sGC Activity Research Articles

Vericiguat in Heart Failure: Characteristics, Scientific Evidence and Potential Clinical Applications.

Despite recent advances in heart failure (HF) management, the risk of death and hospitalizations remains high in the long term. HF is characterized by endothelial dysfunction, inflammation and increased oxidative stress, due to a reduction in the activity of the nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signaling pathway. All these factors contribute to direct damage at the myocardial, vascular and renal level. Vericiguat restores the deficiency in this signaling pathway, through stimulation and activation of sGC, aiming to increase cGMP levels, with a reduction in HF-related oxidative stress and endothelial dysfunction. Two main clinical trials were developed in this setting: the SOCRATES-REDUCED phase II study and the VICTORIA phase III study. They found that vericiguat is safe, well tolerated and effective with an absolute event-rate reduction in patients affected by HF with reduced ejection fraction (HFrEF) and recent cardiac decompensation. In patients with HF with preserved ejection fraction (HfpEF), the SOCRATES-PRESERVED trial demonstrated an improvement in quality of life and health status, but the proven beneficial effects with vericiguat are still limited. Further studies are needed to correctly define the role of this drug in heart failure syndromes. Our paper reviews the potential applications and pharmacological characteristics of vericiguat in HFrEF and HFpEF.

Soluble guanylate cyclase activator BAY 54-6544 improves vasomotor function and survival in an accelerated ageing mouse model.

DNA damage is a causative factor in ageing of the vasculature and other organs. One of the most important vascular ageing features is reduced nitric oxide (NO)soluble guanylate cyclase (sGC)—cyclic guanosine monophosphate (cGMP) signaling. We hypothesized that the restoration of NO‐sGC‐cGMP signaling with an sGC activator (BAY 54–6544) may have beneficial effects on vascular ageing and premature death in DNA repair‐defective mice undergoing accelerated ageing. Eight weeks of treatment with a non‐pressor dosage of BAY 54–6544 restored the decreased in vivo microvascular cutaneous perfusion in progeroid Ercc1 ∆/− mice to the level of wild‐type mice. In addition, BAY 54–6544 increased survival of Ercc1 ∆/− mice. In isolated Ercc1 ∆/− aorta, the decreased endothelium‐independent vasodilation was restored after chronic BAY 54–6544 treatment. Senescence markers p16 and p21, and markers of inflammation, including Ccl2, Il6 in aorta and liver, and circulating IL‐6 and TNF‐α were increased in Ercc1 ∆/− , which was lowered by the treatment. Expression of antioxidant genes, including Cyb5r3 and Nqo1, was favorably changed by chronic BAY 54–6544 treatment. In summary, BAY 54–6544 treatment improved the vascular function and survival rates in mice with accelerated ageing, which may have implication in prolonging health span in progeria and normal ageing.

Prenatal hypothyroidism diminished exogenous NO-mediated diastolic effects in fetal rat thoracic aorta smooth muscle via increased oxidative stress

Maternal hypothyroidism is an important problem of modern healthcare and is reported to increase the risk of cardiovascular diseases in the offspring later in life. However, it is unclear whether hypothyroidism during pregnancy causes vascular damage in the fetal period. We established the prenatal hypothyroidism rat model and collected the fetuses at the 21th day of gestation (GD21). Thyroid hormone concentrations in maternal and offspring blood serum were assessed by enzyme-linked immunosorbent assay (ELISA). The thoracic aortas of the fetuses were isolated for microvessel functional testing and histochemical stainings. qPCR and Western blot were performed to access mRNA and protein expression. We found that the concentrations of thyroid hormones in the serum of pregnant rats and fetuses were significantly suppressed at GD21. The responses of the fetal thoracic aortas to SNP were significantly attenuated in the PTU group. However, no statistical difference was found between the two groups when treated with either inhibitor (ODQ) or activator (BAY58–2667) of sGC. The production of O2−• in the arterial wall was significantly increased in hypothyroid fetuses. Moreover, the level of NADPH oxidase (NOX) was increased, while superoxide dismutase 2 (SOD2) was down-regulated in the PTU group, ultimately contributing to the increased production of superoxide. Additionally, decreased SNP-mediated vasodilation found in fetal vessels was improved by either NOX inhibitor (Apocynin) or SOD mimic (Tempol). These results indicate that increased oxidative stress is probably the cause of the diminished diastolic effect of exogenous NO in the thoracic artery of prenatal hypothyroidism exposed fetuses.

Therapeutic effects of a soluble guanylate cyclase activator, BAY 60-2770, on lower urinary tract dysfunction in mice with spinal cord injury.

We aimed to evaluate the effects of a soluble guanylate cyclase (sGC) activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury (SCI). Mice were divided into the following three groups: spinal cord intact (group A), SCI + vehicle (group B), and SCI + BAY 60-2770 (group C). SCI mice underwent Th8-Th9 spinal cord transection and treatment with BAY 60-2770 (10 mg/kg/day) once daily for 2-4 wk after SCI. We evaluated urodynamic parameters using awake cystometry and external urethral sphincter electromyograms (EMG); mRNA levels of mechanosensory channels, nitric oxide (NO)-, ischemia-, and inflammation-related markers in L6-S1 dorsal root ganglia, the urethra, and bladder tissues; and protein levels of cGMP in the urethra at 4 wk after SCI. With awake cystometry, nonvoiding contractions, postvoid residual, and bladder capacity were significantly larger in group B than in group C. Voiding efficiency (VE) was significantly higher in group C than in group B. In external urethral sphincter EMGs, the duration of notch-like reductions in intravesical pressure and reduced EMG activity time were significantly longer in group C than in group B. mRNA expression levels of transient receptor potential ankyrin 1, transient receptor potential vanilloid 1, acid-sensing ion channel (ASIC)1, ASIC2, ASIC3, and Piezo2 in the dorsal root ganglia, and hypoxia-inducible factor-1α, VEGF, and transforming growth factor-β1 in the bladder were significantly higher in group B than in groups A and C. mRNA levels of neuronal NO synthase, endothelial NO synthase, and sGCα1 and protein levels of cGMP in the urethra were significantly lower in group B than in groups A and C. sGC modulation might be useful for the treatment of SCI-related neurogenic lower urinary tract dysfunction.NEW & NOTEWORTHY This is the first report to evaluate the effects of a soluble guanylate cyclase activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury.

Nitric Oxide Signalling in Descending Vasa Recta after Hypoxia/Re-Oxygenation.

Reduced renal medullary oxygen supply is a key factor in the pathogenesis of acute kidney injury (AKI). As the medulla exclusively receives blood through descending vasa recta (DVR), dilating these microvessels after AKI may help in renoprotection by restoring renal medullary blood flow. We stimulated the NO-sGC-cGMP signalling pathway in DVR at three different levels before and after hypoxia/re-oxygenation (H/R). Rat DVR were isolated and perfused under isobaric conditions. The phosphodiesterase 5 (PDE5) inhibitor sildenafil (10−6 mol/L) impaired cGMP degradation and dilated DVR pre-constricted with angiotensin II (Ang II, 10−6 mol/L). Dilations by the soluble guanylyl cyclase (sGC) activator BAY 60-2770 as well as the nitric oxide donor sodium nitroprusside (SNP, 10−3 mol/L) were equally effective. Hypoxia (0.1% O2) augmented DVR constriction by Ang II, thus potentially aggravating tissue hypoxia. H/R left DVR unresponsive to sildenafil, yet sGC activation by BAY 60-2770 effectively dilated DVR. Dilation to SNP under H/R is delayed. In conclusion, H/R renders PDE5 inhibition ineffective in dilating the crucial vessels supplying the area at risk for hypoxic damage. Stimulating sGC appears to be the most effective in restoring renal medullary blood flow after H/R and may prove to be the best target for maintaining oxygenation to this vulnerable area of the kidney.

Mapping of the sGC Stimulator BAY 41-2272 Binding Site on H-NOX Domain and Its Regulation by the Redox State of the Heme.

Soluble guanylate cyclase (sGC) is the main receptor of nitric oxide (NO) and by converting GTP to cGMP regulates numerous biological processes. The β1 subunit of the most abundant, α1β1 heterodimer, harbors an N-terminal domain called H-NOX, responsible for heme and NO binding and thus sGC activation. Dysfunction of the NO/sGC/cGMP axis is causally associated with pathological states such as heart failure and pulmonary hypertension. Enhancement of sGC enzymatic function can be effected by a class of drugs called sGC “stimulators,” which depend on reduced heme and synergize with low NO concentrations. Until recently, our knowledge about the binding mode of stimulators relied on low resolution cryo-EM structures of human sGC in complex with known stimulators, while information about the mode of synergy with NO is still limited. Herein, we couple NMR spectroscopy using the H-NOX domain of the Nostoc sp. cyanobacterium with cGMP determinations in aortic smooth muscle cells (A7r5) to study the impact of the redox state of the heme on the binding of the sGC stimulator BAY 41-2272 to the Ns H-NOX domain and on the catalytic function of the sGC. BAY 41-2272 binds on the surface of H-NOX with low affinity and this binding is enhanced by low NO concentrations. Subsequent titration of the heme oxidant ODQ, fails to modify the conformation of H-NOX or elicit loss of the heme, despite its oxidation. Treatment of A7r5 cells with ODQ following the addition of BAY 41-2272 and an NO donor can still inhibit cGMP synthesis. Overall, we describe an analysis in real time of the interaction of the sGC stimulator, BAY 41-2272, with the Ns H-NOX, map the amino acids that mediate this interaction and provide evidence to explain the characteristic synergy of BAY 41-2272 with NO. We also propose that ODQ can still oxidize the heme in the H-NOX/NO complex and inhibit sGC activity, even though the heme remains associated with H-NOX. These data provide a more-in-depth understanding of the molecular mode of action of sGC stimulators and can lead to an optimized design and development of novel sGC agonists.

Cinaciguat (BAY-582667) Modifies Cardiopulmonary and Systemic Circulation in Chronically Hypoxic and Pulmonary Hypertensive Neonatal Lambs in the Alto Andino.

Neonatal pulmonary hypertension (NPHT) is produced by sustained pulmonary vasoconstriction and increased vascular remodeling. Soluble guanylyl cyclase (sGC) participates in signaling pathways that induce vascular vasodilation and reduce vascular remodeling. However, when sGC is oxidized and/or loses its heme group, it does not respond to nitric oxide (NO), losing its vasodilating effects. sGC protein expression and function is reduced in hypertensive neonatal lambs. Currently, NPHT is treated with NO inhalation therapy; however, new treatments are needed for improved outcomes. We used Cinaciguat (BAY-582667), which activates oxidized and/or without heme group sGC in pulmonary hypertensive lambs studied at 3,600 m. Our study included 6 Cinaciguat-treated (35 ug kg−1 day−1 x 7 days) and 6 Control neonates. We measured acute and chronic basal cardiovascular variables in pulmonary and systemic circulation, cardiovascular variables during a superimposed episode of acute hypoxia, remodeling of pulmonary arteries and changes in the right ventricle weight, vasoactive functions in small pulmonary arteries, and expression of NO-sGC-cGMP signaling pathway proteins involved in vasodilation. We observed a decrease in pulmonary arterial pressure and vascular resistance during the acute treatment. In contrast, the pulmonary pressure did not change in the chronic study due to increased cardiac output, resulting in lower pulmonary vascular resistance in the last 2 days of chronic study. The latter may have had a role in decreasing right ventricular hypertrophy, although the direct effect of Cinaciguat on the heart should also be considered. During acute hypoxia, the pulmonary vascular resistance remained low compared to the Control lambs. We observed a higher lung artery density, accompanied by reduced smooth muscle and adventitia layers in the pulmonary arteries. Additionally, vasodilator function was increased, and vasoconstrictor function was decreased, with modifications in the expression of proteins linked to pulmonary vasodilation, consistent with low pulmonary vascular resistance. In summary, Cinaciguat, an activator of sGC, induces cardiopulmonary modifications in chronically hypoxic and pulmonary hypertensive newborn lambs. Therefore, Cinaciguat is a potential therapeutic tool for reducing pulmonary vascular remodeling and/or right ventricular hypertrophy in pulmonary arterial hypertension syndrome.

Vericiguat: A Review in Chronic Heart Failure with Reduced Ejection Fraction.

Vericiguat (Verquvo®) is the first oral soluble guanylate cyclase (sGC) stimulator to be approved for the treatment of adults with symptomatic, chronic heart failure with reduced ejection fraction (HFrEF). In the phase III VICTORIA trial, vericiguat added to standard of care (SOC) was associated with a significantly lower risk of the primary composite endpoint of death from cardiovascular (CV) causes or first hospitalization from heart failure (HHF) than placebo added to SOC in adults with chronic HFrEF. The risk of all-cause mortality or first HHF (secondary composite endpoint) and the total number of HHF were also statistically significantly reduced by vericiguat therapy. Vericiguat showed no benefit with respect to the primary endpoint in a subgroup of patients with grossly elevated N-terminal pro-brain natriuretic peptide levels. Vericiguat was generally well tolerated; the most common treatment-related adverse event (AE) was hypotension. AEs of special interest included symptomatic hypotension and syncope, which occurred with low incidences that were similar between treatment groups. Thus, vericiguat is an effective and generally well-tolerated treatment option in patients with symptomatic, chronic HFrEF who have experienced a recent worsening event, expanding the options currently available for chronic HFrEF management.

Treatment with the soluble guanylate cyclase activator BAY 60–2770 normalizes bladder function in an in vivo rat model of chronic prostatitis

Background and purposeChronic pelvic pain syndrome (CPPS) is a common and bothersome condition for which no pharmacological treatment options with acceptable efficacy exist. The aim of this study was to investigate the effects of the soluble guanylate cyclase (sGC) activator BAY 60–2770 and the COX-2 inhibitor celecoxib on bladder function in a rat model of CPPS. Experimental approachForty-eight male Sprague-Dawley rats were intraprostatically injected with either saline, serving as control, or zymosan, to induce prostatitis. On days 8–20, the rats were treated with either dimethylsulphoxide (DMSO; vehicle), celecoxib, BAY 60–2770 or a combination of celecoxib and BAY 60–2770. Thereafter, micturition parameters were assessed in a metabolic cage and urine samples were collected. The following day, cystometry was performed. Subsequently, the urinary bladder and prostate were removed and examined histopathologically. Key resultsInduction of prostatitis led to a significant increase of micturition frequency and corresponding decrease of volume per micturition. These alterations were ameliorated by celecoxib, and completely normalized by BAY 60–2770. Induction of prostatitis led to a significantly increased number of non-voiding contractions, decreased bladder compliance and increased voiding time. These parameters were normalized by treatment with BAY 60–2770, either alone or in combination with celecoxib. The immunohistochemical analysis showed signs of prostate inflammation, but not bladder inflammation. Conclusion and implicationsInduction of prostatitis led to significant impairment in bladder function. These alterations could be prevented by BAY 60–2770, alone or in combination with celecoxib. This is the first study to show that sGC activators could be a promising option for the treatment of CPPS.

Nitric oxide signaling controls collective contractions in a colonial choanoflagellate

Although signaling by the gaseous molecule nitric oxide (NO) regulates key physiological processes in animals, including contractility,1-3 immunity,4,5 development,6-9 and locomotion,10,11 the early evolution of animal NO signaling remains unclear. To reconstruct the role of NO in the animal stem lineage, we set out to study NO signaling in choanoflagellates, the closest living relatives of animals.12 In animals, NO produced by the nitric oxide synthase (NOS) canonically signals through cGMP by activating soluble guanylate cyclases (sGCs).13,14 We surveyed the distribution of the NO signaling pathway components across the diversity of choanoflagellates and found three species that express NOS (of either bacterial or eukaryotic origin), sGCs, and downstream genes previously shown to be involved in the NO/cGMP pathway. One of the species coexpressing sGCs and a bacterial-type NOS, Choanoeca flexa, forms multicellular sheets that undergo collective contractions controlled by cGMP.15 We found that treatment with NO induces cGMP synthesis and contraction in C.flexa. Biochemical assays show that NO directly binds C.flexa sGC1 and stimulates its cyclase activity. The NO/cGMP pathway acts independently from other inducers of C.flexa contraction, including mechanical stimuli and heat, but sGC activity is required for contractions induced by light-to-dark transitions. The output of NO signaling in C.flexa-contractions resulting in a switch from feeding to swimming-resembles the effect of NO in sponges1-3 and cnidarians,11,16,17 where it interrupts feeding and activates contractility. These data provide insights into the biology of the first animals and the evolution of NO signaling.

Activation of the Soluble Guanylate Cyclase Increases 26S Proteasome Activities and Protects against Proteotoxicity in Cardiomyocytes

BackgroundThe ubiquitin‐proteasome system (UPS) mediates the selective degradation of unwanted and unneeded proteins in the cell, playing crucial roles in protein quality control and diverse other cellular processes. Proteasome malfunction and proteotoxicity are implicated in the genesis of a large subset of heart diseases. Our laboratory has previously discovered that activation of the cGMP‐dependent protein kinase (PKG) primes the proteasome. Here we test the hypothesis that by increasing cGMP and PKG activity, soluble guanylate cyclase (sGC) activation activates the proteasome and thereby protects against proteotoxicity in cardiomyocytes.Methods and ResultsCompared with vehicle control (DMSO), treatment with an sGC activator (cinaciguat) in cultured neonatal rat cardiomyocytes (NRCMs) led to increases in the levels of Ser239‐phosphorylated vasodilator‐stimulated phosphoprotein (VASP) (2.17±0.07 vs. 1.0±0.20, p<0.0001), indicative of PKG activation. We then used adenoviral vectors expressing degron CL1‐fused green fluorescent proteins (GFPu, a proven UPS substrate) or red fluorescent proteins (RFP, a stable protein control) as a reporter system for UPS performance. Compared with DMSO, cinaciguat treatment reduced the GFPu/RFP ratio (0.59±0.06 vs. 1.0±0.18, p=0.0135) as revealed by western blotting, and shortened GFPu half‐life in cultured NRCMs as revealed by cycloheximide chase assays. Cinaciguat treatment decreased Thr41/Ser45‐phosphorylated β‐catenin, an endogenous substrate of the UPS (0.60±0.12 vs. 1.0±0.08, p=0.0014). These data demonstrate that sGC activation enhances UPS performance. Moreover, in‐gel proteasome activity assays followed by western blot analyses reveal that cinaciguat treatment increased the chymotrypsin like activities of 26S proteasomes (1.52±0.17 vs. 1.0±0.09, p=0.0019) without changing 26S proteasome assembly, showing that sGC activation enhances UPS performance via activating 26S proteasomes. We next used NRCMs with adenovirus‐mediated overexpression of a human‐disease‐linked misfolded protein CryABR120Gas a cell model of cardiac proteotoxicity. CryABR120G overexpression‐induced increases in LDH leakage and PKCδ cleavage (both indicative of cell injury/death) were significantly attenuated by cinaciguat treatment (LDH in media: 0.71±0.20 vs.1.44±0.26, p<0.0001); further MTT assays consistently showed cinaciguat promoted NRCMs viability (1.32±0.11 vs. 1.02±0.05, p<0.0001).ConclusionsActivation of sGC with cinaciguat increases PKG activity, activates the 26S proteasome, facilitates UPS functioning, and thereby protects against proteotoxicity in cardiomyocytes.

Therapeutic augmentation of NO-sGC-cGMP signalling: lessons learned from pulmonary arterial hypertension and heart failure.

The nitric oxide (NO)-guanylate cyclase (GC)-cyclic guanosine monophosphate (cGMP) pathway plays an important role in cardiovascular, pulmonary and renal function. Phosphodiesterase-5 inhibitors (PDE-5i) inhibit cGMP degradation, whereas both soluble guanylate cyclase (sGC) stimulators and sGC activators directly increase sGC. PDE-5i (e.g. sildenafil, tadalafil) and sGC stimulators (e.g. riociguat, vericiguat) have been extensively used in pulmonary artery hypertension (PAH) and heart failure (HF). PDE-5i have also been used in end-stage HF before and after left ventricular (LV) assist device (LVAD) implantation. Augmentation of NO-GC-cGMP signalling with PDE-5i causes selective pulmonary vasodilation, which is highly effective in PAH but may have controversial, potentially adverse effects in HF, including pre-LVAD implant due to device unmasking of PDE-5i-induced RV dysfunction. In contrast, retrospective analyses have demonstrated that PDE-5i have beneficial effects when initiated post LVAD implant due to the improved haemodynamics of the supported LV and the pleiotropic actions of these compounds. sGC stimulators, in turn, are effective both in PAH and in HF due to their balanced pulmonary and systemic vasodilation, and as such they are preferable to PDE-5i if the use of a pulmonary vasodilator is needed in HF patients, including those listed for LVAD implantation. Regarding the effectiveness of PDE-5i and sGC stimulators when initiated post LVAD implant, these two groups of compounds should be tested in a randomized control trial.

Delphi consensus recommendation for optimization of pulmonary hypertension therapy focusing on switching from a phosphodiesterase 5 inhibitor to riociguat.

Dual combination therapy with a phosphodiesterase‐5 inhibitor (PDE5i) and endothelin receptor antagonist is recommended for most patients with intermediate‐risk pulmonary arterial hypertension (PAH). The RESPITE and REPLACE studies suggest that switching from a PDE5i to a soluble guanylate cyclase (sGC) activator may provide clinical improvement in this situation. The optimal approach to escalation or transition of therapy in this or other scenarios is not well defined. We developed an expert consensus statement on the transition to sGC and other treatment escalations and transitions in PAH using a modified Delphi process. The Delphi process used a panel of 20 physicians with expertise in PAH. Panelists answered three questionnaires on the management of treatment escalations and transitions in PAH. The initial questionnaire included open‐ended questions. Later questionnaires consolidated the responses into statements that panelists rated on a Likert scale from −5 (strongly disagree) to +5 (strongly agree) to determine consensus. The Delphi process produced several consensus recommendations. Escalation should be considered for patients who are at high risk or not achieving treatment goals, by adding an agent from a new class, switching from oral to parenteral prostacyclins, or increasing the dose. Switching to a new class or within a class should be considered if tolerability or other considerations unrelated to efficacy are affecting adherence. Switching from a PDE5i to an SGC activator may benefit patients with intermediate risk who are not improving on their present therapy. These consensus‐based recommendations may be helpful to clinicians and beneficial for patients when evidence‐based guidance is unavailable.

The role of activation of two different sGC binding sites by NO-dependent and NO-independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes.

The mechanoelectrical feedback (MEF) mechanism in the heart that plays a significant role in the occurrence of arrhythmias, involves cation flux through cation nonselective stretch‐activated channels (SACs). It is well known that nitric oxide (NO) can act as a regulator of MEF. Here we addressed the possibility of SAC’s regulation along NO‐dependent and NO‐independent pathways, as well as the possibility of S‐nitrosylation of SACs. In freshly isolated rat ventricular cardiomyocytes, using the patch‐clamp method in whole‐cell configuration, inward nonselective stretch‐activated cation current ISAC was recorded through SACs, which occurs during dosed cell stretching. NO donor SNAP, α1‐subunit of sGC activator BAY41‐2272, sGC blocker ODQ, PKG blocker KT5823, PKG activator 8Br‐cGMP, and S‐nitrosylation blocker ascorbic acid, were employed. We concluded that the physiological concentration of NO in the cell is a necessary condition for the functioning of SACs. An increase in NO due to SNAP in an unstretched cell causes the appearance of a Gd3+‐sensitive nonselective cation current, an analog of ISAC , while in a stretched cell it eliminates ISAC . The NO‐independent pathway of sGC activation of α subunit, triggered by BAY41‐2272, is also important for the regulation of SACs. Since S‐nitrosylation inhibitor completely abolishes ISAC , this mechanism occurs. The application of BAY41‐2272 cannot induce ISAC in a nonstretched cell; however, the addition of SNAP on its background activates SACs, rather due to S‐nitrosylation.ODQ eliminates ISAC , but SNAP added on the background of stretch increases ISAC in addition to ODQ. This may be a result of the lack of NO as a result of inhibition of NOS by metabolically modified ODQ. KT5823 reduces PKG activity and reduces SACs phosphorylation, leading to an increase in ISAC . 8Br‐cGMP reduces ISAC by activating PKG and its phosphorylation. These results demonstrate a significant contribution of S‐nitrosylation to the regulation of SACs.

A novel insight into the molecular mechanism of human soluble guanylyl cyclase focused on catalytic domain in living cells

Human soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. In this work, fluorescent proteins were employed to study the NO-induced sGC molecular mechanism via mutagenesis at the catalytic domain. The conformational change of sGC by mutant α1C595 was investigated in living cells through fluorescence lifetime imaging microscopy (FLIM). The results indicated that the NO-induced conformational change of the catalytic domain of sGC from “open to “closed” upon GTP-binding was regulated by the hydrogen (H)-bonding network of the catalytic domain. The mutation of C595 caused a big conformational change of catalytic domain with H-bond variation, which not only demonstrates the key role of the C595 site in the process of conformational change of the catalytic domain, but also reveals the regulatory mechanism of sGC at the catalytic domain. This finding would guide the design of small-molecule drugs targeting the catalytic domain to modulate sGC activity.

The sGC stimulator BAY-747 and activator runcaciguat can enhance memory in vivo via differential hippocampal plasticity mechanisms

Soluble guanylate cyclase (sGC) requires a heme-group bound in order to produce cGMP, a second messenger involved in memory formation, while heme-free sGC is inactive. Two compound classes can increase sGC activity: sGC stimulators acting on heme-bound sGC, and sGC activators acting on heme-free sGC. In this rodent study, we investigated the potential of the novel brain-penetrant sGC stimulator BAY-747 and sGC activator runcaciguat to enhance long-term memory and attenuate short-term memory deficits induced by the NOS-inhibitor L-NAME. Furthermore, hippocampal plasticity mechanisms were investigated. In vivo, oral administration of BAY-747 and runcaciguat to male Wistar rats enhanced memory acquisition in the object location task (OLT), while only BAY-747 reversed L-NAME induced memory impairments in the OLT. Ex vivo, both BAY-747 and runcaciguat enhanced hippocampal GluA1-containing AMPA receptor (AMPAR) trafficking in a chemical LTP model for memory acquisition using acute mouse hippocampal slices. In vivo only runcaciguat acted on the glutamatergic AMPAR system in hippocampal memory acquisition processes, while for BAY-747 the effects on the neurotrophic system were more pronounced as measured in male mice using western blot. Altogether this study shows that sGC stimulators and activators have potential as cognition enhancers, while the underlying plasticity mechanisms may determine disease-specific effectiveness.

Targeting neurotrophin and nitric oxide signaling to treat spinal cord injury and associated neurogenic bladder overactivity

Purpose or the research:Nearly 300,000 people are affected by spinal cord injury (SCI) with approximately 18,000 new cases annually, according to the National SCI Statistics Center. SCI affects physical mobility and impairs the function of multiple internal organs to cause lower urinary tract (LUT) dysfunctions manifesting as detrusor sphincter dyssynergia (DSD) and neurogenic detrusor overactivity (NDO) with detrimental consequences to the quality of life and increased morbidity. Multiple lines of evidence now support time dependent evolution of the complex SCI pathology which requires a multipronged treatment approach of immediate, specialized care after spinal cord trauma bookended by physical rehabilitation to improve the clinical outcomes. Instead of one size fits all treatment approach, we propose adaptive drug treatment to counter the time dependent evolution of SCI pathology, with three small molecule drugs with distinctive sites of action for the recovery of multiple functions. Principal results:Our findings demonstrate the improvement in the recovery of hindlimb mobility and bladder function of spinal cord contused mice following administration of small molecules targeting neurotrophin receptors, LM11A-31 and LM22B-10. While LM11A-31 reduced the cell death in the spinal cord, LM22B-10 promoted cell survival and axonal growth. Moreover, the soluble guanylate cyclase (sGC) activator, cinaciguat, enhanced the revascularization of the SCI injury site to promote vessel formation, dilation, and increased perfusion. Major conclusions:Our adaptive three drug cocktail targets different stages of SCI and LUTD pathology: neuroprotective effect of LM11A-31 retards the cell death that occurs in the early stages of SCI; and LM22B-10 and cinaciguat promote neural remodeling and reperfusion at later stages to repair spinal cord scarring, DSD and NDO. LM11A-31 and cinaciguat have passed phase I and IIa clinical trials and possess significant potential for accelerated clinical testing in SCI/LUTD patients.

Benign prostatic hyperplasia/obstruction ameliorated using a soluble guanylate cyclase activator.

Benign prostatic hyperplasia (BPH) is a feature of ageing males. Up to half demonstrate bladder outlet obstruction (BOO) with associated lower urinary tract symptoms (LUTS) including bladder overactivity. Current therapies to reduce obstruction, such as α1-adrenoceptor antagonists and 5α-reductase inhibitors, are not effective in all patients. The phosphodiesterase-5 inhibitor (PDE5I) tadalafil is also approved to treat BPH and LUTS, suggesting a role for nitric oxide (NO• ), soluble guanylate cyclase (sGC), and cGMP signalling pathways. However, PDE5I refractoriness can develop for reasons including nitrergic nerve damage and decreased NO• production, or inflammation-related oxidation of the sGC haem group, normally maintained in a reduced state by the cofactor cytochrome-b5-reductase 3 (CYB5R3). sGC activators, such as cinaciguat (BAY 58-2667), have been developed to enhance sGC activity in the absence of NO• or when sGC is oxidised. Accordingly, their effects on the prostate and LUT function of aged mice were evaluated. Aged mice (≥24 months) demonstrated a functional BPH/BOO phenotype, compared with adult animals (2-12 months), with low, delayed voiding responses and elevated intravesical pressures as measured by telemetric cystometry. This was consistent with outflow tract histological and molecular data that showed urethral constriction, increased prostate weight, greater collagen deposition, and cellular hyperplasia. All changes in aged animals were attenuated by daily oral treatment with cinaciguat for 2 weeks, without effect on serum testosterone levels. Cinaciguat had only transient (1 h) cardiovascular effects with oral gavage, suggesting a positive safety profile. The benefit of cinaciguat was suggested by its reversal of an overactive cystometric profile in CYB5R3 smooth muscle knockout mice that mirrors a profile of oxidative dysfunction where PDE5I may not be effective. Thus, the aged male mouse is a suitable model for BPH-induced BOO and cinaciguat has a demonstrated ability to reduce prostate-induced obstruction and consequent effects on bladder function. © 2021 The Pathological Society of Great Britain and Ireland.

FoxO4 controls sGCβ transcription in vascular smooth muscle.

Nitric oxide (NO) binds soluble guanylyl cyclase β (sGCβ) to produce cGMP and relax vascular smooth muscle cells (SMCs) needed for vasodilation. Although the regulation of NO-stimulated sGC activity has been well characterized at the posttranslational level, the mechanisms that govern sGC transcription remain incompletely understood. Recently, we identified Forkhead box subclass O (FoxO) transcription factors as essential for expression of sGC; however, the specific FoxO family member responsible for the expression of sGCβ in SMC remains unknown. Using FoxO shRNA knockdown adenovirus treatment in rat aortic SMCs, we show that FoxO1 or FoxO3 knockdown causes greater than twofold increases in Gucy1a3 and Gucy1b3 mRNA expression, without changes in NO-dependent cGMP production or cGMP-dependent phosphorylation. FoxO4 knockdown produced a 50% decrease in Gucy1a3 and Gucy1b3 mRNA with 70% loss of sGCα and 50% loss of sGCβ protein expression. Knockdown of FoxO4 expression decreased cGMP production and downstream protein kinase G-dependent phosphorylation more than 50%. Triple FoxO knockdown exacerbated loss of sGC-dependent function, phenocopying previous FoxO inhibition studies. Using promoter luciferase and chromatin immunoprecipitation assays, we find that FoxO4 acts as a transcriptional activator by directly binding several FoxO DNA motifs in the promoter regions of GUCY1B3 in human aortic SMCs. Collectively, our data show FoxO4 is a critical transcriptional regulator of sGCβ expression in SMC.NEW & NOTEWORTHY One of the key mechanisms of vascular smooth muscle cell (SMC) dilation occurs through nitric oxide (NO)-dependent induction of soluble guanylyl cyclase (sGC) by means of its β-subunit. Herein, we are the first to identify Forkhead box subclass O protein 4 (FoxO4) as a key transcriptional regulator of GUCY1B3 expression, which codes for sGCβ protein in human and animal SMCs. This discovery will likely have important implications for the future usage of antihypertensive and vasodilatory therapies which target NO production, sGC, or FoxO transcription factors.

Anti-spasmolytic effects of BAY 60-2770, a soluble guanylate cyclase activator, in isolated coronary arteries

This study investigated whether soluble guanylate cyclase (sGC) activators (activating the heme-oxidized and heme-free sGC) have potential as therapeutic drugs for coronary artery spasm. Isolated canine and porcine coronary arteries were suspended in organ chambers for isometric tension recording. The contractile responses of canine coronary arteries to potassium chloride (3×10-2 M), endothelin-1 (3×10-11 to 3×10-8 M), prostaglandin F2α (10-8 to 10-5 M), and 5-hydroxytryptamine (10-9 to 10-6 M) were suppressed by previous exposure to the sGC activator BAY 60-2770 (10-10, 10-9 and 10-8 M). In porcine coronary arteries, the addition of BAY 60-2770 (10-10, 10-9 and 10-8 M) concentration-dependently prolonged the cycle length of 3,4-diaminopyridine (10-2 M)-induced phasic contractions and reduced the peak tension. As for vessel-size-dependent difference in vasoreactivity, BAY 60-2770 (10-12 to 10-7 M) caused a greater relaxation of porcine coronary arteries precontracted with endothelin-1 (3×10-8 M) in small arteries (#9 in AHA classification) than in large arteries (#6 in AHA classification). These findings suggest that sGC activators are beneficial for the treatment of vasospastic angina. In addition, anti-spasmolytic efficacy of sGC activators may be expected to be observed even in microvascular angina.