, two major cardiac serine/threonine phosphatases, 104 phosphatase 2A (PP2A) and 2B (PP2B or calcineurin) were found to associate with cardiac CaV1.2. 105,106 For PP2A, two attachment sites were identified within the C-terminus of the α1 subunit: 107,108 one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds immediately downstream of residues 1965-1971 without competition between these two phosphatases for binding to this rather narrow region. 108 Injection of a peptide that contains residues 1965-1971 and displaces PP2A but not PP2B from endogenous CaV1.2 increases basal and β-AR stimulated cardiac ICa,L. 108 Similarly, inhibition of PP2B with cyclosporin A or the calcineurin auto-inhibitory peptide increases cardiac ICa,L. 109 This indicates that anchoring of PP2A and PP2B on cardiac Cav1.2 negatively regulates LTCC activity, most likely by counterbalancing basal and stimulated phosphorylation that is mediated by PKA and possibly other kinases.

Beta-Adrenergic Regulation of LTCCs in Pathological Situations

CaV1.2 Involvement in Early and Delayed Afterdepolarizations

According to the Coumel's triangle, the production of a clinical arrhythmia requires three ingredients: an arrhythmogenic substrate, a trigger factor and modulation factors of which the most common is the autonomic nervous system. 110 In pathological conditions such as atrial fibrillation (AF), cardiac hypertrophy (CH) or heart failure (HF), these factors are united to trigger fatal cardiac arrhythmias. These pathologies are accompanied with structural changes, electrophysiological remodeling, abnormal β-AR activation at tissue and cellular levels promoting aberrant electrical activities and modulation. Modified excitation-contraction coupling due to altered calcium homeostasis in pathological conditions is a major cause of arrhythmias 111 . As described in the previous paragraph, a fine tuning of the L-type calcium current (i.e. of calcium cycling) and its regulation in discrete sub-cellular compartments is required to achieve a normal cardiomyocyte function. In physiopathological conditions, deregulation of this coupling leads to calcium waves and calcium alternans to promote reentrant arrhythmias and triggered activities. 111,112 At the level of the cardiomyocyte, in AF 113 or in HF 114 , calcium handling is perturbed leading to afterdepolarizations. When action potential duration (APD) is excessively prolonged, early afterdepolarization (EAD) may occur during the repolarization phase while arrhythmogenic delayed afterdepolarization (DAD) take place when the cell is fully returned to its resting potential. Nonreentrant mechanisms involves triggered activities from either EADs or DADs. When APD is prolonged, due to alteration of Na + or K + conductances (as in long QT syndromes or upon antiarrhythmic treatments), ICa,L recovery from inactivation occurs during the plateau phase of the AP which causes EADs. 115 This is particularly true when ICa,L window current is increased upon β-AR activation thus increasing calcium entry during the plateau phase and allowing its reactivation. 115,116 EADs are also promoted by increased calmodulin-dependent protein kinase II (CaMKII) activity in hypertrophy and HF. CaMKII is activated by increased intracellular calcium levels upon β-AR activation via PKA-dependent and independent mechanisms. 117,118 CaMKII in turn phosphorylates CaV1.2 on its CaVβ2a subunit triggering afterdepolarizations. 119,120 However, intracellular calcium overload, by activating the reverse mode of the Na + /Ca 2+ exchange (NCX), generates a depolarizing current during the late AP phase 2 and phase 3, that is believed to act synergistically with ICa,L to induce EADs. 121,122 Similarly, DADs occur at high intracellular calcium load and their incidence is increased by both rapid pacing and β-AR stimulation. 114,123 The main triggering mechanism for DADs is spontaneous calcium release activating a transient inward current (ITI) due to excess diastolic calcium handled by sarcolemal NCX. Large enough DADs can eventually sufficiently depolarize the membrane potential to reach threshold and trigger an AP. Again, increased LTCC activity contributes to DADs occurrence and Ca 2+ evoked arrhythmias. For instance, mutations in the LTCC have been associated with a number of inherited arrhythmia syndromes. 124 Increased LTCC due to CaV1.2 mutations such as depicted in the Thimothy syndrome when reinforced upon β-AR stimulation is a source for intracellular calcium disorders triggering DADs and severe arrhythmias. 125 These observations demonstrate that a fine tuning of the β-AR modulation of calcium entry via CaV1.2 channels in the myocytes is required to maintain proper calcium homeostasis and electrical activity of the heart.

Atrial Fibrillation

Atrial fibrillation (AF) is an extremely common cardiac arrhythmia, most prevalent in elderly people, that is profoundly influenced by the autonomic nervous system especially by the adrenergic component. 126 It is accompanied with APD shortening and intracellular calcium homeostasis disorders. 113,127 Decreased depolarizing ICa,L contributes to such APD shortening by favouring repolarization thus re-entry substrates. If reductions in ICa,L have been consistently observed in atrial myocytes from patients with permanent AF [128][129][130] or from patients in sinus rythm with a high risk of AF, 131,132 the exact mechanism for such downregulation is not clearly established. This reduction might be primarily due to transcriptional down-regulation of the α1C subunit of LTCC via a Ca 2+ -dependent calmodulin-calcineurin-NFAT system at the cost of the APD reduction observed in AF. 133 Besides, it has been also associated with diminished expression of the CaVβ and α2-δ accessory subunits 134 which are essential for α1C trafficking to the plasma membrane. Normal trafficking of CaV1.2 might as well be impaired by a zinc binding protein (ZnT-1) upregulated in AF patients. 135 Upregulation of a microRNA, miR-328, has also been correlated to AF producing downexpression of α1C and CaVβ1. 136 Oxidant stress that occurs in AF may also participate to decrease LTCC activity by inducing S-nitrolysation of α1C 137 and by promoting its β-AR stimulation to trigger arrhythmogenic EADs. 138 While ICa,L amplitude is clearly decreased in AF, its β-AR regulation is not systematically modified. 139 Even though β-AR receptor density is not impaired in AF 140 , polymorphism of β-AR occurs in AF patients, leading to decreased β-AR cascade, 141 that could partially explain the decreased LTCC activity in AF. Another plausible mechanism has been proposed to be a decreased phosphorylation of the CaV1.2 channel due to the increased phosphatase 1 or phosphatase 2A activities observed in AF. 130,142 This might be also influenced by the hyperphosphorylation by PKA of the peptide inhibitor 1 (I-1) that is prominent in AF to relieve its inhibitory effect on PP-1. 142,143 Furthermore, depressed expression of the major PDE4 isoform detected in human atria, PDE4D, correlates with aging a well known favouring factor for AF development. [START_REF] Molina | Cyclic AMP phosphodiesterase type 4 protects against atrial arrhythmias[END_REF] While PDE3 is the main cAMP degrading enzyme in human atria, PDE4 substantially contributes to the enzymatic control of β-AR stimulation of ICa,L in human atria and its inhibition leads to arrhythmias due to dysregulated intracellular calcium homeostasis. [START_REF] Molina | Cyclic AMP phosphodiesterase type 4 protects against atrial arrhythmias[END_REF] All these observations suggest that altered β-AR modulation of CaV1.2 channels happens in AF to contribute to such arrhythmia.

Hypertrophy and Heart Failure

In human Heart Failure (HF), a chronic activation of β-AR induced by the elevation of circulating catecholamine levels occurs contributing to the progression of the disease, 144,145 stressing the necessity for a strict control of β-AR signaling. This hypothesis is further demonstrated in animal models with exacerbated β-AR/cAMP/PKA signaling which develop cardiac hypertrophy and heart failure. [146][147][148] A hallmark of heart failure is β-AR desensitization with a loss of β-AR signaling compartmentation, [START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: The role of cyclic nucleotide phosphodiesterases[END_REF]144 intracellular calcium cycling perturbations, 114 accompanied with profound alterations of the structure of the cardiomyocytes notably a loss of the t-tubules. 149,150 Desensitization of β-AR is promoted by its phosphorylation by the G-protein coupled receptor kinase 2 (GRK2), a serine/threonine kinase up regulated in hypertrophy and HF. 151 Not only the number of functional β-AR is reduced in HF, but their localization is also changed, with a redistribution to cell crests of the β2-AR subtype normally localized in the t-tubules whereas β1-R remain uniformly distributed at the membrane. [START_REF] Nikolaev | ß2-Adrenergic receptor redistribution in heart failure changes cAMP compartmentation[END_REF] Interestingly, 80% of the CaV1.2 channels are located in the t-tubules 152 where they are targeted by the protein BIN-1. 153 The number of t-tubular LTCCs is decreased in failing myocytes, 154,155 a reduction associated with a down-regulation of BIN-1 in human failing cardiomyocytes. 156 This must contribute to the decreased EC coupling 157 that might be also affected by the architectural reorganization of the dyadic cleft. 157,158 Nonetheless, there is no real consensus in the literature concerning a reduction of ICa,L amplitude in failing cardiomyocytes. If the reduction of CaV1.2 channels is well described in animals models for HF, it seems largely accepted that ICa,L amplitude is maintained especially in failing human cardiomyocytes. 159,160 This apparent discrepancy might be explained by an increased open probability of single CaV1.2 channels either due to their increased phosphorylation, 161,162 to altered PKA and phosphatase activities, 155,163 or increased expression of CaVβ ancillary subunits. 164,165 Furthermore, cardiac remodeling induced by chronic activation of β-AR signaling activates CaMKII, 166,167 known to increase the CaV1.2 channel activity 120 and participate in calcium influx remodeling in heart failure. 168 Blunted β-AR stimulation of ICa,L in human failing cardiomyocytes has been consistently reported 162,169 probably due to more abundant heteromeric Gi/oα proteins, [170][171][172] leading to an increased β2-R receptor coupling with Gi antagonizing the β1-AR stimulation of ICa,L. 173 In addition, increased Gi/oα modifies PP1 and PP2A activities, two phosphatases controlling LTCC phosphorylation. 163 GRK2 may also contribute to the remodeling of the β-AR stimulation of ICa,L in HF, since KO mice for this kinase appear more resistant to adverse remodeling following myocardial infarction, but demonstrated an increased basal ICa,L amplitude and blunted β-AR stimulation. 174 Furthermore, over-expression of βARKct, a peptide derived from the GRK2 C-terminus, is able to increase β-AR stimulation of ICa,L independently of PKA in normal and failing cardiomyocytes, by sequestering Gβγ proteins. 175 Moreover, expression of a PDE4 isoform, PDE4B, which modulates β-AR stimulation of ICa,L [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias[END_REF] has been shown to be decreased in a rat model of compensated hypertrophy, 176 suggesting that not only the production but the enzymatic degradation of the cAMP controlling ICa,L is affected in pathological conditions. Thus, in hypertrophy and HF, the β-AR regulation of CaV1.2 channels is altered contributing to a dysregulation of calcium handling promoting calcium induced arrhythmias. Accurate calcium influx is not only a determinant for normal cardiomyocyte function during the excitation-contraction coupling, but recent evidence in the literature highlighted a possible role of CaV1.2 channels in gene transcription and pro-hypertrophic signaling. 177 Few studies stated that chronic increase calcium influx via CaV1.2 channels is deleterious by promoting pro-hypertrophic signaling pathways. For instance, mice overexpressing the α1C subunit of CaV1.2 channels exhibit cardiac growth and cardiomyopathy hypertrophy 178 and similarly, overexpression of CaVβ2a leads to cardiac hypertrophy, by increasing ICa,L, and activation of calcineurin/NFAT and CaMKII/HDAC signaling pathways. 179 These assumptions are confirmed by the fact that LTCC blockers can prevent cardiac remodeling in animal subjected to pressure-overload 180 and by the attenuation of cardiac hypertrophy observed when the expression of CaVβ2 subunit is inhibited. 181 Astonishingly, decreasing the expression of α1C in mice also produces cardiac hypertrophy and HF, but this deleterious effect appears not to be directly connected to the diminished ICa,L amplitude but more possibly to the compensatory neuroendocrine stress induced to balance the decreased contractility. 182 Recently, it has been proposed that PKA phosphorylation of the serine 1700 of α1C relieves its auto-inhibition by inducing the dissociation of its proteolytically cleaved and covalently re-associated distal Cterminus from the proximal C-terminus. [START_REF] Fuller | Molecular mechanism of calcium channel regulation in the fight-or-flight response[END_REF] Interestingly, the C-terminus part of α1C can translocate to the nucleus to act as a transcription factor for its own expression and to activate hypertrophic signaling. 183 A possible role of its C-terminus in hypertrophy and HF is reaffirmed by recent studies showing that deletion of the terminal part of the α1C subunit results in heart failure in vivo. [START_REF] Fu | Deletion of the distal C terminus of Cav1.2 channels leads to loss of ß-adrenergic regulation and heart failure in vivo[END_REF]184 It is tempting to speculate that chronic β-AR stimulation may promote hypertrophy and HF by favouring a permanent dissociation of the C-terminus of the pore forming subunit of CaV1.2 channels to induce cardiac remodeling. Activation curves were derived from current-voltage relations and described by: G/Gmax = 1/{1+exp{(V½,act-V)/k}}. (A and B were obtained in similar recording conditions as described in ref [START_REF] Leroy | Phosphodiesterase 4B in the cardiac L-type Ca 2+ channel complex regulates Ca 2+ current and protects against ventricular arrhythmias[END_REF] . PKA also phosphorylates CaVβ2a on two serine residues 478 and 479. This subunit associates to the giant ahnak cytosqueletal protein that may also be an important factor for the cAMP/PKA regulation of CaV1.2 channels.

Figure 1 :

 1 Figure 1: β-adrenergic modulation of cardiac L-type calcium channels. Modulation of cardiac

Figure 2 :

 2 Figure 2: The effect of β-adrenergic stimulation on ICa,L in adult mice ventricular

Figure 3 :

 3 Figure 3: Subunit structure of cardiac L-type CaV1.2 channel and phosphorylation sites for

Figure 4 :

 4 Figure 4: Opposite effect β3-adrenergic receptor stimulation on LTCC activity in human


BSc Hind Mehel

MS Julius Emons

PhD Christiane Vettel

Katrin Wittköpper

Danilo Seppelt

MS Matthias Dewenter

PhD Susanne Lutz

MD Samuel Sossalla

MD Lars S Maier

BSc Patrick Lechêne

PhD Katrinwittköpper

MS Daniloseppelt

PhD Jérôme Leroy

BSc Florence Lefebvre

BSc Audrey Varin

MD Thomas Eschenhagen

MD Stanley Nattel

MD Dobromir Dobrev

MD Wolfram-Hubertus Zimmermann

PhD Viacheslav O Nikolaev

PhD Grégoirevandecasteele

PhD Rodolphefischmeister
email: rodolphe.fischmeister@inserm.fr

MD Ali El-Armouche
email: ali.el-armouche@med.uni-goettingen.de

Rodolphe Fischmeister

Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts β-Adrenergic Responses in Cardiomyocytes

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Heart failure (HF) is among the most common causes of morbidity and mortality in the Western world. Regardless of the underlying cause, HF patients show a hyper-active sympathetic nervous system with elevated plasma catecholamine levels and chronic activation of the β-adrenergic receptor (β-AR) signaling pathway. This leads to desensitization and attenuated β-AR responsiveness due to decreasedβ1-ARs number and function (1,2). These changes may participate in the progression of HF by further compromising contractile performance. On the other hand, some of the functional abnormalities of failing cardiomyocytes, particularly β-AR desensitization, may also be adaptations that protect from detrimental effects of chronic β-AR stimulation, e.g. arrhythmias and myocardial hypertrophy (2). Accordingly, drugs intended to reverse or bypass β-AR desensitization (catecholamines or PDE inhibitors), cause symptomatic improvement in short term, but demonstratelong-term increased mortality due to sudden cardiac death (3). Conversely, large-scale clinical trials show that β-ARblockers reduce mortality in HF (4)(5)(6).

There is recent evidence that higher myocardial levels of the second messenger cGMP may be beneficial, partly by antagonizing the cAMP-mediated effects of β-AR overstimulation (7)[START_REF] Pokreisz | Ventricular phosphodiesterase-5 expression is increased in patients with advanced heart failure and contributes to adverse ventricular remodeling after myocardial infarction in mice[END_REF][START_REF] Stangherlin | cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes[END_REF]. Cyclic GMP is synthesized by either soluble guanylyl cyclases activated by NO or particulate guanylyl cyclases activated by the natriuretic peptides ANP and BNP [START_REF] Su | Differential effects of cGMP produced by soluble and particulate guanylyl cyclase on mouse ventricular myocytes[END_REF]. In the heart, cAMP and cGMP levels are regulated in a highly specific and stimulus-dependent manner by cyclic nucleotide degrading phosphodiesterases (PDEs) [START_REF] Zaccolo | cAMP and cGMP signaling cross-talk: role of phosphodiesterases and implications for cardiac pathophysiology[END_REF][START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases[END_REF]. Among the PDE superfamily, the PDE2 isoform is unique in being activated by cGMP via binding to the regulatory N-terminal GAF-B domain [START_REF] Francis | Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions[END_REF][START_REF] Martinez | GAF domains: two-billion-year-old molecular switches that bind cyclic nucleotides[END_REF]. cGMP functions as an allosteric regulator of PDE2, and occupancy of this binding-site changes the kinetics for cyclic nucleotide hydrolysis stimulating the hydrolysis of cAMP 5-30 fold. This may provide a negative crosstalk mechanism between cAMP and cGMP signaling pathways. Indeed, we showed previously that cGMP activation of PDE2 locally depletes cAMP in the vicinity of Ltype Ca 2+ channels, thus strongly antagonizing β-AR or cAMP-stimulation of the L-type Ca 2+ current (ICa,L) [START_REF] Hartzell | Opposite effects of cyclic GMP and cyclic AMP on Ca2+ current in single heart cells[END_REF][START_REF] Mery | Nitric oxide regulates cardiac Ca2+ current. Involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation[END_REF][START_REF] Dittrich | Local response of L-type Ca(2+) current to nitric oxide in frog ventricular myocytes[END_REF]. In human atrium, where cAMP turnover is more important, a constitutive PDE2 activity controls basal ICa,L [START_REF] Rivet-Bastide | cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes[END_REF] and activation of this PDE2 by cGMP also decreases stimulated ICa,L [START_REF] Vandecasteele | Cyclic GMP regulation of the L-type Ca(2+) channel current in human atrial myocytes[END_REF]. In neonatal cardiomyocytes, local PDE2 is coupled to a β-ARstimulated pool of adenylyl cyclases and cAMP-dependent protein kinase type-II isoforms, antagonizing β-AR inotropic responses partly via the β3-AR/NO/cGMP axis [START_REF] Stangherlin | cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes[END_REF][START_REF] Mongillo | Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway[END_REF].

However, despite the unique function of PDE2 in cGMP and cAMP signaling and the well-known perturbations in β-AR and NO/natriuretic peptide signaling pathways, it is unknown whether PDE2 has a significant pathophysiological role in failing hearts. Here, we show that myocardial PDE2 is markedly upregulated in HF and that specific PDE2 inhibition restores β-AR responsiveness in diseased cardiomyocytes, whereas PDE2 overexpression blunts catecholamine responsiveness and protects against pathological hypertrophy. These data establish PDE2 as a new member of the group of proteins contributing to the well-known phenomenon of β-AR desensitization in chronic HF and implicates PDE2 as a key downstream element that might be a target for the development of novel anti-adrenergic therapeutic approaches.

Materials and Methods

The study conformed to the principles outlined in the Declaration of Helsinki and all procedures were approved by the ethics committees of our institutions. Patient characteristics and drugs as well as all methods used, including preparation of protein extracts, PDE activity assays, myocyte isolation and adenoviral transductions, cAMP measurements by FRET, Ca 2+ transients and cell shortening (IonOptix ® ), L-type Ca 2+ current measurements, and data analysis and statistics are detailed in the supplemental methods section in the Online Appendix.

Results

Myocardial PDE2 is increased in end-stage HF. We measured PDE2 protein levels in left ventricular (LV) myocardium from patients with terminal HF (EF≤37%) and compared them to non-failing donor hearts (NF, n=6). Each sample was normalized to calsequestrin (CSQ) as internal control. Immunoblots demonstrated greater signal-intensity in the failing hearts (~2fold) with dilated (DCM, n=5) and ischemic (ICM, n=5) cardiomyopathy (Figure 1A and Supplemental Table 1 for patient characteristics). This was independent of -blocker treatment before heart transplantation (Supplemental Figure 1). The protein data were confirmed at a functional level by a radioenzymatic assay in the presence and absence of the specific PDE2 inhibitor BAY 60-7550 (BAY, 100 nM) showing an about 2-fold greater PDE2 activity toward cAMP in failing hearts compared to NF controls (Supplemental Figure 2).

Interestingly, PDE2 expression did not differ between non-failing controls and hypertrophied

LV myocardium from patients with preserved cardiac function (EF≥50%) who underwent aortic valve replacement due to aortic stenosis (AS; n=6; Figure 1A and Supplemental Table 1). Given the well-known limitations of human heart tissue samples including partly uncontrolled clinical parameters, we re-evaluated PDE2 expression in a well-established large animal HF model of dogs subjected to ventricular tachy-pacing (n=9). Immunoblotting confirmed our findings in human HF showing ~2-fold greater PDE2 protein expression in failing dog hearts compared to sham-controls (n=7, Figure 1B).

Increased myocardial PDE2 expression/activity results from excessive adrenergic drive

in vivo. We tested whether PDE2 upregulation is -like β1-AR downregulation -a consequence of pathological β-AR overstimulation. As expected, chronic isoprenaline infusions (ISO; 2.4 µg/g/d for 4 days) in rats induced cardiac hypertrophy (as indicated by an increased heart-tobody weight ratio, Figure 2A) and induction of the fetal gene expression program (as indicated by a >8-fold increase in transcript levels of the cardiac fetal gene ANP, Figure 2B).

In line with our hypothesis, this was accompanied by a ~2-fold increase in PDE2 protein levels (Figure 2C). Next we determined PDE2 mRNA levels by quantitative RT-PCR and found 4fold higher levels in the ISO infused hearts compared to control hearts, indicating that PDE2 is regulated at the transcriptional level (Figure 2D).To determine whether the increased LV PDE2 protein expression is translated into greater activity we analyzed PDE2-dependent cAMP and cGMP hydrolytic activities. As shown in Figure 2E and 2F, relative cAMP-PDE2 activity was 4-fold higher and cGMP-PDE2 activity was 2-fold greater in LV homogenates from the ISO vs. vehicle treated hearts.

Higher cAMP-PDE2 hydrolytic activity in diseased cardiomyocytes. To further evaluate changes in PDE2 at the cardiomyocyte level, ventricular cardiomyocytes were isolated from adult rats either subjected to chronic ISO or vehicle-infusion, and cAMP-PDE2 hydrolytic activity was monitored using the adenovirally expressed Epac2-camps FRET biosensor for cAMP. Acute application of ISO (1 nM) produced a clear increase in cAMP in control cardiomyocytes, which was markedly attenuated in cardiomyocytes from ISO-treated hearts (-70%, Figure 3A), as expected from the known β-AR desensitization in this model. The basal FRET ratios were lower in cells isolated from ISO-treated rats (Figure 3B). Application of BAY had no significant effect on cAMP accumulation in control cardiomyocytes, in line with the fact that PDE2 accounts for only 3% of the total cAMP-hydrolyzing activity under normal conditions [START_REF] Mongillo | Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway[END_REF]. By comparison, application of the non-selective PDE inhibitor IBMX (100 µM) or the adenylyl cyclase-activator forskolin (10 µM) in the continued presence of BAY produced a similar net increase in cAMP (Figure 3B). The absolute cAMP levels (CFP/YFP ratio in Fig. 3B) were lower in ISO cardiomyocytes vs. control (p=0.05). However, in chronically ISO treated cardiomyocytes, application of BAY alone produced a significant increase in cAMP levels (Figure 3B andC). The overall maximal changes in cAMP levels from basal to forskolin/IBMX maximum were similar in both groups, suggesting comparable rates of cAMP production. These data indicate a stronger contribution of PDE2 to cAMP hydrolysis in diseased cardiomyocytes.

Since PDE2 is activated by cGMP, we tested whether higher PDE2 activity in diseased cardiomyocytes could be further enhanced by cGMP synthesis. To this end, we challenged cardiomyocytes with the NO donor sodium nitroprusside (SNP, 50 µM) after acute β-AR stimulation with ISO. SNP reduced the ISO response in chronic ISO cardiomyocytes, but had no significant effect in vehicle-controls (Figure 3 D andE). Therefore, further activation of upregulated PDE2 is still possible in diseased cardiomyocytes. Interestingly, these data point to a predominant effect of the cGMP-induced PDE2 activation over the cGMP-mediated PDE3 inhibition in disease.

To investigate how increased PDE2 expression translates into function, Ca 2+ transients (Fura-2 ratio) and sarcomere shortening were measured in isolated cardiomyocytes with the IonOptix ® system. ISO (1 nM) produced a large increase in Fura-2 ratio and sarcomere shortening in NaCl myocytes, but only a modest increase in cardiomyocytes from ISO-treated hearts (Figure 4) again confirming the β-AR desensitization. BAY alone produced a small but significant effect on both parameters in vehicle-treated cardiomyocytes (Supplemental Table 2). However, in chronically ISO treated cardiomyocytes, BAY produced a 2-fold larger effect (Supplemental Table 2). Importantly, the application of BAY in addition to ISO had no significant effect on vehicle-treated cardiomyocytes, but increased 2-3-fold the ISO effect on Ca 2+ transients and sarcomere shortening in the chronic ISO group (Figure 4B). Thus, PDE2 inhibition partially restores β-AR responsiveness, which indicates that PDE2 is in fact playing a functional role in diseased myocytes.

Overexpression of PDE2 blunts responses to acute β-AR stimulation and protects from

norepinephrine-induced hypertrophy. To dissect further the specific role of upregulated PDE2, we infected adult rat ventricular myocytes with either an adenovirus encoding HAtagged mouse PDE2A2 (Ad-PDE2) or a control adenovirus encoding β-galactosidase (Ad-βGal). Immunoblot detection of overexpressed PDE2 and, at the functional level, higher PDE2 hydrolysis activity toward cAMP and cGMP in Ad-PDE2 infected cardiomyocytes are shown in Supplemental Figure 3. We then determined the consequences for cAMP changes in response to β-AR stimulation. For this, cardiomyocytes co-expressing Epac2-camps and either β-Gal or PDE2 were challenged with a short application of ISO (100 nM, 15 s) either alone or in the presence of BAY (100 nM). As monitored by FRET, PDE2 overexpression markedly decreased intracellular cAMP levels generated by β-AR stimulation. This β-AR response was almost completely restored when the cardiomyocytes were additionally challenged with BAY (Supplemental Figure 4).

Next, we examined whether PDE2 overexpression modifies the β-AR regulation of excitation-contraction coupling. While overexpression of PDE2 had no significant effect on basal ICa,L density (4.8±0.7 pA/pF in Ad-PDE2, n=7 vs. 3.2±0.7 pA/pF in Ad-βGal, n=8), the response to a pulse application of 100 nM ISO was markedly blunted in myocytes infected with Ad-PDE2 (Figure 5A andC). However, inhibition of PDE2 with BAY restored a normal response to ISO (Figure 5B andD). In another series of experiments, sarcomere shortening and Fura-2 ratio were simultaneously recorded (Figure 6A). Under basal conditions, the amplitude and relaxation kinetics of sarcomere shortening were not significantly different between Ad-βGal and Ad-PDE2 cardiomyocytes (Figure 6B andC). Ad-PDE2 cardiomyocytes developed significantly smaller Ca 2+ transients than Ad-βGal, yet with no difference in the relaxation kinetics. When the cells were challenged with ISO (1 nM), Ad-βGal cardiomyocytes responded with a strong increase in both contraction and Ca 2+ transient amplitudes (Figure 6B), accompanied by an acceleration in their relaxation kinetics (Figure 6C). In PDE2-overexpressing cardiomyocytes, however, these responses were markedly attenuated. To test sarcoplasmic reticulum (SR) function at the molecular level, we assayed phosphorylated phospholamban (PLB-P). Immunoblots showed that over-expression of PDE2 does not change the phosphorylation level of PLB under basal conditions, but significantly decreases the P-PLB/total PLB ratio after stimulation with 1 nM ISO (Supplemental Figure 5). These results suggest that PDE2 overexpression does not affect SR function under basal conditions and is consistent with our findings that the β-AR response of Ca 2+ transients and sarcomere shortening was inhibited by PDE2 over-expression without negative effect on basal contractility. Collectively, our findings highlight a direct and potent involvement of PDE2 in β-AR responsiveness in HF.

Cardiac hypertrophy is one of the first stages of stress-induced HF. To examine the effect of upregulated PDE2 on cardiomyocyte hypertrophy, we determined the increase in cell size in cultured adult cardiomyocytes in response to 24-h stimulation with norepinephrine (NOR) or phenylephrine (PHE). As shown in Figure 7, PDE2 overexpression attenuated significantly the NOR-and PHE-induced increase in cell surface area. These data indicate that PDE2 has protective effects under conditions of and/or β-AR induced pathological hypertrophy.

Discussion

The major findings of the present study are: (i) myocardial PDE2 is upregulated in human and experimental HF, (ii) specific PDE2 inhibition restores β-AR responsiveness in diseased cardiomyocytes, (iii) increased PDE2 activity is further enhanced by cGMP stimulation via NO donors and (iv) PDE2 overexpression abolishes β-AR responsiveness without affecting basal contractility and protects against pathological hypertrophy.

PDE2 is upregulated in failing hearts. Consistent with the literature, our study shows rather low protein expression of PDE2 in healthy hearts (see Figure 1) and accordingly inhibition of PDE2 has little effect on cAMP hydrolytic activity in healthy cardiomyocytes (see Figure 3B/C). However, our data indicate that in HF, with enhanced adrenergic drive and marked perturbations in the β-AR and NO/natriuretic peptide signaling, PDE2 becomes upregulated and, by mediating cGMP-dependent cAMP degradation, contributes to the imbalance between these two signaling pathways. This is consistent with recent findings showing disorganization of PKA regulatory subunits and A-kinase anchoring proteins, which showed increased capturing of PKG and PDE2 [START_REF] Aye | Reorganized PKA-AKAP associations in the failing human heart[END_REF]. In contrast, the expression and activity of other cAMPhydrolyzing PDEs (e.g. PDE3) is reduced in human and experimental HF [START_REF] Ding | Functional role of phosphodiesterase 3 in cardiomyocyte apoptosis: implication in heart failure[END_REF][START_REF] Abi-Gerges | Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on beta-adrenergic cAMP signals[END_REF]. PDE regulation in vivo is complex and one transduction signal may affect several PDEs differently.

For instance, cGMP activates PDE2, but inhibits competitively PDE3 [START_REF] Degerman | Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3)[END_REF]. Through the latter mechanism, an increase in cGMP levels inhibits cAMP degradation. Thus, it is likely that under HF conditions both the lower PDE3 and higher PDE2 activity could be a consequence of increased cGMP signaling that is further amplified by increased PDE2 and/or reduced PDE3 expression. In this context it is interesting to note that the cGMP-hydrolyzing PDE5 is also increased in failing human hearts [START_REF] Pokreisz | Ventricular phosphodiesterase-5 expression is increased in patients with advanced heart failure and contributes to adverse ventricular remodeling after myocardial infarction in mice[END_REF]. This may partly compensate for the PDE2/PDE3 alterations.

β-AR signaling is a biologically dynamic system that adapts quickly and reversibly by desensitization in response to overstimulation. Accordingly, β-blockers "resensitize" the failing heart and are associated with normalization of β-ARs (1,2). However, β-blocker therapy did not appear to normalize upregulated myocardial PDE2 expression in our samples, pointing to different regulatory mechanisms of altered β-ARs and PDE2 in HF. In addition, it was intriguing to observe that PDE2 expression was still normal in LV tissue samples from AS patients with myocardial hypertrophy and preserved cardiac function (see Figure 1A).

This indicates an important role for PDE2 in decompensated rather than earlier stages of myocardial remodeling.

PDE2 upregulation and its consequences after chronic excessive β-AR drive. The chronic ISO infusion model recapitulates key features of human HF, particularly pathological

hypertrophy and the well-known phenomenon of β-AR desensitization [START_REF] Wittkopper | Constitutively active phosphatase inhibitor-1 improves cardiac contractility in young mice but is deleterious after catecholaminergic stress and with aging[END_REF][START_REF] El-Armouche | Phosphatase inhibitor-1-deficient mice are protected from catecholamine-induced arrhythmias and myocardial hypertrophy[END_REF]. In this model, we observed an increase in PDE2 protein expression, mRNA transcription and PDE2 cGMP/cAMP hydrolytic activity. Specific PDE2 inhibition partially restored β-AR responsiveness in diseased cardiomyocytes, indicating that PDE2 upregulation contributes significantly to the well-known phenomenon of β-AR desensitization, considered to be a major protective mechanism in HF. A central translational question is whether potentially beneficial PDE2 upregulation in HF can be further amplified by co-activation of cGMP synthesis. Indeed, diseased cardiomyocytes showed a significant decrease in cAMP levels when challenged with the NO donor SNP further validating the feasibility of PDE2 activation in the context of HF (see below).

PDE2 abolishes β-AR mediated inotropic effects via cAMP degradation and reduction

of ICa,L amplitude. In cardiomyocytes isolated from chronic ISO rats, β-AR stimulation of cell shortening and Ca 2+ transients was severely blunted due to PDE2 overexpression (Figure 4). Moreover, adenoviral PDE2 overexpression markedly blunts ISO-induced (i) inotropy, (ii) cAMP generation and (iii) the increase in ICa,L and Ca 2+ transients in adult cardiomyocytes.

Importantly, all effects were reversible upon specific PDE2 inhibition with BAY. This is the first direct evidence that PDE2 overexpression inhibits the increase in ICa,L by reducing cAMP concentrations and indicates that PDE2 regulates a specific cAMP pool that modulates β-AR signaling. Our findings are consistent with previous reports showing indirectly that activation of PDE2 is responsible for the inhibitory effect of intracellular cGMP on ICa,L in frog ventricular [START_REF] Mery | Nitric oxide regulates cardiac Ca2+ current. Involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation[END_REF][START_REF] Mery | Erythro-9-(2-hydroxy-3nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterase in isolated cardiac myocytes[END_REF] and human atrial myocytes [START_REF] Vandecasteele | Cyclic GMP regulation of the L-type Ca(2+) channel current in human atrial myocytes[END_REF] and that via stimulation of sGC with NO donors attenuates β-AR stimulation in frog [START_REF] Mery | Erythro-9-(2-hydroxy-3nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterase in isolated cardiac myocytes[END_REF] and neonatal rat cardiomyocytes [START_REF] Mongillo | Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway[END_REF].

PDE2 does not affect basal contractility but protects against pathological hypertrophy.

NO/natriuretic peptides have been shown to exert both beneficial cGMP-dependent antihypertrophic and potentially detrimental negative inotropic effects on the failing heart.

Both effects have been largely attributed to activation of cGMP-dependent PKG [START_REF] Rosenkranz | Antihypertrophic actions of the natriuretic peptides in adult rat cardiomyocytes: importance of cyclic GMP[END_REF][START_REF] Fiedler | Inhibition of calcineurin-NFAT hypertrophy signaling by cGMP-dependent protein kinase type I in cardiac myocytes[END_REF], with uncertain contribution of cGMP-dependent PDE2 activation. Our findings in PDE2 overexpressing cardiomyocytes suggest no negative effect on basal contractility or ICa,L (Figure 5 and6B), but beneficial effects of PDE2 upregulation in the context of pathological hypertrophy induced by norepinephrine and phenylephrine (Figure 7). The antihypertrophic properties together with the maintained basal contractility suggest that the upregulated PDE2 in HF is beneficial despite limiting the contractile reserve upon β-AR stimulation -a profile that is quite similar to β1-AR downregulation in HF.

Potential limitations.

To study PDE function we evaluated different species including humans. However, the role played by each PDE isoform varies among mammalian species [START_REF] Zaccolo | cAMP and cGMP signaling cross-talk: role of phosphodiesterases and implications for cardiac pathophysiology[END_REF][START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases[END_REF] and accordingly our results may not recapitulate the situation in humans in all details.

In addition, selective PDE2 inhibitors or activators for in vivo use have not been developed and PDE2 knockout mice have not yet been generated to study the precise role of PDE2 in the healthy and diseased heart. Therefore we studied the function of cardiac PDEs using specific inhibitors or adenoviral overexpression in vitro, but this might not reflect the complex regulation of the PDEs in vivo, particularly of PDE2, which is a dual-esterase positively regulated by cGMP.

Novelty and clinical significance. The current therapeutic strategies for HF are complex and only moderately efficient. Clearly, improved HF therapies are necessary, but a lack of understanding of the pathophysiological mechanisms underlying HF has hindered the development of more effective, rational therapeutic approaches. Several studies demonstrated beneficial effects of cGMP stimulation in HF, and multicenter trials are currently testing the therapeutic value of inhibition of cGMP hydrolyzing PDEs (PDE5) or activation of sGC in patient populations [START_REF] Lee | Phosphodiesterases and cyclic GMP regulation in heart muscle[END_REF]. The rationale of these studies is consistent with our hypothesis that augmentation of cGMP signaling may serve as a myocardial brake, blunting the detrimental effects of excessive catecholamines in HF. Despite substantial advances in the field, there are still controversies regarding the therapeutic value of activated cGMP signaling via PDE5 inhibition, and the clinical translation of increasing cGMP synthesis has been limited by hypotension and tolerance development with nitrates and rather neutral responses to natriuretic peptides [START_REF] Lee | Phosphodiesterases and cyclic GMP regulation in heart muscle[END_REF]. To the best of our knowledge,this study is first to show that PDE2 is upregulated in HF and constrains the effects of chronic β-AR stimulation on cardiomyocyte Ca 2+ handling and contractility in HF. Moreover, the upregulated PDE2 expression appears unaffected by β-blocker therapy, but could be further enhanced in diseased cardiomyocytes and protects against hypertrophic stimuli without negatively affecting basal contractility, clearly suggesting that activation of PDE2 could be a novel and feasible approach for the treatment of HF. In addition, cAMP hydrolysis through PDE2 has been shown to mediate the ANP/cGMP-induced decreases of aldosterone production in adrenal glomerulosa cells, suggesting synergistic beneficial effects on the cardio-renal axis [START_REF] Macfarland | High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in cAMP and steroidogenesis in adrenal glomerulosa cells[END_REF]. Overall, our data suggest that increased myocardial PDE2 levels may constitute an important defence mechanism in end-stage HF by antagonizing β-AR/cAMP mediated cardiac toxicity. Thus, augmenting myocardial PDE2 activity in early stages of HF may represent a new subcellular anti-adrenergic therapeutic strategy in HF. Further extensive studies in large-animal HF as well as the development of specific PDE2 activators for the in vivo use is required to define precisely the specific contributions of PDE2 to HF pathophysiology and to validate the therapeutic value of enhanced PDE2 activity in HF patients. *p<0.05 vs. Ad-βGal basal; $ p<0.05 vs. Ad-PDE2 basal; # p<0.05, ## p<0.01, ### p<0.001 Ad-PDE2 vs. Ad-βGal. Statistical significance for the differences between control and ISO was evaluated using paired t-test and unpaired t-test between Ad-PDE2 vs. Ad-βGal. Differences between the groups were assessed by one-way ANOVA followed by Bonferroni's multiple comparison test; *p<0.05 vs. basal, # p<0.05 Ad-EGFP vs. Ad-PDE2. The maximal amplitude of whole-cell I Ca,L was measured as previously described (4).

Currents were not compensated for capacitance and leak currents. Statistics. To assess differences between two or multiple groups the Student t test (Online Fig. 1, 2 and 5) or one-way ANOVA followed by Bonferroni's (Fig 7 ) were used, respectively, if a normal distribution could be assumed. In all other comparisons nonparametric analyses were applied. Differences between multiple groups were assessed by Kruskal-Wallis followed by Dunn's post hoc test (Fig. 4), differences between two groups by the Mann-Whitney test (Fig. 1, 2, 3 and 6) and differences between pairs were assessed by the Wilcoxon matched pair analysis (Fig. 4 and6). Results are presented as means±SEM.

Measurements of Ca

P values of less than 0.05 were considered as statistically significant. Statistical significance for the differences between Control and Bay 60-7550 was evaluated using paired t-test because each cell was successively exposed to Control and then to Bay 60-7550, providing an internal control for each cell: *p<0.05.

Figure 1 PDE2

 1 Figure 1PDE2 is upregulated in failing hearts

Figure 2 PDE2

 2 Figure 2PDE2 in a rat model of chronic isoprenaline (ISO) exposure: elevated expression and activity

Figure 3 Higher PDE2 cAMP hydrolytic activity in hypertrophied and β- adrenergically desensitized cardiomyocytes After 4

 34 Figure 3Higher PDE2 cAMP hydrolytic activity in hypertrophied and βadrenergically desensitized cardiomyocytes

Figure 4 PDE2

 4 Figure 4 PDE2 inhibition increases β-AR stimulation of Ca 2+ transients and sarcomere shortening in hypertrophied and β-adrenergically desensitized cardiomyocytes

Figure 5 PDE2

 5 Figure 5 PDE2 overexpression decreases β-AR stimulation of L-type Ca 2+ current (ICa,L) in cardiomyocytes

Figure 6 PDE2

 6 Figure 6 PDE2 overexpression blunts β-AR stimulation of Ca 2+ transients and sarcomere shortening in cardiomyocytes

Figure 7 PDE2

 7 Figure 7 PDE2 overexpression protects cardiomyocytes against hypertrophy induced by /β-adrenergic stimulation

  Figure1

Figure 7

 7 Figure5

  2+ transients and cell shortening. ARVMs were loaded with 5 µM Fura-2 AM in a Ringer solution containing (in mM): NaCl 121.6, KCl 5.4, MgCl 2 1.8, NaHCO 3 4, NaH 2 PO 4 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10 (pH 7.4), and 1 mM CaCl 2 at room temperature for 15 min. The cells were field stimulated (5 V, 4 ms) at a frequency of 0.5 Hz. Sarcomere length and Fura-2 ratio (measured at 512 nm upon excitation at 340 nm and 380 nm) were simultaneously recorded using an IonOptix® System.Determination of cellular hypertrophy. Twenty four hours after adenoviral infection withAd-GFP or Ad-PDE2-GFP, cells were stimulated with 10 µM norepinephrine (NOR) or 10 µM phenylephrine (PHE) for an additional 24 h in FBS-free MEM. Images of GFP positive cells were taken from different fields in each dish with a cooled charge coupled (CCD) camera (Sensicam PE; PCO, Kelheim, Germany) using the 20x objective of a Nikon TE 300 inverted microscope and 488 nm illumination. Surface area of individual myocytes was determined using Image J software (Wayne Rasband, National Institutes of Health, USA).

  cardiomyopathy, AS: aortic stenosis. Drugs: A: angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, C: calcium-channel-blockers, D: diuretics (including aldosterone antagonists), G: cardiac glycosides N: nitrates, R: antiarrhythmics (except betablockers), O: dopamine/dobutamine, S: steroids, L: clonidine, V: vasopressin, X: β-Blocker, n.d.: not determine.*These samples were used for Fig.1 and Supplemental Fig.1.

Figure 1

 1 Figure 1Immunoblot analysis (A) and quantification (B) of PDE2 protein levels in left ventricular myocardium from patients with end-stage dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM) treated with beta-blockers (BB) compared to ICM patients without beta-blocker therapy (n=6). B, PDE2 was normalized to calsequestrin (CSQ).

Figure 2

 2 Figure 2 PDE2 activity toward cAMP in LV myocardium from patients with end-stage dilated (DCM, n=6) or ischemic cardiomyopathy (ICM, n=4) were compared to non-failing donor hearts (NF, n=6) by radioenzymatic assay and defined as the fraction of total cAMP-PDE activity inhibited by 100 nM BAY 60-7550; p=0.0534 for DCM vs. NF.

Figure 3

 3 Figure 3Characterization of PDE2 overexpression in adult rat ventricular myocytes

Figure 4 PDE2

 4 Figure 4 PDE2 overexpression attenuates ISO-induced cAMP generation

Figure 5 PDE2

 5 Figure 5 PDE2 overexpression attenuates ISO-induced phospholamban phosphorylation at PKA site in adult rat ventricular myocytes

Table 1 : Clinical characteristics of patients.

 1

	Patient ID # Age Gender Diagnosis Drugs	NYHA CI	LVEF
	1	28	M	NF	n.d.			n.d.
	2	34	M	NF	n.d.			n.d.
	3	20	M	NF	n.d.			n.d.
	4	23	F	NF)	n.d.			n.d.
	5	n.d. F	NF)	n.d.			n.d.
	6	58	M	ICM	ACDOX	3-4	1.9	19%
	7*	57	F	ICM	ACDX	n.d.	n.d.	23%
	8	40	M	ICM	ACDX	n.d.	2.5	20%
	9	48	F	ICM	ACDGX	3	2.0	25%
	10*	66	M	ICM	AGDX	3-4	2.4	25%
	11	43	M	ICM	n.d.	n.d	n.d	23%
	12	57	M	ICM	ADG	4	-	15%
	13	64	M	ICM	ADNO	4	3.9	15%
	14	49	M	ICM	ADROS	4	2.1	15%
	15	46	M	ICM	DO	3	2.2	15-20%
	16	55	M	ICM	RD	-	1.7	20%
	17	52	F	ICM	ADRC	3-4	1.7	25%
	18*	53	M	DCM	DRX	3	5.7	37%
	19	31	F	DCM	ACDX	3	n.d	34%
	20	47	M	DCM	n.d.	3-4	2.1	28%
	21	55	M	DCM	ADGO	3	3.5	20%
	22	50	M	DCM	DRGX	3-4	3.0	25%
	23	58	M	DCM	ADX	3-4	1.9	25%
	24	24	M	DCM	ADXO	3-4	2.6	19%
	25	43	M	DCM	ADGXO	4	1.3	20%
	26	80	F	AS	AX			50%
	27	76	F	AS	D			60%
	28	81	F	AS	AX			60%
	29	60	M	AS	AX			65%
	30	57	M	AS	ACX			65%
	31	81	F	AS	AC			60%
	Diagnosis: NF: non-failing donor, ICM: ischemic cardiomyopathy, DCM: dilated

Table 2 : Effects of PDE2 inhibition on basal sarcomere shortening and Ca 2+ transients

 2

		Sarcomere shortening (%)	Fura-2 ratio (%)
	NaCl (n=17)
	Control	2.36 ± 0.74	34.32 ± 2.21
	Bay 60-7550	3.09 ± 0.96 *	37.63 ± 2.64 *
	Chronic ISO (n=16)
	Control	2.04 ± 0.40	36.60 ± 6.02
	Bay 60-7550	3.60 ± 0.92 *	41.01 ± 6.09 *

Acknowledgements

This study was supported by the Deutsche Forschungsgemeinschaft (DFG EL 270/5-1 to AEA and SFB 1002 to SL, LSM, WHZ, VON and AEA), DZHK (German Centre for Cardiovascular Research to LSM, TE, DD, WHZ, AEA), European Union (contract LSHM-CT-2005-018833/EUGeneHeart to AEA, TE, RF), by the Fondation Leducq for the Transatlantic Network of Excellence cycAMP grant 06CVD02 (to RF), and the European-North American Atrial Fibrillation Research Alliance grant 07CVD03 (to DD and SN), by the German Heart Foundation (to AEA), by the Fondation pour la Recherche Médicale (to HM and RF), by ANR 2010 BLAN 1139-01 (to GV) and by the Investment for the Future program ANR-11-IDEX-0003-01 within the LABEX ANR-10-LABX-0033 (to RF).

Methods

All experiments were carried out according to the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J n°L358, 18 December 1986), the local ethics committees guidelines and the French decree n°87-848 of October 19, 1987(J Off Rép Fr, 20 October 1987, pp. 12,245-12,248). Authorizations to perform animal experiments according to this decree were obtained from the Ministère Français de l'Agriculture, de la Pêche et de l'Alimentation (nº D 92-283, December 13, 2012).

Human myocardial tissue.

Failing left ventricular (LV) tissues were obtained from patients with end-stage dilated (DCM) or ischemic cardiomyopathy (ICM) at the time of cardiac transplantation. For comparison non-failing (NF) human LV tissues were obtained from organ donors with no known history of cardiac disease who could not be transplanted for technical reasons. LV tissues (septal "Morrow" resections) also were obtained from adult patients with symptomatic severe aortic stenosis (AS) undergoing aortic valve replacement with significant myocardial hypertrophy but normal ejection fraction. Patient characteristics and medications are summarized in Supplemental Table 1. The study was reviewed and approved by the Ethical Committee of the University Göttingen and all patients gave written informed consent.

Tissue from canine model of pacing-induced HF. Rapid ventricular pacing was used to

induce HF in dogs as previously described (1) and kindly provided from Dr. S. Nattel (Montreal, Quebec, Canada) (2). Briefly, mongrel dogs were instrumented with a corkscrew electrode in the LV attached to a pacemaker implanted in the neck. Hearts were paced at 240 bpm for 2 weeks. Control dogs were instrumented but not paced. Tachypacing-induced hemodynamic changes were typical of a dilated cardiomyopathy with failing hearts demonstrating elevated left ventricular end-diastolic pressure (LVEDP) and decreased dP/dt max , typical of this model. Tachypnea, ascites, pulmonary congestion, and pleural effusion were present. Hearts were removed under anesthesia with morphine (2 mg/kg SC) and α-chloralose (120 mg/kg, i.v.).

Adenoviruses. The mus musculus PDE2A2 isoform was kindly provided by Dr Joe Beavo (Washington University, Seattle, USA). This isoform was encoded into a replication-defective serotype 5 adenovirus (Ad-PDE2) or into a bicistronic replication-defective serotype 5 adenovirus encoding EGFP (Ad-PDE2-GFP) by GeneCust (Evry, France).

Replication-defective serotype 5 adenovirus encoding β-galactosidase (Ad-βGal) or EGFP (Ad-GFP) were obtained from the GVPN (Nantes, France). Cells were infected with an MOI 50-100 in standard culture conditions for a period of 48 h.

Rat isoprenaline infusion model. Male Wistar rats (240-300 g) were treated for 4-5 days with either isoprenaline (2.4 mg/kg/day, ISO) or vehicle (0.9% NaCl, Ctr) administered via osmotic minipumps (Alzet, USA) (3). The rats were sacrificed by cervical dislocation under light isoflurane anesthesia. Hearts were rapidly removed and remaining blood was washed out in ice-cold 0.9% NaCl solution. Ventricular tissue samples were frozen in liquid nitrogen and stored at 80°C until use or ventricular cardiomyocytes were isolated from these hearts for FRET experiments, sarcomere shortening and calcium transient measurements.

Isolation and culture of rat cardiomyocytes. Adult rat ventricular myocytes (ARVMs) were isolated from male Wistar rat heart mounted on a Langendorff apparatus and perfused through the coronaries with collagenase A as described previously (4). Cells were cultured in Minimum Essential Medium (MEM) containing 1.2 mM Ca 2+ , 2.5% fetal bovine serum (FBS), 1% penicillin-streptomycin and 2% HEPES (pH 7.6) and plated on laminin-coated culture dishes (10 μg/ml laminin). ARVMs were left to adhere for 2 h in a 95% O 2 , 5% CO 2 atmosphere at 37°C, before the medium was replaced with FBS-free MEM containing adenovirus according to the experimental setting.

Immunoblot analysis. Protein samples were separated in denaturating acrylamide gels and subsequently transferred onto nitrocellulose or PVDF membranes. After blocking the membranes with 0.5% skimmed milk (AppliChem) or with 3% bovine serum albumin for 1 h, the incubation with anti-ANP (1:200, Santa Cruz), anti-Calsequestrin (1:200, Dianova), anti-PDE2 (1:1000, FabGennix), anti-Phosphorylated PLB at Ser16 (1:500, Badrilla) and anti-total PLB (1:500, Santa Cruz) 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.