Pathological cardiac remodeling involves cell-type-specific regulatory changes that require integrative analysis across all cardiac cell populations. Here, we present a protocol for isolating single nuclei from fresh-frozen murine cardiac ventricular tissue to enable the integrated analysis of gene expression and chromatin accessibility. We describe steps for mechanical homogenization, sequential filtration, sucrose cushion purification, and fluorescence-activated nuclei sorting (FANS). This protocol enables multiomic analysis across various cardiac cell types and supports epigenomic studies of gene regulation.

Cyclic guanosine 3',5'-monophosphate (cGMP) signalling in cardiomyocytes is intricately organized within cellular compartments, influencing several physiological and pathophysiological processes in the heart. We review how compartmented cGMP signalling is regulated and can be monitored within cardiomyocytes, emphasizing recent advances and methodologies. Three different guanylyl cyclases (GCs), two particulate (GC-A and GC-B) and one soluble (sGC), produce distinct cGMP pools, giving rise to differential signalling in cardiomyocytes. Phosphodiesterases (PDEs) maintain intracellular cGMP gradients. The use of intracellular biosensors, particularly those based on fluorescence resonance energy transfer (FRET), has enabled visualization of cGMP dynamics with high spatial and temporal resolution. These tools, which are constantly being improved, allow the observation of localized cGMP signals and have revealed discrete signalling compartments regulated by the subcellular localization of GCs and PDEs. Understanding these complex signalling networks in cardiomyocytes of healthy and diseased hearts can guide potential therapeutic approaches to treat cardiac dysfunction, including hypertrophy and heart failure. In conclusion, new tools are refining our understanding of compartmented cGMP signalling, which may lead to novel strategies for targeted therapeutic interventions in cardiovascular diseases.

Millions of people are affected by atrial fibrillation (AF) and heart failure with preserved ejection fraction (HFpEF), two disorders frequently found simultaneously. However, the interrelationship between these intertwined disorders is poorly understood, partly owing to the lack of preclinical models. We aimed to evaluate whether a recently developed mouse model of HFpEF could also be used as a model of AF and, potentially, to study the co-occurrence and interrelationship between the two conditions. Mice were fed a dietary regimen of high-fat diet and Nω-nitro-l-arginine methyl ester in the drinking water to induce HFpEF. Twenty-four-hour ECG recordings acquired by telemetry were analysed for autonomic imbalance. After 24 h ECG recording, mice received isoprenaline, and a further 1 h of recording was assessed for chronotropic incompetence, susceptibility to atrial arrhythmia and conduction impairment. Evaluation of diastolic function was achieved by transcarotid catheterization and histological analysis performed on the hearts. Resting heart rate was significantly increased after 3 weeks of the dietary regimen, with a trend observed as early as 1 week. Premature atrial contractions, sinus pauses and atrioventricular blocks occurred significantly after 3 weeks of the dietary regimen. Significant diastolic dysfunction, chronotropic incompetence and higher occurrence of AF after isoprenaline stimulation were observed in the HFpEF group at 6 weeks of the dietary regimen. Our study revealed that sinoatrial node and atrial dysfunction precede the simultaneous occurrence of AF, diastolic dysfunction and chronotropic incompetence. This mouse HFpEF model might be helpful for studying the interdependence between AF and HFpEF.

The natriuretic peptides ANP, BNP and CNP activate transmembrane guanylyl cyclases (GC) that producecyclic GMP (cGMP). We have previously employed targeted FRET-based biosensors to demonstrate that thenatriuretic peptides have differential effects in cardiomyocytes and intact heart, where CNP activates GC-B thatincreases cGMP near troponin I and phospholamban (PLB), enhancing relaxation, while activation of GC-A withANP/BNP modestly increases cGMP only near PLB and do not enhance relaxation, suggesting spatiallyrestricted cGMP signaling from GC-A and GC-B. Using a biosensor targeted to the outer mitochondrialmembrane (OMM), we have found that GC-A and GC-B increase cGMP at the OMM and reduce cardiomyocyteapoptosis. To understand this spatial cGMP signaling from GC-A and GC-B, we combined targeted variants ofthe cGMP scavenger SponGee with our targeted FRET-based cGMP biosensors in cardiac H9c2 cells.Activation of GC-B increased cytosolic cGMP, which was modestly reduced by the lipid raft (Lyn-SponGee),non-raft (SponGee-Kras) or OMM-targeted (OMM-SponGee) cGMP scavengers compared to the untargetedSponGee. At the OMM, cGMP increase from GC-B was reduced in content and kinetics only by the OMMSponGee,while cGMP increase from GC-A was reduced by SponGee-Kras and the untargeted SponGee.Our results indicate that GC-A and GC-B are differentially organized on the plasma membrane and that cGMPreaching the OMM could have different origin. Using cGMP scavengers can therefore be used to deciphersignaling from different areas of the plasma membrane to various subcellular locations in cardiac cells.

In 2023, it was 50 years since the first statin was isolated from fungi and its structure determined. This finding is a fascinating parallel story to the discovery of penicillin.

Natriuretic peptides are important regulators in the cardiovascular and renal system with pleiotropic effects. Atrial (ANP) and brain natriuretic peptide (BNP) activate the natriuretic peptide receptor A (NPR-A), causing production of cyclic guanosine monophosphate (cGMP). Various designer natriuretic peptides have been developed and investigated for treatment of heart failure and hypertension, but their major limitations are that they have short half-life and are not available for oral administration. Our novel approach is to find small molecular drugs to help and treat patients with cardiovascular diseases (CVDs), through targeting NPR-A. We have identified small molecular allosteric enhancers of NPR-A, 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, a pre-study in an animal model was performed to test the formulation and assure the lead compound is well tolerated by the animals.

Natriuretic peptides (NPs) increase cGMP, show beneficial cardiovascular effects and regulate energy metabolism in other tissues. However, little is known about their direct effect on cardiac mitochondria and cardiomyocyte apoptosis. Here, we examined whether NPs increase cGMP around mitochondria and alter apoptosis in cardiomyocytes. We constructed a novel FRET-based biosensor with high selectivity towards cGMP and found that ANP and CNP increase cGMP at the outer mitochondrial membrane. Moreover, ANP and CNP increased phosphorylation of the pro-apoptotic protein Drp1 and CNP prevented fragmentation of mitochondria. Stimulating cardiomyocytes with ANP or CNP reduced apoptosis, caspase 9 activation and cytochrome c release, suggesting that NPs decrease apoptosis through the intrinsic pathway that involves mitochondria. We suggest that cGMP increase in the outer mitochondrial membrane microdomain that inhibits the pro-apoptotic protein Drp1, leading to reduced mitochondrial fragmentation and thereby reduced apoptosis.

Despite the proven effects of statins in preventing cardiovascular disease, their diabetogenic effect has caused concern. The mechanism of this diabetogenic effect is unknown. We suggest a novel mechanism that may contribute to the diabetogenic effect of statins, through an effect of statins that has apparently escaped previous consideration. Briefly, by inhibiting HMG-CoA reductase, statins may cause accumulation of acetate, which through FFA2 and FFA3 stimulation may inhibit insulin secretion.

Background and Purpose: Guanylyl cyclase-A (GC-A), activated by endogenous atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), plays an important role in the regulation of cardiovascular and renal homeostasis and is an attractive drug target. Even though small molecule modulators allow oral administration and longer half-life, drug targeting of GC-A has so far been limited to peptides. Thus, in this study we aimed to develop small molecular activators of GC-A.Experimental Approach: Hits were identified through high-throughput screening and optimized by in silico design. Cyclic GMP was measured in QBIHEK293A cells expressing GC-A, GC-B or chimerae of the two receptors using AlphaScreen technology. Binding assays were performed in membrane preparations or whole cells using I-125-ANP. Vasorelaxation was measured in aortic rings isolated from Wistar rats.Key Results: We have identified small molecular allosteric enhancers of GC-A, which enhanced ANP or BNP effects in cellular systems and ANP-induced vasorelaxation in rat aortic rings. The mechanism of action appears novel and not mediated through previously described allosteric binding sites. In addition, the selectivity and activity depend on a single amino acid residue that differs between the two similar receptors GC-A and GC-B.Conclusion and Implications: We describe a novel allosteric binding site on GC-A, which can be targeted by small molecules to enhance ANP and BNP effects. These compounds will be valuable tools in further development and proof-of-concept of GC-A enhancement for the potential use in cardiovascular therapy.

ABSTRACT Natriuretic peptide receptor (NPR)-A (also known as NPR-A, NPR1 or guanylyl cyclase-A, GC-A) is an attractive but challenging target to activate with small molecules. GC-A is activated by endogenous atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), and this activation leads to the production of 3’,5’-cyclic guanosine monophosphate (cGMP). This system plays an important role in the regulation of cardiovascular and renal homeostasis. However, utilization of this receptor as a drug target has so far been limited to peptides, even though small molecule modulators allow oral administration and longer half-life. We have identified small molecular allosteric enhancers of GC-A, which strengthened ANP or BNP activation in various in vitro and ex vivo systems. These compounds do not mediate their actions through previously described allosteric binding sites or via known mechanisms of action. In addition, their selectivity and activity are dependent on only one amino acid in GC-A. Our findings show that there is a novel allosteric binding site on GC-A, which can be targeted by small molecules that increase the signaling effects of ANP and BNP.

Abstract 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.

Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through β-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure.