Abstract

The cAMP\PKA pathway regulates a wide range of cellular processes in the heart. cAMP compartmentalization has been demonstrated by FRET-based sensors in cardiac cell, although studies so far have suffered from limited spatial resolution of the imaging approach. The local control of cAMP signals in distinct signalling hubs is of critical importance to achieve specific functions. We developed a new set of tools, based on a novel FRET-tag, named INF2, for quantitative imaging of cAMP capable of dissecting compartmentalized cAMP signalling. Due to its novel design, the INF2 sensor can be targeted to individual macromolecular complexes by fusion to targeting proteins, retaining FRET efficiency independently of the protein it is fused to. These targeted sensors allow accurate quantification and comparison of cAMP fluctuations confined to specific signalling hubs. Expression of the targeted sensors in neonatal and in adult rat cardiomyocytes shows the expected specific targeting. The FRET response to catecholamine stimulation of INF2 fusions to AKAP79, AKAP18d and TnI, three proteins involved in the regulation of excitation-contraction coupling, shows that cAMP signals are uniquely regulated in the microenvironment surrounding these three hubs. Moreover the FRET signal generated by these sensors upon PGE2 and GLP-1 stimulation shows different cAMP responses in different plasma membrane compartments. Such differences are abolished by pretreatment with the PDE inhibitor IBMX, emphasizing the key role of phosphodiesterases in the control of the local cAMP levels. Our results indicate that these new tools have the potential to dissect of cAMP microdomains with unprecedented spatial resolution.