Cyclic nucleotide-mediated signalling is highly compartmentalised in cardiac myocytes, and the roles of individual phosphodiesterases in regulating cAMP- and cGMP-mediated signalling depend upon their intracellular localisation. While PDE3A and PDE3B are both expressed in cardiac myocytes (PDE3A more abundantly [
25]), their intracellular distributions are distinct, with PDE3A localised mainly to the sarcoplasmic reticulum and PDE3B to T tubules in proximity to mitochondria [
26] (
Figure 1). PDE3 activity is also associated with nuclear membranes in cardiac myocytes [
27], though the specific isoforms have not been delineated. Furthermore, in subcellular preparations from cardiac muscle, PDE3A1 is recovered solely in microsomal fractions, while PDE3A2 and PDE3A3 are recovered in cytosolic as well as microsomal fractions [
16]. This corresponds to studies in cells transfected with PDE3A1- and PDE3B-derived constructs that show that the N-terminal hydrophobic loops in these isoforms direct the insertion of these proteins into lipid membranes [
17,
18].
3.1. PDE3A
The precise intracellular localisation of PDE3 isoforms depends upon their interactions with anchoring, scaffold, and adaptor proteins that recruit the enzyme to multiprotein signalling complexes [
28,
29,
30,
31,
32,
33]. Localised A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signalling proteins—adenylyl cyclases, phosphatases, Epacs, PDEs and other effector molecules—to ‘signalosomes’ that allow selective phosphorylation of individual PKA substrates [
31,
32,
33,
34].
PDE3 has long been known to be associated with the sarcoplasmic reticulum of cardiac myocytes [
35,
36]. Confocal microscopy studies more recently demonstrated co-localisation of PDE3A with SERCA2, AKAP18, phospholamban, and desmin in the Z-bands of cardiac myocytes [
37,
38]. PDE3A was found to be a constituent of a multiprotein complex in the sarcoplasmic reticulum containing AKAP18, phospholamban, and SERCA2 [
38,
39]. Addition of cAMP to microsomes from human heart results in the phosphorylation of phospholamban by endogenous PKA; this leads to a dissociation of phospholamban from SERCA2 and an increase in SERCA2 activity, and this effect is potentiated by PDE3 inhibition [
38] (
Figure 2). Although PDE3 and PDE4 have both been found to co-immunoprecipitate with AKAP-based signalosomes from human and mouse myocardium and modulate effects of cAMP on
L-type Ca
2+ channels, ryanodine-sensitive Ca
2+ channels, and SERCA2 [
40,
41,
42,
43], only PDE3 inhibition potentiates the PKA-mediated phosphorylation of phospholamban and the consequent stimulation of SERCA2 activity [
37]. The role of PDE3A in modulating these effects is the likely explanation for the inotropic actions of PDE3 inhibition.
Furthermore, while PDE3 isoforms regulate the phosphorylation of other proteins through the cAMP/PKA pathway, they are themselves substrates for protein kinases that modulate their catalytic activity and protein–protein interactions [
19,
21,
44,
45,
46,
47]. The incorporation of PDE3A into the SERCA2 complex is an example. In co-immunoprecipitation experiments, phosphorylation of endogenous PDE3A by PKA increases its interactions with SERCA2, caveolin-3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 [
38]. Studies with recombinant proteins showed that phosphorylation of PDE3A by PKA increased its co-immunoprecipitation with SERCA2a and AKAP18, suggesting that PDE3A interacts directly with both proteins in a phosphorylation-dependent manner [
38]. Deletion of the N-terminal region of PDE3A1/PDE3A2 blocked PKA-induced phosphorylation of PDE3A and its interaction with recombinant SERCA2. Of particular interest is the sequence RRRRSSS (amino acids 288–294 of the open reading frame, which are found only in PDE3A1), which provides three serines that can be phosphorylated in vitro by different kinases under different conditions [
19]. The introduction of serine-to-alanine substitutions at S292-4 identified this sequence as the principal site responsible for regulating the interactions of PDE3A1 with SERCA2 (
Figure 1).
The absence of the S292-4 site from PDE3A2 and PDE3A3 establishes a PDE3A1-specific mechanism for recruitment of PDE3A to the SERCA2 complex. The protein–protein interactions of PDE3 isoforms are distinct in other ways as well, as has been described for the phosphorylation-dependent interactions of PDE3A1, PDE3A2, and PDE3B with 14-3-3 [
20,
21,
48,
49,
50]. The common sequence of PDE3A1 and PDE3A2 includes two 14-3-3-binding sites: S428, a PKC site; and S312, a PKA/PKB (alternatively referred to Akt) site that resembles S318, a 14-3-3-binding site in PDE3B [
48,
49] (
Figure 1). In vitro, PDE3A1 is preferentially phosphorylated by PKA at S312, whereas PDE3A2 is preferentially phosphorylated by PKC at S428; in preparations from human hearts, PDE3A1 is phosphorylated primarily at S312, while PDE3A2 is phosphorylated primarily at S428 [
50]. Furthermore, in transfected HEK293 cells, the phosphorylation-dependent interactomes of PDE3A1 and PDE3A2 are distinct [
50]: PDE3A1 interacts with the 5-HT
4(b) receptor, for example, while PDE3A2, PDE3A3, and PDE3B do not [
51]. These unique protein–protein interactions may provide opportunities for isoform-specific targeting of the protein–protein interactions of individual isoforms.
In cardiac myocytes, PDE3A is also part of a multiprotein complex containing the unconventional AKAP PI3Kγ [
52]. While PI3Kγ directly binds the RII subunit of the PKA holoenzyme, how PDE3A contacts the complex is still unclear, though a likely intermediate is the PI3Kγ interactor p84/p87 [
53]. Within this complex, PKA exerts a negative feedback regulation by phosphorylating and activating the different associated PDE isoforms, including PDE3A [
39]. In line with the role of PDE3A (and PDE4A/B) in controlling SERCA2, loss of the scaffold function of PI3Kγ leads to cAMP elevation and PKA-mediated hyperphosphorylation of phospholamban and
L-type Ca
2+ channels [
39,
54] (
Figure 3, left panel). In hearts lacking PI3Kγ and subjected to pressure overload, this effect causes cAMP elevation, contractile dysfunction, and increased mortality due to lethal arrhythmic events such as sustained ventricular tachycardia [
39] (
Figure 3, right panel). Interestingly, this abnormally increased cAMP accumulation appears to be selectively associated with signalling through β-adrenergic receptors localised to T tubules [
55]. This would suggest that the PI3Kγ PKA/PDE complex is part of an ‘insulating’ system that blocks ‘leakage’ of cAMP from this compartment to the sarcoplasmic reticulum [
39]. In heart failure, the expression of various elements of the PI3Kγ PKA/PDE3A-containing complex change their stoichiometric ratios, with reduced expression of p84/87 and coincident increased expression of PI3Kγ. This causes a dysfunctional cAMP regulation potentially contributing to the increase in ventricular arrhythmias associated with heart failure [
52].
3.2. PDE3B
In contrast to PDE3A, PDE3B is mainly located in caveolin-3-rich areas of the plasma membrane and, in cardiac myocytes, in the T tubules (
Figure 1). This indicates that the two PDE3 genes are specifically involved in the regulation of spatially and functionally distinct pools of cAMP. In line with this view, while PDE3A is involved in the regulation of contractility, PDE3B appears more connected to the regulation of metabolism. PDE3B is also known to have a role in the liver, pancreatic β cells, and in the brown and white adipose tissue where it is involved in the control of the anti-lipolytic effect of insulin, generally in contrast to β-adrenergic signalling [
56,
57]. In the heart, the specific inactivation of PDE3A causes alterations in basal cardiac contractility, while genetic ablation of PDE3B has no major impact on contractility but protects the myocardium from ischemic damage [
26,
37]. Interestingly, the protective effect of PDE3 inhibitors in ischemic heart preconditioning had been known for several years, but the precise nature of the PDE3 isoform only emerged with studies in knockout mice that excluded a role of PDE3A. The specific localisation of PDE3B in T tubules, and particularly both in dyads and in close proximity to mitochondria, suggests that this particular phosphodiesterase plays a role in modulating energy metabolism. Elevation in cAMP concentration in this location might mediate cardioprotective signals that improve mitochondrial function and energy supply during ischaemia/reperfusion, where mitochondrial Ca
2+ overload and consequent mitochondrial permeability transition (MPT) pore opening, oxidative stress, and apoptosis contribute to injury [
58]. In hearts lacking PDE3B, mitochondria are enriched in Bcl-2, produce lower amounts of reactive oxygen species, and show more numerous contacts with T tubules. In response to ischaemia, mitochondria from PDE3B-deficient cardiac myocytes are more resistant to Ca
2+-induced opening of the MPT pore, and associate with caveolin-3-enriched membrane subfractions containing cardioprotective proteins. The recruitment of these cardioprotective proteins to these subfractions is PKA-dependent, and can be reproduced in wild-type mice by PDE3 inhibition [
26].
The nature of the localization signal that keeps PDE3B in such spatially and functionally distinct cellular subdomains remains unknown. Various reports indicate that PDE3B can be recruited to different multiprotein complexes with distinct properties. In adipocytes, for example, treatment with insulin increases the phosphorylation of PDE3B associated with internal membranes, promoting its interactions with IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 (p85-subunit of phosphoinositide 3-kinase), PKB (protein kinase B), HSP-90 (heat-shock protein 90), 14-3-3, and a 50 kD protein [
46,
47,
56,
59,
60]. Conversely, treatment with β
3-adrenergic receptor agonists increases the phosphorylation of PDE3B associated with caveolin-1 in caveolae, promoting interactions with β
3-adrenergic receptors, PKA-RII (PKA regulatory subunit), and hormone-sensitive lipase [
46,
61]. Whether these associations also occur in cardiac myocytes is not yet clear. PDE3B has been reported to weakly associate in a complex containing the unconventional AKAP PI3Kγ, especially with overexpression in HEK293 cells [
53,
62]. Nonetheless, the significant effects on contractility and rhythm detected in mice lacking PI3Kγ indicate that the determinant of the cardiac phenotype is the ability of PI3Kγ to form more stable complexes with other phosphodiesterases, including PDE3A and PDE4A/B [
39].
Of note, however, is that PDE3B can be phosphorylated not only by PKA but also by PKB, the main effector of the PI3K pathway. Although the weak interaction between PI3Kγ and PDE3B reported in the heart awaits further experimental confirmation, severe reduction of the G protein-coupled receptor-driven PI3K pathway by the concomitant loss of PI3Kγ and PI3Kβ has been shown to lead to reduced PDE3B phosphorylation and activity in neurons [
63]. Nonetheless, whereas the specific association of PI3Kγ with β-adrenergic signalling in T tubules supports at least a spatial co-localisation of both PI3K and PDE3B [
39,
53], experiments in knockout mice indicate that PI3Kγ and PDE3B might be part of different functional complexes. On the other hand, the association of PI3Kγ with PKA triggers a negative feedback loop where PKA phosphorylates and inhibits PI3Kγ itself. Interestingly, loss of PDE3B in the heart leads to an unexpectedly strong elevation of cAMP [
26], which likely remains spatially confined due to the normal activity of the other phosphodiesterases. Whether this elevated cAMP and PKA activity influences PI3Kγ catalytic activity and, indirectly, the PKB signalling axis is yet to be determined. As enhanced PI3Kγ signalling is involved in the downregulation of β-adrenergic receptor density on the cell surface and, generally, in cardiac myocyte decompensation under stress, PDE3B inhibition could reduce a detrimental signal [
64]. Inhibitors specifically distinguishing PDE3B from PDE3A are not currently available, but specific targeting of PDE3B may be therapeutically useful for ischaemia/reperfusion injury.