Wito Richter

Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are differentially expressed and regulated in rat tissue

Cyclic nucleotide PDE4 (phosphodiesterase 4) inhibitors are being developed as potent anti-inflammatory drugs for use in chronic lung diseases, but the complexity of the PDE4 family has hampered this process. The four genes comprising the PDE4 family, PDE4A, PDE4B, PDE4C and PDE4D, are all expressed as multiple splice variants. The most widely used criterion to identify PDE4 variants expressed endogenously is their migration on SDS/PAGE. However, when a PDE4D3-selective antibody was used for immunoprecipitation, the pattern of expression obtained did not confirm the expression predicted by SDS/PAGE. This observation, together with the recent discovery of additional PDE4D transcripts, prompted us to re-evaluate the pattern of expression of these variants. The nine rat PDE4D splice variants, PDE4D1 to PDE4D9, were cloned, their electrophoretic properties compared, and their in vivo mRNA and protein levels determined. Using this approach, we found that the pattern of distribution of the PDE4D splicing variants is more complex than previously reported. Multiple variants co-migrate in single immunoreactive bands, and variant-selective antibodies were necessary to discriminate between splice variants. Tissues that were thought to express only PDE4D3, express three closely related proteins, with PDE4D8 and PDE4D9 as the predominantly expressed forms. In addition, activation of cAMP signalling produces phosphorylation and activation of variants other than PDE4D3, and expression of PDE4D mRNA does not always correlate with the pattern of protein expression. As PDE4 inhibitors have different affinities for distinct PDE4D splicing variants, our results indicate that a better definition of the pattern of PDE4 expression is required for target validation.

Signaling from β1‐ and β2‐adrenergic receptors is defined by differential interactions with PDE4

β1‐ and β2‐adrenergic receptors (βARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by β1AR but not β2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that β1AR forms a signaling complex with a cAMP‐specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β‐arrestin/PDE complex reported previously. The β1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the β2AR is a prerequisite for the recruitment of a complex consisting of β‐arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of β1‐ and β2‐adrenoceptor signaling.

Spatiotemporal Dynamics of β-Adrenergic cAMP Signals and L-Type Ca2+ Channel Regulation in Adult Rat Ventricular Myocytes Role of Phosphodiesterases

Steady-state activation of cardiac β-adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer–based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca2+ channel current (ICa,L) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. ICa,L amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for ≈5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 &mgr;mol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 &mgr;mol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-l-methylxanthine (300 &mgr;mol/L), cAMP signals and ICa,L declined with a time constant >10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to ≈50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct β-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4.

Dimerization of the Type 4 cAMP-specific Phosphodiesterases Is Mediated by the Upstream Conserved Regions (UCRs)

The cAMP-specific PDE4 family consists of four genes, each expressed as several splice variants. These variants are termed long and short forms depending on the presence or absence of two unique N-terminal domains called upstream conserved regions 1 and 2 (UCR1 and 2). UCR1 and UCR2 have been shown to form a module necessary for the activation of PDE4 upon phosphorylation by the cAMP-dependent kinase (PKA). Here we have uncovered PDE4 oligomerization as a novel function for the UCR1/UCR2 module. Using several different approaches including gel filtration, sucrose density gradient centrifugation, pull-down of differentially tagged PDE constructs, and yeast two-hybrid assay, we show that the long PDE4 splice variant PDE4D3 behaves as a dimer, whereas the short splice variant PDE4D2 is a monomer. Internal deletions of either the C-terminal portion of UCR1 or the N-terminal portion of UCR2 abolishes dimerization of PDE4D3 indicating that both domains are involved in this intermolecular interaction. The dimerization, however, is structurally distinguishable from a previously described intramolecular interaction involving the same domains. PKA phosphorylation and site-directed mutagenesis shown to ablate the latter do not interfere with dimerization. Therefore, dimerization of the long PDE4 forms may be an additional function of the UCR domains that further explains differences in the regulatory properties between the long and short PDE4 splice variants.

Dynamic regulation of cystic fibrosis transmembrane conductance regulator by competitive interactions of molecular adaptors.

Disorganized ion transport caused by hypo- or hyperfunctioning of the cystic fibrosis transmembrane conductance regulator (CFTR) can be detrimental and may result in life-threatening diseases such as cystic fibrosis or secretory diarrhea. Thus, CFTR is controlled by elaborate positive and negative regulations for an efficient homeostasis. It has been shown that expression and activity of CFTR can be regulated either positively or negatively by PDZ (PSD-95/discs large/ZO-1) domain-based adaptors. Although a positive regulation by PDZ domain-based adaptors such as EBP50/NHERF1 is established, the mechanisms for negative regulation of the CFTR by Shank2, as well as the effects of multiple adaptor interactions, are not known. Here we demonstrate a physical and physiological competition between EBP50-CFTR and Shank2-CFTR associations and the dynamic regulation of CFTR activity by these positive and negative interactions using the surface plasmon resonance assays and consecutive patch clamp experiments. Furthermore whereas EBP50 recruits a cAMP-dependent protein kinase (PKA) complex to CFTR, Shank2 was found to be physically and functionally associated with the cyclic nucleotide phosphodiesterase PDE4D that precludes cAMP/PKA signals in epithelial cells and mouse brains. These findings strongly suggest that balanced interactions between the membrane transporter and multiple PDZ-based adaptors play a critical role in the homeostatic regulation of epithelial transport and possibly the membrane transport in other tissues.

Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice

β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca²⁺-induced Ca²⁺ release via phosphorylation of L-type Ca²⁺ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR-dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca²⁺ current (ICa,L) is increased only in Pde4b-/- mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca²⁺ transients, cell contraction, and spontaneous Ca²⁺ release events were increased in Pde4b-/- and Pde4d-/- myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b-/- mice but not in WT mice. These results identify PDE4B in the CaV1.2 complex as a critical regulator of ICa,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca²⁺-induced Ca²⁺ release in cardiomyocytes.

Decreased Expression and Activity of cAMP Phosphodiesterases in Cardiac Hypertrophy and Its Impact on β-Adrenergic cAMP Signals

Rationale: Multiple cyclic nucleotide phosphodiesterases (PDEs) degrade cAMP in cardiomyocytes but the role of PDEs in controlling cAMP signaling during pathological cardiac hypertrophy is poorly defined. Objective: Evaluate the &bgr;-adrenergic regulation of cardiac contractility and characterize the changes in cardiomyocyte cAMP signals and cAMP-PDE expression and activity following cardiac hypertrophy. Methods and Results: Cardiac hypertrophy was induced in rats by thoracic aortic banding over a time period of 5 weeks and was confirmed by anatomic measurements and echocardiography. Ex vivo myocardial function was evaluated in Langendorff-perfused hearts. Engineered cyclic nucleotide-gated (CNG) channels were expressed in single cardiomyocytes to monitor subsarcolemmal cAMP using whole-cell patch-clamp recordings of the associated CNG current (ICNG). PDE variant activity and protein level were determined in purified cardiomyocytes. Aortic stenosis rats exhibited a 67% increase in heart weight compared to sham-operated animals. The inotropic response to maximal &bgr;-adrenergic stimulation was reduced by ≈54% in isolated hypertrophied hearts, along with a ≈32% decrease in subsarcolemmal cAMP levels in hypertrophied myocytes. Total cAMP hydrolytic activity as well as PDE3 and PDE4 activities were reduced in hypertrophied myocytes, because of a reduction of PDE3A, PDE4A, and PDE4B, whereas PDE4D was unchanged. Regulation of &bgr;-adrenergic cAMP signals by PDEs was blunted in hypertrophied myocytes, as demonstrated by the diminished effects of IBMX (100 &mgr;mol/L) and of both the PDE3 inhibitor cilostamide (1 &mgr;mol/L) and the PDE4 inhibitor Ro 201724 (10 &mgr;mol/L). Conclusions: &bgr;-Adrenergic desensitization is accompanied by a reduction in cAMP-PDE and an altered modulation of &bgr;-adrenergic cAMP signals in cardiac hypertrophy.

Conserved expression and functions of PDE4 in rodent and human heart

PDE4 isoenzymes are critical in the control of cAMP signaling in rodent cardiac myocytes. Ablation of PDE4 affects multiple key players in excitation–contraction coupling and predisposes mice to the development of heart failure. As little is known about PDE4 in human heart, we explored to what extent cardiac expression and functions of PDE4 are conserved between rodents and humans. We find considerable similarities including comparable amounts of PDE4 activity expressed, expression of the same PDE4 subtypes and splicing variants, anchoring of PDE4 to the same subcellular compartments and macromolecular signaling complexes, and downregulation of PDE4 activity and protein in heart failure. The major difference between the species is a fivefold higher amount of non-PDE4 activity in human hearts compared to rodents. As a consequence, the effect of PDE4 inactivation is different in rodents and humans. PDE4 inhibition leads to increased phosphorylation of virtually all PKA substrates in mouse cardiomyocytes, but increased phosphorylation of only a restricted number of proteins in human cardiomyocytes. Our findings suggest that PDE4s have a similar role in the local regulation of cAMP signaling in rodent and human heart. However, inhibition of PDE4 has ‘global’ effects on cAMP signaling only in rodent hearts, as PDE4 comprises a large fraction of the total cardiac PDE activity in rodents but not in humans. These differences may explain the distinct pharmacological effects of PDE4 inhibition in rodent and human hearts.

Cyclic AMP compartments and signaling specificity: role of cyclic nucleotide phosphodiesterases.

### Introduction The concept that cAMP has restricted access to different pools of intracellular effectors was articulated more than 30 years ago. Observations made by [Corbin et al. (1977)][1] demonstrated that the two isoenzymes of the major intracellular cAMP target, PKA I and II, are located in

Cyclic adenosine monophosphate phosphodiesterase type 4 protects against atrial arrhythmias.

OBJECTIVES This study was designed to examine whether a cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE), PDE4, is expressed in human atrium and contributes to the control of electrical stability. BACKGROUND Atrial fibrillation is accompanied by a profound remodeling of membrane receptors and alterations in cAMP-dependent regulation of Ca(2+) handling. Being responsible for cAMP hydrolysis, PDEs are likely to play a role in this setting. In the rodent heart, PDE4 contributes up to 60% of total cAMP-hydrolytic activity. However, its role in the human heart remains controversial. METHODS L-type Ca(2+) current and spontaneous Ca(2+) release were recorded in isolated human atrial myocytes. Intracellular cAMP was measured by live cell imaging using a fluorescence resonance energy transfer-based sensor. Contractile force and arrhythmias were recorded in human atrial trabeculae. PDE activity was measured in human atrial tissue from patients in sinus rhythm and permanent atrial fibrillation. RESULTS PDE4 is expressed in human atrial myocytes and accounts for approximately 15% of total PDE activity. PDE4D represents the major PDE4 subtype. PDE4 inhibition increased intracellular cAMP and L-type Ca(2+) current and dramatically delayed their decay after a brief β-adrenergic stimulation. PDE4 inhibition also increased the frequency of spontaneous Ca(2+) release at baseline, as well as the contractile response and the incidence of arrhythmias in human atrial strips during β-adrenergic stimulation. Total PDE activity decreased with age, and the relative PDE4 activity was lower in patients with permanent atrial fibrillation than in age-matched sinus rhythm controls. CONCLUSIONS PDE4 is critical in controlling cAMP levels and thereby Ca(2+) influx and release in human atrial muscle, hence limiting the susceptibility to arrhythmias.