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Abstract Background cAMP is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular Ca2+ levels. While imaging of calcium is commonly used in neuroscience applications, probing for cAMP levels has not yet been performed in living vertebrate neuronal tissue before. Results Using a strictly neuron-restricted promoter we virally transduced neurons in the organotypic brainstem slices which contained pre-Bötzinger complex, constituting the rhythm-generating part of the respiratory network. Fluorescent cAMP sensor Epac1-camps was expressed both in neuronal cell bodies and neurites, allowing us to measure intracellular distribution of cAMP, its absolute levels and time-dependent changes in response to physiological stimuli. We recorded [cAMP]i changes in the micromolar range after modulation of adenylate cyclase, inhibition of phosphodiesterase and activation of G-protein-coupled metabotropic receptors. [cAMP]i levels increased after membrane depolarisation and release of Ca2+ from internal stores. The effects developed slowly and reached their maximum after transient [Ca2+]i elevations subsided. Ca2+-dependent [cAMP]i transients were suppressed after blockade of adenylate cyclase with 0.1 mM adenylate cyclase inhibitor 2'5'-dideoxyadenosine and potentiated after inhibiting phosphodiesterase with isobutylmethylxanthine and rolipram. During paired stimulations, the second depolarisation and Ca2+ release evoked bigger cAMP responses. These effects were abolished after inhibition of protein kinase A with H-89 pointing to the important role of phosphorylation of calcium channels in the potentiation of [cAMP]i transients. Conclusion We constructed and characterized a neuron-specific cAMP probe based on Epac1-camps. Using viral gene transfer we showed its efficient expression in organotypic brainstem preparations. Strong fluorescence, resistance to photobleaching and possibility of direct estimation of [cAMP] levels using dual wavelength measurements make the probe useful in studies of neurons and the mechanisms of their plasticity. Epac1-camps was applied to examine the crosstalk between Ca2+ and cAMP signalling and revealed a synergism of actions of these two second messengers.

Sulfonylureas (SUs) are a class of antidiabetic drugs widely used in the management of diabetes mellitus type 2. They promote insulin secretion by inhibiting the ATP-sensitive potassium channel in pancreatic β-cells. Recently, the exchange protein directly activated by cAMP (Epac) was identified as a new class of target proteins of SUs that might contribute to their antidiabetic effect, through the activation of the Ras-like guanosine triphosphatase Rap1, which has been controversially discussed. We used human embryonic kidney (HEK) 293 cells expressing genetic constructs of various Förster resonance energy transfer (FRET)-based biosensors containing different versions of Epac1 and Epac2 isoforms, alone or fused to different phosphodiesterases (PDEs), to monitor SU-induced conformational changes in Epac or direct PDE inhibition in real time. We show that SUs can both induce conformational changes in the Epac2 protein but not in Epac1, and directly inhibit the PDE3 and PDE4 families, thereby increasing cAMP levels in the direct vicinity of these PDEs. Furthermore, we demonstrate that the binding site of SUs in Epac2 is distinct from that of cAMP and is located between the amino acids E443 and E460. Using biochemical assays, we could also show that tolbutamide can inhibit PDE activity through an allosteric mechanism. Therefore, the cAMP-elevating capacity due to allosteric PDE inhibition in addition to direct Epac activation may contribute to the therapeutic effects of SU drugs.

Aims: Na+-activated Slack potassium (K+) channels are increasingly recognized as regulators of neuronal activity, yet little is known about their role in the cardiovascular system. Slack activity increases when intracellular Na+ concentration ([Na+]i) reaches pathophysiological levels. Elevated [Na+]i is a major determinant of the ischemia and reperfusion (I/R)-induced myocardial injury, thus we hypothesized that Slack plays a role under these conditions., Methods: and results: K+ currents in cardiomyocytes (CMs) obtained from wildtype (WT) but not from global Slack knockout (KO) mice were sensitive to electrical inactivation of voltage-sensitive Na+-channels. Live-cell imaging demonstrated that K+ fluxes across the sarcolemma rely on Slack, while the depolarized resting membrane potential in Slack-deficient CMs led to excessive cytosolic Ca2+ accumulation and finally to hypoxia/reoxygenation-induced cell death. Cardiac damage in an in vivo model of I/R was exacerbated in global and CM-specific conditional Slack mutants and largely insensitive to mechanical conditioning maneuvers. Finally, the protection conferred by mitochondrial ATP-dependent K+ channels required functional Slack in CMs., Conclusions: Collectively, our study provides evidence for Slack's crucial involvement in the ion homeostasis of no or low O2-stressed CMs. Thereby, Slack activity opposes the I/R-induced fatal Ca2+-uptake to CMs supporting the cardioprotective signaling widely attributed to mitoKATP function., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Aims: Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains., Methods and Results: HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia., Conclusion: Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains., Competing Interests: Conflict of interest: A.G. and E.H. are cofounders and shareholders of Kither Biotech, a pharmaceutical company developing PI3K inhibitors for respiratory diseases, not in conflict with the content of this manuscript., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

White and brown adipocytes are central mediators of lipid metabolism and thermogenesis, respectively. Their function is tightly regulated by all three β-adrenergic receptor (β-AR) subtypes which are coupled to the production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). While known for decades in other cell types, compartmentation of adipocyte β-AR/cAMP signaling by spatial organization of the pathway and by cAMP degrading phosphodiesterases (PDEs) as well as its role in the regulation of lipolysis is only beginning to emerge. Here, we provide a short overview of recent findings which shed light on compartmentalized signaling using live cell imaging of cAMP in adipocytes and discuss possible future directions of research which could open up new avenues for the treatment of metabolic disorders., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Flavonoid aglycones are secondary plant metabolites that exhibit a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and antiplatelet effects. However, the precise molecular mechanisms underlying their inhibitory effect on platelet activation remain poorly understood. In this study, we applied flow cytometry to analyze the effects of six flavonoid aglycones (luteolin, myricetin, quercetin, eriodictyol, kaempferol, and apigenin) on platelet activation, phosphatidylserine externalization, formation of reactive oxygen species, and intracellular esterase activity. We found that these compounds significantly inhibit thrombin-induced platelet activation and decrease formation of reactive oxygen species in activated platelets. The tested aglycones did not affect platelet viability, apoptosis induction, or procoagulant platelet formation. Notably, luteolin, myricetin, quercetin, and apigenin increased thrombin-induced thromboxane synthase activity, which was analyzed by a spectrofluorimetric method. Our results obtained from Western blot analysis and liquid chromatography-tandem mass spectrometry demonstrated that the antiplatelet properties of the studied phytochemicals are mediated by activation of cyclic nucleotide-dependent signaling pathways. Specifically, we established by using Förster resonance energy transfer that the molecular mechanisms are, at least partly, associated with the inhibition of phosphodiesterases 2 and/or 5. These findings underscore the therapeutic potential of flavonoid aglycones for clinical application as antiplatelet agents., Competing Interests: The authors declare no conflicts of interest.

Aims: Despite massive efforts, we remain far behind in our attempts to identify effective therapies to treat heart failure with preserved ejection fraction (HFpEF). Diastolic function is critically regulated by sarcoplasmic/endoplasmic reticulum (SR) calcium ATPase 2a (SERCA2a), which forms a functional cardiomyocyte (CM) microdomain where 3',5'-cyclic adenosine monophosphate (cAMP) produced upon β-adrenergic receptor (β-AR) stimulation leads to phospholamban (PLN) phosphorylation and facilitated Ca2+ re-uptake., Methods and Results: To visualize real-time cAMP dynamics in the direct vicinity of SERCA2a in healthy and diseased myocytes, we generated a novel mouse model on the leprdb background that stably expresses the Epac1-PLN Förster resonance energy transfer biosensor. Mice homozygous for the leprdb mutation (db/db) developed obesity and type 2 diabetes and presented with a HFpEF phenotype, evident by mild left ventricular hypertrophy and elevated left atria filling pressures. Live cell imaging uncovered a substantial β2-AR subtype stimulated cAMP response within the PLN/SERCA2a microdomain of db/db but not healthy control (db/+) CMs, which was accompanied by increased PLN phosphorylation and accelerated calcium re-uptake. Importantly, db/db CMs also exhibited a desensitization of β1-AR stimulated cAMP pools within the PLN/SERCA2a microdomain, which was accompanied by a blunted lusitropic effect, suggesting that the increased β2-AR control is an intrinsic compensatory mechanism to maintain PLN/SERCA2a-mediated calcium dynamics and cardiac relaxation. Mechanistically, this was due to a local loss of cAMP-degrading phosphodiesterase 4 associated specifically with the PLN/SERCA2a complex., Conclusion: These newly identified alterations of cAMP dynamics at the subcellular level in HFpEF should provide mechanistic understanding of microdomain remodelling and pave the way towards new therapies., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

The ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling (ECC) by signaling in discrete subcellular microdomains. Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations of PDE4 activity in human hearts has been shown to result in arrhythmias and heart failure. Here, we sought to systematically investigate specific roles of PDE4B and PDE4D in the regulation of cAMP dynamics in three distinct subcellular microdomains, one of them located at the caveolin-rich plasma membrane which harbors the L-type calcium channels (LTCCs), as well as at two sarco/endoplasmic reticulum (SR) microdomains centered around SR Ca 2+ -ATPase (SERCA2a) and cardiac ryanodine receptor type 2 (RyR2). Transgenic mice expressing Förster Resonance Energy Transfer (FRET)-based cAMP-specific biosensors targeted to caveolin-rich plasma membrane, SERCA2a and RyR2 microdomains were crossed to PDE4B-KO and PDE4D-KO mice. Direct analysis of the specific effects of both PDE4 subfamilies on local cAMP dynamics was performed using FRET imaging. Our data demonstrate that all three microdomains are differentially regulated by these PDE4 subfamilies. Whereas both are involved in cAMP regulation at the caveolin-rich plasma membrane, there are clearly two distinct cAMP microdomains at the SR formed around RyR2 and SERCA2a, which are preferentially controlled by PDE4B and PDE4D, respectively. This correlates with local cAMP-dependent protein kinase (PKA) substrate phosphorylation and arrhythmia susceptibility. Immunoprecipitation assays confirmed that PDE4B is associated with RyR2 along with PDE4D. Stimulated Emission Depletion (STED) microscopy of immunostained cardiomyocytes suggested possible co-localization of PDE4B with both sarcolemmal and RyR2 microdomains. In conclusion, our functional approach could show that both PDE4B and PDE4D can differentially regulate cardiac cAMP microdomains associated with calcium homeostasis. PDE4B controls cAMP dynamics in both caveolin-rich plasma membrane and RyR2 vicinity. Interestingly, PDE4B is the major regulator of the RyR2 microdomain, as opposed to SERCA2a vicinity, which is predominantly under PDE4D control, suggesting a more complex regulatory pattern than previously thought, with multiple PDEs acting at the same location.

T-cell activation is a pivotal process of the adaptive immune response with 3',5'-cyclic adenosine monophosphate (cAMP) as a key regulator of T-cell activation and function. It governs crucial control over T-cell differentiation and production of pro-inflammatory cytokines, such as IFN-γ. Intriguingly, levels of intracellular cAMP differ between regulatory (Treg) and conventional T-cells (Tcon). During cell-cell contact, cAMP is transferred via gap junctions between these T-cell subsets to mediate the immunosuppressive function of Treg. Moreover, the activation of T-cells via CD3 and CD28 co-stimulation leads to a transient upregulation of cAMP. Elevated intracellular cAMP levels are balanced precisely by phosphodiesterases (PDEs), a family of enzymes that hydrolyze cyclic nucleotides. Various PDEs play distinct roles in regulating cAMP and cyclic guanosine monophosphate (cGMP) in T-cells. Research on PDEs has gained growing interest due to their therapeutic potential to manipulate T-cell responses. So far, PDE4 is the best-described PDE in T-cells and the first PDE that is currently targeted in clinical practice to treat autoimmune diseases. But also, other PDE families harbor additional therapeutic potential. PDE2A is a dual-substrate phosphodiesterase which is selectively upregulated in Tcon upon activation. In this Mini-Review, we will highlight the impact of cAMP regulation on T-cell activation and function and summarize recent findings on different PDEs regulating intracellular cAMP levels in T-cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bielenberg, Kurelic, Frantz and Nikolaev.)

The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aims: Cardiomyopathies (CMPs) are a heterogeneous group of diseases that are defined by structural and functional abnormalities of the cardiac muscle. Dilated cardiomyopathy (DCM), the most common CMP, is defined by left ventricular dilation and impaired contractility and represents a common cause of heart failure. Different phenotypes result from various underlying genetic and acquired causes with variable effects on disease development and progression, prognosis, and response to medical treatment. Current treatment algorithms do not consider these different aetiologies, due to lack of insights into treatable drivers of cardiac failure in patients with DCM. Our study aims to precisely phenotype and genotype the various subtypes of DCM and hereby lay the foundation for individualized therapy., Methods and Results: The Geno- And Phenotyping of PrImary Cardiomyopathy (GrAPHIC) is a currently ongoing prospective observational monocentric cohort study that recruits patients with DCM after exclusion of other causes such as coronary artery disease, valvular dysfunction, myocarditis, exposure to toxins, and peripartum CMP. Patients are enrolled at our heart failure outpatient clinic or during hospitalization at the University Hospital Hamburg. Clinical parameters, multimodal imaging and functional assessment, cardiac biopsies, and blood samples are obtained to enable an integrated genomic, functional, and biomarker analysis., Conclusions: The GrAPHIC will contribute to a better understanding of the heterogeneous nature of primary CMPs focusing on DCM and provide improved prognostic approaches and more individualized therapies., (© 2023 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

In mouse cardiomyocytes, the expression of two subfamilies of the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase 1 (PDE1)-PDE1A and PDE1C-has been reported. PDE1C was found to be the major subfamily in the human heart. It is a dual substrate PDE and can hydrolyze both 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Previously, it has been reported that the PDE1 inhibitor ITI-214 shows positive inotropic effects in heart failure patients which were largely attributed to the cAMP-dependent protein kinase (PKA) signaling. However, the role of PDE1 in the regulation of cardiac cGMP has not been directly addressed. Here, we studied the effect of PDE1 inhibition on cGMP levels in adult mouse ventricular cardiomyocytes using a highly sensitive fluorescent biosensor based on Förster resonance energy transfer (FRET). Live-cell imaging in paced and resting cardiomyocytes showed an increase in cGMP after PDE1 inhibition with ITI-214. Furthermore, PDE1 inhibition and PDE1A knockdown amplified the cGMP-FRET responses to the nitric oxide (NO)-donor sodium nitroprusside (SNP) but not to the C-type natriuretic peptide (CNP), indicating a specific role of PDE1 in the regulation of the NO-sensitive guanylyl cyclase (NO-GC)-regulated cGMP microdomain. ITI-214, in combination with CNP or SNP, showed a positive lusitropic effect, improving the relaxation of isolated myocytes. Immunoblot analysis revealed increased phospholamban (PLN) phosphorylation at Ser-16 in cells treated with a combination of SNP and PDE1 inhibitor but not with SNP alone. Our findings reveal a previously unreported role of PDE1 in the regulation of the NO-GC/cGMP microdomain and mouse ventricular myocyte contractility. Since PDE1 serves as a cGMP degrading PDE in cardiomyocytes and has the highest hydrolytic activities, it can be expected that PDE1 inhibition might be beneficial in combination with cGMP-elevating drugs for the treatment of cardiac diseases.

Butyrophilin (BTN)-3A and BTN2A1 molecules control the activation of human Vγ9Vδ2 T cells during T cell receptor (TCR)-mediated sensing of phosphoantigens (PAg) derived from microbes and tumors. However, the molecular rules governing PAg sensing remain largely unknown. Here, we establish three mechanistic principles of PAg-mediated γδ T cell activation. First, in humans, following PAg binding to the intracellular BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the extracellular V-domain of BTN3A2/BTN3A3. Moreover, the localization of both protein domains on different chains of the BTN3A homo-or heteromers is essential for efficient PAg-mediated activation. Second, the formation of BTN3A homo-or heteromers, which differ in intracellular trafficking and conformation, is controlled by molecular interactions between the juxtamembrane regions of the BTN3A chains. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and the division of labor in BTN proteins improves our understanding of PAg sensing and elucidates a mode of action that may apply to other BTN family members., (© 2023. The Author(s).)

Background: β 1 AR (beta-1 adrenergic receptor) and β 2 AR (beta-2 adrenergic receptor)-mediated cyclic adenosine monophosphate signaling has distinct effects on cardiac function and heart failure progression. However, the mechanism regulating spatial localization and functional compartmentation of cardiac β-ARs remains elusive. Emerging evidence suggests that microtubule-dependent trafficking of mRNP (messenger ribonucleoprotein) and localized protein translation modulates protein compartmentation in cardiomyocytes. We hypothesized that β-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation., Methods: The localization pattern of β-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on β-AR mRNA localization was studied using vinblastine, and its effect on receptor localization and function was evaluated with immunofluorescent and high-throughput Förster resonance energy transfer microscopy. An mRNA protein co-detection assay identified plausible β-AR translation sites in cardiomyocytes. The mechanism by which β-AR mRNA is redistributed post-heart failure was elucidated by single molecule fluorescence in situ hybridization, nanobiopsy, and high-throughput Förster resonance energy transfer microscopy on 16 weeks post-myocardial infarction and detubulated cardiomyocytes., Results: β 1 AR and β 2 AR mRNAs show differential localization in cardiomyocytes, with β 1 AR found in the perinuclear region and β 2 AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of β 2 AR transcripts toward the perinuclear region. The close proximity between β 2 AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of β 2 AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both β 1 AR and β 2 AR mRNAs are redistributed toward the cell periphery, similar to what is seen in cardiomyocytes undergoing drug-induced detubulation. This suggests that t-tubule remodeling contributes to β-AR mRNA redistribution and impaired β 2 AR function in failing hearts., Conclusions: Asymmetrical microtubule-dependent trafficking dictates differential β 1 AR and β 2 AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 β-ARs on the plasma membrane. The localization pattern is altered post-myocardial infarction, resulting from transverse tubule remodeling, leading to distorted β 2 AR-mediated cyclic adenosine monophosphate signaling., Competing Interests: Disclosures None.

The renin-angiotensin-aldosterone system (RAAS) is one of the key players in the regulation of blood volume and blood pressure. Dysfunction of this system is connected with cardiovascular and renal diseases. Regulation of RAAS is under the control of multiple intracellular mechanisms. Cyclic nucleotides and phosphodiesterases are the major regulators of this system since they control expression and activity of renin and aldosterone. In this review, we summarize known mechanisms by which cyclic nucleotides and phosphodiesterases regulate renin gene expression, secretion of renin granules from juxtaglomerular cells and aldosterone production from zona glomerulosa cells of adrenal gland. We also discuss several open questions which deserve future attention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gambaryan, Mohagaonkar and Nikolaev.)

Aims: Atrial fibrillation (AF) is associated with altered cAMP/PKA signaling and an AF-promoting reduction of L-type Ca2+-current (ICa,L), the mechanisms of which are poorly understood. Cyclic-nucleotide phosphodiesterases (PDEs) degrade cAMP and regulate PKA-dependent phosphorylation of key calcium-handling proteins, including the ICa,L-carrying Cav1.2α1C subunit. The aim was to assess whether altered function of PDE type-8 (PDE8) isoforms contributes to the reduction of ICa,L in persistent (chronic) AF (cAF) patients., Methods and Results: mRNA, protein levels, and localization of PDE8A and PDE8B isoforms were measured by RT-qPCR, western blot, co-immunoprecipitation and immunofluorescence. PDE8 function was assessed by FRET, patch-clamp and sharp-electrode recordings. PDE8A gene and protein levels were higher in paroxysmal AF (pAF) vs. sinus rhythm (SR) patients, whereas PDE8B was upregulated in cAF only. Cytosolic abundance of PDE8A was higher in atrial pAF myocytes, whereas PDE8B tended to be more abundant at the plasmalemma in cAF myocytes. In co-immunoprecipitation, only PDE8B2 showed binding to Cav1.2α1C subunit which was strongly increased in cAF. Accordingly, Cav1.2α1C showed a lower phosphorylation at Ser1928 in association with decreased ICa,L in cAF. Selective PDE8 inhibition increased Ser1928 phosphorylation of Cav1.2α1C, enhanced cAMP at the subsarcolemma and rescued the lower ICa,L in cAF, which was accompanied by a prolongation of action potential duration at 50% of repolarization., Conclusion: Both PDE8A and PDE8B are expressed in human heart. Upregulation of PDE8B isoforms in cAF reduces ICa,L via direct interaction of PDE8B2 with the Cav1.2α1C subunit. Thus, upregulated PDE8B2 might serve as a novel molecular mechanism of the proarrhythmic reduction of ICa,L in cAF., Competing Interests: Conflict of interest The authors have no competing interests. R.B.S. reports grants from European Union Horizon 2020, the German Ministry of Research and Education, ERACOSysMed3 and personal fees from BMS/Pfizer, outside this work., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes.

Lipolysis of stored triglycerides is stimulated via β-adrenergic receptor (β-AR)/3',5'-cyclic adenosine monophosphate (cAMP) signaling and inhibited via phosphodiesterases (PDEs). In type 2 diabetes, a dysregulation in the storage/lipolysis of triglycerides leads to lipotoxicity. Here, we hypothesize that white adipocytes regulate their lipolytic responses via the formation of subcellular cAMP microdomains. To test this, we investigate real-time cAMP/PDE dynamics at the single-cell level in human white adipocytes with a highly sensitive florescent biosensor and uncover the presence of several receptor-associated cAMP microdomains where cAMP signals are compartmentalized to differentially regulate lipolysis. In insulin resistance, we also detect cAMP microdomain dysregulation mechanisms that promote lipotoxicity, but regulation can be restored by the anti-diabetic drug metformin. Therefore, we present a powerful live-cell imaging technique capable of resolving disease-driven alterations in cAMP/PDE signaling at the subcellular level and provide evidence to support the therapeutic potential of targeting these microdomains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The rate of calcium cycling and calcium transient amplitude are critical determinants for the efficient contraction and relaxation of the heart. Calcium-handling proteins in the cardiac myocyte are altered in heart failure, and restoring the proper function of those proteins is an effective potential therapeutic strategy. The calcium-handling proteins or their regulators are phosphorylated by a cAMP-dependent kinase (PKA), and thereby their activity is regulated. A-Kinase Anchoring Proteins (AKAPs) play a seminal role in orchestrating PKA and cAMP regulators in calcium handling and contractile machinery. This cAMP/PKA orchestration is crucial for the increased force and rate of contraction and relaxation of the heart in response to fight-or-flight. Knockout models and the few available preclinical models proved that the efficient targeting of AKAPs offers potential therapies tailor-made for improving defective calcium cycling. In this review, we highlight important studies that identified AKAPs and their regulatory roles in cardiac myocyte calcium cycling in health and disease., Competing Interests: The authors declare no conflicts of interest.

G-protein coupled receptors (GPCR) regulate 3',5'-cyclic adenosine monophosphate (cAMP) levels in T cells. cAMP as ubiquitous second messenger is crucial for adequate physiology of T cells by mediating effector T cell (Teff) function as well as regulatory T cell (Treg)-mediated immunosuppression. Several GPCRs have been identified to be crucial for Teff and Treg function. However, the role of the orphan, constitutively active Gs-coupled GPCR GPR52 is unknown. Here we show that GPR52 regulates cAMP levels in T cells but does not affect T cell function. We found that stimulation of transfected HEK cells or primary T cells with a GPR52 agonist results in a rise of intracellular cAMP. However, neither Gpr52 deficiency nor pharmacological modulation of GPR52 by antagonists or agonists affected T cell activation, differentiation, and proliferation or Treg-mediated immunosuppression. Moreover, Gpr52 deletion did not modify the clinical disease course of experimental autoimmune encephalomyelitis (EAE). Our results demonstrate that a modulation of cAMP levels in T cells does not inevitably result in altered T cell function. While we could not identify an obvious role of GPR52 in in vitro T cell assays and in vivo CNS autoimmunity, it might regulate T cell function in a different context or affect the function of other GPR52-expressing cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Krieg, Sonner, Kurelic, Engler, Scharenberg, Bauer, Nikolaev and Friese.)

Background: Aldosterone is a critical pathological driver for cardiac and renal diseases. We recently discovered that mutant atrial natriuretic peptide (MANP), a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP in vivo. MANP and natriuretic peptide (NP)-augmenting therapy sacubitril/valsartan are under investigations for human hypertension treatment. Understanding the elusive mechanism of aldosterone inhibition by NPs remains to be a priority. Conflicting results were reported on the roles of the pGC-A (particulate guanylyl cyclase A receptor) and NP clearance receptor in aldosterone inhibition. Furthermore, the function of PKG (protein kinase G) and PDEs (phosphodiesterases) on aldosterone regulation are not clear., Methods: In the present study, we investigated the molecular mechanism of aldosterone regulation in a human adrenocortical cell line H295R and in mice., Results: We first provided evidence to show that pGC-A, not NP clearance receptor, mediates aldosterone inhibition. Next, we confirmed that MANP inhibits aldosterone via PDE2 (phosphodiesterase 2) not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer experiments. Further, the inhibitory effect is mediated by a reduction of intracellular Ca2+ levels. We then illustrated that MANP directly reduces aldosterone synthase CYP11B2 (cytochrome p450 family 11 subfamily b member 2) expression via PDE2. Last, in PDE2 knockout mice, consistent with in vitro findings, embryonic adrenal CYP11B2 is markedly increased., Conclusions: Our results innovatively explore and expand the NP/pGC-A/3',5', cyclic guanosine monophosphate (cGMP)/PDE2 pathway for aldosterone inhibition by MANP in vitro and in vivo. In addition, our data also support the development of MANP as a novel ANP analog drug for aldosterone excess treatment.

Obesity and type 2 diabetes (T2D) are on trend to become a huge burden across all ages. They cause harm to almost every organ, especially the heart. For decades, the incidence of heart failure with impaired diastolic function (or called heart failure with preserved ejection fraction, HFpEF) has increased sharply. More and more studies have uncovered obesity and T2D to be closely associated with HFpEF. The sarcoplasmic/endoplasmic reticulum calcium ATPase2a (SERCA2a) microdomain is a key regulator of calcium reuptake into the sarcoplasmic reticulum (SR) during diastole. 3',5'-cyclic adenosine monophosphate (cAMP) and its downstream effector cAMP dependent protein kinase (PKA) act locally within the SERCA2a microdomain to regulate the phosphorylation state of the small regulatory protein phospholamban (PLN), which forms a complex with SERCA2a. When phosphorylated, PLN promotes calcium reuptake into the SR and diastolic cardiac relaxation by disinhibiting SERCA2a pump function. In this review, we will discuss previous studies investigating the PLN/SERCA2a microdomain in obesity and T2D in order to gain a greater understanding of the underlying mechanisms behind obesity- and T2D-induced diastolic dysfunction, with the aim to identify the current state of knowledge and future work that is needed to guide further research in the field.

Aims: Guanylyl cyclase-B (GC-B; natriuretic peptide receptor-B, NPR-B) stimulation by C-type natriuretic peptide (CNP) increases cGMP and causes a lusitropic and negative inotropic response in adult myocardium. These effects are not mimicked by NPR-A (GC-A) stimulation by brain natriuretic peptide (BNP), despite similar cGMP increase. More refined methods are needed to better understand the mechanisms of the differential cGMP signalling and compartmentation. The aim of this work was to measure cGMP near proteins involved in regulating contractility to understand compartmentation of cGMP signalling in adult cardiomyocytes., Methods and Results: We constructed several fluorescence resonance energy transfer (FRET)-based biosensors for cGMP subcellularly targeted to phospholamban (PLB) and troponin I (TnI). CNP stimulation of adult rat cardiomyocytes increased cGMP near PLB and TnI, whereas BNP stimulation increased cGMP near PLB, but not TnI. The phosphodiesterases PDE2 and PDE3 constrained cGMP in both compartments. Local receptor stimulation aided by scanning ion conductance microscopy (SICM) combined with FRET revealed that CNP stimulation both in the t-tubules and on the cell crest increases cGMP similarly near both TnI and PLB. In ventricular strips, CNP stimulation, but not BNP, induced a lusitropic response, enhanced by inhibition of either PDE2 or PDE3, and a negative inotropic response. In cardiomyocytes from heart failure rats, CNP increased cGMP near PLB and TnI more pronounced than in cells from sham-operated animals., Conclusion: These targeted biosensors demonstrate that CNP, but not BNP, increases cGMP near TnI in addition to PLB, explaining how CNP, but not BNP, is able to induce lusitropic and negative inotropic responses., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca 2+ -induced Ca 2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca 2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following β-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca 2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas β-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca 2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca 2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca 2+ -induced Ca 2+ release, whereas β-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: It remains unclear as to whether polycystic ovary syndrome (PCOS) is an additional risk factor in the development of left ventricular (LV) hypertrophy in obese women. In the current study, we provide clarity on this issue by rigorously analysing patient LV geometry beyond the basic clinical measures currently used. Importantly, the cohort contained only normotensive patients that would normally be deemed low risk with no further intervention required., Methods: The study comprised 24 obese women with PCOS and 29 obese Control women. Transthoracic echocardiography was used to evaluate LV structure/function. Basic clinical and metabolic data were collected for each participant consisting of age, BMI, blood pressure, fasting glucose, LDL-C, HLD-C, cholesterol and triglyceride levels. Exclusion criteria; BMI < 30 g/m2, type 2 diabetes, hypertension., Results: Both groups exhibited concentric remodelling of the LV posterior wall at a prevalence of ~20%, this associated with grade 1 diastolic dysfunction. Estimated LV mass/height2.7 was increased patients with PCOS (45 ± 2.2 vs 37 ± 1.6) with 33% exhibiting LV mass/height2.7 above ASE guidelines, compared to 7% in Controls. Furthermore, 25% of patients with PCOS were characterised with concentric hypertrophy, an alteration in LV geometry that was not observed in the Control group., Conclusions: To our knowledge, this is the first study to assess LV geometric patterns in obese women with PCOS. The results suggest that obese women with PCOS are at greater risk of concentric hypertrophy than obese only women and provide justification for additional cardiovascular risk assessment in normotensive obese/PCOS women., Competing Interests: he authors have declared that no competing interests exist.

Background: Neutrophil extracellular traps (NETs)-as double-edged swords of innate immunity-are involved in numerous processes such as infection, inflammation and tissue repair. Research on neutrophil granulocytes is limited because of their short lifetime of only a few hours. Several attempts have been made to prolong the half-life of neutrophils using cytokines and bacterial products and have shown promising results. These long-term surviving neutrophils are reported to maintain phagocytic activity and cytokine release; however, little is known regarding their capability to release NETs., Methods: We analysed the prolongation of neutrophil survival in vitro under various culture conditions using granulocyte colony-stimulating factor (G-CSF), lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) by flow cytometry and a viability assay. Additionally, we assessed NET formation following stimulation with phorbol 12-myristate 13-acetate (PMA) by immunofluorescence staining, myeloperoxidase (MPO)-DNA sandwich-ELISA and fluorometric assays for cell-free DNA (cfDNA), neutrophil elastase (NE) and myeloperoxidase (MPO)., Results: Untreated neutrophils could form NETs after stimulation with PMA for up to 24 h. Incubation with LPS extended their ability to form NETs for up to 48 h. At 48 h, NET release of neutrophils cultured with LPS was significantly higher compared to that of untreated cells; however, no significantly different enzymatic activity of NE and MPO was observed. Similarly, incubation with G-CSF resulted in significantly higher NET release at 48 h compared to untreated cells. Furthermore, NETs showed significantly higher enzymatic activity of NE and MPO after incubation with G-CSF. Lastly, incubation with TNF-α had no influence on NET release compared to untreated cells although survival counts were altered by TNF-α., Conclusions: G-CSF, LPS or TNF-α each at low concentrations lead to prolonged survival of cultured neutrophils, resulting in considerable differences in NET formation and composition. These results provide new information for the use of neutrophils in long-term experiments for NET formation and provide novel insights for neutrophil behaviour under inflammatory conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kolman, Pagerols Raluy, Müller, Nikolaev, Trochimiuk, Appl, Wadehn, Dücker, Stoll, Boettcher, Reinshagen and Trah.)

Cachexia is associated with poor prognosis in chronic heart failure patients, but the underlying mechanisms of cachexia triggered disease progression remain poorly understood. Here, we investigate whether the dysregulation of myokine expression from wasting skeletal muscle exaggerates heart failure. RNA sequencing from wasting skeletal muscles of mice with heart failure reveals a reduced expression of Ostn, which encodes the secreted myokine Musclin, previously implicated in the enhancement of natriuretic peptide signaling. By generating skeletal muscle specific Ostn knock-out and overexpressing mice, we demonstrate that reduced skeletal muscle Musclin levels exaggerate, while its overexpression in muscle attenuates cardiac dysfunction and myocardial fibrosis during pressure overload. Mechanistically, Musclin enhances the abundance of C-type natriuretic peptide (CNP), thereby promoting cardiomyocyte contractility through protein kinase A and inhibiting fibroblast activation through protein kinase G signaling. Because we also find reduced OSTN expression in skeletal muscle of heart failure patients, augmentation of Musclin might serve as therapeutic strategy., (© 2022. The Author(s).)

Transgenic mice play a significant role in modern biomedical research. In addition to mechanistic studies of a specific gene and protein function, transgenic mice are used as an exciting tool for in vivo or in situ analysis of fluorescent biosensors, which are capable of directly reporting second messenger levels and biochemical processes in real time and living cells. In this chapter, we present a detailed protocol for the generation of plasmid vectors and transgenic mice ubiquitously or constitutively expressing cytosolic and targeted Förster resonance energy transfer (FRET)-based biosensors for the second messengers 3',5'-cyclic adenosine and guanosine monophosphates. These tools and techniques hold great potential for the analysis of second messenger dynamics in physiologically relevant systems., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Heart failure with preserved ejection fraction (HFpEF) will soon take over as the predominant form of heart failure. This is largely driven by the continuing increased incidences of obesity and type 2 diabetes (T2D), which promote HFpEF in the absence of pressure overload stresses. With beta-blockers showing little effectiveness in treating obesity/T2D HFpEF and with no HFpEF-targeted drugs currently available, we are in urgent need of a better understanding of how obesity/T2D HFpEF develops and how we may treat this condition. An exciting emerging field aiming to do this focuses on the investigation of 3',5'-cyclic adenosine monophosphate (cAMP) microdomains in the heart. The previous work has largely focused on the investigation of cAMP microdomain remodelling in heart failure with reduced ejection fraction (HFrEF), with this work uncovering potential new targets for intervention strategies that otherwise would have been overlooked when studying changes in cAMP dynamics at the whole-cell level. In this review, we aimed to discuss current advancements in our understanding of cAMP microdomain remodelling in HFrEF vs that in obesity/T2D-associated HFpEF, with particular focus on the unresolved questions and limitations we face in being able to translate this knowledge., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

3',5'-cyclic guanosine monophosphate (cGMP) is a druggable second messenger regulating cell growth and survival in a plethora of cells and disease states, many of which are associated with hypoxia. For example, in myocardial infarction and heart failure (HF), clinical use of cGMP-elevating drugs improves disease outcomes. Although they protect mice from ischemia/reperfusion (I/R) injury, the exact mechanism how cardiac cGMP signaling is regulated in response to hypoxia is still largely unknown. By monitoring real-time cGMP dynamics in murine and human cardiomyocytes using in vitro and in vivo models of hypoxia/reoxygenation (H/R) and I/R injury combined with biochemical methods, we show that hypoxia causes rapid but partial degradation of cGMP-hydrolyzing phosphodiesterase-3A (PDE3A) protein via the autophagosomal-lysosomal pathway. While increasing cGMP in hypoxia prevents cell death, partially reduced PDE3A does not change the pro-apoptotic second messenger 3',5'-cyclic adenosine monophosphate (cAMP). However, it leads to significantly enhanced protective effects of clinically relevant activators of nitric oxide-sensitive guanylyl cyclase (NO-GC). Collectively, our mouse and human data unravel a new mechanism by which cardiac cGMP improves hypoxia-associated disease conditions., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

3',5'-cyclic adenosine monophosphate (cAMP) is well-known for its diverse immunomodulatory properties, primarily inhibitory effects during T cell activation, proliferation, and production of pro-inflammatory cytokines. A decrease in cAMP levels, due to the hydrolyzing activity of phosphodiesterases (PDE), is favoring inflammatory responses. This can be prevented by selective PDE inhibitors, which makes PDEs important therapeutic targets for autoimmune disorders. In this study, we investigated the specific roles of PDE2A and PDE3B in the regulation of intracellular cAMP levels in different mouse T cell subsets. Unexpectedly, T cell receptor (TCR) activation led to a selective upregulation of PDE2A at the protein level in conventional T cells (Tcon), whereas no changes were detected in regulatory T cells (Treg). In contrast, protein expression of PDE3B was significantly higher in both non-activated and activated Tcon subsets as compared to Treg, with no changes upon TCR engagement. Live-cell imaging of T cells expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, has enabled cAMP measurements in real time and revealed stronger responses to the PDE2A inhibitors in activated vs non-activated Tcon. Importantly, stimulation of intracellular cGMP levels with natriuretic peptides led to an increase of cAMP in non-activated and a decrease of cAMP in activated Tcon, suggesting that TCR activation changes the PDE3B-dependent positive to PDE2A-dependent negative cGMP/cAMP cross-talk. Functionally, this switch induced higher expression of early activation markers CD25 and CD69. This constitutes a potentially interesting feed-forward mechanism during autoimmune and inflammatory responses that may be exploited therapeutically., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MS declared a past co-authorship with one of the authors VON to the handling editor., (Copyright © 2021 Kurelic, Krieg, Sonner, Bhaiyan, Ramos, Frantz, Friese and Nikolaev.)