Introduction
Protein kinase D (PKD) is a serine/threonine kinase that consists of an N-terminal regulatory domain (containing two cysteine-rich zinc finger-like motifs and a pleckstrin homology domain) and a C-terminal catalytic domain [
20,
34]. PKD has been shown to be activated in vitro by diacylglycerol (DAG) and 12-phorbol 13-myristate ester (PMA) [
35]. In addition, phosphorylation of Ser744 and Ser748 in the kinase activation loop of PKD by protein kinase C (PKC) results in DAG-independent catalytic activity [
42]. Although PKD can act either in parallel with or downstream of PKC, previous studies suggest that the latter is the principal mechanism of PKD activation in various cell types [
7,
39,
42].
Previously, we have shown that PKD regulates myofilament Ca
2+ sensitivity, most likely through its phosphorylation of cardiac troponin I (cTnI) [
8,
15], which we have confirmed recently using mouse myocardium expressing non-phosphorylatable cTnI [
5]. In addition, work from the Olson laboratory [
14,
37] has shown that PMA and the α
1-adrenergic receptor (AR) agonist phenylephrine (PE) induce cardiac hypertrophy in neonatal rat ventricular myocytes by a PKD-dependent mechanism, which involves the direct phosphorylation and subsequent nuclear export of histone deacetylase 5 (HDAC5). A role for PKD in HDAC5 redistribution has been supported by studies in failing rabbit and human myocardium [
6], and recent evidence from mice with cardiac-specific PKD deletion indicate that PKD is necessary for the full manifestation of pathological cardiac remodelling in response to stress [
13]. Taken together, these findings indicate that PKD plays multiple important roles in cardiac myocytes, making it imperative to better understand the mechanisms that regulate its activity in this cell type.
We have previously investigated the roles of PKC isoforms and cAMP-dependent protein kinase (PKA) in the regulation of PKD activity in adult rat ventricular myocytes (ARVM) [
17]. We found that PKCε plays a predominant role in endothelin-1 (ET1)-induced PKD activation and such activation is inhibited by a PKA-mediated pathway. This counter regulation of PKD by PKA could have important implications, both under normal circumstances where PKA activity is likely to suppress PKD activation and circumstances where PKA activation is impaired, such as in heart failure [
22]. Myocardial PKA activity is thought to be controlled in a localised manner through several mechanisms, including the binding of PKA regulatory subunits to A-kinase anchoring proteins (AKAPs) [
12] and the maintenance of discrete cAMP pools by the breakdown of cAMP by compartmentalised phosphodiesterase (PDE) isoforms [
4,
27,
36,
40]. The principal PDE isoforms that target cAMP in cardiac myocytes are believed to be PDE2, PDE3 and PDE4 [
38], and it has been suggested that these isoforms have distinct roles in regulating PKA activity in different subcellular compartments, and thereby control different downstream events [
21,
26‐
28,
31,
38,
41].
The principal aim of the present study was to determine if the PKA activity that inhibits PKD activation in ARVM is under the control of distinct PDE isoform(s) in a compartmentalised manner. Our findings indicate that in the presence of basal adenylate cyclase activity, PDE3 and PDE4 regulate the PKA activity that inhibits PKD activation in a redundant manner. However, we find no evidence for a specific role for PDE3 and PDE4 in this process, since PKA-mediated inhibition of PKD activation (which is likely to occur at the sarcolemma) and PKA-mediated phosphorylation of cTnI (at the sarcomere) and phospholamban (PLB; at the sarcoplasmic reticulum) were similarly regulated by these PDE isoforms.
Discussion
The major findings of this study are that in adult cardiac ventricular myocytes: (1) PDE3 and PDE4 regulate the PKA activity that inhibits ET1-induced PKD activation; (2) the PKA activities that are responsible for PLB phosphorylation at the sarcoplasmic reticulum and cTnI phosphorylation within the sarcomere are also regulated by the same PDE isoforms, PDE3 and PDE4; (3) PDE3 and PDE4 appear to operate in a redundant manner in the above process, such that their individual inhibition has little impact but their combined inhibition markedly increases PKA activity; (4) PKA activation, either by non-selective PDE inhibition or by β-AR stimulation, inhibits PKD activation not only by ET1 but also by α1-AR stimulation, suggesting that the pertinent inhibitory crosstalk mechanism regulates PKD activation by multiple GqPCR signalling pathways.
The marked inhibitory effect of the non-selective PDE inhibitor IBMX on ET1-induced PKD activation is consistent with our earlier study that first reported a PKA-mediated inhibitory crosstalk mechanism in PKD regulation [
17], and additionally suggests that PDE inhibition produces a sufficient increase in the PKA activity that inhibits PKD activation, even in the absence of receptor-mediated adenylate cyclase stimulation. The data we have obtained with isoform-selective PDE inhibitors indicate that PDE3 and PDE4 are the principal isoforms that regulate the pertinent PKA activity, and that these PDE isoforms operate in a redundant manner, such that inhibition of both is required to achieve marked inhibition of PKD activation. Upon exposure of myocytes to ET1, PKC-mediated PKD activation is likely to occur at the sarcolemma, following the recruitment of novel PKC isoforms and PKD itself to this compartment through the interaction of their cysteine-rich zinc finger domains with DAG within the lipid bilayer and DAG-mediated PKC activation [
2]. On the basis of our current findings, it is reasonable to speculate that PDE3 and PDE4 are responsible for regulating cAMP levels and therefore PKA activity at a sarcolemmal compartment where PKD activation occurs. Consistent with such a possibility, in unstimulated ARVM, the combined inhibition of PDE3 and PDE4 has been shown to increase the activity of a sarcolemmal PKA target, the L-type Ca
2+ channel [
38], while the individual inhibition of PDE2, PDE3 or PDE4 was without effect [
31,
38].
On the other hand, our findings regarding the phosphorylation of known PKA targets in other myocyte compartments argue against specific roles for PDE3 and PDE4 in regulating PKA activity exclusively at a sarcolemmal compartment. We have found that the combined inhibition of PDE3 and PDE4 was sufficient and necessary also for inducing marked phosphorylation of cTnI and PLB at their PKA-targeted residues and producing significant functional effects on myocyte contraction, relaxation and Ca
2+ transients, indicating that PDE3 and PDE4 regulate PKA activities additionally at sarcomeric and sarcoplasmic reticular compartments. In this context, it is worth noting that PDE3 and PDE4 are responsible for the vast majority of the total PDE activity in rat cardiac myocytes [
26,
31]. It is possible, therefore, that the similar effects of the various PDE inhibitors on PKD activation and cTnI and PLB phosphorylation in our studies reflect their quantitative effects on total cellular PDE activity, rather than indicating specific roles for PDE3 and PDE4 in regulating PKA activity in defined subcellular compartments. Nevertheless, compartment-specific roles of PDE isoforms may become more evident in the presence of receptor-mediated stimulation of cAMP generation by adenylate cyclase; indeed, while combined inhibition of PDE3 and PDE4 was required to increase L-type Ca
2+ channel activity in unstimulated ARVM [
38], PDE4 inhibition alone was sufficient to potentiate the induction of such channel activity by sub-maximal β-AR stimulation [
23,
31,
38]. An interesting corollary finding in our study was that combined PDE3 and PDE4 inhibition and β-AR stimulation each produced comparable effects on myocyte protein phosphorylation (Fig.
4) and function (Fig.
5), suggesting the presence of considerable basal adenylate cyclase activity in this preparation.
In the course of the present study, we have observed a differential impact of PKA activation, either by PDE inhibition or by β-AR stimulation, on the appearance of two phosphoprotein moieties that are detected by the phospho-Ser498 HDAC5 antibody. ET1 or PE stimulation markedly increased the phosphorylation of both 140-kDa moiety and 105-kDa moiety, but the phosphorylation status of the 105-kDa protein tracked more faithfully the changes in PKD phosphorylation that arose from PKA activation, consistent with this protein being a downstream target of activated PKD. Although the identities of the two phosphoproteins cannot be ascertained from the present study, it is notable that the motif around the PKD target site in HDAC5, Ser498 (PLSRTQ
SSPL; underlining indicates the phosphorylated residue) [
19], is quite well conserved among other members of the class II HDAC family (e.g. Ser630 in HDAC4, PLSRAQ
SSPA; Ser457 in HDAC7, PLSRTQ
SSPA; Ser449 in HDAC9, PLNRTQ
SAPL). Since the phospho-Ser498 HDAC5 antibody was raised against a synthetic phosphopeptide derived from the HDAC5 sequence around Ser498, such homology suggests that the 140- and 105-kDa phosphoproteins detected by this antibody are likely to be different HDAC isoforms. It would be important to determine how the phosphorylation and nuclear export of specific HDAC isoforms are modulated by the PKA-mediated inhibition of PKD activation that we have delineated in our previous [
17] and current work, and to investigate the impact of such inhibitory crosstalk on HDAC-regulated transcriptional reprogramming processes that facilitate cardiac hypertrophy and remodelling upon neurohormonal stimulation [
3]. In this context, there is evidence in both adult [
32] and neonatal [
29] cardiac myocytes that the pro-hypertrophic consequences of α
1-AR stimulation are attenuated by β-AR stimulation through a PKA-mediated pathway, although the molecular mechanism(s) underlying this phenomenon have not been deciphered. Furthermore, recent evidence suggests that in failing myocardium, in which the downregulation of β-AR-mediated signalling pathways is a common phenomenon [
25], ET1-induced nuclear export of HDAC5 is potentiated, at least in part through a PKD-mediated pathway [
6]. These findings support the possibility that PKA-mediated inhibition of PKD activation is of physiological significance, particularly in the context of cardiac hypertrophy and failure.
The upstream molecular mechanism(s) that are responsible for PKA-mediated inhibition of PKD activation in cardiac myocytes remain unknown. Our observation that PKA activation, either by PDE inhibition or by β-AR stimulation, inhibits PKD activation both by ET1 and by PE indicates that the inhibitory crosstalk mechanism regulates PKD activation by multiple G
qPCR signalling pathways, and suggests that this mechanism must target one or more components that are common to such pathways. Putative PKA targets within the pertinent inhibitory crosstalk mechanism include phospholipase C (PLC) isoforms, since in non-cardiac cells PLC-β
2 and -β
3 have been shown to be directly phosphorylated by PKA, resulting in their inactivation [
1,
24]. Furthermore, regulator of G protein signalling (RGS) 4 and G protein-coupled receptor kinase 2 (which contains an RGS domain) have also been shown to be directly phosphorylated by PKA, with such phosphorylation increasing their affinity for Gα
q and thereby inhibiting G
qPCR-mediated stimulation of PLC activity [
18]. If such proximal inhibitory mechanisms were operative in cardiac myocytes, however, they would be expected to attenuate not only PKD activation but also additional G
qPCR-mediated signalling events.
The present study has provided novel information regarding the mechanisms that regulate PKA-mediated inhibition of PKD activation in cardiac myocytes, particularly by revealing the roles of PDE3 and PDE4 and by showing that the inhibitory crosstalk process affects PKD activation by multiple GqPCR-mediated pathways. Further work is required to determine both the downstream functional consequences and the upstream molecular mechanisms of such crosstalk.