Phosphodiesterases as Crucial Regulators of Cardiomyocyte cAMP in Health and Disease

Abstract

Cyclic nucleotides are ubiquitous second messengers, which regulate cellular functions by acting in discrete subcellular microdomains. Cardiac phosphodiesterases (PDEs) are indispensable for -adrenoceptor signaling regulation by restricting and maintaining distinct cAMP microdomains. In the mammalian myocardium, at least five PDE families (PDE1, 2, 3, 4, 8) contribute to cAMP breakdown, each with unique binding affinities and regulatory properties. Moreover, different PDE families and isoforms localize to distinct functional cAMP microdomains, making them promising targets to modulate cell function. However, the use of selective PDE inhibitors to treat heart failure is problematic due to a high risk of tachyarrhythmias and sudden cardiac death. Furthermore, progressive heart failure is accompanied by severely altered PDE expression patterns. Despite considerable insights into cardiac cAMP handling in general, exact mechanisms of cAMP regulation by PDEs inside functionally relevant microdomains are still poorly understood. This work firstly describes compartmentalized functions of cardiac cAMP and uncovers that atrial natriuretic peptide can augment catecholamine-stimulated contractility in order to increase heart function in early cardiac hypertrophy. Real-time cAMP analysis of distinct β1- and β2-adrenoceptor-associated sarcolemmal cAMP microdomains using a novel targeted Förster resonance energy transfer (FRET)-based biosensor, pmEpac1, reveals that this effect is brought about by spatial redistribution of cGMP-sensitive phosphodiesterases 2 and 3 between β-adrenoceptor subtype-specific cAMP microdomains. While whole-cell PDE protein levels and activities are still unaffected at this early disease stage, differential subcellular PDE localization leads to altered cGMP/cAMP-crosstalk, which shifts the balance between β1- and β2-adrenoreceptor-mediated effects on cardiac function. These findings point towards a novel functionally relevant adaptation mechanism, which occurs early during disease and might compensate for a loss of heart function by redistribution of cGMPregulated PDEs between distinct membrane microdomains, thereby modulating the functionally relevant ANPcGMP / -adrenoceptor-cAMP crosstalk at the sarcolemma of adult cardiomyocytes. Secondly, a previously unappreciated PDE4 inhibitory side effect of atropine, a clinically relevant muscarinic receptor blocker, was uncovered in this work. This mechanism can, at least in part, explain incidences of tachycardia, which are frequently observed upon atropine administration and have been attributed solely to the antagonism at cardiac muscarinic M2-receptors. However, in isolated mouse cardiomyocytes expressing the FRET-based cAMP biosensor Epac1-camps, even upon Gi-protein inactivation with pertussis toxin or in M2- receptor knockout cells, atropine increases isoproterenol pre-stimulated cAMP levels, similar to the effects of PDE inhibitors. Furthermore, in intact wild type and M2-receptor deficient hearts, it leads to increased beating frequency. Detailed analysis of atropine-mediated changes in cAMP handling using FRET approaches and in vitro assays show that atropine indeed inhibits PDE4 activity. Therefore, inhibition of PDE4 by atropine may be responsible at least for some of its multiple side effects.