Despite recent advances in pharmacological treatment, chronic heart failure (HF) is associated with significant morbidity and mortality, and further treatment options are needed. Intact nitric oxide (NO)-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signalling is a prerequisite of cardiovascular health. cGMP produced by NO/NO-sGC acts as a second messenger molecule via various downstream targets, which influence a broad spectrum of critical physiological parameters. Impairment of this cascade in the cardiovascular system is considered an important pathomechanism in HF. This review examines pharmacological therapies that act through the NO-sGC-cGMP signalling pathway. We will focus on the molecular mode(s) of action of NO-independent but haem-dependent sGC stimulators, and will examine evidence from preclinical studies demonstrating cardiovascular benefits of these therapies and their increasing number of effects on other susceptible tissues and organs, which together could contribute to clinical outcomes in HF. The sGC stimulator vericiguat may be considered, in addition to standard therapy, for adults with symptomatic HF with reduced ejection fraction following a worsening event. The findings from pivotal clinical trials that led to these recommendations will be outlined and classified in terms of their significance for different subpopulations. These include the Phase 3 VICTORIA and VICTOR trials. Finally, further research areas and ongoing studies designed to address existing gaps in our knowledge regarding vericiguat and related drugs will be highlighted.

Resident cardiac fibroblast–derived (RCF-derived) cardiac myofibroblasts (CMFs) contribute to myocardial repair but also drive adverse ventricular remodeling and contractile dysfunction after myocardial infarction (MI). The sodium-activated potassium channel Slick (Slo2.1) has been described in cardiomyocyte (CM) mitochondria; however, transcriptomic analyses indicate higher Slick expression in RCFs/CMFs. Here, we investigated the role of Slick in cardiac fibroblast function and post-MI remodeling. Using live-cell imaging and whole-cell patch-clamp recordings, we found that plasma membrane Slick channels in RCFs and CMFs regulated potassium (K + ) efflux and modulated store-operated calcium entry (SOCE), particularly in CMFs. Global Slick KO and conditional CMF-specific KO hearts exhibited reduced fibrosis and preserved left ventricular function after ischemia/reperfusion injury. This cardioprotection was associated with diminished CMF activation and proliferation, reduced inflammation, and improved CM survival after MI. Collectively, these findings identify fibroblast Slick channels as regulators of SOCE-dependent fibrogenesis and demonstrate that their deletion mitigates maladaptive remodeling and functional decline after MI.

Natriuretic peptide receptor 2 (Npr2; also termed guanylyl cyclase B) is a transmembrane guanylyl cyclase that is highly abundant in nociceptors. Here, we investigated the role of production of cyclic GMP (cGMP) by Npr2 in pain processing. Adult mice with a deletion of Npr2 specifically in nociceptive sensory neurons exhibited deficits in noxious heat sensing, which can activate the nonselective cation channels TRPV1 and TRPA1. In parallel, Npr2-deficient mice showed a reduction in TRPV1-mediated nocifensive behavior and Ca2+ influx into sensory neurons. Furthermore, Npr2-deficient mice had considerably reduced hypersensitivity after hindpaw injection of TRPA1 and TRPV1 activators or after hindpaw injection of complete Freund adjuvant, a model of persistent inflammatory pain. These results indicate that Npr2 contributes to the pain sensitization that can lead to chronic pain. Patch-clamp recordings revealed that the endogenous Npr2 ligand, C-type natriuretic peptide (CNP), enhanced the excitability of nociceptive sensory neurons through Npr2. CNP/Npr2 signaling led to the phosphorylation of cysteine-rich LIM-only protein 4 (CRP4), a substrate of cGMP-dependent protein kinase I. Behavioral and electrophysiological analyses using CRP4-deficient mice revealed that CRP4 limited CNP/Npr2-mediated pain sensitization. Our findings reveal a role for CNP/Npr2 signaling in sensory neurons in acute nociceptive and chronic pain and suggest that CRP4 is a downstream target that attenuates pain sensitization.

Background Treatment with inhaled nitric oxide (NO) improves rates of survival and neurologic outcomes in a mouse model of resuscitation after cardiac arrest. The effect of NO is dependent on the soluble guanylyl cyclase/cyclic guanosine monophosphate (NO-sGC-cGMP) pathway. NO-sGC is a potential target for drugs to modulate NO-dependent signaling in conditions that include ischemia-associated inflammation. The objective of this study was to determine whether CYR119, a stimulator of NO-sGC that can penetrate the central nervous system, improves outcomes after resuscitation from cardiac arrest. Methods Adult C57BL/6J wild-type mice of both sexes were subjected to potassium chloride-induced cardiac arrest and cardiopulmonary resuscitation. Fifteen minutes after the return of spontaneous circulation, mice were randomized to receive subcutaneous injections of either CYR119 or vehicle alone. The length of survival after the procedure and degree of neurological dysfunction were assessed. A composite outcome measure was used to define a good outcome as survival with good neurological function, while a poor outcome was defined as either death or exhibiting poor neurologic function. In addition, mRNA levels of inflammatory cytokines in the brain and a plasma marker of kidney injury were measured. Results CYR119 significantly improved 10-day survival (35 % in CYR119-treated mice; 15 % in vehicle-treated mice) and the likelihood of achieving a good outcome, demonstrating an association between treatment and both survival and neurological recovery. CYR119-treated mice also exhibited reduced transcript levels of TNF⍺ and IL-1β in the hippocampus and cortex, respectively, and lower plasma creatinine levels. Conclusion The current study revealed that CYR119 substantially improved the likelihood of survival with good neurologic function in mice resuscitated from cardiac arrest. The beneficial effects of post-arrest treatment with CYR119 were associated with decreased mRNA expression of inflammatory cytokines in the brain and decreased plasma creatinine levels, suggestive of renal protection. These findings support the potential of CYR119 as a therapeutic strategy for post-cardiac arrest recovery.

Differentiation of cardiac fibroblasts (CFs) into myofibroblasts (CMFs) is considered a critical event in response to the maladaptive cardiac remodeling triggered by angiotensin II (Ang II). Active CMFs are proliferative and contribute to the production of extracellular matrix and matricellular proteins such as periostin, to myocardial fibrosis, and thus to muscle stiffness. Although previous studies provided substantial evidence for the antifibrotic signaling elicited by NO/NP-cGMP-cGKI, the role of this axis in modulating CMF function(s) in vivo remains unclear. To address this, Ang II was delivered through osmotic minipumps into tamoxifen-induced CMF-specific cGKI knockout (cmfKO) and littermate control (CTR) male mice. CMF-restricted Cre activity in periostin+ cells resulted in an effective depletion of the cGKI protein observed in myocardial sections and in primary CF/CMF protein lysates obtained from Ang II- and tamoxifen-treated cmfKO. Although both genotypes responded identically to Ang II in terms of blood pressure (BP) and cardiac enlargement, cmfKO hearts showed significantly increased cardiomyocyte cross-sectional areas and developed a marked increase in myocardial fibrosis. Moreover, non-invasive echocardiography revealed a structure-related distortion of global systolic function and longitudinal deformation capacity in cmfKO versus CTR. Consistent with the results obtained in vivo, we observed a higher proliferation rate of CF/CMF derived from Ang II-treated cmfKO hearts compared with respective CTR cells, as well as an increase in cardiomyocyte apoptosis in the absence of cGKI in periostin+ CMF. Our data confirm that endogenous cGKI function in periostin+ CMFs counteracts the Ang II-induced morphologic and structural changes that impair cardiomyocyte survival ultimately causing loss of heart function in mice.

The voltage- and calcium (Ca2+)-activated potassium (K+) channel of large conductance (BKCa) is aberrantly expressed in various breast cancer (BC) subtypes, including estrogen receptor (ER)-positive tumors. Increased proliferation of BC cells in response to tamoxifen (TAM) and its metabolites (TAM+M) has been shown to rely on the cell's BKCa status. However, the mechanism by which TAM+M impact on BKCa activity to promote malignancy is yet not clear. By examining murine MMTV-PyMT tumor-derived BC cells and human BC cell lines with a genetically encoded K+ ion indicator and electrophysiological recordings, we identified BKCa-dependent intracellular K+ signals and currents provoked by treatment with clinically relevant TAM+M in an ER-independent manner. In line with this, genetical or pharmacological blockade of BKCa significantly diminished the TAM+M-induced modulation of BKCa K+ currents and consequently also the drop of intracellular K+ ions in BC cells. Changes in the K+ balance subsequently triggered intra- and extracellular Ca2+ mobilization, which was in turn stimulated by the TAM+M-BKCa axis. Our results highlight that BKCa "oncochannels" may modulate the response of BC cells to TAM+M. Activation of the TAM+M-BKCa axis causes significant changes in K+ and Ca2+ ion homeostasis, which ultimately contributes to the outcome of endocrine-based BC pharmacotherapy.

BACKGROUND AND PURPOSE:Lipolysis is tightly regulated by pro-lipolytic β-adrenoceptor signalling, which activates the cAMP/PKA pathway, and by antilipolytic hormones like insulin and FGF1, which counter-regulate lipolysis through cAMP-degrading phosphodiesterases (PDEs). While the spatial compartmentalization of cAMP signalling is recognized, comparisons between distinct cAMP pools remain under-investigated in adipocytes. Moreover, the dynamics of cAMP around lipid droplets (LD) where lipolysis occurs, are particularly intriguing. Thus, we studied whether adipose FGF1/PDE4D and insulin/PDE3B pathways regulate distinct cAMP microdomains to execute their antilipolytic actions. EXPERIMENTAL APPROACH:We evaluated the role of subcellular cAMP pools in lipolysis regulation by PDEs, or antilipolytic hormones, by utilizing EPAC1-based FRET cAMP biosensors specifically designed to localize in the cytoplasm or at the plasma membrane of living cells. Additionally, we developed the first LD-associated cAMP biosensor by fusing the lipid droplet-associated protein perilipin-1 to the EPAC1-based probe. KEY RESULTS:We identified previously unrecognized cAMP pools surrounding LDs that are distinct from cytoplasmic cAMP and resistant to PDE inhibition or antilipolytic stimuli. PDE4D exhibits a stronger effect on all three cAMP pools investigated than PDE3B. FGF1 mainly inhibits the cAMP in the initiation of the signalling at the plasma membrane, whereas insulin targets mainly cytoplasmic cAMP pools. CONCLUSION AND IMPLICATIONS:The discovery of LD-associated cAMP as a distinct subcellular pool suggests that cAMP signalling in adipocytes is more compartmentalized than previously recognized. The distinct pathways by which FGF1 and insulin regulate adipose cell cAMP levels highlight that antilipolytic signalling is not uniform, refining our understanding of lipolysis regulation.

BACKGROUND AND PURPOSE:Hexokinase 2 (HK2) is a key enzyme linked to high tumour cell proliferation. Its inhibitors such as 3-bromopyruvic acid (3-BP) induce cancer cell death, highlighting HK2 modulation as potential anti-cancer treatment. However, standard chemotherapies often cause the emergence of senescent cancer cells, which goes along with cell metabolic reprogramming and treatment failure. This study explores whether targeting HK2 can induce cancer cell senescence and whether metabolic changes in senescent cancer cells are tied to the cellular HK2 status. EXPERIMENTAL APPROACH:The expression of hexokinase 1 (HK1) and HK2 was assessed using immunoblot and immunofluorescence analysis in cell lines and in primary murine breast cancer (BC) cells. The senescence-inducing potential of HK2 inhibition and the effect of chemotherapy-induced senescence on HK1 and HK2 expression were assessed. Cell-based approaches were complemented by analysing single-cell RNA sequencing data from BC patients. KEY RESULTS:BC cell sensitivity to HK2 inhibition did not correlate with HK2 expression levels. Consistently, senescence was linked to a decrease in HK2 and an increase in HK1 expression. Moreover, genetic knockdown of HK2 induced senescence, indicating that a change in the HK2/HK1 ratio drives, rather than results, from cellular senescence. This shift in HK2/HK1 ratio was confirmed in single-cell RNA sequencing data of BC biopsies. CONCLUSIONS AND IMPLICATIONS:Expressional shifts in the HK2/HK1 ratio may serve as a novel marker for BC cell senescence. Whereas targeting HK2 shows promise in untreated cancers, senescence-inducing anti-cancer therapies may limit the effectiveness of HK2-targeted treatments in pre-treated cancer patients.

A balanced activity of cGMP signaling contributes to the maintenance of cardiovascular homeostasis. Vascular smooth muscle cells (VSMCs) can generate cGMP via three ligand-activated guanylyl cyclases, the NO-sensitive guanylyl cyclase, the atrial natriuretic peptide (ANP)-activated GC-A, and the C-type natriuretic peptide (CNP)-stimulated GC-B. Here, we study natriuretic peptide signaling in murine VSMCs and atherosclerotic lesions. Correlative profiling of pathway activity and VSMC phenotype at the single-cell level shows that phenotypic modulation of contractile VSMCs to chondrocyte-like plaque cells during atherogenesis is associated with a switch from ANP/GC‑A to CNP/GC‑B signaling. Silencing of the CNP/GC-B axis in VSMCs results in an increase of chondrocyte-like plaque cells. These findings indicate that the CNP/GC-B/cGMP pathway is a marker and atheroprotective regulator of modulated VSMCs, limiting their transition to chondrocyte-like cells. Overall, this study highlights the plasticity of cGMP signaling in VSMCs and suggests analogies between CNP-dependent remodeling of bone and blood vessels. The second messenger 3’,5’-cyclic guanosine monophosphate (cGMP) is important for the maintenance of cardiovascular homeostasis. Here, correlative single-cell profiling of cGMP dynamics and vascular cell phenotype reveals a druggable atheroprotective mechanism and suggests parallels between remodeling processes in bone and atherosclerotic arteries.

Increasing evidence suggests that adipose tissue plays a key role in the development, progression, and treatment of the globally epidemic disease type 2 diabetes (T2D). For example, adipose tissue dysfunction, lipotoxicity, and insulin resistance (IR) are major contributors and targets for the treatment of T2D. We previously identified the Fibroblast growth factor 1 (FGF1)/Phosphodiesterase 4D (PDE4D) pathway, which lowers plasma glucose concentration by suppressing lipolysis in adipose tissue and ultimately regulating hepatic glucose production in obese insulin-resistant mice. While phosphorylation of PDE4D is critical for its activity, the upstream signaling mechanisms remain unclear. In this study, we identified p21-activated kinases (PAKs) as regulator of PDE4D phosphorylation and suppression of lipolysis by FGF1. Inhibition of PAK-induced cAMP accumulation prevented antilipolytic function of FGF1, and reversed suppression of lipolysis caused by PDE4D overexpression, linking PAKs to the regulation of cAMP by PDE4D in murine adipocytes in vitro. Chronic inhibition of PAKs decreased lipid accumulation in both mouse and human adipocyte cultures, lowered expression of adipogenic markers, and induced IR, suggesting a previously unidentified role of PAKs in adipocyte function and differentiation. We conclude that PAKs play a crucial role in regulating the FGF1/PDE4D antilipolytic pathway, adipogenesis and IR, thereby highlighting their potential as therapeutic targets for T2D.

Synaptic plasticity, a crucial determinant of learning and memory consolidation, is determined by dynamic modulation of synaptic transmission efficiency. Previous studies highlighted the involvement of 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase I (cGKI) in synaptic plasticity and cognitive functions; its postsynaptic role, however, remains insufficiently characterized and requires further elucidation. We explore cGKI's contribution to activity-dependent hippocampal synaptic transmission and hippocampus-dependent memory formation using CA1 pyramidal neuron-specific knockout mice (CA1-cGKI-KO) and litter-matched controls (CA1-cGKI-CTRL). Hippocampal CA1-specific cGKI depletion was confirmed by Western blot and immunofluorescence. In accordance with a significant function of cGKI in spatial memory formation, CA1-cGKI-KO were incapable to develop goal-directed target localization strategies in the Morris Water Maze coupled with deficits in memory acquisition. These learning impairments were consistent with significantly hampered electrically-induced CA3 to CA1 Schaffer-collateral long-term potentiation (LTP) in CA1-cGKI-KO brain slices. This finding was completed by reduced phosphorylation of the AMPAR subunit GluA1 at S845 in slices from CA1-cGKI-cKO compared to CA1-cGKI-CTRL after chemically induced LTP (cLTP). Additional application of the cGMP-elevating pharmacological agents cinaciguat and vardenafil during cLTP augmented GluA1 S845 phosphorylation exclusively in CA1-cGKI-CTRL, but not CA1-cGKI-cKO. Furthermore, cLTP increased the neuronal Ca 2+-oscillation frequency recorded using FURA-2AM in primary hippocampal neurons. This effect was amplified by cinaciguat, vardenafil and 8-Br-cGMP. Based on these findings, we suggest that cGKI regulates hippocampal synaptic plasticity by controlling oscillatory Ca 2+ influx during LTP. This suggests cGMP/cGKI has the potential to serve as a pharmacological target for the treatment of cognitive impairment.

Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20% O2) or physioxic (5% O2) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6% vs. 32% mosaicism),higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition.