Protein Kinase G Activity Research Articles

Apelin and Acetylcholine Activate Distinct Nitric Oxide Signaling Pathways to Cause Endothelium‐Dependent Relaxation of Isolated Coronary Arteries

The peptide, apelin, is expressed in fat cells, endothelial cells, and CNS neurons. Increasing evidence (e.g. inotropic and vasomotor effects) supports a role for apelin in the regulation of the cardiovascular system. Apelin is reported to cause vasodilation in peripheral arteries but the underlying molecular mechanisms are not well understood. In the present study, we investigated the role of nitric oxide (NO) in apelin‐induced relaxation of isolated rat coronary arteries, and compared it with the classic endothelium‐dependent vasodilator, acetylcholine. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and RT‐PCR analysis (n=3). Immunofluorescence staining and confocal microscopy demonstrated the presence of APJ receptors on endothelial and smooth muscle cells in the coronary arterial wall. In freshly isolated coronary endothelial cells, apelin (10−6 M) caused an increase in DAF‐2 fluorescence that was abolished by nitro‐l‐arginine (3 × 10−5 M; NLA) and F13A (10−7 M), an APJ receptor antagonist, consistent with an increase in NO production. In coronary arterial rings contracted with 5‐HT (10−7 M), apelin caused endothelium‐dependent relaxations (pD2=6.90 ± 0.1; Emax=45 ± 6%; n=9) that were abolished in the presence of NLA, F13A, or iberiotoxin (10−7 M; IBTx), as well as in rings contracted with a depolarizing concentration of K+ (40 mM). Neither ODQ (10−5 M) nor DT‐2 (10−6 M), a protein kinase G (PKG) inhibitor, had any effect on apelin‐induced relaxations, and apelin (10−6 M) itself had no effect on intracellular cGMP accumulation in coronary arteries (0.09 ± 0.03 vs 0.05 ± 0.01 pmol/mg; n=4; p>0.05). By contrast, endothelium‐dependent relaxations to acetylcholine (pD2=6.89 ± 0.1; Emax=97 ± 2%; n=8) were inhibited by NLA, ODQ, and DT‐2. Acetylcholine (10−6 M) increased DAF‐2 fluorescence in isolated endothelial cells, and increased cGMP accumulation (0.08 ± 0.04 vs 0.26 ± 0.02 pmol/mg; n=4; p<0.05) in coronary arteries in an NLA‐ and ODQ‐sensitive manner. Patch clamp studies in freshly isolated coronary smooth muscle cells demonstrated that the NO donor, DEA NONOate (10−6 M) caused an increase in large conductance, calcium‐activated K (BKCa) current (peak current density (+80mV) = 53.02 ± 10.4 vs. 90.42 ± 8.6 pA/pF without and with DEA NONOate, respectively; n=6; p<0.05), that was inhibited by IBTx (peak current density = 33.8 ± 5.3 pA/pF; n=6; p<0.05 vs DEA NONOate alone) but not by ODQ (peak current density = 90.2 ± 7.9 pA/pF; n=6; p>0.05 vs DEA NONOate alone). Taken together, the data demonstrate that, in coronary arteries, endothelium‐derived NO causes arterial relaxation via two distinct signaling pathways: 1) which is activated by apelin, is sensitive to changes in membrane potential and requires activation of BKCa channels; this pathway is independent of cGMP formation and may be due to direct activation of BKCa channels by NO; and 2) which is activated by acetylcholine, is cGMP‐dependent and is mediated by activation of PKG. Differential regulation of distinct NO‐signaling pathways may provide opportunities for developing novel therapeutic strategies for the treatment of the coronary arterial dysfunction that often occurs in cardiovascular disease.Support or Funding InformationSupported by a grant from the NIH National Heart, Lung and Blood Institute (HL124338)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Type 1 Phosphodiesterse Confers Therapeutic Benefit to Proteinopathy‐based HFpEF in Mice

Heart Failure with preserved Ejection Fraction (HFpEF) has overtaken Heart Failure with reduced Ejection Fraction (HFrEF) and represents more than 50% of heart failure cases, yet no pharmacological therapy with mortality benefits has been identified. Targeted clearance of misfolded proteins by the ubiquitin‐proteasome system (UPS) plays a pivotal role in protein quality control. UPS function insufficiency contributes to HF genesis, making UPS priming a potential therapeutic strategy for treatment of HF. Previously we reported that protein kinase G (PKG) positively regulates cardiac UPS functioning while others showed protein kinase A (PKA) stimulates the proteasome. Thus, we hypothesize that simultaneous activation of PKG and PKA improves cardiac UPS function and thereby benefits HF treatment. We found that myocardial protein and mRNA levels of phosphodiesterase 1 (PDE1), a major cardiac PDE that degrades both PKG and PKA, were markedly elevated in CryABR120G transgenic (tg) mice which are a classic model of cardiac proteinopathy with HFpEF as the major manifestation of its cardiac dysfunction. To test whether PDE1 inhibition primes cardiac UPS and thereby ameliorates cardiac proteotoxicity, we performed cell cultures and mouse in vivo experiments taking advantage of a proven surrogate UPS substrate (GFPdgn) and a bona fide misfolded protein (CryABR120G), which demonstrates that pharmacological PDE1 inhibition significantly improves cardiac UPS performance and enhances degradation of misfolded proteins. We then treated CryABR120G tg mice with PDE1 inhibitor LSN2790158 (3 mg/kg/day × 28 days) or vehicle control via osmotic mini‐pumps, starting at 4 months of age when HFpEF has clearly developed in the tg mice as reflected by significant decreases in left ventricular (LV) end‐diastolic volume, stroke volume, and cardiac output and increases in end‐diastolic LV posterior wall thickness along with unchanged EF (>50%), compared to littermate non‐tg mice. Echocardiography revealed that the PDE1 inhibitor treatment resulted in striking improvement of HFpEF. And Kaplan‐Meier survival analysis shows a significant delay of premature death in the PDE1 inhibition group vs. vehicle‐treated group. Additionally, PDE1 inhibition group showed significant reduction of NP‐40 insoluble but SDS soluble fractions of CryAB that represent a misfolded species of CryAB degraded by the proteasome, suggesting that the cardioprotective effect of PDE1 inhibition is, at least in part, due to enhanced removal of misfolded proteins. Our data provide compelling evidence for the first time that PDE1 inhibition shall be explored as a new therapeutic strategy for HFpEF and heart disease with increased proteotoxic stress.Support or Funding InformationThis work was supported in part by NIH grants HL072166, HL085629, and HL131667 and American Heart Association Predoctoral Fellowship grant 16PRE27790059; the PDE1 inhibitor LSN2790158 was provided by Eli Lilly and CompanyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelium-derived hyperpolarizing factor and protein kinase G Iα activation: H2O2 versus S-nitrosothiols

Protein kinase G (PKG) Iα mediates the cyclic guanosine monophosphate-mediated vasodilatory effects induced by NO. Endothelium-derived hyperpolarizing factors (EDHFs), like H2O2 can activate PKGIα in a cyclic guanosine monophosphate-independent manner, but whether this is true for all EDHFs (e.g., S-nitrosothiols) is unknown. Here, we investigated the contribution of PKGIα to bradykinin-, H2O2-, L-S-nitrosocysteine-, and light-induced relaxation in porcine coronary arteries, making use of the fact that thioredoxin reductase inhibition with auranofin or 1-chloro-2,4-dinitrobenzene potentiates PKGIα. Thioredoxin reductase inhibition potentiated bradykinin and H2O2, but not L-S-nitrosocysteine or light. The relaxations by the latter 2 and bradykinin, but not those by H2O2, were prevented by the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Yet, after S-nitrosothiol depletion with ethacrynic acid, thioredoxin reductase inhibition also potentiated light-induced relaxation, and this was prevented by the Na+-K+ ATPase inhibitor ouabain. This indicates that photorelaxation depends on sGC activation by S-nitrosothiols, while only after S-nitrosothiol depletion oxidized PKGIα comes into play, and acts via Na+-K+ ATPase. In conclusion, both bradykinin- and light-induced relaxation of porcine coronary arteries depend, at least partially, on oxidized PKGIα, and this does not involve sGC. H2O2 also acts via oxidized PKGIα in an sGC-independent manner. Yet, S-nitrosothiol-induced relaxation is PKGIα-independent. Clearly, PKG activation does not contribute universally to all EDHF responses, and targeting PKGIα may only mimick EDHF under certain conditions. It is therefore unlikely that PKGIα activators will be universal vasodilators.

Prevention of PKG-1α Oxidation Suppresses Antihypertrophic/Antifibrotic Effects From PDE5 Inhibition but not sGC Stimulation.

Stimulation of sGC (soluble guanylate cyclase) or inhibition of PDE5 (phosphodiesterase type 5) activates PKG (protein kinase G)-1α to counteract cardiac hypertrophy and failure. PKG1α acts within localized intracellular domains; however, its oxidation at cysteine 42, linking homomonomers, alters this localization, impairing suppression of pathological cardiac stress. Because PDE5 and sGC reside in separate microdomains, we speculated that PKG1α oxidation might also differentially influence the effects from their pharmacological modulation. Knock-in mice expressing a redox-dead PKG1α (PKG1αC42S) or littermate controls (PKG1αWT) were subjected to transaortic constriction to induce pressure overload and treated with a PDE5 inhibitor (sildenafil), sGC activator (BAY602770 [BAY]), or vehicle. In PKG1αWT controls, sildenafil and BAY similarly enhanced PKG activity and reduced pathological hypertrophy/fibrosis and cardiac dysfunction after transaortic constriction. However, sildenafil failed to protect the heart in PKG1αC42S, unlike BAY, which activated PKG and thereby facilitated protective effects. This corresponded with minimal PDE5 activation in PKG1αC42S exposed to transaortic constriction versus higher activity in controls and little colocalization of PDE5 with PKG1αC42S (versus colocalization with PKG1αWT) in stressed myocytes. In the stressed heart and myocytes, PKG1α C42-disulfide formation contributes to PDE5 activation. This augments the pathological role of PDE5 and so in turn enhances the therapeutic impact from its inhibition. PKG1α oxidation does not change the benefits from sGC activation. This finding favors the use of sGC activators regardless of PKG1α oxidation and may help guide precision therapy leveraging the cyclic GMP/PKG pathway to treat heart disease.

The anti-diabetic drug dapagliflozin induces vasodilation via activation of PKG and Kv channels

AimConsidering the clinical efficacy of dapagliflozin in patients with type 2 DM and the pathophysiological relevance of Kv channels for vascular reactivity. We investigate the vasodilatory effect of dapagliflozin and related mechanisms using phenylephrine (Phe)-induced contracted aortic rings. Material and methodsArterial tone measurement was performed in aortic smooth muscle. Key findingsApplication of dapagliflozin induced vasodilation in a concentration-dependent manner. Pre-treatment with the BKCa channel inhibitor paxilline, the KATP channel inhibitor glibenclamide, and the Kir channel inhibitor Ba2+ did not change dapagliflozin-induced vasodilation. However, application of the Kv channels inhibitor 4-AP effectively inhibited dapagliflozin-induced vasodilation. Application of the Ca2+ channel inhibitor nifedipine and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor thapsigargin did not alter the vasodilatory effect of dapagliflozin. Moreover, the adenylyl cyclase inhibitor SQ 22536 and the protein kinase A (PKA) inhibitor KT 5720 had no effect on dapagliflozin-induced vasodilation. Although guanylyl cyclase inhibitors, NS 2028 and ODQ, did not reduce the vasodilatory effect of dapagliflozin, the protein kinase G (PKG) inhibitor KT 5823 effectively inhibited dapagliflozin-induced vasodilation. The vasodilatory effect of dapagliflozin was not affected by elimination of the endothelium. Furthermore, pretreatment with the nitric oxide synthase inhibitor L-NAME or the small-conductance Ca2+-activated K (SKCa) channel inhibitor apamin did not change the vasodilatory effect of dapagliflozin. SignificanceWe concluded that dapagliflozin induced vasodilation via the activation of Kv channels and PKG, and was independent of other K+ channels, Ca2+ channels, intracellular Ca2+, and the endothelium.

Nitric oxide-cGMP-PKG signaling in the bed nucleus of the stria terminalis modulates the cardiovascular responses to stress in male rats

The bed nucleus of the stria terminalis (BNST) constitutes an important component of neural substrates of physiological and behavioral responses to aversive stimuli, and it has been implicated on cardiovascular responses evoked by stress. Nevertheless, the local neurochemical mechanisms involved in BNST control of cardiovascular responses during aversive threats are still poorly understood. Thus, the aim of the present study was to assess the involvement of activation in the BNST of the neuronal isoform of the enzyme nitric oxide synthase (nNOS), as well as of signaling mechanisms related to nitric oxide effects such as soluble guanylate cyclase (sGC) and protein kinase G (PKG) on cardiovascular responses induced by an acute session of restraint stress in male rats. We observed that bilateral microinjection of either the nonselective NOS inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME), the selective nNOS inhibitor Nω-Propyl-L-arginine (NPLA) or the sGC inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) into the BNST enhanced the tachycardic response and decreased the drop in tail cutaneous temperature evoked by acute restraint stress, but without affecting the increase on blood pressure. Bilateral BNST treatment with the selective PKG inhibitor KT5823 also facilitated the heart rate increase and decreased the drop in cutaneous temperature, in addition to enhancing the blood pressure increase. Taken together, these results provide evidence that NO released from nNOS and activation of sGC and PKG within the BNST play an inhibitory influence on tachycardia to stress, whereas this signaling mechanism mediates the sympathetic-mediated cutaneous vasoconstriction.

New dimeric cGMP analogues reduce proliferation in three colon cancer cell lines

Activation of the cGMP-dependent protein kinase G (PKG) can inhibit growth and/or induce apoptosis in colon cancer. In this study we evaluated the effects on cell viability, cell death and proliferation of novel dimeric cGMP analogues, compared to a monomeric compound. Three colon cancer cell lines, which only express isoform 2 of PKG, were treated with these novel cGMP analogues and responded with increased PKG activity. cGMP analogues reduced cell viability in the three cell lines and this was due to a cytostatic rather than cytotoxic effect. These findings suggest that activation of PKG2 can be a therapeutic target in the treatment of colon cancer and, most importantly, that dimeric cGMP analogues can further improve the beneficial effects previously observed with monomeric cGMP analogues.

Sildenafil improves blood perfusion in steroid-induced avascular necrosis of femoral head in rabbits via a protein kinase G-dependent mechanism

ObjectiveThe aim of the study were to evaluate the effect of sildenafil against avascular necrosis of femoral head (ANFH) in a rabbit model, and to study the role of protein kinase G (PKG) pathway and vascular endothelial growth factor (VEGF) in ANFH. MethodsThree weeks after inducing ANFH with methylprednisolone injection, 45 female adult New Zealand white rabbits were divided into three groups and treated as follows: group SI received daily intraperitoneal sildenafil with a dose of 10 mg/kg per day; group SD received daily sildenafil identically to group SI plus auricular vein injection DT3 (a specific PKG inhibitor); group NS received only normal saline. The blood perfusion function in the femoral head was measured by perfusion MRI and ink artery infusion. Bilateral femora heads were examined histopathologically for the presence of osteonecrosis; VEGF of tissue was examined by Western blot analysis; cGMP level and PKG activity were also measured. ResultsThe incidence of ANFH in SI group was significantly lower than that observed in NS and SD groups (p < 0.05). VEGF in SI group was increased compared to NS group. cGMP level and PKG activity were also significantly different between NS and SI group (p < 0.05). However, these effects of sildenafil in SD group were all markedly inhibited by the administration of DT3 compared to SI group. ConclusionSildenafil appear to increase the perfusion of femoral head by up-regulating VEGF through PKG pathway. The increased perfusion of femoral head could prevent ANFH.

Abstract 62: Sildenafil Inhibits Neointimal Hyperplasia After Angioplasty, and Reduces Platelet Aggregation

Background: Restenosis and stent thrombosis after stent implantation are one of the devating issues in interventional cardiology. Sildenafil has shown its effect in reducing cardiac hypertrophy as well as improving erectile dysfunction through protein kinase G (PKG) activation. Some studies have demonstrated that PKG occupies a central switching role in modulating vascular smooth muscle cell (VSMC) phenotype in response to vascular injury. In this study, we investigated the effects of PKG activation by sidenafil on platelet aggregation and neointimal hyperplasia. Methods and Results: In terms of restenosis after vascular injury, sildenafil significantly reduced neointimal hyperplasia in rat carotid arteries compared to control group. This effect of sildenafil was accompanied by the reduction of viability, cell cycle progression, and migration of VSMCs. This was also confirmed in the injured arteries in vivo. Further studies showed that the increased PKG activity by sildenafil inhibited PDGF-stimulated phenotype change of VSMCs from a contractile to a synthetic form. Conversely, the use of PKG inhibitor or gene transfer of dominant-negative PKG reversed the effects of sildenafil, resulting in the increased viability of VSMCs and neointimal formation. In addition, the mice treated with sidenafil showed the facilitated re-endothelialization, compared to control group. Furthermore, we confirmed the effect of sidenafil through PKG activation using cGK-KO mice. Interestingly, sildenafil significantly inhibited platelet aggregation induced by ADP or thrombin. This effect was reversed by PKG inhibitor, suggesting that sildenafil inhibits platelet aggregation via PKG pathway. Furthermore, assays for VASP phosphorylation and P-selectin activation showed the same inhibitory effect of sildenafil on platelet activation. Conclusions: This study showed that sildenafil inhibits not only platelet aggregation, but also neointimal hyperplasia through the PKG pathway. These findings suggest that sildenafil could be a promising candidate drug of drug-eluting stents for the prevention of restenosis without other complications such as stent thrombosis.

Abstract 222: Inhibition of Phosphodiesterase 1 Confers Striking Therapeutic Benefit to HFpEF in Mice

Heart Failure with preserved Ejection Fraction (HFpEF) is the most prevalent HF but with virtually no effective treatment proven. Targeted removal of misfolded proteins by the ubiquitin-proteasome system (UPS) is pivotal to protein quality control. UPS function insufficiency contributes to HF genesis, making UPS priming an attractive strategy to treat HF. Previously we reported that protein kinase G (PKG) positively regulates cardiac UPS functioning while others showed PKA stimulates the proteasome. Thus, we propose that simultaneous activation of PKG and PKA might be a powerful strategy to improve cardiac UPS function and thereby benefit HF treatment. Here we provide the first evidence that strongly supports this proposition. We found that myocardial protein and mRNA levels of phosphodiesterase 1 (PDE1), a major cardiac PDE that degrades both PKG and PKA, were markedly elevated in CryAB R120G transgenic (tg) mice which are a classic model of cardiac proteinopathy with HFpEF as the major manifestation of its cardiac dysfunction. To test whether PDE1 inhibition (PDE1I) primes cardiac UPS and thereby ameliorates cardiac proteotoxicity, we performed cell cultures and mouse in vivo experiments taking advantage of a proven surrogate UPS substrate (GFPdgn) and a bona fide misfolded protein (CryAB R120 ), which demonstrates that pharmacological PDE1I significantly improves cardiac UPS performance and enhances degradation of the misfolded protein. We then treated CryAB R120G tg mice with PDE1 inhibitor LSN2790158 (3mg/kg/day x 28 days) or vehicle control via osmotic mini-pumps, starting at 4 months of age when HFpEF has clearly developed in the tg mice as reflected by significant decreases in left ventricular (LV) end-diastolic volume, stroke volume, and cardiac output and increases in end-diastolic LV posterior wall thickness along with unchanged EF, compared to littermate non-tg mice. Echocardiography revealed that that 4 weeks of PDE1 inhibitor treatment resulted in drastic improvement of HFpEF and Kaplan-Meier survival analysis shows striking delay of premature death in the PDE1I group vs. vehicle-treated group. Taken together, our data provide compelling evidence for the first time that PDE1I shall be explored as a new therapeutic strategy for HFpEF.

Sulfhydration-associated phosphodiesterase 5A dimerization mediates vasorelaxant effect of hydrogen sulfide.

The study was designed to examine if the vasorelaxant effect of hydrogen sulfide was mediated by sulfhydration-associated phosphodiesterase (PDE) 5A dimerization. The thoracic aorta of rat was separated and the vasorelaxant effects were examined with in vitro vascular perfusion experiments. The dimerization and sulfhydration of PDE 5A and soluble guanylatecyclase (sGC) were measured. PDE 5A and protein kinase G (PKG) activities were tested. Intracellular cGMP content was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that NaHS relaxed isolated rat vessel rings at an EC50 of (1.79 ± 0.31)×10−5mol/L, associated with significantly increased PKG activity and cGMP content in vascular tissues. Sulfhydration of sGC β1 was increased, while the levels of sGC αβ1 dimers were apparently decreased after incubation with NaHS in vascular tissues. Moreover, PDE 5A homodimers were markedly decreased, and accordingly the PDE 5A activity demonstrated by the content of 5′-GMP was significantly decreased after incubation with NaHS or GYY4137. Mechanistically, both NaHS and GYY4137 significantly enhanced the PDE 5A sulfhydration in vascular tissues. DTT partially abolished the effects of NaHS on PDE 5A activity, cGMP content and vasorelaxation. Therefore, the present study for the first time suggested that H2S exerted vasorelaxant effect probably via sulfhydration-associated PDE 5A dimerization.

Type 5 phosphodiesterase regulates glioblastoma multiforme aggressiveness and clinical outcome.

Expression of type 5 phosphodiesterase (PDE5), a cGMP-specific hydrolytic enzyme, is frequently altered in human cancer, but its specific role in tumorigenesis remains controversial. Herein, by analyzing a cohort of 69 patients affected by glioblastoma multiforme (GBM) who underwent chemo- and radiotherapy after surgical resection of the tumor, we found that PDE5 was strongly expressed in cancer cells in about 50% of the patients. Retrospective analysis indicated that high PDE5 expression in GBM cells significantly correlated with longer overall survival of patients. Furthermore, silencing of endogenous PDE5 by short hairpin lentiviral transduction (sh-PDE5) in the T98G GBM cell line induced activation of an invasive phenotype. Similarly, pharmacological inhibition of PDE5 activity strongly enhanced cell motility and invasiveness in T98G cells. This invasive phenotype was accompanied by increased secretion of metallo-proteinase 2 (MMP-2) and activation of protein kinase G (PKG). Moreover, PDE5 silencing markedly enhanced DNA damage repair and cell survival following irradiation. The enhanced radio-resistance of sh-PDE5 GBM cells was mediated by an increase of poly(ADP-ribosyl)ation (PARylation) of cellular proteins and could be counteracted by poly(ADP-ribose) polymerase (PARP) inhibitors. Conversely, PDE5 overexpression in PDE5-negative U87G cells significantly reduced MMP-2 secretion, inhibited their invasive potential and interfered with DNA damage repair and cell survival following irradiation. These studies identify PDE5 as a favorable prognostic marker for GBM, which negatively affects cell invasiveness and survival to ionizing radiation. Moreover, our work highlights the therapeutic potential of targeting PKG and/or PARP activity in this currently incurable subset of brain cancers.

Prevention of the development of heart failure with preserved ejection fraction by the phosphodiesterase-5A inhibitor vardenafil in rats with type 2 diabetes.

AimsHeart failure with preserved ejection fraction (HFpEF) has a great epidemiological burden. The pathophysiological role of cyclic guanosine monophosphate (cGMP) signalling has been intensively investigated in HFpEF. Elevated levels of cGMP have been shown to exert cardioprotective effects in various cardiovascular diseases, including diabetic cardiomyopathy. We investigated the effect of long‐term preventive application of the phosphodiesterase‐5A (PDE5A) inhibitor vardenafil in diabetic cardiomyopathy‐associated HFpEF.Methods and resultsZucker diabetic fatty (ZDF) rats were used as a model of HFpEF and ZDF lean rats served as controls. Animals received vehicle or 10 mg/kg body weight vardenafil per os from weeks 7 to 32 of age. Cardiac function, morphology was assessed by left ventricular (LV) pressure–volume analysis and echocardiography at week 32. Cardiomyocyte force measurements were performed. The key markers of cGMP signalling, nitro‐oxidative stress, apoptosis, myocardial hypertrophy and fibrosis were examined. The ZDF animals showed diastolic dysfunction (increased LV/cardiomyocyte stiffness, prolonged LV relaxation time), preserved systolic performance, decreased myocardial cGMP level coupled with impaired protein kinase G (PKG) activity, increased nitro‐oxidative stress, enhanced cardiomyocyte apoptosis, and hypertrophic and fibrotic remodelling of the myocardium. Vardenafil effectively prevented the development of HFpEF by maintaining diastolic function (decreased LV/cardiomyocyte stiffness and LV relaxation time), by restoring cGMP levels and PKG activation, by lowering apoptosis and by alleviating nitro‐oxidative stress, myocardial hypertrophy and fibrotic remodelling.ConclusionsWe report that vardenafil successfully prevented the development of diabetes mellitus‐associated HFpEF. Thus, PDE5A inhibition as a preventive approach might be a promising option in the management of HFpEF patients with diabetes mellitus.

Activation of Protein Kinase G (PKG) Reduces Neointimal Hyperplasia, Inhibits Platelet Aggregation, and Facilitates Re-endothelialization.

In spite of its great success in reducing restenosis, drug-eluting stent (DES) has unfavorable aspects such as stent thrombosis and delayed re-endothelialization. We examined the effects of PKG activation by Exisulind on neointimal formation, platelet aggregation, and re-endothelialization. Exisulind significantly reduced VSMCs viability, cell cycle progression, migration, and neointimal hyperplasia after vascular injury in rat carotid arteries. Interestingly, in contrast to the effect on VSMC viability, Exisulind did not reduce the viability of endothelial cells. Increased PKG activity by Exisulind inhibited PDGF-stimulated phenotype change of VSMCs from a contractile to a synthetic form. Conversely, the use of PKG inhibitor or gene transfer of dominant-negative PKG reversed the effects of Exisulind, resulting in the increased viability of VSMCs and neointimal formation. In addition, Exisulind facilitated the differentiation of peripheral blood mononuclear cells to endothelial lineage via PKG pathway, while inhibiting to VSMCs lineage, which was correlated with the enhanced re-endothelialization in vivo. Finally, Exisulind reduced platelet aggregation, which was mediated via PKG activation. This study demonstrated that Exisulind inhibits neointimal formation and platelet aggregation while increasing re-endothelialization via PKG pathway. These findings suggest that Exisulind could be a promising candidate drug of DES for the prevention of restenosis without other complications.

Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance.

Helional induces Ca2+ decrease and serotonin secretion of QGP-1 cells via a PKG-mediated pathway.

The secretion, motility and transport by intestinal tissues are regulated among others by specialized neuroendocrine cells, the so-called enterochromaffin (EC) cells. These cells detect different luminal stimuli, such as mechanical stimuli, fatty acids, glucose and distinct chemosensory substances. The EC cells react to the changes in their environment through the release of transmitter molecules, most importantly serotonin, to mediate the corresponding physiological response. However, little is known about the molecular targets of the chemical stimuli delivered from consumed food, spices and cosmetics within EC cells. In this study, we evaluated the expression of the olfactory receptor (OR) 2J3 in the human pancreatic EC cell line QGP-1 at the mRNA and protein levels. Using ratiofluorometric Ca(2+) imaging experiments, we demonstrated that the OR2J3-specific agonist helional induces a transient dose-dependent decrease in the intracellular Ca(2+) levels. This Ca(2+) decrease is mediated by protein kinase G (PKG) on the basis that the specific pharmacological inhibition of PKG with Rp-8-pCPT-cGMPS abolished the helional-induced Ca(2+) response. Furthermore, stimulation of QGP-1 cells with helional caused a dose-dependent release of serotonin that was comparable with the release induced by the application of a direct PKG activator (8-bromo-cGMP). Taken together, our results demonstrate that luminal odorants can be detected by specific ORs in QGP-1 cells and thus cause the directed release of serotonin and a PKG-dependent decrease in intracellular Ca(2.)

Abstract 131: PDE1 Inhibition Improves Cardiac Protein Quality Control

Protein quality control (PQC) functions to minimize the level and toxicity of misfolded proteins in the cell. PQC relies on molecular chaperones and the targeted degradation of misfolded proteins. The latter is currently known to require the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP). Virtually all cardiovascular diseases end up heart failure (HF), the leading cause of death of our society. UPS function insufficiency is implicated in the genesis of a large subset of HF, making cardiac PQC enhancement via promoting UPS and ALP function a promising therapeutic strategy to treat HF. Previously, we have demonstrated that stimulating protein kinase G (PKG) genetically or via inhibition of the type 5 phosphodiesterase (PDE5) improves UPS performance, facilitates the removal of misfolded proteins in cardiomyocytes and slows down the progression of cardiac proteinopathy in a transgenic mouse model (CryAB R120G ). PKA has also been shown to enhance proteasomal function. Our preliminary studies reveal that myocardial protein levels of PDE1A, which suppresses both PKG and PKA, are remarkably elevated in the CryAB R120G mice. Hence we hypothesize that PDE1 inhibition (PDE1I) stimulates cardiac proteasomes via PKG and PKA activation and thereby protects against cardiac proteotoxicity. To test our hypothesis, we took advantage of a proven surrogate UPS substrate (GFPu or GFPdgn) as well as a bona fide misfolded protein (CryAB R120G ) that is known to induce cardiac proteinopathy in human and mice. In cultured cardiomyocytes, PDE1 inhibitor LSN2790158 dose- and time-dependently decreased GFPu. Cycloheximide (CHX) chase assays further confirmed that PDE1I shortened the half-life of GFPu, indicative of improved UPS performance. Furthermore, PDE1I promoted the degradation of CryAB R120G . Our in vivo findings revealed that GFPdgn mice treated with LSN2790158 (3mg/kg, i.p.) displayed a significant reduction of myocardial GFPdgn protein but not mRNA levels. Taken together, our data strongly indicate that PDE1I improves cardiac UPS performance and PDE1 represents a potential target to treat cardiac diseases with elevated proteotoxicity.

Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as a plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.

Abstract 556: The Soluble Guanylyl Cyclase Activator Bay 60-2770 Inhibits Arterial Smooth Muscle Cell Migration in Protein Kinase G-dependent Manner

Atherosclerotic lower extremity peripheral artery disease (PAD) is among the most prevalent, morbid and mortal of all cardiovascular disorders. Pathologic arterial smooth muscle (ASM) cell migration is a major component of atherogenic PAD and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of PAD and other vascular growth disorders. In this study we hypothesized that the novel, heme-independent soluble guanylyl cyclase activator BAY 60-2770 (BAY) inhibits ASM cell migration through phosphorylation of the protein kinase G (PKG) target and actin-binding protein vasodilator-stimulated phosphoprotein (VASP). In a rat model of injury-induced arterial growth, BAY significantly reduced neointima formation and luminal narrowing compared to vehicle (Veh)-treated control arteries after 2 weeks. Using rat and human ASM cells BAY significantly attenuated cell migration, reduced G:F actin, and increased cyclic GMP content, PKG activity and phosphorylated VASP at Ser239 (pVASP.S239) compared to Veh controls. Using site-directed mutagenesis, both full-length VASP-overexpressing (wild type, WT) and VASP.S239 phosphorylation-resistant mutants showed significantly reduced cell migration compared to naïve controls, however, there was no effect on cell migration between either VASP transfected group in the presence of BAY. Interestingly, both VASP mutants showed significantly increased PKG activity compared to naïve cells, and in turn pharmacologic PKG blockade in the presence of BAY fully reversed the inhibitory effect of BAY alone on cell migration. These data suggest BAY has capacity to inhibit ASM cell migration through cyclic GMP/PKG/VASP signaling yet through mechanisms independent of pVASP.S239. Findings from this study implicate BAY via cyclic GMP/PKG/VASP as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as PAD.

Pulmonary Arteries Show Differences in the Effects of Angiotensin II Stimulation of Mitochondrial Superoxide on Regulation of Heme Biosynthesis and Soluble Guanylate Cyclase Expression

It is well established that Angiotensin II (AngII) increases mitochondrial superoxide levels through the AT1 receptor. Ferrochelatase (FECH), the mitochondrial heme‐generating enzyme, has an iron‐sulfur cluster required for its stability and activity, which could potentially be disrupted by superoxide. We have observed that 24 hr organoid culture of bovine coronary arteries (BCAs) with AngII caused a depletion of mitochondrial superoxide dismutase‐2 (SOD‐2), FECH and soluble guanylate cyclase (sGC), as well as an increase in deficiency in heme (detected by depletion of cytochrome oxidase subunit‐4 (COx4), but not catalase). The heme precursor δ‐aminolevulinic acid (ALA) prevents these effects of AngII through a mechanism that appears to involve promoting protoporphyrin IX (PpIX) accumulation and its stimulation of sGC/cGMP‐activation of protein kinase G (PKG). Thus, in this study we investigated the effects of AngII on 24 hr organoid cultured endothelium‐denuded bovine pulmonary arteries (BPAs). AngII increased mitochondrial (MitoSox/HPLC) and extra‐mitochondrial/cytosolic superoxide (DHE/HPLC) and protoporphyrin IX (PpIX) levels (detected by its surface fluorescence), and it depleted SOD‐2 (western blot) and Cox4 (western blot) protein expression. However AngII did not deplete protein expression of FECH, sGC and catalase (western blot). ALA prevented AngII‐elicited increases in mitochondrial superoxide, and depletion of SOD‐2 and COx4. Furthermore, co‐organoid culture of BPAs with AngII in presence of cGMP‐independent activation of PKG (DHEA (dehydroepiandrosterone)) and cGMP‐mediated activation of PKG (8‐bromo‐cGMP) reverses the increases in mitochondrial and extra‐mitochondrial superoxide induced by AngII in BPA. The increase in PpIX and depletion of heme, but not FECH, suggests that increased mitochondrial superoxide in AngII‐treated BPA is impairing the availability of iron for heme generation from PpIX. Since agents which bind the heme site of sGC appear to protect it from depletion when the sGC heme is oxidized, the observed AngII‐elicited increase in PpIX in BPA may prevent the depletion of sGC. In contrast, AngII did not increase PpIX in BCA, and the depletion of both FECH and heme appears to be a more dominant factor in AngII‐treated BCA. While increased PKG stimulation by PpIX generation from ALA may function to attenuate AngII‐induced increases in mitochondrial superoxide and depletion of heme in both BPA and BCA, and the increase in PpIX observed in BPA treated with AngII may function to prevent the depletion of sGC, and perhaps FECH.Support or Funding InformationSupported by NIH grant: R01HL115124