Regulation Of Phosphodiesterase Research Articles

Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination.

We identified Rho-related BTB domain containing 1 (RhoBTB1) as a key regulator of phosphodiesterase 5 (PDE5) activity, and through PDE5, a regulator of vascular tone. We identified the binding interface for PDE5 on RhoBTB1 by truncating full-length RhoBTB1 into its component domains. Co-immunoprecipitation analyses revealed that the C-terminal half of RhoBTB1 containing its two BTB domains and the C-terminal domain (B1B2C) is the minimal region required for PDE5 recruitment and subsequent proteasomal degradation via Cullin-3 (CUL3). The C-terminal domain was essential in recruiting PDE5 as constructs lacking this region could not participate in PDE5 binding or proteasomal degradation. We also identified Pro353 and Ser363 as key amino acid residues in the B1B2C region involved in CUL3 binding to RhoBTB1. Mutation of either of these residues exhibited impaired CUL3 binding and PDE5 degradation, although the binding to PDE5 was preserved. Finally, we employed ascorbate peroxidase 2 (APEX2) proximity labeling using a B1B2C-APEX2 fusion protein as bait to capture unknown RhoBTB1 binding partners. Among several B1B2C-binding proteins identified and validated, we focused on SET domain containing 2 (SETD2). SETD2 and RhoBTB1 directly interacted, and the level of SETD2 increased in response to pharmacological inhibition of the proteasome or Cullin complex, CUL3 deletion, and RhoBTB1-inhibition with siRNA. This suggests that SETD2 is regulated by the RhoBTB1-CUL3 axis. Future studies will determine whether SETD2 plays a role in cardiovascular function.

Neuroprotective Effects of Theobromine in permanent bilateral common carotid artery occlusion rat model of cerebral hypoperfusion.

Cerebral hypoperfusion (CH) is a common underlying mechanism of dementia disorders linked to aberrations in the neurovascular unit. Hemodynamic disturbances adversely affect cellular energy homeostasis that triggers a sequence of events leading to irrevocable damage to the brain and neurobehavioral discrepancies. Theobromine is a common ingredient of many natural foods consumed by a large population worldwide. Theobromine has shown health benefits in several studies, attributed to regulation of calcium homeostasis, phosphodiesterase, neurotransmission, and neurotrophins. The current study evaluated the neuroprotective potential of theobromine against CH in the permanent bilateral common carotid artery occlusion (BCCAO) prototype. Wistar rats were distributed in Sham-operated (S), S + T100, CH, CH + T50, and CH + T100 groups. Animals received permanent BCCAO or Sham treatment on day 1. Theobromine (50, 100mg/kg) was given orally in animals subjected to BCCAO for 14 days daily. CH caused neurological deficits (12-point scale), motor dysfunction, and memory impairment in rats. Treatment with theobromine significantly attenuated neurological deficits and improved sensorimotor functions and memory in rats with CH. In biochemistry investigation of the entire brain, findings disclosed reduction in brain oxidative stress, inflammatory intermediaries (tumor necrosis factor-α, interleukin-1β and - 6, nuclear factor-κB), markers of cell demise (lactate dehydrogenase, caspase-3), acetylcholinesterase activity, and improvement in γ-aminobutyric acid quantity in rats that were given theobromine for 14 days daily after CH. Histopathological analysis substantiated attenuation of neurodegenerative changes by theobromine. The findings of this study indicated that theobromine could improve neurological scores, sensorimotor abilities, and memory in CH prototype.

Post-translational regulation of Tyrosyl-DNA phosphodiesterase (TDP1 and TDP2) for the repair of the trapped topoisomerase-DNA covalent complex

DNA topoisomerases are essential enzymes that regulate DNA topology, the transmission of genetic materials, and gene expressions both in the nucleus and mitochondria. Trapped topoisomerases (Top1 and Top2) in covalent complexes with DNA (Topoisomerase cleavage complexes; Topcc) are detrimental DNA lesions that perturb active genome integrity and trigger cell death. Comprehensive research on the recently discovered enzymes TDP1 and TDP2 exemplify their spectacular role in repairing trapped Topcc as well as in a myriad of diverse DNA lesions. Posttranslational modifications (PTMs), play critical roles in regulating the optimal function of the DNA Damage Response (DDR) proteins. This review summarizes the mechanistic aspects of DNA damage induced by trapped Topcc during transcription and their role in human diseases. We have also highlighted the pivotal role of PTMs in fine-tuning the intricate and multilayered regulatory processes of TDP1 and TDP2 molecular networks for the repair of trapped Topcc.

Abstract P144: Local Signaling And Cardiac Hypertrophy:specificity In The Face Of Pleiotropy

Rationale: Most likely, the overall myocyte cell growth during the pathological state of cardiac hypertrophy is regulated by a local muscle A-kinase anchoring protein β (mAKAPβ) complex. The mAKAPβ may act as a significant coordinator of myocyte hypertrophic signals. It may critically integrate the hypertrophic signals due to β-adrenergic and leukemia inhibitory factor (LIF)/gp-130 receptor stimulation. Observations suggest that mAKAPβ signalosome may act as a gate-keeper for regulating nuclear factor of activated T cells (NFATc) activity and nuclear localization of this complex might be directly linked to the induction of cardiac hypertrophy. The mAKAPβ complex might function through modulating the profiles of a local cyclic adenosine 3’,5’ monophosphate (cAMP) microdomain at perinuclear region in cardiomyocytes. The acute stimulation of cAMP may be beneficial for the heart, whereas chronic stimulation might cause damage. The transition between the chronic and acute function of cAMP is probably modulated by the ability of myocytes to tightly regulate the cAMP levels in local microdomains across the cell. Any dysfunction in this process may lead to net accumulation and a global rise of cAMP levels, leading to deleterious effects on the heart. Objective: cAMP is a single messenger but delivers multiple messages in myocytes. How is this managed? Here, we aim to investigate a key question that how mAKAPβ signalosome might ensure the microdomain specificity despite the pleiotropic nature of the second messenger. Methods and Results: Our results may explain how, in the context of hypertrophy, mAKAPβ complex coordinates the interactions between two coupled cAMP-induced feedback loops and LIF-induced activation of the MAPK pathway. Our results may also explain that mAKAPβ complex functions through anchoring protein kinase A (PKA) and ERK5 in the signalosome thus, modulating the bidirectional regulation of phosphodiesterase and hence the control of localized cAMP metabolism as well as the shape and temporal profile of the second messenger in a specific domain. Conclusion: Here, we propose a mechanistic model which suggests that stress-induced reprofiling of cAMP flux at discrete cellular locations may lead to cardiovascular disease.

Epigenomic, Transcriptomic, and Protective Effect of Carotenoid Fucoxanthin in High Glucose-Induced Oxidative Stress in Mes13 Kidney Mesangial Cells

Diabetic nephropathy (DN) is the major cause of kidney related diseases in patients induced by high glucose (HG) affecting around 40% of type 1 and 2 diabetic patients. It is characterized by excessive inflammation inducing factors, reactive oxygen species (ROS) overproduction, and potential epigenomic related changes. Fucoxanthin (FX), a carotenoid found in brown seaweed, has a structure which includes an allenic bond and a 5,6-monoepoxide in the molecule, with strong antioxidant and anti-inflammatory activity. However, understanding of the impact of FX on DN was lacking. In this study we tested the early effects of high glucose (HG) on mouse mesangial kidney Mes13 cells, a potential in vitro cell culture model of DN. Our results show that HG induced oxidative stress on kidney mesangial Mes13 cells, while FX treatment attenuates the oxidative stress by decreasing the ROS, demonstrated by flow cytometry. Next, we utilized next-generation sequencing (NGS) to profile the HG-induced early epigenomic and transcriptomic changes in this in vitro DN model and the protective effects of FX. Differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were analyzed using R software in HG and FX treated groups. Differential regulation of signaling pathways was studied using Reactome Pathway Analysis in the comparison. DEG analysis shows that novel biomarkers with specific pathways, including interleukin regulation, Toll-like receptor pathway, and PKA phosphorylation pathways, were found to be modulated by the FX treatment. TGF β 1i1 (TGFB 1i1), MAP-3-kinase-13(MAP3K13) involved in crucial cellular processes including glucose metabolism, phosphodiesterase regulation was methylated in HG, which was demethylated with FX treatment. Integrated transcriptomic and CpG methylome analysis of DEGs and DMRs revealed that genes like adenylate cyclase (Adcy7), calponin 1 (CNN1), potassium voltage-gated channel interacting protein 2 (KCNIP2), phosphatidylinositol-4-phosphate 5-kinase type 1 β (PIP5K1B), and transmembrane protein with EGF-like and two follistatin-like domains 1 (TMEFF1), which were modulated by FX in HG-exposed Mes13 cells, potentially modulate ion channel transport and glucose metabolism. In summary, our current study shows that novel early epigenomic and transcriptomic biomarkers were altered during the disease progression of HG-induced DN and that FX modified these alterations potentially contributing to the protective effects of mesangial cells from the HG-induced oxidative stress and damage.

Structural Analysis of the Regulatory GAF Domains of cGMP Phosphodiesterase Elucidates the Allosteric Communication Pathway

Regulation of photoreceptor phosphodiesterase (PDE6) activity is responsible for the speed, sensitivity, and recovery of the photoresponse during visual signaling in vertebrate photoreceptor cells. It is hypothesized that physiological differences in the light responsiveness of rods and cones may result in part from differences in the structure and regulation of the distinct isoforms of rod and cone PDE6. Although rod and cone PDE6 catalytic subunits share a similar domain organization consisting of tandem GAF domains (GAFa and GAFb) and a catalytic domain, cone PDE6 is a homodimer whereas rod PDE6 consists of two homologous catalytic subunits. Here we provide the x-ray crystal structure of cone GAFab regulatory domain solved at 3.3 Å resolution, in conjunction with chemical cross-linking and mass spectrometric analysis of conformational changes to GAFab induced upon binding of cGMP and the PDE6 inhibitory γ-subunit (Pγ). Ligand-induced changes in cross-linked residues implicate multiple conformational changes in the GAFa and GAFb domains in forming an allosteric communication network. Molecular dynamics simulations of cone GAFab revealed differences in conformational dynamics of the two subunits forming the homodimer and allosteric perturbations on cGMP binding. Cross-linking of Pγ to GAFab in conjunction with solution NMR spectroscopy of isotopically labeled Pγ identified the central polycationic region of Pγ interacting with the GAFb domain. These results provide a mechanistic basis for developing allosteric activators of PDE6 with therapeutic implications for halting the progression of several retinal degenerative diseases.

Probing the Structural Dynamics of the Catalytic Domain of Human Soluble Guanylate Cyclase

In the nitric oxide (NO) signaling pathway, human soluble guanylate cyclase (hsGC) synthesizes cyclic guanosine monophosphate (cGMP); responsible for the regulation of cGMP-specific protein kinases (PKGs) and phosphodiesterases (PDEs). The crystal structure of the inactive hsGC cyclase dimer is known, but there is still a lack of information regarding the substrate-specific internal motions that are essential for the catalytic mechanism of the hsGC. In the current study, the hsGC cyclase heterodimer complexed with guanosine triphosphate (GTP) and cGMP was subjected to molecular dynamics simulations, to investigate the conformational dynamics that have functional implications on the catalytic activity of hsGC. Results revealed that in the GTP-bound complex of the hsGC heterodimer, helix 1 of subunit α (α:h1) moves slightly inwards and comes close to helix 4 of subunit β (β:h4). This conformational change brings loop 2 of subunit β (β:L2) closer to helix 2 of subunit α (α:h2). Likewise, loop 2 of subunit α (α:L2) comes closer to helix 2 of subunit β (β:h2). These structural events stabilize and lock GTP within the closed pocket for cyclization. In the cGMP-bound complex, α:L2 detaches from β:h2 and establishes interactions with β:L2, which results in the loss of global structure compactness. Furthermore, with the release of pyrophosphate, the interaction between α:h1 and β:L2 weakens, abolishing the tight packing of the binding pocket. This study discusses the conformational changes induced by the binding of GTP and cGMP to the hsGC catalytic domain, valuable in designing new therapeutic strategies for the treatment of cardiovascular diseases.

A compendium of G-protein-coupled receptors and cyclic nucleotide regulation of adipose tissue metabolism and energy expenditure.

With the ever-increasing burden of obesity and Type 2 diabetes, it is generally acknowledged that there remains a need for developing new therapeutics. One potential mechanism to combat obesity is to raise energy expenditure via increasing the amount of uncoupled respiration from the mitochondria-rich brown and beige adipocytes. With the recent appreciation of thermogenic adipocytes in humans, much effort is being made to elucidate the signaling pathways that regulate the browning of adipose tissue. In this review, we focus on the ligand-receptor signaling pathways that influence the cyclic nucleotides, cAMP and cGMP, in adipocytes. We chose to focus on G-protein-coupled receptor (GPCR), guanylyl cyclase and phosphodiesterase regulation of adipocytes because they are the targets of a large proportion of all currently available therapeutics. Furthermore, there is a large overlap in their signaling pathways, as signaling events that raise cAMP or cGMP generally increase adipocyte lipolysis and cause changes that are commonly referred to as browning: increasing mitochondrial biogenesis, uncoupling protein 1 (UCP1) expression and respiration.

Circadian regulation of phosphodiesterase 6 genes in zebrafish differs between cones and rods: Implications for photopic and scotopic vision

A correlation is known to exist between visual sensitivity and oscillations in red opsinand rhodopsin gene expression in zebrafish, both regulated by the clock gene. This indicates that an endogenous circadian clock regulates behavioural visual sensitivity, apart from the regulation exerted by the pineal organ. However, the specific mechanisms for cones (photopic vision) and rods (scotopic vision) are poorly understood. In this work, we performed gene expression, cosinor and immunohistochemical analyses to investigate other key genes involved in light perception, encoding the different subunits of phosphodiesterase pde6 and transducin GαT, in constant lighting conditions and compared to normal light-dark conditions. We found that cones display prominent circadian oscillations in mRNA levels for the inhibitory subunit gene pde6ha that could contribute to the regulation of photopic sensitivity by preventing overstimulation in photopic conditions. In rods, the mRNA levels of the inhibitory subunit gene pde6ga oscillate under normal conditions and dampen down in constant light but continue oscillating in constant darkness. There is an increase in total relative expression for pde6gb in constant conditions. These observations, together with previous data, suggest a complex regulation of the scotopic sensitivity involving endogenous and non-endogenous components, possibly present also in other teleost species. The GαT genes do not display mRNA oscillations and therefore may not be essential for the circadian regulation of photosensitivity. In summary, our results support different regulation for the zebrafish photopic and scotopic sensitivities and suggest circadian regulation of pde6ha as a key factor regulating photopic sensitivity, while the regulatory mechanisms in rods appear to be more complex.

Mitogen-activated protein kinase regulation of the phosphodiesterase RegA in early Dictyostelium development.

Mitogen-activated protein kinase (MAPK) regulation of cAMP-specific phosphodiesterase function has been demonstrated in mammalian cells and suspected to occur in other eukaryotes. Epistasis analysis in the soil amoeba Dictyostelium discoideum suggests the atypical MAPK Erk2 downregulates the function of the cAMP-specific phosphodiesterase RegA to regulate progression of the developmental life cycle. A putative MAPK docking motif located near a predicted MAPK phosphorylation site was characterized for contributions to RegA function and binding to Erk2 because a similar docking motif has been previously characterized in the mammalian PDE4D phosphodiesterase. The overexpression of RegA with alterations to this docking motif (RegAD-) restored RegA function to regA- cells based on developmental phenotypes, but low-level expression of RegAD- from the endogenous regA promoter failed to rescue wild-type morphogenesis. Co-immunoprecipitation analysis indicated that Erk2 associates with both RegA and RegAD-, suggesting the docking motif is not required for this association. Epistasis analysis between regA and the only other Dictyostelium MAPK, erk1, suggests Erk1 and RegA can function in different pathways but that some erk1- phenotypes may require cAMP signalling. These results imply that MAPK downregulation of RegA in Dictyostelium is accomplished through a different mechanism than MAPK regulation of cAMP-specific phosphodiesterases in mammalian cells and that the regulation in Dictyostelium does not require a proximal MAPK docking motif.

Comparative transcriptomic and proteomic analysis of Arthrobacter sp. CGMCC 3584 responding to dissolved oxygen for cAMP production

Arthrobacter sp. CGMCC 3584 is able to produce high yields of extracellular cyclic adenosine monophosphate (cAMP), which plays a vital role in the field of treatment of disease and animal food, during aerobic fermentation. However, the molecular basis of cAMP production in Arthrobacter species is rarely explored. Here, for the first time, we report the comparative transcriptomic and proteomic study of Arthrobacter cells to elucidate the higher productivity of cAMP under high oxygen supply. We finally obtained 14.1% and 19.3% of the Arthrobacter genome genes which were up-regulated and down-regulated notably, respectively, with high oxygen supply, and identified 54 differently expressed proteins. Our results revealed that high oxygen supply had two major effects on metabolism: inhibition of glycolysis, pyruvate metabolism, nitrogen metabolism, and amino acid metabolism (histidine, branched-chain amino acids and glutamate metabolism); enhancement of the tricarboxylic acid cycle and purine metabolism. We also found that regulation of adenylate cyclase and phosphodiesterase was not significant under high oxygen supply, suggesting efficient cAMP export might be important in cAMP production. These findings may contribute to further understanding of capacities of Arthrobacter species and would be highly useful in genetic regulation for desirable production.

Enhanced anticancer efficacy of histone deacetyl inhibitor, suberoylanilide hydroxamic acid, in combination with a phosphodiesterase inhibitor, pentoxifylline, in human cancer cell lines and in-vivo tumor xenografts.

Vorinostat [suberoylanilide hydroxamic acid (SAHA)], a histone deacetylase inhibitor, shows limited clinical activity against solid tumors when used alone. The methyl xanthine drug, pentoxifylline (PENT), has been described to have antitumor properties. The aim of this study was to look for the enhanced anticancer activities of both agents when used in combination at doses lower than their respective efficacy dose when used alone. We investigated the antitumor potential of this novel combination in vitro and in vivo. The combination index was assessed for these two drugs to look for synergistic antiproliferative activity against a broad spectrum of human cancer cell lines. Consistent additive to synergistic interactions were observed in HCT116 cells when PENT was combined with SAHA at all drug tested concentrations. The combination of SAHA and PENT induces chromatin condensation and apoptosis downstream of the pan histone deacetylase inhibition and phosphodiesterase regulation, leading to subsequent cell cycle arrest at their lower tested concentrations. Further, the ability of this combination to inhibit angiogenesis, both in vitro and in vivo, was examined and a significant inhibition in tube formation in HUVEC cells and neovascularization of Matrigel plug was observed. A significant inhibition in tumor growth was observed in severe combined immunodeficient mice bearing HCT116 (colon) and PC3 (prostate) human xenografts treated with SAHA (30 mg/kg, intraperitoneal) in combination with PENT (60 mg/kg, intraperitoneal), with no loss in body weight and 100% survival. In conclusion, these findings indicate the enhanced anticancer activity of SAHA in combination with PENT both in vitro and in vivo.

Ginsenoside Rg5 attenuates hepatic glucagon response via suppression of succinate-associated HIF-1α induction in HFD-fed mice.

Ginsenosides regulate glucose homeostasis. This study investigated the effect of ginsenoside Rg5 (Rg5) on the hepatic glucagon response, focusing on the regulation of metabolism. Mice fed a high-fat diet (HFD) showed increased hepatic glucose production (HGP). We observed the effects of Rg5 on hepatic fatty acid oxidation and glucagon response. The regulation of phosphodiesterase (PDE) 4B by succinate was also investigated in hepatocytes. Rg5 inhibited endogenous glucose production in HFD-fed mice. Rg5 reduced cyclic AMP (cAMP) accumulation and inhibited transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) by dephosphorylation of the cAMP response element-binding transcription factor in the liver, demonstrating the inhibitory effect on hepatic glucagon response. HFD feeding increased succinate accumulation in the liver due to the reversal of succinate dehydrogenase activation and triggered hypoxia-inducible factor-1α (HIF-1α) induction. Succinate prevented cAMP degradation by inactivating PDE4B, thereby increasing cAMP accumulation in response to glucagon. Knockdown of HIF-1α with small interfering RNA diminished the effect of succinate, indicating that HIF-1α was essential for succinate to inactivate PDE4B. Rg5 inhibited succinate accumulation in hepatocytes by combating fatty acid oxidation, and thus reduced cAMP accumulation by blocking succinate/HIF-1α induction. Rg5 reduced HGP as a consequence of the inhibition of the glucagon response. Succinate acted as a metabolic signal to enhance the hepatic glucagon response. Rg5 reduced hepatic succinate accumulation by combating fatty acid oxidation and attenuated the hepatic glucagon response by suppressing succinate/HIF-1α induction, suggesting that succinate-associated HIF-1α induction in hepatocytes might be a therapeutic target in the treatment of diabetes.

Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation.

Phosphodiesterase inhibitor, pentoxifylline enhances anticancer activity of histone deacetylase inhibitor, MS-275 in human breast cancer in vitro and in vivo

MS-275, a histone deacetylase inhibitor (HDACi), is undergoing clinical trials for treatment of various cancers. Pentoxifylline, a nonselective phosphodiesterase (PDE) inhibitor, has been shown to increase the effectiveness of antitumor chemotherapy. In the present study, the potential anti-cancer activity of MS-275 in combination with pentoxifylline in panel of cell lines and human breast cancer xenograft model were examined.A Panel of cancer cell lines were treated with MS-275 and pentoxifylline to determine their impact on cellular proliferation, cell cycle regulation, apoptosis, anti-angiogenesis. The in vivo activities of MS-275 and pentoxifylline were assessed in a Matrigel plug angiogenesis model and human breast cancer (MDA-MB-231) xenograft model.Combination of MS-275 with pentoxifylline showed enhanced anti-proliferative activity in a panel of cancer cell lines (HCT 116, MCF-7, PC3 and MDA-MB-231). Apoptotic studies performed using, Hoechst staining and cell cycle analysis reveal that this combination at the lower concentrations induces apoptosis downstream of the HDAC inhibition and PDE regulation. Further, combination showed enhanced antiangiogenic activity in Matrigel tube formation assay using HUVECs and in Matrigel plug assay in vivo. A significant inhibition (P<0.001) of tumor growth was observed in mice bearing MDA-MB-231 breast cancer xenograft treated with the combination of MS-275 (5mg/kg p.o.) and pentoxifylline (60mg/kg i.p.) than treatments alone, without much signs of toxicity.Taken together, our study demonstrated enhanced anticancer activity of MS-275 and pentoxifylline combination both in vitro and in vivo with reduced toxicity. However, further studies are required to understand the mechanism for this combination effect.

Phosphodiesterase regulation of alcohol drinking in rodents

Alcohol use disorders are chronically relapsing conditions characterized by persistent drinking despite the negative impact on one's life. The difficulty of achieving and maintaining sobriety suggests that current treatments fail to fully address the underlying causes of alcohol use disorders. Identifying additional pathways controlling alcohol consumption may uncover novel targets for medication development to improve treatment options. One family of proteins recently implicated in the regulation of alcohol consumption is the cyclic nucleotide phosphodiesterases (PDEs). As an integral component in the regulation of the second messengers cyclic AMP and cyclic GMP, and thus their cognate signaling pathways, PDEs present intriguing targets for pharmacotherapies to combat alcohol use disorders. As activation of cAMP/cGMP-dependent signaling cascades can dampen alcohol intake, PDE inhibitors may provide a novel target for reducing excessive alcohol consumption, as has been proposed for PDE4 and PDE10A. This review highlights preclinical literature demonstrating the involvement of cyclic nucleotide-dependent signaling in neuronal and behavioral responses to alcohol, as well as detailing the capacity of various PDE inhibitors to modulate alcohol intake. Together these data provide a framework for evaluating the potential utility of PDE inhibitors as novel treatments for alcohol use disorders.

Carbon monoxide modulates electrical activity of murine myocardium via cGMP-dependent mechanisms.

Carbon monoxide (CO) is critical in cell signaling, and inhalation of gaseous CO can impact cardiovascular physiology. We have investigated electrophysiological effects of CO and their potential cGMP-dependent mechanism in isolated preparations of murine myocardium. The standard microelectrode technique was used to record myocardial action potentials (APs). Exogenous CO (0.96 × 10(-4)-4.8 × 10(-4) M) decreased AP duration in atrial and ventricular tissue and accelerated pacemaking activity in sinoatrial node. Inhibitors of heme oxygenases (zinc and tin protoporphyrin IX), which are responsible for endogenous CO production, induced the opposite effects. Inhibitor of soluble guanylate cyclase (sGC), ODQ (10(-5) M) halved CO-induced AP shortening, while sGC activator azosidnone (10(-5) M-3 × 10(-4) M) and cGMP analog BrcGMP (3 × 10(-4) M) induced the same effects as CO. To see if CO effects are attributed to differential regulation of phosphodiesterase 2 (PDE2) and 3 (PDE3), we used inhibitors of these enzymes. Milrinone (2 × 10(-6) M), selective inhibitor of cGMP-downregulated PDE3, blocked CO-induced rhythm acceleration. EHNA(2 × 10(-6) M), which inhibits cGMP-upregulated PDE2, attenuated CO-induced AP shortening, but failed to induce any positive chronotropic effect. Our findings indicate that PDE2 activity prevails in working myocardium, while PDE3 is more active in sinoatrial node. The results suggest that cardiac effects of CO are at least partly attributed to activation of sGC and subsequent elevation of cGMP intracellular content. In sinoatrial node, this leads to PDE3 inhibition, increased cAMP content, and positive chronotropy, while it also causes PDE2 stimulation in working myocardium, thereby enhancing cAMP degradation and producing AP shortening. Thus, CO induces significant alterations of cardiac electrical activity via cGMP-dependent mechanism and should be considered as a novel regulator of cardiac electrophysiology.

Roles of Phosphodiesterases in Cyclic Nucleotide Cross-Talk in Cardiac Myocytes

The cyclic nucleotides (cNs) cAMP and cGMP regulate the response of cardiac myocytes to both external and internal stimuli. Distinctly-regulated phosphodiesterases (PDEs) control the degradation of these cNs. As a result of their regulation by cNs, PDEs also facilitate communication between the β-adrenergic and Nitric Oxide (NO)/cGMP/Protein Kinase G (PKG) signaling pathways, which regulate the synthesis of cAMP and cGMP, respectively. Activation of the β-adrenergic pathway potentiates cardiac contractility, whereas activation of the NO/cGMP/PKG pathway reduces it. As a result, the balance between cNs plays a critical role in regulating contraction. The phenomena where cAMP influences the dynamics of the cGMP pathway, and vice versa, are commonly referred to as cN cross-talk. However, the cross-talk response and the individual role of each PDE isoenzyme in shaping this response remain to be fully characterized. We have developed a computational model of the cN cross-talk network that mechanistically integrates the β-adrenergic and NO/cGMP/PKG pathways via regulation of PDEs by both cNs. The individual model components and the entire network model replicate experimentally observed activation-response relationships and temporal dynamics. The model predicts that under sub-maximal β-adrenergic stimulation, an increase of PDE2 and a decrease of PDE3 cAMP hydrolysis rates under concomitant NO stimulation results in a net cAMP accumulation, leading to the observed NO-mediated potentiation of Protein Kinase A (PKA) activation. In addition, under concomitant β-adrenergic stimulation, due to cGMP accumulation from increased PDE5 and decreased PDE3 cGMP hydrolysis rates, PKG can be further activated beyond the level achieved under NO alone. By defining cN cross-talk reactions based on the binding affinity of cNs to specific PDE domains and the associated hydrolysis rates, we pin-pointed the principal mechanisms driving the cross-talk response within this non-linear, tightly-coupled reaction system.

Regulation of the Cardiac β-Adrenergic Pathway via Camp-Cgmp Competition

In the cardiac myocyte, the second messenger cAMP is synthesized by the β-adrenergic signaling pathway upon sympathetic activation. It activates Protein Kinase A (PKA) mediated phosphorylation of downstream targets that are critical to the control of cardiac contractility. The dynamics of cAMP are also controlled indirectly by cGMP-mediated regulation of phosphodiesterase (PDE) isoenzymes. It is not yet clear how the cGMP signal is transduced by the PDEs to regulate cAMP. To better understand this, we have developed mechanistically detailed models of PDEs 1 - 4, the primary cAMP hydrolyzing PDEs in cardiac myocytes, and integrated them into an existing model of the β-adrenergic signaling pathway. Our PDE models are based upon experimental studies performed on purified PDE enzymes which show that cyclic nucleotides (cNs) bind competitively to the domains of PDEs 1, 2, and 3. PDE4 is regulated by PKA but does not interact appreciably with cGMP. Our individual PDE models reproduce cAMP hydrolysis rates as regulated by various cGMP concentrations, and the fully integrated model also replicates experimentally observed cAMP dose-response relationships and temporal dynamics. Our model shows that PDE2 is critical to the regulation of cAMP signals, especially during increased stimulation of the β-adrenergic pathway. In addition, it reveals that high levels of cGMP out-compete cAMP for catalytic sites of PDEs 1, 2, and 3, and suppress their cAMP hydrolysis rates. This leads to a net accumulation of cAMP despite the negative feedback of the cAMP-driven increases in rate of PDE4-mediated cAMP hydrolysis. These results provide insights into how PDEs serve as an integration point for cN signals and how cN interactions regulate β-adrenergic response.

Abstract 20459: Epigenetic Activation of Phosphodiesterase Subtypes Lead to Compromised Beta-Adrenergic Signaling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes From Dilated Cardiomyopathy Patients

Introduction: Familial dilated cardiomyopathy (DCM) has been modeled by human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, the mechanisms of compromised signaling transduction and contractile function in DCM iPSC-CMs are still not well understood. Methods and Results: Beating iPSC-CMs were generated from healthy individuals and DCM patients. RNA-seq and real-time PCR showed strict regulation of the main beta-adrenergic signaling proteins in iPSC-CMs during maturation. Confocal imaging of spontaneous calcium activity and hydrogel-based traction force microscopy (TFM) technology demonstrated beta-adrenergic stimulation induced both inotropic and chronotropic regulation in the contractility of iPSC-CMs. Following extended in vitro maturation of iPSC-CMs, we observed a transition in the beta-adrenergic receptor (beta-AR) subtype dependence from beta-2 AR dominance at early stage (day 30) to beta-1/2 AR co-existence at late stage (day 60). Comparison of the beta-adrenergic responsiveness between iPSC-CMs from DCM patients and their familial controls showed compromised beta-adrenergic signaling in DCM cells. Microarray data and expression profiling indicated up-regulated phosphodiesterases (PDE) 2A, 3A and 5A in DCM iPSC-CMs, which impaired cAMP generation and blunted the beta-adrenergic response. By blocking PDE2A, 3A or 5A, beta-adrenergic signaling reactivity and contractile function in DCM iPSC-CMs were both greatly improved. To further elucidate underlying mechanism of PDE regulation, we conducted chromatin immunoprecipitation (CHIP) assays, which showed significant up-regulation of activation histone marker and down-regulation of repressive histone marker in the PDE promoters during maturation process of DCM iPSC-CMs, which closely recapitulated the epigenetic modulation in the ventricle tissues harvested from DCM patients. Conclusions: Patient-specific DCM iPSC-CMs recapitulated impaired beta-adrenergic responsiveness and contractility in diseased heart. Studies on iPSC-CM model revealed a novel epigenetic mechanism that underlies PDE subtype specific regulation and signaling deficiency in DCM pathogenesis, which may serve as a new therapeutic target in the future.