The ubiquitous second messenger cAMP is an important regulator of cardiac function and disease. In cardiomyocytes, cAMP signalling is organized in discrete subcellular microdomains which are often involved in calcium cycling and lead to temporal and spatial segregation of subcellular cAMP responses. To directly visualize cAMP dynamics in such microdomains, we generated two novel targeted fluorescence resonance energy transfer (FRET)-based biosensors and expressed them in cardiomyocytes of transgenic mice under the alpha-myosin heavy chain promoter. The first sensor reports local cAMP dynamics associated with the sarcolemmal T-tubular microdomains and the second one can visualize cAMP directly at the sarcoplasmic reticulum calcium ATPase 2 (SERCA2) Echocardiography, immunostaining and cell fractionation analysis confirmed the absence of any adverse cardiac phenotype and the proper localization of the sensors in transgenic mouse hearts. Local cAMP signals were measured in single freshly isolated adult ventricular myocytes and compared to cAMP dynamics in the bulk cytosolic compartment. FRET measurements revealed the predominant role of phosphodiesterase type 4 (PDE4) in the regulation of basal cAMP levels in the SERCA2 compartment and PDE3 in the regulation of the beta2-adrenergic receptor stimulated cAMP pools at the T-tubules. In failing cardiomyocytes, isolated from mouse hearts 8 weeks after transverse aortic constriction, these local PDE4 and PDE3 activities were severely reduced which led to specific subcellular changes in cAMP compartmentation In conclusion, through generation of novel FRET biosensors and transgenic models, we were able to get direct insights into localized cAMP signaling in various subcellular microdomains and to unravel its changes in a cardiac disease model.