12th International Conference on cGMP

Guanylyl cyclase C (GUCY2C) is a transmembrane receptor that produces cGMP in intestinal epithelial cells. GUCY2C is universally overexpressed by colorectal cancers (CRC) and a subset of gastroesophageal and pancreatic adenocarcinomas, with expression maintained in metastases. Compartmentalized expression, near-universal tumor expression, and functional roles in opposing the oncogenic phenotype make GUCY2C an ideal target for cancer immunotherapy. We have pursued GUCY2C-targeted vaccines to prevent recurrence in patients with resected gastrointestinal malignancies.

Our first-generation vaccine, Ad5-GUCY2C-PADRE, employed an adenovirus serotype 5 (Ad5) vector encoding the extracellular domain of GUCY2C fused to the PADRE T-helper epitope. A phase 1 trial demonstrated safety and the ability to generate GUCY2C-specific T-cell responses, but efficacy was limited by widespread pre-existing Ad5 neutralizing antibodies (NAbs). To overcome this barrier, we developed Ad5.F35-GUCY2C-PADRE, a chimeric vector replacing the Ad5 fiber with that of serotype 35. A phase 2A dose-finding trial demonstrated that this second-generation vaccine is safe, overcomes pre-existing Ad5 NAbs, and induces robust dose-dependent GUCY2C-specific T-cell responses associated with improved recurrence-free survival. Results from this trial will be presented.

Building on these findings, we are now advancing a heterologous prime-boost strategy combining Ad5.F35-GUCY2C-PADRE with a Listeria monocytogenes-based vaccine (Lm-GUCY2C) to further amplify and diversify the antitumor immune response. Here, we will present an overview of GUCY2C-targeted vaccine development and clinical results.

Heart failure, Hypertension and Chronic Kidney Disease remain major contributors to morbidity and mortality, and often overlap within individuals. Novel approaches to modulate vascular tone and renovascular homeostasis may address unmet medical need in all three conditions. Natriuretic peptides signal through natriuretic peptide receptor 1 (NPR1), a membrane-bound guanylyl cyclase, to promote vasodilation and relieve venous congestion, but recombinant peptide infusions have been limited by short duration of effect. In a human genetic analysis of more than 700,000 individuals, lifelong exposure to coding variants in NPR1 was associated with changes in blood pressure and heart failure risk, supporting therapeutic NPR1 modulation. REGN5381 is an investigational monoclonal agonist antibody that allosterically activates NPR1 by inducing an active-like receptor conformation. In preclinical studies, REGN5381 produced haemodynamic effects that preferentially target the venous vasculature, reducing venous pressure and systolic blood pressure in normotensive and hypertensive models. In healthy volunteers, a single intravenous dose produced expected haemodynamic changes consistent with lower venous pressures, with general tolerability and no serious treatment-emergent adverse events reported across dose groups; blood pressure reductions persisted for several days in mildly hypertensive adults. Beyond haemodynamics, NPR1 agonism with REGN5381 improved renal outcomes across multiple preclinical chronic kidney disease models, including reductions in urinary albumin and injury biomarkers; combination with enalapril further improved biomarkers, histopathology, and suppression of fibrotic and inflammatory gene programs. Together, these findings support NPR1 agonism as an approach to selectively lower venous pressures, preserve renal function and potentially address cardiorenal diseases.

C-type natriuretic peptide (CNP) has therapeutic potential in heart failure with preserved ejection fraction (HFpEF) due to its broad range of beneficial effects on cardiovascular structure and function. This study presents the design of a CNP analogue ( 65) for once-weekly subcutaneous administration. The design of 65incorporated five strategic substitutions and an engineered fatty acid protractor to enhance the chemical stability, improve the pharmacokinetics, and lower the isoelectric point (pI). Low pI was found to be essential for minimizing injection site reactions and improving subcutaneous bioavailability. 65demonstrated a promising pharmacokinetic profile for once-weekly treatment and an improved bioavailability compared to high pI CNP analogs. Furthermore, 65 was engineered for solubility at pH 6.5 to enable stability in liquid formulation. In vitro and in vivoassessments supported the therapeutic potential in HFpEF. 65is currently under clinical investigation in Phase 1.

The public health burden related to the morbidity and mortality of hypertension is enormous and increasing. In 2023, WHO reported that 1.3 billion patients worldwide are diagnosed with hypertension (HT), from which 10‐15% suffer from resistant HT (RHT). RHT is a frequent disease, and its prevalence continues to rise, additionally it causes premature morbidity and death, since currently there are limited therapeutic options. Thus, the development of novel drugs that can control the elevated blood pressure in these patients is urgent and of high social and clinical impact for several millions of people worldwide. Natriuretic peptides are important in the regulation of blood pressure. Activation of natriuretic peptide receptor A (NPR-A; GC-A) by endogenous atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) via production of cyclic guanosine monophosphate (cGMP) is associated with several beneficial cardiovascular and renal effects. Our novel approach is to find a small molecule to treat RHT, through a novel mechanism of action, targeting NPR-A. Through several rounds of screening, we have found small molecular allosteric modulators which increase the efficacy and potency of BNP and ANP in their ability to activate NPR‐A and generate cGMP. These compounds were characterized as NPR-A-selective allosteric enhancers, and their activity is dependent on one unique amino acid in NPR-A. Finally, in an animal model, we have also obtained preliminary evidence for their efficacy in vivo.

Following the VICTORIA trial, vericiguat was approved for patients with worsening HFrEF (those with recent hospitalizations or need for IV diuretics). The subsequent VICTOR trial focused on a more stable, ambulatory population with no recent worsening. In total, 11,155 patients, representing a broad range of clinical severity were enrolled in the two trials. VICTORIA (n=5,050) was conducted from 2016–2019 and enrolled high-risk patients with recent HFrEF worsening. VICTOR (n=6,105) was conducted 2021–2025 and enrolled lower-risk patients with no recent worsening and more contemporary background therapy. Baseline Characteristics of the combined population included median age 68 years; 23.7% were female, mean LVEF 29.8%, 69.9% of participants were NYHA Class II, and 30.1% were Class III–IV. Median baseline NT-proBNP was 1864 pg/mL; 88.7% of patients had levels <6000 pg/mL. Use of quadruple therapy was significantly higher in the VICTOR trial, reflecting the 7-year interval between trial initiations.

The pooled analysis demonstrated a consistent benefit for vericiguat across multiple clinical endpoints. Vericiguat reduced the risk of CV death or HF hospitalization by 9% compared to placebo (Vericiguat: 25.9%, Placebo: 27.9%; Hazard Ratio (HR): 0.91 [95% CI 0.85–0.98]). Significant risk reductions were observed in all individual components: CV Death: HR 0.89; First HF Hospitalization: HR 0.92; All-Cause Death: HR 0.90

The treatment effect for the primary endpoint emerged around 4 months, while the mortality benefit became apparent after 8 months. Importantly, there was no evidence of intertrial heterogeneity, meaning the drug's effect was consistent regardless of whether patients were from the high-risk VICTORIA or lower-risk VICTOR cohorts. A major finding involved the relationship between baseline NT-proBNP levels and treatment success. Patients with NT-proBNP <6000 pg/mL derived the most significant benefit. In this subgroup, vericiguat reduced the primary composite risk with an HR of 0.86. The Number Needed to Treat (NNT) for one year to prevent one composite event was 29 for the <6000 pg/mL group, compared to 37 in the overall pooled population.

Vericiguat was generally well-tolerated with a safety profile consistent with previous reports. Serious adverse events occurred in 27.7% of the vericiguat group vs. 29.2% of the placebo group. Discontinuation rates due to adverse events were low and balanced (7.8% vericiguat vs. 6.9% placebo). Symptomatic hypotension (11.8% vs. 9.8%) and anemia (8.5% vs. 6.7%) were more common with vericiguat but rarely led to treatment cessation.

Overall vericiguat provides incremental clinical benefit across the broad HFrEF spectrum, even for patients already receiving contemporary quadruple therapy (beta-blockers, MRAs, ARNIs, and SGLT2 inhibitors). Its once-daily dosing and favorable safety profile (no laboratory monitoring required) support its use as an additional therapeutic option.

Inhaled mosliciguat is an investigational, potential first-in-class, soluble guanylate cyclase (sGC) activator designed for lung-targeted delivery via dry powder inhalation. sGC is a key enzyme in the nitric oxide (NO) signaling pathway, where NO binds to the prosthetic heme group of sGC to stimulate cyclic guanosine monophosphate (cGMP) production, promoting vasodilation and potentially reducing inflammation, apoptosis, and fibrosis. Under conditions of oxidative stress, such as pulmonary hypertension (PH), sGC may lose its heme moiety, forming heme-free ‘apo-sGC,’ which is poorly responsive to NO and results in impaired cGMP production. Inhaled mosliciguat, an sGC activator, binds directly to the heme pocket of sGC to increase cGMP production, independent of NO. Mosliciguat’s ability to restore activity to pathologic NO-insensitive apo-sGC may enable more effective cGMP production under conditions of reduced NO production and oxidative stress.

Mosliciguat exhibited potent vasodilatory properties in preclinical studies in PH animal models. In the Phase 1b ATMOS trial, single-dose inhaled mosliciguat reduced mean peak pulmonary vascular resistance by up to 38% in participants with either pulmonary arterial hypertension or chronic thromboembolic pulmonary hypertension and was well tolerated, with no clinically relevant systemic adverse events. In healthy volunteers, single-dose inhaled mosliciguat increased pulmonary arterial volume with greater dilation of small diameter vessels as measured by quantitative high resolution computed tomography. Two Phase 2 trials are evaluating mosliciguat’s safety and efficacy in interstitial lung disease-associated PH: PHocus (NCT06635850), a global, randomized, double-blinded, placebo-controlled trial, and PHactor (NCT07333183), an open-label study assessing tolerability and safety on top of inhaled treprostinil.

Patients with diabetes or the metabolic syndrome are at increased risk for coronary artery disease, in part due to impaired endothelial nitric oxide (NO) bioavailability and reduced downstream soluble guanylate cyclase (sGC)-cGMP signaling. Coronary endothelial function (CEF) can be assessed noninvasively by MRI as the percent change in coronary artery cross-sectional area (CSA) during isometric handgrip exercise (IHE), an endothelial NO–dependent stress. We hypothesized that pharmacologic enhancement of sGC signaling with vericiguat would improve CEF in patients with cardiometabolic disease and coronary endothelial dysfunction.

We conducted a randomized, double-blind, placebo-controlled study in 32 patients with diabetes or the metabolic syndrome and impaired baseline CEF, randomized 2:1 to vericiguat or placebo. CEF was assessed by MRI at baseline and after six weeks of study drug administration following titration to 10 mg/day or maximally tolerated dose.

Mean age was 60.6±8.6 years and 40.6% were women. There were no baseline group differences in clinical characteristics or CEF. In the placebo group, CSA during IHE did not significantly change from baseline to follow-up (-7.4±8.5% to +1.4±12.6%; p = 0.0512). In contrast, the vericiguat group demonstrated significant improvement in CSA during IHE from -6.8±6.7% to +3.1±8.5% (p=0.0002). Achieved vericiguat dose was independently associated with improvement in CEF (β=6.34; p=0.0497). There were no significant group differences in adverse events.

Vericiguat improved MRI-assessed coronary endothelial function in patients with cardiometabolic disease, providing in vivo human evidence that enhancement of NO-sGC-cGMP signaling improves coronary vascular function.

Loss‑of‑function mutations in soluble guanylyl cyclase (sGC) disrupt NO–cGMP signaling and are associated with a range of cardiovascular and cerebrovascular diseases. Although sGC stimulators and activators are clinically established cGMP‑enhancing drug classes, their capacity to restore activity of disease‑associated sGC mutants, and the extent to which individual mutants respond differentially to these compounds, remains poorly defined.

Here, we systematically analyzed the enzymatic activity of multiple disease‑associated sGC-α₁ subunit mutants using biochemical activity assays with representative sGC stimulators and activators. Purified wild‑type and mutant sGC enzymes were evaluated under defined assay conditions, enabling direct comparison of compound‑dependent activation profiles across mutants. The sGC activator BAY 60‑2770 and the sGC stimulator BAY 41‑2272 were used as pharmacological tool compounds.

Both compound classes enhanced cGMP formation across the mutant panel, but with pronounced mutation‑specific differences in efficacy and maximal activation. Several mutants showed robust responsiveness to sGC activators, consistent with impaired heme‑dependent regulation, whereas others retained sensitivity to sGC stimulators, indicating partial preservation of NO/heme signaling competence. Importantly, BAY 60‑2770 and BAY 41‑2272 enhanced mutant enzymes to markedly different extents, revealing distinct pharmacological response patterns that could not be predicted from basal activity alone.

These findings demonstrate that disease‑associated sGC mutations are not uniformly loss‑of‑function but instead display distinct, pharmacologically addressable activity profiles, supporting mutation‑informed therapeutic stratification and providing a mechanistic basis for mutant‑selective modulation of the NO-sGC-cGMP pathway.

Nitric oxide (NO) is a central signaling molecule in vascular and immune biology whose therapeutic applications extend beyond vasodilation. At high intermittent concentrations, inhaled NO exhibits antimicrobial activity and modulates pulmonary physiology through mechanisms that likely involve both direct nitrosative chemistry and host-mediated signaling pathways.

Preclinical studies demonstrate dose-dependent bacterial killing associated with oxidative and nitrosative stress, including disruption of microbial respiration, DNA damage, and biofilm dispersal. In a large-animal model of multidrug-resistant pneumonia, intermittent inhaled NO at 300 ppm reduced pulmonary bacterial burden and improved lung function, while lung tissue analyses suggested alterations in soluble guanylate cyclase (sGC)–dependent cyclic GMP (cGMP) signaling . These findings raise the possibility that, beyond direct antimicrobial effects, NO may influence epithelial, vascular, or immune responses via the sGC–cGMP pathway. However, the relative contribution of cGMP-dependent signaling versus direct redox-mediated microbial toxicity remains to be fully elucidated.

Parallel phase I human studies demonstrate that intermittent high-dose delivery is feasible with continuous monitoring of methemoglobin and nitrogen dioxide, without cumulative toxicity. Recent breath-by-breath quantification of NO absorption and methemoglobin kinetic modeling further provide tools to link delivered dose to systemic uptake and potential downstream signaling.

Together, these data support a framework in which inhaled NO acts as a spatially confined redox mediator with possible engagement of sGC–cGMP pathways. Defining the balance between direct antimicrobial chemistry and host signaling modulation represents a critical next step in advancing precision gas therapeutics.

Reference:Yu B,et al.Sci Transl Med. 2026. PMID: 41564156

Nitric oxide (NO) signaling relies on it activating cGMP production by the sGC heterodimer. To mature to functional form in mammalian cells, GAPDH supplies heme to the apo-sGCβ-Hsp90 complex, which then undergoes ATP-dependent heme insertion and falls apart, allowing sGCa to bind and form a heterodimer. Physiologic NO stimulates heme allocation into apo-sGCβ by this process to increase the cell’s level of functional heterodimer. Utilizing purified apo-sGCβ and GAPDH reporter proteins whose heme contents are indicated by fluorescence, we found that heme transfer from GAPDH into apo-sGCβ is kinetically coupled and limited by the ability of the apo-sGCβ to incorporate heme. Hsp90 negatively or positively influences heme transfer kinetics depending on ATP, while NO promotes heme transfer into apo-sGCβ in part by speeding GAPDH heme dissociation. Remarkably, the intake of ferric heme by apo-sGCβ depends on it being reduced to ferrous heme within the protein, suggesting Cys residue redox involvement. In HEK2943 cells, this same heme redox transition was required for apo-sGCβ to incorporate GAPDH heme and mature to a functional heterodimer. Mutagenesis in apo-sGCβ revealed that Cys78 was unique in enabling the reduction needed for ferric heme incorporation. Cys78 was not required for apo-sGCβ to incorporate ferrous heme or ferrous heme-NO. No other Cys residue or intracellular thiols could substitute for Cys78. Our findings illuminate sGC maturation by clarifying roles of Hsp90, NO, and sGCβ Cys residues in regulating sGC heme incorporation, reduction, heterodimer formation, and its consequent capacity for biological cGMP production.

Development of small molecule soluble guanylate cyclase (sGC) stimulators for the treatment of cardiovascular and NO-signaling related disorders remains an area of intense research and development. The mammalian isoform of sGC that is the target of therapeutics such as Adempas® is a heterodimer composed of α1- and β1-subunits. The C-terminus of each subunit contains a catalytic domain leading to an active site that is composed of residues from both subunits. The catalytic domains also form a pseudosymmetric site that contains residues known to be involved in nucleotide binding but lack the complete complement of amino acids required for catalysis. Sequence analysis shows that each subunit also contains well-defined PAS-like domain, and a predicted coiled-coil region. The N-termini of the α1- and β1-subunits are homologous to the H-NOX (Heme-Nitric oxide/OXygen) family of proteins. The N-terminus of β1-subunit contains a ferrous heme cofactor that functions as a selective receptor for NO. Activation of sGC can be brought about by NO, stimulators such as Adempas® or a combination of both. In the past 10 years, understanding of the structural states of sGCs has grown tremendously. Structures of basal and full activity conformations have been reported . Yet continued work reveals sGCs are not limited to these conformational end states. Subtle mutations can affect ligand-induced activation profiles without affecting the structures of these end states. Further, the stimulator and activator paradigm of sGC is far from a dichotomy of end states reflects more a flexible conformational landscape that influences catalytic activity.

Pulmonary arterial hypertension is a progressive disease with a poor prognosis. The mechanisms underlying the cardiovascular alterations are incompletely understood but involve increased pulmonary arterial tone, vascular remodeling and right ventricular pressure overload. Beta arrestin 1 and 2 are ubiquitously expressed proteins that regulate G protein signaling and scaffold non-receptor signaling complexes. As beta arrestins have been shown to regulate airway tone, we investigated the role of beta arrestin 1 in the pulmonary vasculature and right heart using complementary in vitro, ex vivo and in vivo models. We found that beta arrestin 1 is a central regulator of soluble guanylyl cyclase (sGC) activity in pulmonary smooth muscle cells and also modulates cardiomyocyte and right ventricular function. Thus, beta arrestin 1 is a potential therapeutic target in pulmonary hypertension.

Little is known how reduced nitric oxide (NO) bioavailability in HFpEF affects the cardiac lymphatic system and drives disease progression. Bulk RNA-sequencing from humans and animal models shows the NO scavenger α-globin (Hba) is significantly upregulated in HFpEF hearts. In a mouse model of HFpEF (cardiometabolic heart failure; CMHF), we observed a similar increase in Hba in cardiac lymphatic endothelial cells (LECs). We hypothesized LEC Hba upregulation disrupts lymphatic function to drive pathological remodeling. To test this, we generated Prox1-CreERT2/Hba1fl/fl mice (LEC Hba KO) to delete Hba in LECs. Echocardiography confirmed CMHF-induced diastolic dysfunction, increased wall thickness, and myocyte hypertrophy, particularly in lymphatic-rich regions; these phenotypes were rescued in CMHF LEC Hba KO mice. Although serum NO metabolites were reduced in CMHF mice, this was independent of Hba genotype, implicating local NO modulation. In CMHF hearts, myocyte pPLN(S16) and pVASP(S239) were decreased, but were significantly increased in CMHF LEC Hba KO hearts. To probe immunomodulatory effects, we treated primary LECs with the NO scavenger PTIO and performed bulk RNA-seq: LEC identity markers were downregulated, lymphatic density was reduced in CMHF hearts (rescued by LEC Hba KO), and T cell–activating gene programs were enriched. Functionally, DETA-NONOate reduced, while PTIO increased, T cell adhesion to LECs. Flow cytometry showed CMHF increased cardiac CD4+ and CD8+ T cell accumulation and activation, both abolished by LEC Hba deletion. Together, these data suggest LEC Hba promotes cardiac remodeling by enhancing LEC–T cell interactions and/or limiting LEC-derived NO signaling to myocyte cGMP pathways.

Introduction: Persistent suboptimal-controlled hypertension drives adverse cardiac remodelling, often progressing to heart failure. Dietary nitrate (NO ₃^-) supplementation reduces blood pressure (BP) in hypertensive patients.However, whether it prevents or delays worsening cardiovascular outcomes remains unknown.

Methods : A matched nested cohort analysis was conducted within the NITRATE-TOD trial (NCT03088514) that recruited 149 hypertensive patients (88 males, 61 females; 56.2±14.1 years) with left ventricular hypertrophy (LVH, n=69) and those without LVH but with uncontrolled BP despite medications (non-LVH, n=80). Patients received either NO ₃^--rich beetroot juice (~6 mmol NO ₃^-/day) or NO₃^--depleted placebo for 16-weeks. Propensity‑score matching identified 31 LVH and 31 non‑LVH patients per treatment arm for analysis (age and sex matched). Blood, saliva and urine were collected, and flow-mediated dilation (FMD) assessed pre- and post-supplementation to assess changes in NO bioavailability, inflammatory markers and endothelial function.

Results: At baseline, LVH patients exhibited greater LV mass, elevated hsCRP and nitrite (NO₂^-) levels with a trend towards worse FMD versus non-LVH. NO ₃^-concentration across all biofluids increased after NO₃^-supplementation. Plasma [NO₂^-] increased ~5-fold in non-LVH patients (P=0.01) but not in the LVH patients. Statistically significant changes in plasma [uric acid], [hsCRP] and [cGMP] were not observed, although [cGMP] trended to increase with NO₃^- treatment in LVH. FMD trended to improve in the non-LVH cohort only after NO₃^-supplementation (0.91%,P =0.09).

Conclusion: Sixteen weeks of daily NO ₃^-supplementation showed an improved endothelial function trend coupled with increased NO₂^-levels in non-LVH patients, suggesting greater therapeutic benefit at earlier stages of hypertension-related pathogenesis.

The kidney contributes to blood pressure control by water and sodium handling. Nitric oxide (NO) is a crucial regulator of medullary blood flow and natriuresis through a vasculotubular crosstalk. NO is produced in multiple cell types including endothelial cells (ECs), epithelial cells, and pericytes. The specific role of EC eNOS in vasculotubular crosstalk is unknown. The study aims to identify the role of EC eNOS in controlling renal function. We compared EC eNOS knockout (KO), EC eNOS knockin (KI) and their respective wildtype (WT) and global eNOS KO (gKO) littermates. As assessed by Millar, EC eNOS KOs and gKOs were hypertensive as compared to WTs, while reactivation of eNOS in EC eNOS KIs rescued the phenotype. AngII increased blood pressure in WTs, but not in EC eNOS KOs, as determined via radiotelemetry. EC eNOS KOs and gKOs lacked carbachol-induced vasodilation in isolated perfused kidney, which was restored in EC eNOS KIs. EC eNOS KOs showed decreased sodium and urine excretion after AngII, as compared to WTs. EC eNOS KIs showed a preserved sodium excretion after AngII as compared to gKOs. AngII increased glomerular filtration rate (GFR) in WTs, while EC eNOS KOs lacked the compensatory GFR response to AngII. gKOs showed decreased GFR after AngII, while the reactivation of eNOS in EC rescued GFR in EC eNOS KIs. EC eNOS KOs showed increased urinary albumin/creatinine ratio as compared to WTs, after AngII. In conclusion, EC eNOS modulates sodium and urine excretion and regulates GFR, thus contributing to blood pressure control.

Introduction: cGMP-dependent protein kinases (PKGs) are main effectors in cGMP signalling. cGMP binding to the cyclic nucleotide binding domains (CNB-A and CNB-B) drives the conformational changes associated with kinase activation. Increased cGMP sensitivity in PKGI mutants has been implicated in thoracic aortic diseases (TAD). Therefore, PKG kinase activity modulation through antagonistic cGMP analogues could provide a basis for therapeutic interference. However, despite identical CNB sequences, current cGMP analogues, such as Rp-8-Br-PET-cGMPS, produce a paradoxical agonistic effect on PKG Iα, but not Iβ.

Results: To investigate the mode of action of Rp-8-Br-PET-cGMPS on the PKG I splice variants, CNB-A and -B binding-deficient mutants were generated. The site specificity of analogue binding and modulation of kinase activity were quantified. Besides SPR, the novel switchSENSE technology was employed to elucidate conformational changes evoked by Rp-8-Br-PET-cGMPS binding. The PKG inhibitor Rp-8-Br-PET-cGMPS had a partial agonistic effect on PKG Iα that was not observed for PKG Iβ. CNB-B binding-deficient PKG I mutants did not show activation upon Rp-8-Br-PET-cGMPS binding, suggesting that agonistic effects are driven by the CNB-B. Furthermore, switchSENSE confirmed that Rp-8-Br-PET-cGMPS binding to the CNB-A does not induce conformational changes.

Conclusions: Our data highlights substantial differences in CNB site-specificity. Rp-8-Br-PET-cGMPS binds the CNB-A of PKG I in an open conformation, without inducing the active-like state. In contrast, agonistic effects are driven by the CNB-B. The difference between the PKGI splice variants is based on allosteric influences of the N-terminus, particularly by the stabilization of the autoinhibitory domain in the catalytic cleft.

The cGMP-dependent protein kinase 1 (PKGI) is a central effector of cGMP and mediates many (patho-)physiological processes, e.g. in the vascular and gastrointestinal tract. Alternative splicing generates two isoforms (PKGIα and PKGIβ) being identical in their regulatory and catalytic domains but differing in their N-terminal leucine zipper region. PKGIβ interacts with inositol 1,4,5-triphosphate receptor associated cGMP-kinase substrate 1 (IRAG1), anchoring PKGIβ to the endoplasmic reticulum. There, both proteins form a macro-complex with IP₃-receptor type 1, regulating the Ca²⁺-efflux into the cytoplasm in a cGMP-dependent manner.

Using single-molecule FRET measurements, we investigate the molecular dynamics of PKGI by site-specifically engineering fluorescent dyes into PKGIβ via bioorthogonal reactions, a method called SLAM-FRET. Labelling glutamate E472 within the small lobe of the catalytic domain (CAT) shows decreasing relative FRET values after adding cGMP, indicating a widening of both CATs upon transitioning into the active conformation. By introducing function-altering mutations (e.g. the naturally occurring constitutively active variant R192Q, I79G within the autoinhibitory domain, and D517A within the ATP-binding pocket) and comparing them to the wild-type protein, allows us to investigate conformational differences causing the observed phenotype. Additionally to the structural analyses, we perform functional studies using hVASP-phosphorylation.

We observe a cGMP-dependent conformational change in PKGIβ for all mutants used, which, at high [cGMP], results in a similar distance between both CATs. The constitutively active mutants already show a greater distance of the CATs in absence of cGMP, whereas the catalytically inactive mutant exhibits a conformational change similar to the wild-type but at lower [cGMP].

Myocardial infarction is one of the leading causes of mortality worldwide. Rupture of an atherosclerotic plaque leads to formation of a thrombus, occluding the coronary artery. The lack of blood and therefore oxygen and nutrient supply result in damage and death of cardiomyocytes (CMs). Recanalization of the artery is essential to restore blood flow, which ultimately reduces mortality. However, sudden return of blood causes a rapid influx of oxygen, generating a destructive burst of reactive oxygen species (ROS). Hence, both myocardial ischemia and reperfusion injury (I/R) contribute to tissue damage. The cGMP pathway, via its main effector cGMP-dependent protein kinase Iα (cGKIα), may protect the heart from I/R injury and consequences like heart failure, but the pathway is also highly sensitive to ROS-dependent modifications. Moreover, specific interactors of cGMP/cGKIα in CMs as well as their impact on I/R-induced injury remain insufficiently defined.

To fill this knowledge gap, we examined total heart and CM lysates from α MHC^Cre based CM-specific cGKI conditional knockout (cKO) and control (Ctrl) mice that remained either without intervention or underwent an acute I/R protocol. Using LC-MS/MS analysis, we ultimately identified known as well as potential new CM-specific interactors of the cGMP/cGKIα pathway. Comparing healthy and I/R-exposed myocardium, we discovered an injury-induced cGKIα-multiprotein complex.

Overall, our study reveals potential new components and regulatory functions of the cGMP pathway in CMs under healthy conditions and cardiac I/R stress. Next, we will investigate if formation of specific cGKIα complexes during I/R are sensitive to treatment with cGMP-elevating drugs.

Natriuretic peptide receptor 2 (Npr2; guanylyl cyclase B) is highly expressed in nociceptors; however, its relevance to pain processing remains poorly understood. Here we demonstrate that mice with a specific deletion of Npr2 in nociceptive sensory neurons exhibit deficits in the sensing of noxious heat as well as a reduction in TRPV1-mediated nocifensive behavior and neuronal calcium influx. Furthermore, Npr2-deficient mice developed considerably reduced hyperalgesia after intraplantar injection of algogens and proinflammatory compounds. Patch-clamp recordings revealed that the endogenous Npr2 ligand, C-type natriuretic peptide (CNP), enhances the excitability of nociceptive sensory neurons via Npr2. Mechanistically, high-content imaging showed that CNP/Npr2 is coupled with the cGMP-dependent protein kinase I substrate, cysteine-rich LIM-only protein 4, which limits pain sensitization. Our findings reveal an unanticipated role for CNP/Npr2/cGMP signaling in acute nociceptive and chronic pain.

Guanylyl cyclase A (GC-A; also known as NPR1/NPR-A/ANPR) is the principal receptor for atrial natriuretic peptide (ANP) and a central regulator of cardiovascular homeostasis, yet the mechanism by which extracellular ligand binding is transmitted across the membrane to activate intracellular cGMP synthesis has remained incompletely understood. We recently determined cryo-EM structures of full-length human GC-A in the absence and presence of ANP, revealing a compaction–twist activation mechanism in which ANP stabilizes a rearranged extracellular-domain dimer and propagates conformational changes through the juxtamembrane linker, transmembrane helix, kinase-homology domain, coiled-coil, and catalytic guanylyl cyclase domain. Here, we extend this work to two agonistic anti–GC-A antibodies, XX16 and REGN5308. Cellular cGMP assays show that both antibodies activate GC-A, with XX16 displaying greater potency in the absence of ANP, whereas REGN5308 is more effective under sub-saturating ANP conditions. Cryo-EM structures of full-length GC-A bound to these antibodies reveal two distinct activation modes. XX16 binds near the extracellular-domain dimer interface and, even without ANP, stabilizes an active receptor conformation closely resembling the hormone-bound state. By contrast, REGN5308 binds the top of the extracellular domain and populates multiple conformational states; in the absence of ANP it favors an inactive-like structure, whereas ANP enables binding of a second Fab and stabilizes the active state. Accelerated molecular dynamics simulations and ligand-binding experiments further show that both antibodies retard ANP dissociation, with XX16 exerting the stronger stabilizing effect. In a mouse model of obesity-induced hypertension, XX16 significantly lowers blood pressure. Together, these findings define distinct structural and energetic routes for hormone- and antibody-mediated activation of GC-A and provide a framework for therapeutic targeting of receptor guanylyl cyclases.

Rationale and aims: Preeclampsia, or gestational hypertension, is predominantly due to maternal vascular endothelial dysfunction and impaired placental angiogenesis/development. C-type natriuretic peptide (CNP) promotes vascular homeostasis in females and stimulates angiogenesis via activation of the natriuretic peptide receptor C (NPR-C), independent of cyclic GMP. We aimed to determine if CNP treatment or NPR-C activation could attenuate preeclampsia in mice.

Methods and Results: Preeclampsia was induced using the nitric oxide synthase (NOS) inhibitor, nitro-L-arginine methyl ester (L-NAME, 100 mg/kg/day) from gestational day (GD) 7. Preeclamptic mice had smaller blood pressure reductions to bradykinin (p=0.03, 2-way ANOVA) but responded robustly to acute CNP (max. -mmHg: control -6.8±1.7 vs. L-NAME -18.6±3.1). L-NAME also led to foetal growth restriction (p=0.006, n=14-17). Chronic CNP infusion from GD 8-18 reduced MABP (mmHg: L-NAME + saline 98.5±1.4, L-NAME + CNP 86.8±2.1, p<0.0001, n=7) and improved foetal size, which was linked with restored placental angiogenesis. However, NPR-C activation via treatment with cANF4-23 from GD 8-18 in preeclamptic mice led to only a mild reduction in blood pressure (92.4±1.6 mmHg, p=0.038, n=8) and no improvement in fetal or placental parameters.

Conclusions: Treatment with CNP can reverse hyperension in mice, mediated in part by the NPR-C receptor. CNP also has beneficial effects on placental angiogenesis and foetal development, but these appear to be NPR-C independent, suggesting a role for NPR-B and cyclic GMP in the protective response. Targeting of CNP signaling could provide a new therapeutic option for preeclampsia.

Heart rate (HR) is controlled by the pacemaker activity of the sinoatrial node (SAN), which leads to subsequent electrical activation of the atria. Aberrant regulation of SAN and atrial structure and function can lead to arrythmias including bradycardia and atrial fibrillation (AF). The role of CNP and NPR-B signaling in the SAN and atria were assessed using NPR-B heterozygous (NPR-B^+/-) mice, which exhibit a ~50% reduction in NPR-B expression and reduced cGMP levels in SAN and atrial myocytes. NPR-B^+/- mice exhibit slow HR and impaired SAN conduction in association with reduced SAN myocyte spontaneous action potential (AP) firing. SAN AP firing was impaired in NPR-B^+/- mice due to reductions in I_fand I_Ca,Lcurrents in association with lower cGMP and increased hydrolysis of cAMP by phosphodiesterase 3. NPR-B^+/- mice also exhibited increased susceptibility to AF due to increased cGMP-dependent phosphodiesterase 2 activity, which results in enhanced, proarrhythmic β-adrenergic receptor signaling. This results in increases in AP duration, I_Ca,L, spontaneous sarcoplasmic reticulum Ca²⁺release events, and delayed afterdepolarizations in NPR-B ^+/- atrial myocytes. Finally, chronic treatment with CNP in a mouse model of hypertensive heart disease protected against SAN and atrial electrical and structural remodeling, bradycardia, and atrial fibrillation. These data demonstrate that CNP, acting via NPR-B/cGMP signaling, plays an essential role in maintaining normal HR and SAN function, as well as atrial electrical function and Ca²⁺ homeostasis. These data further demonstrate that CNP can protect against SAN and atrial dysfunction and arrhythmogenesis in hypertensive heart disease.

Pulmonary fibrosis (PF) is a progressive, often fatal lung disease with limited therapeutic options. Its pathogenesis is driven by inflammatory infiltration and fibroblast-to-myofibroblast transition, resulting in excessive extracellular matrix deposition. While profibrotic pathways are well characterized, endogenous counter-regulatory mechanisms remain poorly understood. Preclinical data suggest that C-type natriuretic peptide (CNP) may act as such an antifibrotic regulator.

In cultured lung fibroblasts and precision cut lung slices from patients with PF, CNP exhibited antifibrotic effects, supporting clinical relevance. To unravel whether endogenous paracrine CNP counteracts inflammation-driven PF, mice with fibroblast-restricted KO of guanylyl-cyclase-B (GC-B), its cGMP-synthesizing receptor were studied. Fibroblast GC-B-KO mice had enhanced bleomycin-induced lung inflammation, with increased expression of proinflammatory, profibrotic cytokines. However, subsequent lung fibrosis was not exacerbated. Mechanistically, inflammation impaired CNP signaling in myofibroblasts through downregulation of GC-B and its downstream effector, cGMP-dependent protein kinase I (cGKI), as well as upregulation of the cAMP/cGMP-degrading phosphodiesterase 2 (PDE2). Consistently, in lung tissue from patients with idiopathic PF (IPF), GC-B and cGKI expression was reduced and PDE2 expression was enhanced. Single-cell RNA-sequencing data further confirmed diminished GC-B and cGKI expression in stromal cells from patients with interstitial lung disease and IPF. Despite these molecular changes, a single subcutaneous injection of the recently developed long-acting CNP analog, MS-[Gln ^6,14]CNP-38 (1), prevented experimental lung inflammation and fibrosis.

In summary, inflammatory remodeling of lung myofibroblasts impairs the antifibrotic effects of endogenous CNP. Pharmacological restoration of this signaling axis with a long-acting CNP analog represents a promising therapeutic strategy for PF.

Since the original discovery that ANP establishes an axis between the heart and the kidneys, many additional physiological ANP functions have been characterized ^1,2. Via ANP and its guanylyl cyclase (GC-A) receptor, the heart networks with neurohumoral systems and organs adjusting blood pressure and metabolism ². Moreover, within the heart, ANP mediates local communications of cardiomyocytes with endothelial cells and pericytes. This lecture illustrates our novel findings on these circuits and open questions. One critical function of the ANP/GC-A system is sensing and regulating intravascular volume. Increased atrial wall tension evoked by volume expansion releases ANP to reset homeostasis through coordinated endothelial and renal effects. Intriguingly, in the kidney, GC-A is mainly expressed in peritubular endothelial and interstitial cells and barely in tubular epithelia ³, emphasizing that the mechanisms accounting for ANP-evoked natriuresis are still enigmatic. Notably, GC-A and cyclic GMP-regulated proteins (PDE2A and cGKI) are highly expressed in adrenal zona glomerulosa (zG), suggesting that natriuretic effects of ANP are partly mediated by inhibiting aldosterone synthesis. Indeed, our studies of novel genetic mouse models and human adrenal samples reveal that ANP mediates physiological heart-adrenal circuits moderating aldosterone release acutely (through PDE2A) and chronically (through cGKI). The latter prevents hypervolemic hypertension under conditions of salt/volume load.

Patients with therapy-resistant hypertension often have high aldosterone levels, which enhances their cardiovascular risk. Blunted ANP/GC-A signaling contributes to excessive zG growth and aldosterone production and reinforcing this pathway may benefit such patients. This seminar therefore concludes summarizing recent setbacks and advances in the field of NP-based therapeutics.

C-type natriuretic peptide (CNP) is a key stimulator of endochondral bone growth and is already used clinically for the treatment of achondroplasia. We previously showed that CNP activates autonomous Ca²⁺ entry in growth plate chondrocytes via transient receptor potential melastatin 7 (TRPM7) channels, thereby promoting matrix production and bone elongation. In the present study, we asked whether this growth-promoting pathway could be enhanced pharmacologically by targeting cyclic guanosine monophosphate (cGMP) degradation. Based on expression analyses, we focused on phosphodiesterase 3B (PDE3B) in the growth plate and examined the effects of the PDE3 inhibitor cilostazol using live-cell imaging, biochemical assays, histology, ex vivo bone culture, and in vivo skeletal analyses. Cilostazol increased cellular cGMP levels and activated plasma membrane K⁺ channels, most likely through protein kinase G-dependent mechanisms, leading to membrane hyperpolarization. This change is expected to increase the driving force for Ca²⁺ influx and thereby facilitate TRPM7-mediated Ca²⁺ signaling. Consistent with this mechanism, cilostazol promoted elongation of cultured bones and increased body size in juvenile mice. These findings identify PDE3 inhibition as a pharmacological strategy to enhance CNP/cGMP-dependent Ca²⁺ signaling and support the potential repurposing of PDE3 inhibitors for skeletal disorders associated with short stature.

The cyclic guanosine monophosphate (cGMP) signaling pathway is a fundamental regulator of energy homeostasis, yet the specific contribution of its primary degrading enzyme, phosphodiesterase type 5 (PDE5A), to systemic metabolism remains to be fully elucidated. Growing experimental and clinical evidences indicate that enhancement of cGMP signaling through PDE5 inhibition promotes metabolic remodeling in peripheral tissues, thereby improving systemic metabolic control. Recent findings from mouse models further support a direct metabolic role of PDE5. Mice lacking Pde5a display enhanced thermogenic capacity, increased energy expenditure, and moderate browning of white adipose tissue, accompanied by reduced hepatic lipid accumulation and improved systemic metabolic profile. Under metabolic stress conditions, Pde5a deficiency confers protection against diet-induced obesity and metabolic dysfunction, resulting in improved glucose tolerance and enhanced insulin sensitivity. Mechanistically, these effects are driven by a novel functional interplay between PDE5A and PDE3B, establishing a sustained cAMP-cGMP cross-regulation within adipose tissue. Multi-omics analysis revealed an unprecedented systemic shift in glucose handling, characterized by enhanced gluconeogenesis and developmental metabolic reprogramming. These data provide a mechanistic rationale for the cardioprotective benefits observed in landmark studies involving PDE5 inhibitors, explaining how PDE5 inhibition counteracts maladaptive cardiac hypertrophy. Moreover, these findings identify PDE5A as a key regulator of systemic energy balance. By bridging the gap between preclinical models and clinical trial data, they establish a rationale for using PDE5 inhibitors as potential adjuvant therapies for metabolic diseases.

Natriuretic peptides (NPs) increase cGMP, show beneficial cardiovascular effects and regulate mitochondrial activity in other tissues. However, little is known about their direct effect on cardiac mitochondria and cardiomyocyte apoptosis. Here, we examined whether GC-A and GC-B stimulation by ANP and CNP, respectively, regulates mitochondrial cGMP and apoptosis in cardiomyocytes. We developed and applied novel FRET-based biosensors with high cGMP selectivity targeted to mitochondrial microdomains, such as the outer mitochondrial membrane. In adult rat cardiomyocytes, ANP and CNP increased mitochondrial cGMP. The CNP-induced cGMP exceeded that detected in the cytosol. Inhibition of PDE2 further augmented mitochondrial cGMP, an effect not observed in the cytosol. In addition, PDE activity assays revealed significantly higher PDE2 activity in mitochondrial compared to non-mitochondrial fractions, indicating that PDE2 is a key regulator of mitochondrial cGMP. In addition, ANP and CNP increased phosphorylation of the pro-apoptotic GTPase Drp1, reduced mitochondrial fragmentation and attenuated activation of the intrinsic apoptotic pathway, shown by decreased caspase 9 activation and cytochrome c release. These data demonstrate that natriuretic peptides increase mitochondrial cGMP, that is constrained by local PDE2 activity, and inhibit Drp1-dependent mitochondrial fission and thereby limit cardiomyocyte apoptosis.

During development, ventrally born cortical interneurons undertake a complex migration process to reach the developing cortex, where they will form and integrate functional circuits. This process critically relies on a small, microtubule-based antenna called the primary cilium (PC), to re-orient tangentially migrating cortical interneurons radially, towards their target destination. The PC is established as a signalling hub concentrating a variety of different membrane receptors and signalling molecules, including the cGMP and cAMP second messengers. However, PC-elicited signalling, and how it affects migration, remains ill characterised. To address this challenging question, we have developed an innovative toolset of scavengers and optogenetic constructs allowing the specific and local modulation of cGMP or cAMP levels within the PC of migrating interneurons. We first show that specific ciliary cGMP or cAMP buffering dysregulates the cell polarity and directed migration of in vitro migrating interneurons in an opposite manner: while ciliary cAMP buffering stabilises the cell polarity and leads to sustained directed migration, ciliary cGMP buffering induces frequent changes in polarity and direction of migration. Remarkably, photo-activation experiments reveal that increasing ciliary cAMP levels phenocopies ciliary cGMP buffering, suggesting that opposite ciliary second messenger levels induce an opposite regulation of cell polarity. Finally,ex vivo approaches in brain organotypic slices confirm the physiological relevance of such signals, by recapitulating the antagonistic effect of ciliary cAMP and cGMP on the direction of migration. Taken together, our work uncovers the ciliary cAMP/cGMP ratio as the steering wheel of cortical interneurons during their migration to the developing cortex.

The complexity and specificity of cellular processes, such as signal transduction and metabolism, require spatial microcompartmentation and dynamic modulation of the underlying biochemical activities. We hypothesize that cellular biochemical activities are spatially organized into an “activity architecture” and reorganization and restructuring of this activity architecture lead to disease. In this talk, I will introduce a series of genetically encoded or chemigenetic fluorescent biosensors that we have developed to monitor biochemical events in living cells, including a suite of new cGMP-dependent protein kinase activity reporters (GKARs). I will then present a couple of studies where we combine quantitative fluorescence imaging with targeted perturbations as well as biochemical and functional assays to probe the spatiotemporal regulation of GPCR and kinase signaling.

Genetic and pharmacological data indicate that a balanced activity of the NO-cGMP signaling pathway is critical for cardiovascular homeostasis in humans. Our laboratory discovered that NO-induced cGMP production in platelets is strongly shear-dependent. This finding did not only delineate an elegant strategy of nature to prevent thrombosis without causing dangerous bleeding but set up the new concept of mechanosensitive cGMP signaling (Mechano-cGMP). Our recent data indicate that Mechano-cGMP also exists in vascular smooth muscle cells (VSMCs). We used live-cell cGMP imaging to analyze the flow/force-dependency of NO-induced cGMP signals in primary VSMCs and isolated blood vessels from transgenic cGMP sensor mice. Mechano-cGMP was observed in individual VSMCs upon exposing them to fluid shear stress or local pressure puffs, possibly mimicking the deformation and stretching that the cells experience in their natural environment. Co-mapping of cGMP signals, cell stiffness, and protein expression revealed that Mechano-cGMP in VSMCs is associated with high actin content and cell stiffness. Moreover, we were able to trigger Mechano-cGMP in intact carotid arteries by increasing the intravascular pressure. We hypothesize that Mechano-cGMP may be a general principle underlying vascular health and that it may be disrupted in diseases such as hypertension, atherosclerosis, and cancer.

The cyclic guanosine monophosphate (cGMP) signalling pathway is a central regulator of vascular tone and vascular smooth muscle cell (VSMC) function, primarily mediated through the alpha isoform of cGMP-dependent protein kinase I (PKG Iα), encoded by PRKG1. Dysregulation of this pathway has been increasingly implicated in vascular pathologies, including hereditary thoracic aortic aneurysm and dissection (TAAD), a life-threatening condition often affecting younger patients and lacking targeted therapeutic options.

In our recently published study, an activating PRKG1 variant (V219I), associated with inherited aortopathy, was investigated for its impact on cGMP-dependent signalling and VSMC biomechanics. To model this genetic disease, the mutation was introduced via CRISPR-Cas9 genome editing into a control human induced pluripotent stem cell (hiPSC) line (UKEi001-A) and differentiated into VSMCs. The variant results in constitutive PKG Iα activation, thereby amplifying cGMP signalling independent of physiological control. Mutant VSMCs exhibit increased cell size, enhanced deformability, and disrupted actin cytoskeletal organization, reflecting profound alterations in cellular mechanical properties. These findings are consistent with excessive PKG-mediated phosphorylation of cytoskeletal targets, directly linking aberrant cGMP signalling to structural instability at the cellular level. Moreover, our data suggest that hyperactive PKG Iα initiates a cascade in which cellular softening represents a primary defect, followed by maladaptive extracellular matrix remodelling, ultimately increasing susceptibility to aortic dilation and dissection.

Collectively, this work highlights the critical importance of tightly regulated cGMP signalling in maintaining vascular integrity and identifies chronic PKG Iα activation as a pathogenic mechanism and potential therapeutic target in hereditary aortopathies.

Hereditary retinal degeneration (RD) conditions, such as Retinitis Pigmentosa and Leber’s Amaurosis, are untreatable disorders leading to photoreceptor cell death and blindness. These diseases usually start with the degeneration of rod photoreceptors, causing the subsequent loss of cone photoreceptors and severe vision decline. cGMP (cyclic guanosine-3’, 5’-monosphosphate) signaling pathways have emerged as critical disease drivers common to different types of RD. Elevated cGMP levels within photoreceptors result in the over-activation of cGMP-dependent protein kinase G (PKG), leading to photoreceptor cell death. Therefore, allosteric inhibition of PKG by cGMP analogs that compete with cGMP has emerged as a promising therapeutic approach for RD. Specifically, the cGMP analog Rp-8-Br-PET-cGMPS (CN003) preserves in vivo retinal function and reduces photoreceptor degeneration in differentRD models, emerging as a lead compound for RD-type diseases. However, translating leads like CN003 into drugs requires optimizing their potency while preserving selectivity for PKG vs. other retinal kinases and cGMP-effectors.Using Nuclear Magnetic Resonance (NMR) and docking, we aim to determine the molecular mechanism underlying the allosteric inhibition of PKG by CN003 and other cGMP analogs. Our study shows that, upon binding to CN003, PKG adopts an intermediate conformation resembling its inactive state. The CN003’s inhibitory mechanism targets key allosteric hotspots of PKG, including the capping lid, keeping the kinase inactive, while retaining high affinity. Using a ligand-substituent cycle approach, we identified which CN003 substitutions are key for inhibiting PKG and/or enhancing binding affinity, a crucial step toward identifying a pharmacophore model for selective and potent PKG allosteric inhibition.

A growing body of evidence suggests that transient receptor potential canonical (TRPC) 3 and 6 channels contribute to pathological cardiac remodeling. However, we previously demonstrated that activation of TRPC6 enhances β-adrenoceptor (βAR)-stimulated positive inotropy and prevents the development of chronic heart failure in mice via modulation of Zn²⁺ dynamics. In this study, we investigated whether TRPC6-mediated Zn²⁺ influx suppresses sympathetic overactivity–induced chronic heart failure. Chronic βAR stimulation by intraperitoneal administration of isoproterenol (ISO) for 4 weeks induced myocardial dysfunction in wild-type (WT), TRPC3 knockout (C3KO), and TRPC6 knockout (C6KO) mice. Notably, C6KO mice, but not C3KO mice, exhibited more severe cardiac fibrosis than WT mice, suggesting an anti-fibrotic role of TRPC6. Based on the Zn²⁺ permeability of TRPC6 and sequence differences in the pore region between TRPC3 and TRPC6, we generated Zn²⁺ permeation-deficient TRPC6 knock-in (C6KYD) mice. Zinpyr-1 imaging revealed that the TRPC6 activator PPZ2 increased intracellular Zn²⁺ levels in ISO-treated WT hearts, whereas this effect was absent in ISO-treated C6KYD hearts. Consistently, PPZ2 attenuated TGFβ–induced fibrosis in fibroblasts from WT mice, but not C6KYD mice. Furthermore, the anti-fibrotic effect of TRPC6 was abolished by a PKG inhibitor. TRPC6-mediated Zn²⁺ influx promoted PKG dimerization and activation, indicating that PKG signaling is a key downstream mediator of the TRPC6–Zn²⁺ axis. Collectively, these findings demonstrate that the TRPC6–Zn²⁺–PKG signaling pathway plays a critical role in suppressing cardiac fibrosis and protecting against sympathetic overactivity–induced heart failure.

Endothelial dysfunction, a hallmark of type 2 diabetes (T2D), is caused by reduced NO (nitric oxide) bioavailability. Exercise restores endothelial function in T2D by enhancing resilience, but underlying mechanism remains unclear. Under physiological conditions, exercise activates eNOS (endothelial NO synthase), promoting NO-cGMP-dependent vasodilation. However, in T2D, where eNOS uncoupling and excess superoxide (O₂^-) contribute to oxidative stress, exercise may exert vascular benefits through alternative pathways. One such mechanism involves the exercise-induced upregulation of antioxidant superoxide dismutases (SODs), which convert O₂^-to hydrogen peroxide (H ₂O₂). This process may enable H₂O₂-mediated vasodilation, either through NO-cGMP-dependent or -independent pathways. However, the precise mechanisms underlying exercise-induced vasodilation in T2D remain poorly understood. Extracellular SOD (SOD3) is a major secretory copper (Cu)-containing antioxidant enzyme in the vasculature, whose activity requires the Cu transporter ATP7A. SOD3 is secreted by vascular smooth muscle cells and anchors at the endothelial surface. In T2D vessels, ATP7A expression is downregulated, leading to reduced SOD3 function, increased O₂^-levels, and impaired NO-dependent vasodilation. Surprisingly, exercise in T2D increases the ATP7A-SOD3 axis, facilitating SOD3-mediated outside-in H₂O₂(rather than NO) to directly oxidize and activates protein kinase G (PKG)1a via sulfenylation (CysOH formation) at Cys42 in a cGMP-independent manner. This in turn restores endothelium-dependent vasodilation. Our findings identify SOD3-mediated PKG1a oxidation as a potential therapeutic target for endothelial dysfunction in T2D.

Cyclic guanosine monophosphate (cGMP) is a foundational second messenger that governs vascular tone, neuronal signaling, phototransduction, and diverse aspects of tissue homeostasis. Decades of work on guanylyl cyclases, phosphodiesterases, and protein kinase G have established cGMP signaling as a paradigm for spatially organized, tightly regulated intracellular communication. However, cGMP represents only one node within a much broader and evolutionarily conserved network of cyclic nucleotide signaling systems.

In this talk, I will place cGMP within the larger framework of cyclic dinucleotide biology across domains of life. Cyclic nucleotides function as versatile and evolutionarily conserved signaling modules across life—from bacterial cyclic dinucleotide–based defense systems and phage countermeasures to mammalian innate immunity mediated by the cGAS–STING pathway and 2′3′-cGAMP. Despite distinct biological contexts, these systems share a common architectural logic: a sensor-activated cyclase generates a diffusible cyclic nucleotide that engages dedicated receptors and effectors to execute rapid, and often decisive, cellular responses.

By drawing mechanistic and conceptual parallels between cGMP and cGAMP producers, receptors, and downstream effectors, this presentation will offer a unified perspective on cyclic nucleotide signaling—highlighting shared chemical principles, regulatory strategies, and evolutionary continuity from phage immunity to human physiology.

Cyclic octasulfur (S₈) is a product of sulfur respiration in bacteria. According to the endosymbiotic theory, mitochondria have evolved from sulfur bacteria, and thus it may exist in mitochondria. Using a new mass spectrometry method, we found S₈ in mammalian mitochondria at levels similar to bacteria, as concentrated in lipid droplets of mouse and human adipocytes, while bacteria store S₈ in sulfur granules. We identified that endothelial nitric oxide synthase (eNOS) and other NOS enzymes effectively produce S₈ when provided appropriate donors like glutathione trisulfide (GSSSG) together with NADPH. eNOS, activated by Ca²⁺ ionophore A23187 and vascular endothelial growth factor (VEGF), exhibited a significant association and localization on the surface of lipid droplets. This led to the high yield accumulation of S₈ in lipid droplets of endothelial cells. Our results suggest that S₈ acts as a reservoir for reactive hydropersulfides, protecting cells from ferroptosis by countering lipid peroxidation. Persulfides serve as antioxidants and thus protect cells from lipid peroxidation-driven cell death - ferroptosis. Indeed, depletion of S₈ from adipocytes and endothelial cells caused lipid oxidation and ferroptosis. In contrast, supplementation with solubilized S₈ prevented ferroptosis. Our findings suggest that a previously unrecognized mammalian S₈ pool protects cells against oxidative damage and may also help even cancer cells to survive. In summary, S₈ exhibits an evolutionary conservation as a source of antioxidants and a potent regulator of lipid peroxidation. Consequently, it emerges as a crucial cytoprotective molecule, biosynthesized endogenously.

Nitric oxide (NO) has long been viewed as the principal endothelium-derived vasodilator through activation of soluble guanylyl cyclase (GC1) and cGMP-dependent signaling in vascular smooth muscle. However, accumulating evidence indicates that this canonical NO-GC1-cGMP pathway predominates in large conduit arteries, whereas regulation of vascular tone and blood pressure in the resistance arteries relies largely on endothelium-derived hyperpolarization (EDH) with NO acting as a nitrosative signal rather than a freely diffusing gas. Our most recent studies in resistance arteries suggest that GC1 is a key player in shaping endothelial excitability by regulating nitrosative signal via its ability to transfer S-nitroso groups to other proteins (transnitrosation). We created a mouse model which lacks GC1 transnitrosation activity by replacing a single cysteine (C610) in α-GC1 subunit (KI C610S). The mesenteric arteries of this mouse model display impaired acetylcholine-dependent vasorelaxation associated with significantly reduced endothelial Ca²⁺ influx and KCa channel activation. In sharp contrast, NO-dependent vasorelaxation of the mesenteric arteries in the KI C610S remains intact. Identification of the downstream targets of GC1 transnitrosation activity that modulate EDH-dependent vasodilation is currently under investigation.

Cyclic nucleotides (cNMPs), particularly cGMP and cAMP, have constituted a fundamental component of cellular signaling research in animals for several decades. Although their presence in plants was postulated as early as the 1970s, the concept of a functional role for cNMPs in plant biology remained marginal for a long time. Low concentrations of these molecules, methodological challenges in their detection, and the apparent absence of classical cyclase homologs or effectors known from animal systems fostered skepticism regarding their existence and physiological relevance. Consequently, despite these obstacles, discoveries in cNMP signaling in plants lagged rapidly advancing knowledge derived from animal models for many years. However, over the last two decades, this field has undergone a significant reassessment. The identification of plant guanylyl and adenylyl cyclases with atypical structures and their enzymatic activity within complex, multidomain, and multifunctional proteins revealed a distinct molecular architecture of the cNMP pathway in plant cells. Rather than employing the classical, specialized enzymes and effectors characteristic of animal systems, plants utilize modular solutions that integrate cyclic nucleotide production with signal perception and cellular response. This suggests functional convergence rather than simple evolutionary conservation of pathway components.

Currently, cyclic nucleotides are considered essential components of signaling networks that regulate plant responses to stress factors and growth and developmental processes. cGMP and cAMP function in concert with key mediators such as Ca²⁺, reactive oxygen species (ROS), and nitric oxide (NO), forming integrated signaling pathways with high plasticity. Increasing evidence indicates that, despite structural differences, the organizational logic of these pathways, rapid secondary messenger production, signal localization, and precise degradation, exhibits remarkable similarities across biological kingdoms.

This presentation will outline the historical development of research on cyclic nucleotides and their metabolism in plants, the current state of knowledge regarding their biosynthesis, degradation, perception, and putative biological functions, and the methodological challenges associated with their quantification and data interpretation. Comparative aspects will also be discussed to better understand how diverse organisms utilize a shared class of molecules to regulate complex adaptive processes. In this context, cyclic nucleotides in plants emerge not as a marginal evolutionary relic but as a long-underappreciated yet fundamental element integrating environmental signals with cellular responses.