P611Elucidating compartmentation of receptor-mediated cGMP signalling regulating inotropic responses in cardiac myocytes

Abstract

Purpose: To determine how inotropic responses are regulated in heart failure (HF) through signalling pathways increasing cAMP and cGMP is crucial for understanding how new drugs can be of benefit to patients. Myocardial levels of C-type natriuretic peptide (CNP) are increased in HF. CNP activates the guanylyl cyclase NPR-B receptor and increases cGMP. Our group has previously shown that CNP induces a negative inotropic and positive lusitropic response in failing hearts through cGMP-mediated phosphorylation of phospholamban and troponin I. In addition, we have shown that activating these receptors enhances the positive inotropic response to β1-adrenergic and 5-HT4 receptors via a mechanism where increased cGMP levels inhibit phosphodiesterase 3 (PDE3) and thereby the degradation of cAMP from these receptors. Neither of these effects were observed when activating other NPR receptors with ANP and BNP, indicating a tightly compartmented response of both the NPR-B, β1- and 5-HT4 receptors. We therefore hypothesize that cGMP is increased in distinct subcellular compartments in cardiac cells. In the current work, we want to clarify the organization of functional cGMP compartments, importance of PDEs and how cGMP regulates inotropic responses. Methods: We have constructed several intracellular FRET-based sensors for cGMP targeted to different subcellular compartments that allow us to measure the local levels of cGMP in single cardiac myocytes. Results and conclusions: In the current work, we have constructed new FRET-based cGMP sensors, located to specific anchoring and structural proteins regulating inotropic responses, thus enabling measurement of local changes in cGMP concentrations after stimulation of the different natriuretic peptide receptors. Understanding compartmented cGMP signaling would help explain how natriuretic peptides can regulate calcium handling (phospholamban) and calcium sensitivity (troponin I) of cardiac myocytes. FRET = Fluorescence resonance energy transfer