Compartmentalized cGMP signalling in cardiomyocyte hypertrophy and heart failure

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The ubiquitous second messenger cGMP regulates multiple cellular processes including smooth and heart muscle contractility, pathological cardiac growth (hypertrophy) and remodelling. In our previous work, we uncovered that hypertrophy is associated with relocation of the cGMP-regulated phosphodiesterases (PDEs) 2 and 3 between beta-adrenoceptor (beta-AR)-associated submembrane microdomains. This newly identified molecular mechanism allows augmentation of catecholamine-stimulated cardiac contractility by atrial natriuretic peptide (ANP) through its receptor GC-A located in T-tubules of cardiomyocytes, where we could also localise functional beta3-ARs. This microdomain might bring together distinct guanylyl cyclases (GC-A/B and NO-GC) to generate an important local pool of cGMP which regulates contraction and pathological remodelling. cGMP microdomain remodelling is a stage-dependent process which involves dynamic changes of cGMP/cAMP cross-talk due to PDE and beta-AR redistribution. Based on our previous work and recently established mouse lines, we will now continue to analyse changes of submembrane cGMP signalling in hypertrophy and heart failure. Using Förster resonance energy transfer and scanning ion conductance microscopy in pm-DE5 mouse cells (expressing a cGMP sensor targeted to T-tubules and caveolin-rich membrane microdomains), we will study whether hypertrophy leads to changes of PDE2-dependent GC-A/cGMP and PDE3-dependent GC-B/cGMP microdomain regulation in the T-tubules vs cell crests. Potentially, GC-A desensitisation and redistribution at the later transition to chronic disease will be analysed together with other FOR 2060 projects. Secondly, we will use pm-DE5 and pm-Epac1-camps animals bred with beta3-AR transgenic mice to study functional interactions of beta3-AR and GC-A receptors in the T-tubular compartment and the regulation of cGMP/cAMP cross-talk by their respective, presumably distinct cGMP pools in the context of disease. Finally, findings on cGMP dynamics obtained in single isolated cardiomyocytes will be further verified using newly established FRET imaging in intact Langendorff perfused hearts with transgenic sensor expression. In this experimental setting, cell type-specific effects in cardiomyocytes, cardiac fibroblasts and potentially also in endothelial cells can be studied and the role of cell-cell communication in the context of pharmacological treatments (such as by sildenafil or drugs acting via NO-GC) can be evaluated. The long-term goal of this project is to identify new druggable targets at the level of subcellular cGMP microdomains, which can be used to prevent cardiac hypertrophy and heart failure progression.