Kjetil Wessel Andressen

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.

Background and Purpose: Heart failure with preserved ejection fraction (HFpEF) is a significant global health burden, with limited treatment options. In cardiomyocytes, C-type natriuretic peptide (CNP) stimulation of the guanylyl cyclase B receptor (GC-B) increases cGMP, leading to faster relaxation and decreased diastolic stiffness. We therefore examined whether long-term administration of CNP could prevent diastolic dysfunction in a mouse model of HFpEF. Experimental Approach: C57BL/6N mice were subjected to a combination of a high-fat diet (HFD) and the nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) in their drinking water to induce HFpEF. Osmotic pumps delivering either CNP or vehicle were implanted for a duration of 6 weeks. Tail-cuff plethysmography, transthoracic echocardiography, pressure volume loop acquisition and morphometrical analysis was used to investigate cardiac function. Key Results: After 6 weeks of CNP treatment, echocardiography and invasive hemodynamic analysis showed that cardiac diastolic function parameters, such as e’, E/e’, end diastolic pressure and end diastolic pressure volume relationship were all markedly ameliorated compared to vehicle-treated HFpEF mice. Vehicle-treated HFpEF mice displayed progressed cardiomyocyte hypertrophy accompanied by increased left ventricular wall thickness and myocardial fibrosis, while those changes were significantly suppressed in CNP-treated mice. Conclusion and Implications: The results demonstrate that CNP treatment prevents cardiac dysfunction and reduces myocardial hypertrophy and fibrosis in HFpEF mice, suggesting its prospective effectiveness for HFpEF treatment.

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.

The therapeutic benefit of stimulating the cGMP pathway as a form of treatment to combat heart failure, as well as other fibrotic pathologies, has become well established. However, the development and signal compartmentation of this crucial pathway has so far been overlooked. We studied how the three main cGMP pathways, namely, nitric oxide (NO)-cGMP, natriuretic peptide (NP)-cGMP, and β₃-adrenoreceptor (AR)-cGMP, mature over time in culture during cardiomyocyte differentiation from human pluripotent stem cells (hPSC-CMs). After introducing a cGMP sensor for Förster Resonance Energy Transfer (FRET) microscopy, we used selective phosphodiesterase (PDE) inhibition to reveal cGMP signal compartmentation in hPSC-CMs at various times of culture. Methyl-β-cyclodextrin was employed to remove cholesterol and thus to destroy caveolae in these cells, where physical cGMP signaling compartmentalization is known to occur in adult cardiomyocytes. We identified PDE3 as regulator of both the NO-cGMP and NP-cGMP pathway in the early stages of culture. At the late stage, the role of the NO-cGMP pathway diminished, and it was predominantly regulated by PDE1, PDE2, and PDE5. The NP-cGMP pathway shows unrestricted locally and unregulated cGMP signaling. Lastly, we observed that maturation of the β₃-AR-cGMP pathway in prolonged cultures of hPSC-CMs depends on the accumulation of caveolae. Overall, this study highlighted the importance of structural development for the necessary compartmentation of the cGMP pathway in maturing hPSC-CMs.

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.

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.

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.

Significance: G protein-coupled receptors (GPCRs) are targeted by a large fraction of approved drugs and regulate many important cellular processes. Conventional signaling by GPCRs is triggered when agonist-activated receptors associate with heterotrimeric G proteins. We found that serotonin 5-HT ₇ receptors couple to G _s proteins in an unconventional manner, in which agonist binding instead promotes dissociation of preexisting inactive 5-HT ₇ –G _s complexes. Therefore, agonists can initiate signaling via two distinct mechanisms, by promoting the association of active receptors and G proteins and by promoting dissociation of inactive receptors and G proteins.