PDE4DIP in health and diseases

Abstract

Cyclic-AMP (cAMP), the first second messenger to be identified, is synthesized, and is universally utilized as a second messenger, and plays important roles in integrity, and function of organs, including heart. Through its coupling with other intracellular messengers, cAMP facilitates excitation-contraction coupling, increases heart rate and conduction velocity. It is degraded by a class of enzymes called cAMP-dependent phosphodiesterase (PDE), with PDE3 and PDE4 being the predominant isoforms in the heart. This highly diverse class of enzymes degrade cAMP and through anchoring proteins generates dynamic microdomains to target specific proteins and control specific cell functions in response to various stimuli. The impaired function of the anchoring protein either by inherited genetic mutations or acquired injuries results in altered intracellular targeting, and blunted responsiveness to stimulating pathways and contributes to pathological cardiac remodeling, cardiac arrhythmias and reduced cell survival. Recent genetic studies provide compelling evidence for an association between the variants in the anchoring protein PDE4DIP and atrial fibrillation, stroke, and heart failure.

1. cAMP and the importance of its compartmentalization

3′,5′ cyclic adenosine monophosphate (cAMP) was the first molecule identified as a second messenger involved in hormonal response [1]. The discovery of cAMP was a long and difficult process. It was Sutherland who had hypothesized that the hormones, referred to as first messengers, trigger the synthesis of a “second messenger” on the inner surface of the target cell’s membrane, upon binding to its receptor on its external surface.

Discovery of cAMP, as the second messenger was, however, the birth of signal transduction. After decades of research, it was discovered that cAMP resulted from the binding of ligands or first messengers to G-protein coupled receptor (GPCRs) leading to activation of adenylyl cyclase that catalyzes the conversion of ATP to cAMP. This is turn activates the effector proteins, including cAMP-dependent protein kinases, i.e. PKAs. PKA is a serine/threonine kinase that phosphorylates a wide range of protein substrates with the R-R/K-X-S/T-Y motif. Other known effectors include, exchange protein activated by cAMP (EPAC), which is a Rap1 guanine-nucleotide exchange factor directly activated by cAMP and cyclic nucleotide-gated channels (CNGC) [2]. The level of cAMP within cells is controlled by its rate of synthesis from ATP by adenylyl cyclases (ACs) and its rate of degradation to 5′-AMP by cyclic nucleotide phosphodiesterases (PDEs). Response of the cells to cAMP activation, however, was noted to be different between first messengers. This observation led to the discovery that different signals lead to cAMP accumulation and activation of PKAs in different subcellular compartments. This in turn activates PKA substrates [3]. The discovery of cAMP was followed by the identification of a second intracellular messenger, cyclic guanosine monophosphate (cGMP) in rat urine [4]. In this same study, PDE was identified as an enzyme that inactivates cAMP, and was shown to be activated by magnesium ions and inhibited by caffeine. PDEs degrade the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) by breaking their diester bonds.

2. Phosphodiesterases (PDEs)

PDE represent a superfamily of enzymes encoded by 21 different genes based on sequence similarity, mode of regulation, and preference for cAMP or cGMP as substrate, and are subgrouped into 12 families that include at least 21 genes and 100 unique isoforms. Of these PDE families, PDE4, PDE7, and PDE8 are cAMP specific [5], PDE5, PDE6 and PDE9 are cGMP specific and PDE1, PDE2, PDE3,PDE10,and PDE 11 are dual specific. The existence of multiple transcription initiation sites and alternatively spliced forms of many of mRNAs has resulted in more than 100 different forms of PDE localized in different subcellular compartments as part of complexes or signalosomes that are composed of scaffolding proteins, adenylyl cyclase, A-kinase anchoring proteins, cAMP effectors (PKA, EPAC, CNGC, popeye domain containing genes), and distinct PDEs, resulting in targeted cAMP degradation and the creation of localized intracellular cAMP gradients. Except for PDE6, 9, 10 and 11 most PDEs have some degree of expression in the heart. The predominant phosphodiesterases in the myocardium and vascular smooth muscle are PDE3 and PDE4, which regulate for example cardiac excitation-contraction coupling and smooth muscle contractile tone [6].

3. The compartmentalization of phosphodiesterases

The development of FRET (Förster Resonance Energy Transfer)reporters, based on cAMP binding domains of EPAC [7] or the regulatory subunit of PKAs, enabled visualization of cAMP microdomains following cell stimulation. This technique provided an opportunity for the visualization of the compartmentalized signal and the recognition of PDEs as enzymes regulating the spatial and temporal dynamics of cAMP signaling and the creation of cAMP microdomains. In other words, the discrete position of PDEs create cAMP gradients in many different cellular locations causing altered phosphorylation of specific effector. Compartmentalization of proteins that degrade cAMP and are activated by its effectors is critical for normal cell function [8,9]. Although the use of subcellular targeted FRET reporter sensors have helped to confirm the role of PDEs in regulating the temporal and spatial control of cAMP during signal transduction, this approach has limited ability to link specific PDEs to regulation of downstream effector molecules.

A substantial amount of work has been undertaken by pharmacologists in the U.K. to identify isoenzyme selective targets by characterizing tissue expression, subcellular distribution and modulation of tissue function by selective inhibitors. This includes studies focusing on PDE expression in cardiac and airways tissues [10] and delineating the role of PDE4 and its various subtypes [11].

4. The PDEs and the application of their inhibitors in cardiovascular system

4.1. PDE3

PDE3 has been shown to be associated with the sarcoplasmic reticulum and the dominant PDE isoform involved in cAMP breakdown and regulation of inotropy [12,13]. PDE3 has tenfold higher affinity for cAMP compared to cGMP, which acts as its competitive inhibitor [14]. PDE3A forms a protein complex with SERCA2a, phospholamban and A-kinase anchoring protein 18 (AKAP18) in a PKA-phosphorylation dependent manner [15] and targets a pool of cAMP to regulate calcium cycling through sarcoplasmic reticulum [16]. As a result of its high expression in both vasculature and airways, PDE3 has been identified as a potential therapeutic target in cardiovascular disease and asthma. Accordingly, PDE3 inhibitors have subsequently been shown to relax vascular and airway smooth muscle, and inhibit platelet aggregation [17]. PDE3 inhibitors such as milrinone are also positive inotropic agents, and were initially used for the treatment of chronic heart failure. However, chronic treatment with milrinone was found to be associated with increased mortality and was discontinued [18]. It was later shown that the density of β-adrenoceptors is lower [19] while protein and mRNA levels of inhibitory G-proteins are increased in terminally failing hearts than in controls [20]. Accordingly, reduced cAMP production in failing hearts was reported [13]. These findings led to the administration of beta blockers in patients with heart failure, and gradual discontinuation of adrenergic drugs, except for acute indications. The only exception is cilostazol, another PDE3 inhibitor, which is in use for the treatment of peripheral vascular disease.

Interestingly, a 15-base pair (bp) region in the exon 4 of PDE3A gene has been identified as a mutational hotspot for hypertension with brachydactyly [21]. The mutations cause gain of function activity and are associated with an increase in vascular smooth muscle cell proliferation and vascular remodeling in mice [22].

4.2. PDE4

PDE4 is the second most abundant PDE in cardiovascular tissues [23]. PDE4, is a cAMP-specific PDE and the largest PDE subfamily with over 35 different isoforms and as such, the most widely characterized PDE isoenzyme. It is the predominant isoenzyme in the majority of inflammatory cells, and the airways smooth muscle and is implicated in inflammatory airways disease. PDE4 inhibitor roflumilast has been approved for the treatment of chronic obstructive lung disease [24–26]. The molecular structure, compartmentalization and function have been extensively investigated. PDE4 cAMP-hydrolyzing activity has been localized to the transverse tubule/sarcoplasmic reticulum junctional space that is involved in excitation-contraction coupling [27]. There are 4 PDE genes (A-D) with more than 16 isoforms [28]. PDE4A may play a role in the regulation of anxiety and emotional memory. Rolipram (antidepressant and anti-inflammatory) and Cilomilast (anti-inflammatory) – inhibits PDE4A isoforms 1, 2, 6, and 7.

PDE4D is highly expressed in the heart. Recent work in transgenic mice has implicated PDE4 in control of cardiac force and rhythm. Accordingly, selective PDE4 inhibitors augment catecholamine-stimulated cAMP levels and induce arrhythmias in human atrial preparations [29]. Through alternative splicing and the use of multiple transcription initiation sites, the PDE4D gene encodes nine variants (PDE4D1–9) with identical catalytic domains and carboxyterminals and yet unique aminoterminals, which is critical for their subcellular localization. PDE4D hydrolyzes cAMP and regulates its levels within cardiac myocytes where it forms complexes with proteins that mediate sympathetic signaling in the heart, including β-adrenergic receptors [30]. The activity of PDE4D has been also localized to the transverse (T) tubule/sarcoplasmic reticulum (SR) junctional space thus mediating cAMP/calcium homeostasis that is involved in excitation–contraction coupling [27,31,32]. The phosphodiesterase 4D3 (PDE4D3) levels in the RyR2 complex are reduced in failing human hearts, contributing to PKA-hyperphosphorylated and leaky RyR2 channels that promote cardiac dysfunction and cause arrhythmias [33]. Co-compartmentalization of both PKA and PDE4D is critical for sustained specificity of adrenergic signaling to subcellular locations, contractility of cardiomyocytes, and timely termination of the second messenger response [34]. Accordingly, PDE4D gene inactivation in mice has been shown to cause progressive cardiomyopathy, accelerated heart failure after myocardial infarction, and cardiac arrhythmias [33]. These data suggest that PDE4D deficiency may contribute to heart failure and arrhythmias by promoting defective regulation of the RyR2 channel. Interestingly, the use of antidepressant Rolipram, a cAMP-PDE inhibitor that enhances noradrenergic neurotransmission in the central nervous system is associated with increased atrial fibrillation (AF). Co-compartmentalization of both PKA and PDE4D is critical for sustained specificity of adrenergic signaling to subcellular locations, contractility of cardiomyocytes, and timely termination of the second messenger response [34].

5. Phosphodiesterase 4D interacting protein (PDE4DIP)

PDE4DIP AKA Myomegalin (MMGL) is a key component of AKAP-PKA-PDE4D signaling complex that interacts with PDE4D, PKA [35] and several other proteins to form a multiprotein complex and anchors the sequestering components of the cAMP-dependent pathway to Golgi and/or centrosomes [36] (Figure1). The gene encoding the protein is localized on chromosome 1 and has at least 13 splice variants generating 13 isoforms.

Fig. 1.

Schematic figure demonstrating the microdomain of PDE4D, PDE4DIP, cAMP on Golgi apparatus. βAR denotes beta adrenergic, PDE, Phosphodiesterases, PDE4DIP, Phosphodiesterase 4D Interacting Protein.

5.1. The protein structure

PDE4DIP is a large 2324-amino acid protein composed of alpha-helical and coiled-coil structures, with domains shared with microtubule-associated proteins, and a leucine zipper identical to the Drosophila centrosomin [36]. The transcripts of 7.5–8 kilobases are present in most tissues, whereas a splicing variant is expressed as a 230–250 kDa protein in rat heart and skeletal muscle. Immunocyto-chemistry and transfection studies have localized PDE4D and myomegalin to the Golgi/centrosomal area in cultured cells, and in sarcomeric structures of skeletal muscle. It was shown that PDE4DIP is involved in phosphorylation of the sarcomeric proteins cMyBPC and cTNI and hence, lays an important role in regulation of cardiac contractility [37]. Interestingly, it was shown that the siRNA-mediated knockdown of PDE4DIP leads to reduction of cMyBPC levels under conditions of adrenergic stress, likely through reduced phosphorylation and increased degradation [37].

5.2. The protein function

At functional levels PDE4DIP has been shown to interact with AKAP9 on the cis-Golgi networks, an interaction that appeared as necessary for the stability of both proteins (Fig. 1, schematic figure). Disrupting PDE4DIP expression affected endoplasmic reticulum (ER)-to-Golgi trafficking and caused Golgi fragmentation. Further, PDE4DIP is associated with γ-tubulin complexes and the microtubule plus-end tracking protein EB1 (AKA. MAPRE1), and is required for the Golgi localization of these proteins [38]. PDE4DIP loss has been shown to dislocate PDE4D3 from the centrosome, leading to local PKA overactivation and inhibition of the Hh signaling [39].

5.3. Disease association of the genetic variants

5.3.1. Mutations associated with atrial fibrillation (AF), cardiomyopathy and stroke

The genetic screening of independent kindreds with AF and conduction disease by whole exome sequencing (WES) in our laboratory led to the identification of novel PDE4DIP mutations (Fig. 2) that segregated with the disease [40]. The PDE4DIP mutation in the largest family, resulting in alanine substitution by threonine at 123 codon (PDE4DIP^A123T) was further characterized in vitro by our group. The effect of PDE4DIP^A123T on intracellular cAMP signaling was explored using a FRET-based sensor. Isoproterenol was used to stimulate the endogenous β-adrenergic receptors. The FRET sensor imaging showed increased cAMP levels in response to isoproterenol stimulation in cells transfected with the mutant compared with wildtype PDE4DIP. Under normal circumstances, the rise of cAMP in response to isoproterenol triggers PDE4D activation and simultaneous desensitization of the β2AR receptor via phosphorylation by PKA and G-protein coupled receptor serine/threonine kinases (GRKs) at distinct phosphorylation sites [41]. PKA-mediated phosphorylation of the β2AR at Ser345/346 by PKA causes a switch from stimulatory Gs to inhibitory Gi protein [42,43]. On the other hand, prolonged β2AR stimulation results in its GRK-mediated phosphorylation at Ser 355/356, and termination of G protein-mediated signaling by binding the receptor to β-arrestin, which facilitate its endocytosis and desensitization [44,45] (Fig. 1). In addition, PDE4D binds to β-arrestin and is subsequently recruited to the plasma membrane to regulate PKA mediated phosphorylation of β2AR [46–48]. The examination of the PKA and GRK phosphorylation sites on the β2AR revealed an increase in phosphorylation at PKA phosphorylation site and a reduction at the GRK phosphorylation site in cells expressing PDE4DIP^A123T compared with wild type PDE4DIP in response to isoproterenol stimulation. In addition, the examination of the PKA phosphorylation of desmin by immunofluorescent microscopy revealed decreased phosphorylation at its serine 60 site in PDE4DIP^A123T cells. The reduced p-desmin in the mutants suggested that PDE4DIP^A123T mutation causes loss of compartmentalization of both PDE4D and PKA, resulting in increased PKA phosphorylation of β2AR but reduced phosphorylation of desmin. Desmin is also known to be a target of cAMP-dependent kinases [49,50], which regulates its function and assembly [51,52]. Interestingly, our studies in C2C12 cells showed that p-desmin was lower in the mutant PDE4DIP compared with wildtype PDE4DIP after β adrenergic activation. This suggests that PDE4DIP directs PKA to desmin, but loss of PKA compartmentalization by PDE4DIP^pA123T led to increased β2AR and reduced desmin phosphorylation. These findings were highly significant since dysregulation of PDE4D and altered desmin phosphorylation have both been linked to cardiomyopathy and arrhythmias [33,53,54]. AF is considered to be a major risk factor for ischemic stroke and is often diagnosed after the diagnosis of stroke. It is therefore a likely cause of cryptogenic stroke.

Fig. 2.

PDE4DIP mutations in familial AF kindreds. Only Coiled coil domains 1, 3, 8 and 12 are shown.

Interestingly, in a NHLBI Exome Sequence Project a protein-coding variant in PDE4DIP (rs1778155) was associated with an increased risk of stroke by 2.15;(P = 2.63 × 10–8) [55]. The direction and effect size of the variant on risk was consistent in both all-stroke and small-vessel stroke categories (OR, 2.03; meta-analysis P = 7.96 × 10⁻⁶). Subsequently, the investigators analyzed single-nucleotide polymorphisms in PDE4DIP in an independent set of affected sibpairs from SWISS and unrelated ESP African American participants, which showed significantly increased allele sharing identity by descent (P = 1.16 × 10⁻⁴) in 16 of 19 large-vessel concordant pairs with a LOD score of 6.07 (OR, 2.41; MAF, 0.302; P = 2.18 × 10⁻⁵) and a false discovery rate of P = 6.39 × 10⁻³. Genetic variants in PDE4D, the primary interacting partner of PDE4DIP, have also been linked to ischemic stroke in Icelandic families [56]. A subsequent meta-analysis of nine case-control studies of 3808 stroke cases and 4377 controls confirmed a significant association between stroke and certain PDE4DIP genetic variants [57]. The association between PDE4DIP genetic variants and ischemic stroke was confirmed by a larger meta-analysis of 7 studies, withapproximately 12,000 cases and 15,000 controls who were typed for 6 single-nucleotide polymorphisms in PDE4D. In the meta-analysis, PDE4D rs702553 had a significant association with ischemic stroke. The mechanisms of how altered PDE4DIP expression causes ischemic stroke is not clear, but identification of mutations by descend in this gene in patients with AF suggest that some of the patients with stroke may have underlying AF. Interestingly, rs2477088 in PDE4DIP gene has been associated with macrovascular complications of diabetes [58].

5.3.2. Mutations associated with malignant disorders

Somatic mutations in PDE4DIP gene have been described in patients with drug resistant prostate cancer [59], endometriosis-associated ovarian cancer [60] and familial squamous lung cancer cancers [61]. Mutations in PDE4DIP gene have been also discovered in non-small cell cancer of the lung with leptomeningeal metastasis [62]. Germline indels and single nucleotide variants in PDE4DIP gene have been reported in patients with leukemia [63].

This function of PDE4DIP is critical for stabilization at the leading edge of migrating cells and its impact on tumor cell motility and proliferation and plays an important role in tumor progression [64]. Loss of PDE4DIP has also been shown to suppress the proliferation of granule neuron precursors and block the growth of medulloblastoma in a mouse model [39], underscoring its role as an attractive target for the treatment of malignant disorders. Interestingly, myomegalin antibodies have been detected in the serum of patients with esophageal squamous cell carcinoma (SCC) and are associated with a favorable prognosis [65]. Normal chromosome 1q21 has up to 3 copies of PDE4DIP [66]. Interestingly, pineoblastoma have been shown to have up to 8 copies of the gene [67]. Of note, PDE4DIP gene contains a single DUF(Domains of unknown function)1220 domain [68]. DuF1220 domain exhibits the largest.

increase in copy number in the human genome, whereby its copy number has been associated with pathological brain sizes (same reference). Overall, these findings suggest that PDE4DIP may play a role in tumor progression by enhancing tumor cell proliferation and migration and is an attractive target for the treatment of diverse malignant tumors.

Finally, mutations in PDE4DIP gene have been associated with acrodysostosis, an autosomal-dominant disorder characterized by facial dysostosis, severe brachydactyly with cone-shaped epiphyses, short stature, intellectual disability, and resistance to multiple hormones [69].

In conclusion, PDE4DIP has all characteristics of c-AMP/PDE anchoring protein and plays an important role in cellular signaling and the integrity and function of cardiac myocytes and is an attractive target for the treatment of AF and heart failure.