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CaV1.2 and β -Adrenergic Regulation of Cardiac Function Overview of the β-adrenergic regulation of
L-type calcium channels
Jérôme Leroy, Rodolphe Fischmeister
To cite this version:
Jérôme Leroy, Rodolphe Fischmeister. CaV1.2 and β-Adrenergic Regulation of Cardiac Function Overview of theβ-adrenergic regulation of L-type calcium channels. Cardiac Electrophysiology, from Bench to Bedside, 6th Edition, Eds Zipes & Jalife, Elsevier. 2013;Chapter 37:pp. 371-381., pp.371-381, 2013. �hal-02896475�
Ca
V1.2 and β-Adrenergic Regulation of Cardiac Function J
ÉRÔMEL
EROY ANDR
ODOLPHEF
ISCHMEISTERINSERM, UMR-S 769, LabEx LERMIT, University Paris-Sud, Faculty of Pharmacy, Châtenay-Malabry, France
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.1 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 cloned2 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.3,4
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.5 PKA phosphorylation of CaV1.2 channels results in a shift of the channel from the gating mode 1 to mode 2.6 As a result, ICa,L density is increased ~2-3 fold and its voltage dependence slightly shifted towards hyperpolarized potentials (figure 2A). Voltage steady- state 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 β- adrenergic 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.7 Because β-AR activation enhances calcium entry, it also accelerates ICa,L inactivation,8 and calcium-dependent inactivation becomes the main mechanism by which the channel inactivates.9 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.4 Like other high voltage gated calcium channels, CaV1.2 associates with a largely extracellular disulfide-linked α2-δsubunit of 170 kDa.10 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.11 Four genes encode four CaVβ subunits (β1-4) but CaVβ2 is thought to be the main isoform expressed in the heart.12 Both α2-δ13, and CaVβ14 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.15
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).2 Despite the fact that α1C is a substrate for phosphorylation by PKA in vitro,16,17 several attempts to
mimic the adrenergic stimulation of CaV1.2 channels in expression systems have failed.18-20 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.21 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)22 allowing its phosphorylation at serine 1928 when β-ARs are stimulated.23 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,17 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.24 Later on, the role of serine 1928 in ICa,L upregulation by β-AR has been challenged. A first study showed that a DHP- resistant 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.25 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.26 Therefore, while phosphorylation of serine 1928 within the C-terminus of CaV1.2 definitely occurs when β-AR are stimulated17,21,23 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.27 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.28 Thus, the authors concluded that the CaVβ2a
ancillary subunit is the main target for PKA to mediate the βAR 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.14 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.29 Interestingly, ahnak polymorphism occurs, and the genetic variant generated interferes with the βAR stimulation of ICa,L by reducing the CaVβ2 interaction with ahnak.29 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.30 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 channel31) 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.30 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 AKAP15.32
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.33 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.34,35 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.36,37 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.36
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 β1- and 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.33 However, they exert opposite effects on hypertrophy38,39 and apoptosis,40,41 and their respective contribution varies significantly depending on the cardiac tissue, the pathophysiological state, the age or the developmental stage.42 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.43 Another difference between the two β-AR subtypes is their location at the cell surface, with β2-ARs present in the caveolae/lipid rafts44,45 of the transverse-tubular structure46 and β1-ARs distributed throughout both caveolae/lipid rafts and nonlipid raft membrane domains44 and in both plasma and t-tubular membranes.46 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.47 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.43,48,49
The β3-AR differs from β1-and β2-AR subtypes in its molecular structure and pharmacological functions.50 Expression of β3-AR was demonstrated in human myocardium both at the mRNA51,52 and protein level.52-55 Interestingly, β3-AR activation produces a negative inotropic effect in human endomyocardial biopsies from transplanted hearts50,51 and
in left ventricular samples from failing and non-failing explanted hearts50,54 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).56 This is reminiscent of the contractile effects of the serotonin 5-HT4 receptors57, which are also coupled to increases in force of contraction58 or ICa,L59 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.60,61 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.62 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.63 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 2,64,65 Epac,66 adenylyl cyclases 67,68 and PDEs.69 Recently, it has been demonstrated that the phosphoinositide 3-kinase p110, that was shown recently tether
PKA,70 orchestrates multi-protein complexes including different PDEs to control cardiac CaV1.2 phosphorylation during -AR stimulation.71 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.61,66,72
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.73 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.35,60
Cardiac cAMP PDEs degrading belong to 5 families (PDE1-4 and PDE8) which can be distinguished by distinct enzymatic properties and pharmacology.74 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.75-78 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 PDE2.79 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.78,80 In rodents, PDE3 and PDE4 are the major contributors to the total cAMP-hydrolytic activity34,81 and PDE4 is dominant to modulate β-AR regulation of cAMP levels.49,82-84 Multiple PDE4 variants associate with β- ARs,85-87 RyR2,88 SERCA2,89,90 ICa,L,91 and IKs72 to exert local control of ECC. In larger mammals, PDE3 activity is dominant in microsomal fractions92-94 and PDE3 inhibitors exert a potent positive inotropic effect.95 Selective inhibition of PDE3 with milrinone has been shown to improve cardiac contractility in patients with congestive heart failure.96 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 cytoplasm92 but PDE4 is also present in microsomal fractions, where it accounts for ~20% of the activity.93 Recent studies have indicated that PDE4 is expressed in human ventricle where, similar to rodents, it associates with β-ARs, RyR2 and phospholamban.81,88 Moreover, PDE4 is the main PDE modulating LTCC activity in rodent cardiomyocytes77,84 and PDE4 was recently shown to control ICa,L, ECC and arrhythmias in human atrium.97
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.98 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.92 In canine ventricular myocytes, increase in particulate but not total cAMP correlated with increase in Ca2+ transient amplitude and decay kinetics.99 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.84
While PDE4 regulates ICa,L in cardiomyocytes,77,84 the molecular identity of the PDE4 regulating the LTCC was only recently unveiled.91 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 Ca2+ transients.91 In vivo, upon β-AR stimulation, catheter-mediated burst pacing triggers ventricular tachycardia in Pde4b-/- mice but not in wild-type.91 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 Ca2+ that enters the cell via the LTCCs, prevents Ca2+ 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 re- entrant arrhythmias and triggered activities.111,112 At the level of the cardiomyocyte, in AF113 or in HF114, 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+/Ca2+ 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 Ca2+ 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 AF128-130 or from
patients in sinus rythm with a high risk of AF,131,132 the exact mechanism for such down- regulation is not clearly established. This reduction might be primarily due to transcriptional down-regulation of the α1C subunit of LTCC via a Ca2+-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 subunits134 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 down- expression of α1C and CaVβ1.136 Oxidant stress that occurs in AF may also participate to decrease LTCC activity by inducing S-nitrolysation of α1C137 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 AF140, 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.97 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.97 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.146-148A hallmark of heart failure is β-AR desensitization with a loss of β-AR signaling compartmentation,35,144 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.46 Interestingly, 80% of the CaV1.2 channels are located in the t-tubules152 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 coupling157 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 activity120 and participate in calcium influx remodeling in heart failure.168 Blunted β-AR stimulation of ICa,L
in human failing cardiomyocytes has been consistently reported162,169 probably due to more abundant heteromeric Gi/oα proteins,170-172 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,L91 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 hypertrophy178 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-overload180 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 C- terminus from the proximal C-terminus.30 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.32,184 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.
References
1. Bers DM. Cardiac excitation-contraction coupling. Nature 2002;415(6868):198-205.
2. van der Heyden MA, Wijnhoven TJ, Opthof T . Molecular aspects of adrenergic modulation of cardiac L-type Ca2+ channels. Cardiovasc Res 2005;65(1):28-39.
3. Hartzell HC, Méry P-F, Fischmeister R, Szabo G. Sympathetic regulation of cardiac calcium current is due exclusively to cAMP-dependent phosphorylation. Nature 1991;351(6327):573-576.
4. Catterall WA. Voltage-gated calcium channels. Cold Spring Harb Perspect Biol 2011;3(8):a003947.
5. Hess P, Lansman JB, Tsien RW. Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists. Nature
1984;311(5986):538-544.
6. Yue DT, Herzig S, Marban E. ß-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes. Proc Natl Acad Sci USA 1990;87(2):753- 7.
7. Cens T, Rousset M, Leyris JP, Fesquet P, Charnet P. Voltage- and calcium-dependent inactivation in high voltage-gated Ca2+ channels. Prog Biophys Mol Biol 2006;90(1- 3):104-17.
8. Argibay JA, Fischmeister R, Hartzell HC. Inactivation, reactivation and pacing dependence of calcium current in frog cardiocytes: correlation with current density. J Physiol 1988;401(201-226.
9. Findlay I. Physiological modulation of inactivation in L-type Ca2+ channels: one switch. J Physiol 2004;554(Pt 2):275-283.
10. Davies A, Hendrich J, Van Minh AT , Wratten J, Douglas L, Dolphin AC. Functional biology of the alpha(2)delta subunits of voltage-gated calcium channels. Trends
Pharmacol Sci 2007;28(5):220-8.
11. Dolphin AC. Beta subunits of voltage-gated calcium channels. J Bioenerg Biomembr 2003;35(6):599-620.
12. Colecraft HM, Alseikhan B, Takahashi SX, Chaudhuri D, Mittman S,
Yegnasubramanian V, Alvania RS, Johns DC, Marban E, Yue DT. Novel functional properties of Ca2+ channel ß subunits revealed by their expression in adult rat heart cells. J Physiol 2002;541(2):435-452.
13. Fuller-Bicer GA, Varadi G, Koch SE, Ishii M, Bodi I, Kadeer N, Muth JN, Mikala G, Petrashevskaya NN, Jordan MA, Zhang SP, Qin N, Flores CM, Isaacsohn I, Varadi M, Mori Y, Jones WK, Schwartz A. Targeted disruption of the voltage-dependent calcium channel alpha2/delta-1-subunit. Am J Physiol Heart Circ Physiol 2009;297(1):H117- 24.
14. Miriyala J, Nguyen T, Yue DT, Colecraft HM. Role of Cavß subunits, and lack of functional reserve, in protein kinase A modulation of cardiac Cav1.2 channels. Circ Res 2008;102(7):e54-64.
15. Yang L, Katchman A, Morrow JP, Doshi D, Marx SO. Cardiac L-type calcium channel (Cav1.2) associates with gamma subunits. FASEB J 2011;25(3):928-36.
16. Puri TS, Gerhardstein BL, Zhao XL, Ladner MB, Hosey MM. Differential effects of subunit interactions on protein kinase A- and C-mediated phosphorylation of L-type calcium channels. Biochemistry (Mosc) 1997;36(31):9605-9615.
17. De Jongh KS, Murphy BJ, Colvin AA, Hell JW, Takahashi M, Catterall WA. Specific phosphorylation of a site in the full-length form of the 1 subunit of the cardiac L- type calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. Biochemistry (Mosc) 1996;35(32):10392-402.
18. Perez-Reyes E, Yuan WL, Wei XY, Bers DM. Regulation of the cloned L-type
cardiac calcium channel by cyclic-AMP-dependent protein kinase. FEBS Lett 1994;342(2):119-123.
19. Zong XG, Schreieck J, Mehrke G, Welling A, Schuster A, Bosse E, Flockerzi V, Hofmann F. On the regulation of the expressed L-type calcium channel by cAMP- dependent phosphorylation. Pflügers Arch 1995;430(3):340-347.
20. Mikala G, Klockner U, Varadi M, Eisfeld J, Schwartz A , Varadi G. cAMP-dependent phosphorylation sites and macroscopic activity of recombinant cardiac L-type calcium channels. Mol Cell Biochem 1998;185(1-2):95-109.
21. Gao TY, Yatani A, Dell'Acqua ML, Sako H, Green SA, Dascal N, Scott JD, Hosey MM. cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 1997;19(1):185- 196.
22. Hulme JT, Lin TW, Westenbroek RE, Scheuer T, Catterall WA. ß-adrenergic
regulation requires direct anchoring of PKA to cardiac Cav1.2 channels via a leucine zipper interaction with A kinase-anchoring protein 15. Proc Natl Acad Sci USA 2003;100 (22):13093-8.
23. Hulme JT, Westenbroek RE, Scheuer T, Catterall WA. Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Cav1.2 channels during ß1-adrenergic regulation. Proc Natl Acad Sci USA 2006;103(44):16574-9.
24. Hulme JT, Yarov-Yarovoy V, Lin TW , Scheuer T, Catterall WA. Autoinhibitory control of the Cav1.2 channel by its proteolytically processed distal C-terminal domain. J Physiol 2006;576(Pt 1):87-102.
25. Ganesan AN, Maack C, Johns DC, Sidor A, O'Rourke B. ß-Adrenergic stimulation of L-type Ca2+ channels in cardiac myocytes requires the distal carboxyl terminus of alpha1C but not serine 1928. Circ Res 2006;98(2):e11-8.
26. Lemke T, Welling A, Christel CJ, Blaich A, Bernhard D, Lenhardt P, Hofmann F , Moosmang S. Unchanged ß-adrenergic stimulation of cardiac L-type calcium channels in Cav1.2 phosphorylation site S1928A mutant mice. J Biol Chem
2008;283(50):34738-44.
27. Gerhardstein BL, Puri TS, Chien AJ, Hosey MM. Identification of the sites
phosphorylated by cyclic AMP-dependent protein kinase on the ß2 subunit of L-type voltage-dependent calcium channels. Biochemistry (Mosc) 1999;38(32):10361-70.
28. Bunemann M, Gerhardstein BL, Gao TY, Hosey MM. Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the ß2 subunit. J Biol Chem 1999;274(48):33851-33854.
29. Haase H, Alvarez J, Petzhold D, Doller A, Behlke J, Erdmann J, Hetzer R, Regitz- Zagrosek V, Vassort G, Morano I. Ahnak is critical for cardiac CaV1.2 calcium channel function and its ß-adrenergic regulation. FASEB J 2005;19(14):1969-77.
30. Fuller MD, Emrick MA, Sadilek M, Scheuer T, Catterall WA. Molecular mechanism of calcium channel regulation in the fight-or-flight response. Sci Signal
2010;3(141):ra70.
31. Hulme JT, Konoki K, Lin TW, Gritsenko MA, Camp DG 2nd, Bigelow DJ, Catterall WA. Sites of proteolytic processing and noncovalent association of the distal C- terminal domain of Cav1.1 channels in skeletal muscle. Proc Natl Acad Sci USA 2005;102( 14):5274-9.
32. Fu Y, Westenbroek RE, Yu FH, Clark JP 3rd, Marshall MR, Scheuer T, Catterall WA.
Deletion of the distal C terminus of Cav1.2 channels leads to loss of ß-adrenergic regulation and heart failure in vivo. J Biol Chem 2011;286(14):12617-26.
33. Xiang YK. Compartmentalization of beta-adrenergic signals in cardiomyocytes. Circ Res 2011;109(2):231-44.
34. Rochais F, Abi-Gerges A, Horner K, Lefebvre F, Cooper DMF, Conti M, Fischmeister R, Vandecasteele G. A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes.
Circ Res 2006;98(8):1081-1088.
35. Fischmeister R, Castro LRV, Abi-Gerges A, Rochais F, Jurevičius J, Leroy J, Vandecasteele G. Compartmentation of cyclic nucleotide signaling in the heart: The role of cyclic nucleotide phosphodiesterases. Circ Res 2006;99(8):816-828.
36. Perino A, Ghigo A, Scott JD, Hirsch E. Anchoring proteins as regulators of signaling pathways. Circ Res 2012;111(4):482-92.
37. Diviani D, Maric D, Lopez IP, Cavin S, Del Vescovo CD . A-kinase anchoring proteins: Molecular regulators of the cardiac stress response. Biochim Biophys Acta 2012;
38. Morisco C, Zebrowski DC, Vatner DE, Vatner SF, Sadoshima J. ß-Adrenergic cardiac hypertrophy is mediated primarily by the ß1-subtype in the rat heart. J Mol Cell Cardiol 2001;33(3):561-573.
39. Ahmet I, Krawczyk M, Heller P, Moon C, Lakatta EG, Talan MI. Beneficial effects of chronic pharmacological manipulation of ß-adrenoreceptor subtype signaling in rodent dilated ischemic cardiomyopathy. Circulation 2004;110(9):1083-90.
40. Communal C, Singh K, Sawyer DB, Colucci WS. Opposing effects of ß1- and ß2- adrenergic receptors on cardiac myocyte apoptosis - Role of a pertussis toxin-sensitive G proteins. Circulation 1999;100(22):2210-2212.
41. Zaugg M, Xu WM, Lucchinetti E, Shafiq SA, Jamali NZ, Siddiqui MAQ. ß- Adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes. Circulation 2000;102(3):344-350.
42. Brodde OE, Bruck H, Leineweber K. Cardiac adrenoceptors: Physiological and
pathophysiological relevance. J Pharmacol Sci 2006;100(323-337.
43. Chen-Izu Y, Xiao RP, Izu LT, Cheng H, Kuschel M, Spurgeon H, Lakatta EG. Gi- dependent localization of 2-adrenergic receptor signaling to L-type Ca2+ channels.
Biophys J 2000;79(5):2547-2556.
44. Xiang Y, Rybin VO, Steinberg SF, Kobilka B. Caveolar localization dictates
physiologic signaling of β2-adrenoceptors in neonatal cardiac myocytes. J Biol Chem 2002;277(37):34280-34286.
45. Calaghan S, Kozera L, White E. Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte. J Mol Cell Cardiol 2008;45(1):88-92.
46. Nikolaev VO, Moshkov A, Lyon AR, Miragoli M, Novak P, Paur H, Lohse MJ, Korchev YE, Harding SE, Gorelik J. ß2-Adrenergic receptor redistribution in heart failure changes cAMP compartmentation. Science 2010;327(5973):1653-7.
47. Balijepalli RC, Foell JD, Hall DD, Hell JW, Kamp TJ. Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for ß2- adrenergic regulation. Proc Natl Acad Sci USA 2006;103(19):7500-7505.
48. Xiao RP, Lakatta EG. ß1-Adrenoceptor stimulation and ß2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2+, and Ca2+ current in single rat ventricular cells. Circ Res 1993;73(286-300.
49. Nikolaev VO, Bunemann M, Schmitteckert E, Lohse MJ, Engelhardt S. Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching ß1-adrenergic but locally confined ß2-adrenergic receptor-mediated signaling. Circ Res 2006;99(10):1084-1091.
50. Rozec B, Gauthier C. ß3-Adrenoceptors in the cardiovascular system: Putative roles in human pathologies. Pharmacol Ther 2006;111(652-673.
51. Gauthier C, Tavernier G, Charpentier F, Langin D, Lemarec H. Functional β3- adrenoceptor in the human heart. J Clin Invest 1996;98(556-562.
52. Moniotte S, Vaerman J, Kockx MM, Larrouy D, Langin D, Noirhomme P, Balligand J. Real-time RT-PCR for the detection of beta-adrenoceptor messenger RNAs in small human endomyocardial biopsies. J Mol Cell Cardiol 2001;33(12):2121-2133.
53. Chamberlain PD, Jennings KH, Paul F, Cordell J, Berry A, Holmes SD, Park J, Chambers J, Sennitt MV, Stock MJ, Cawthorne MA, Young PW, Murphy GJ. 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. Int J Obes Relat Metab Disord 1999;23(10):1057-1065.
54. Moniotte S, Kobzik L, Feron O, Trochu JN, Gauthier C, Balligand JL. Upregulation of β3-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium. Circulation 2001;103(12):1649-1655.
55. De Matteis R, Arch JR, Petroni ML, Ferrari D, Cinti S, Stock MJ.
Immunohistochemical identification of the β3-adrenoceptor in intact human adipocytes and ventricular myocardium: effect of obesity and treatment with ephedrine and caffeine. Int J Obes Relat Metab Disord 2002;26(11):1442-1450.
56. Skeberdis VA, Gendvilienë V, Zablockaitë D, Treinys R, Mačianskienë R, Bogdelis A, Jurevičius J, Fischmeister R . ß3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current. J Clin Invest 2008;118(3219-3227.
57. Schoemaker RG, Du XY, Bax WA, Bos E, Saxena PR. 5-Hydroxytryptamine stimulates human isolated atrium but not ventricle. Eur J Pharmacol 1993;230(103- 105.
58. Jahnel U, Rupp J, Ertl R, Nawrath H. Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle. Naunyn - Schmiedebergs Arch Pharmacol 1992;346(482-485.
59. Ouadid H, Seguin J, Dumuis A, Bockaert J, Nargeot J. Serotonin increases calcium current in human atrial myocytes via the newly described 5-hydroxytryptamine4
receptors. Mol Pharmacol 1992;41(346-351.
60. Scott JD, Santana LF. A-kinase anchoring proteins: getting to the heart of the matter.
Circulation 2010;121(10):1264-71.
61. Kritzer MD, Li J, Dodge-Kafka K, Kapiloff MS. AKAPs: The architectural underpinnings of local cAMP signaling. J Mol Cell Cardiol 2011;
62. Jones BW, Brunet S, Gilbert ML, Nichols CB, Su T, Westenbroek RE, Scott JD, Catterall WA, McKnight GS. Cardiomyocytes from AKAP7 knockout mice respond normally to adrenergic stimulation. Proc Natl Acad Sci U S A 2012;109(42):17099- 104.
63. Nichols CB, Rossow CF, Navedo MF, Westenbroek RE, Catterall WA, Santana LF, McKnight GS. Sympathetic stimulation of adult cardiomyocytes requires association of AKAP5 with a subpopulation of L-type calcium channels. Circ Res
2010;107(6):747-56.
64. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (Ryanodine receptor): Defective regulation in failing hearts. Cell 2000;101(4):365- 376.
65. Marx SO, Kurokawa J, Reiken S, Motoike H, D'Armiento J, Marks AR, Kass RS.
Requirement of a macromolecular signaling complex for β adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. Science 2002;295(5554):496- 499.
66. Dodge-Kafka KL, Soughayer J, Pare GC, Carlisle Michel JJ, Langeberg LK, Kapiloff MS, Scott JD. The protein kinase A anchoring protein mAKAP co-ordinates two
integrated cAMP effector pathways. Nature 2005;437(574-578.
67. Efendiev R, Samelson BK, Nguyen BT, Phatarpekar PV, Baameur F, Scott JD, Dessauer CW. AKAP79 interacts with multiple adenylyl cyclase (AC) isoforms and scaffolds AC 5 and 6 to AMPA receptors. J Biol Chem 2010;285(19):14450-8.
68. Bauman AL, Soughayer J, Nguyen BT, Willoughby D, Carnegie GK, Wong W, Hoshi N, Langeberg LK, Cooper DM, Dessauer CW, Scott JD. Dynamic regulation of cAMP synthesis through anchored PKA-adenylyl cyclase V/VI complexes. Mol Cell
2006;23(6):925-931.
69. Dodge KL, Khouangsathiene S, Kapiloff MS, Mouton R, Hill EV, Houslay MD, Langeberg LK, Scott JD. mAKAP assembles a protein kinase A/PDE4
phosphodiesterase cAMP signaling module. EMBO J 2001;20(8):1921-1930.
70. Perino A, Ghigo A, Ferrero E, Morello F, Santulli G, Baillie GS, Damilano F, Dunlop AJ, Pawson C, Walser R, Levi R, Altruda F, Silengo L, Langeberg LK, Neubauer G, Heymans S, Lembo G, Wymann MP, Wetzker R, Houslay MD, Iaccarino G, Scott JD, Hirsch E. Integrating cardiac PIP3 and cAMP signaling through a PKA anchoring function of p110gamma. Mol Cell 2011;42(1):84-95.
71. Ghigo A, Perino A, Mehel H, Zahradnikova AJr, Morello F, Leroy J, Nikolaev VO, Damilano F, Cimino J, De Luca E, Richter W, Westenbroek R, Catterall WA, Zhang J, Yan C, Conti M, Gomez AM, Vandecasteele G, Hirsch E, Fischmeister R. PI3Kγ Protects against catecholamine-induced ventricular arrhythmia through PKA-mediated regulation of distinct phosphodiesterases. Circulation 2012;PMID: 23008439 (in press).
72. Terrenoire C, Houslay MD, Baillie GS, Kass RS. The cardiac IKs potassium channel macromolecular complex includes the phosphodiesterase PDE4D3. J Biol Chem 2009;284(14):9140-6.
73. Rich TC, Fagan KA, Nakata H, Schaack J, Cooper DMF, Karpen JW. Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. J Gen Physiol 2000;116(2):147-161.
74. Mika D, Leroy J, Vandecasteele G, Fischmeister R. PDEs create local domains of cAMP signaling. J Mol Cell Cardiol 2012;52(323-329.
75. Méry P-F, Pavoine C, Pecker F, Fischmeister R. Erythro-9-(2-hydroxy-3-
nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterase in isolated cardiac myocytes. Mol Pharmacol 1995;48(1):121-130.
76. Rivet-Bastide M, Vandecasteele G, Hatem S, Verde I, Benardeau A, Mercadier JJ, Fischmeister R. cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes. J Clin Invest 1997;99(11):2710-2718.
77. Verde I, Vandecasteele G, Lezoualc'h F, Fischmeister R. Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+
current in rat ventricular myocytes. Br J Pharmacol 1999;127(1):65-74.
78. Vandecasteele G, Verde I, Rucker-Martin C, Donzeau-Gouge P, Fischmeister R.
Cyclic GMP regulation of the L-type Ca2+ channel current in human atrial myocytes. J Physiol 2001;533(2):329-340.
79. Dittrich M, Jurevicius J, Georget M , Rochais F, Fleischmann BK, Hescheler J, Fischmeister R. Local response of L-type Ca2+ current to nitric oxide in frog ventricular myocytes. J Physiol 2001;534(1):109-121.
80. Kirstein M, Rivet-Bastide M, Hatem S, Bénardeau A, Mercadier JJ, Fischmeister R.
Nitric oxide regulates the calcium current in isolated human atrial myocytes. J Clin Invest 1995;95(2):794-802.
81. Richter W, Xie M, Scheitrum C, Krall J, Movsesian MA, Conti M. Conserved expression and functions of PDE4 in rodent and human heart. Basic Res Cardiol
2011;106(2):249-62.
82. Mongillo M, McSorley T, Evellin S, Sood A, Lissandron V , Terrin A, Huston E , Hannawacker A, Lohse MJ, Pozzan T, Houslay MD, Zaccolo M. Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases.
Circ Res 2004;95(1):65-75.
83. Rochais F, Vandecasteele G, Lefebvre F, Lugnier C, Lum H, Mazet J-L, Cooper DMF, Fischmeister R. 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. J Biol Chem 2004;279(50):52095-52105.
84. Leroy J, Abi-Gerges A, Nikolaev VO, Richter W, Lechęne P , Mazet J-L, Conti M , Fischmeister R, Vandecasteele G. Spatiotemporal dynamics of ß-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: Role of phosphodiesterases. Circ Res 2008;102(9):1091-1100.
85. Baillie GS, Sood A, McPhee I, Gall I, Perry SJ, Lefkowitz RJ, Houslay MD. β- Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates β-
adrenoceptor switching from Gs to G i. Proc Natl Acad Sci USA 2003;100(3):941-945.
86. Richter W, Day P, Agraval R, Bruss MD, Granier S, Wang YL, Rasmussen SGF , Horner K, Wang P, Lei T, Patterson AJ, Kobilka BK, Conti M . Signaling from ß1- and ß2-adrenergic receptors is defined by differential interactions with PDE4. Embo J 2008;27(2):384-393.
87. De Arcangelis V, Liu R, Soto D, Xiang Y. Differential association of
phosphodiesterase 4D isoforms with ß2-adrenoceptor in cardiac myocytes. J Biol Chem 2009;284(49):33824-32.
88. Lehnart SE, Wehrens XHT, Reiken S, Warrier S, Belevych AE, Harvey RD, Richter W, Jin SLC, Conti M, Marks A. Phosphodiesterase 4D deficiency in the ryanodine receptor complex promotes heart failure and arrhythmias. Cell 2005;123(1):23-35.
89. Kerfant BG, Zhao D, Lorenzen-Schmidt I, Wilson LS, Cai S, Chen SR, Maurice DH, Backx PH. PI3KY is required for PDE4, not PDE3, activity in subcellular
microdomains containing the sarcoplasmic reticular calcium ATPase in cardiomyocytes. Circ Res 2007;101(4):400-8.
90. Beca S, Helli PB, Simpson JA, Zhao D, Farman GP, Jones P, Tian X, Wilson LS, Ahmad F, Chen SR, Movsesian MA, Manganiello V, Maurice DH, Conti M, Backx PH. Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca2+ release and cardiac contractility, independently of L-type Ca2+ current. Circ Res
2011;109(9):1024-30.
91. Leroy J, Richter W, Mika D, Castro LRV, Abi-Gerges A, Xie M, Scheitrum C, Lefebvre F, Schittl J, Westenbroek R, Catterall WA, Charpentier F, Conti M, Fischmeister R, Vandecasteele G. Phosphodiesterase 4B in the cardiac L-type Ca2+
channel complex regulates Ca2+ current and protects against ventricular arrhythmias. J Clin Invest 2011;121(7):2651-61.
92. Weishaar RE, Kobylarz-Singer DC, Steffen RP, Kaplan HR. Subclasses of cyclic AMP-specific phosphodiesterase in left ventricular muscle and their involvement in regulating myocardial contractility. Circ Res 1987;61(539-547.
93. Lugnier C, Muller B, Lebec A, Beaudry C, Rousseau E. Characterization of indolidan- sensitive and rolipram-sensitive cyclic nucleotide phosphodiesterases in canine and human cardiac microsomal fractions. J Pharmacol Exp Ther 1993;265(1142-1151.
94. Smith CJ, Huang R, Sun D, Ricketts S, Hoegler C, Ding JZ, Moggio RA, Hintze TH.
Development of decompensated dilated cardiomyopathy is associated with decreased
gene expression and activity of the milrinone-sensitive cAMP phosphodiesterase PDE3A. Circulation 1997;96(9):3116-3123.
95. Osadchii OE. Myocardial phosphodiesterases and regulation of cardiac contractility in health and cardiac disease. Cardiovasc Drugs Ther 2007;21(171-94.
96. Monrad ES, Baim DS, Smith HS, Lanoue AS, Silverman KJ , Gervino EV, Grossman W. Assessment of long-term therapy with milrinone and the effects of milrinone withdrawal. Circulation 1986;73(3 Pt 2):III205-12.
97. Molina CE, Leroy J, Xie M, Richter W, Lee I-O, Maack C, Rucker-Martin C,
Donzeau-Gouge P, Verde I, Hove-Madsen L, Barriga M, Conti M, Vandecasteele G, Fischmeister R. Cyclic AMP phosphodiesterase type 4 protects against atrial
arrhythmias. J Am Coll Cardiol 2012;59(24):2182-2190.
98. Rapundalo ST, Solaro RJ, Kranias EG. 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. Circ Res 1989;64(104-111.
99. Hohl CM, Li Q. Compartmentation of cAMP in adult canine ventricular myocytes - Relation to single-cell free Ca2+ transients. Circ Res 1991;69(1369-1379.
100. Jurevicius J, Fischmeister R. cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by ß-adrenergic agonists. Proc Natl Acad Sci USA 1996;93(1):295-299.
101. Zaccolo M, Pozzan T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 2002;295(5560):1711-1715.
102. Nikolaev VO, Lohse MJ. Monitoring of cAMP synthesis and degradation in living cells. Physiology (Bethesda) 2006;21(86-92.
103. Ono K, Fozzard HA. Phosphorylation restores activity of L-type calcium channels
