Manuela Zaccolo - Cardiac Sciences - 'Nanodomain signalling in the heart'

cAMP and its effector PKA are key regulators of cardiac function and defective cAMP/PKA signalling is a hallmark of heart failure (HF) and genetic cardiomyopathies. This signalling pathway is also at the core of current therapies, which however remain unsatisfactory and need improvement. Current therapeutic strategies largely ignore signalling processes occurring in cardiomyocytes at the subcellular level. We use FRET-based imaging approaches to measure cAMP/PKA signalling in real-time and with high spatio-temporal resolution. Using this approach we were able to directly show that cAMP/PKA signalling is highly compartmentalised within subcellular nanodomains (1, 2), with different sites affecting different functions (2). In a recent study we found that adrenergic stimulation generates pools of cAMP with different amplitude and kinetics at the plasmalemma and at the myofilaments and that such local regulation is disrupted in HF (2). Local PKA activity is dictated by local [cAMP], controlled at each specific site by particular adenylyl cyclases (AC) and phosphodiesterases (PDE) isoforms. Local phosphorylation of targets results from the balance of local PKA and phosphatase (PP) activity. All these components can potentially be manipulated to affect local signalling. Compartmentalization of signalling provides a unique opportunity to intervene therapeutically with increased precision by selectively targeting individual nanodomains to affect only the desired function. Recently, we have conducted an integrated PDE phospho-interactome ananlysis that unveiled multiple novel and non-obvious cAMP nanodomain under specific regulation of PDE isoforms (4). We are currently validating these data and characterising the function reglated by these novel nanodomans.The overall aim of our work is to build a detailed map of cAMP nanodomains in cardiac myocytes . The map will be used as a blueprint to assess alterations in pathological conditions to gain novel mechanistic understanding of pathological processes at the molecular level. This information will guide development of new strategies for targeted therapeutic interventions.The project will test novel FRET-based reporters targeted to specific subcellular sites in cardiac myocytes to establish local cAMP dynamics at key signalling nodes that participate in the regulation of cardiac myocyte function. The work will involve biochemical and genetic approaches to study cAMP signalling at these sites in animal models of HF and in human cardiac myocytes differentiated from inducible pluripotent stem cells.