The cAMP signaling pathway plays a critical role in regulating many different aspects of cardiac myocyte function, including gene transcription, cell metabolism, and excitation-contraction coupling. However, not all G-protein coupled receptors that stimulate cAMP production produce the same responses. Subcellular compartmentation of cAMP is essential to explain how different receptors can utilize the same diffusible second messenger to elicit unique functional responses. Furthermore, disruption of cAMP compartmentation has been linked to various disease states, including cardiac hypertrophy, heart failure, and arrhythmias. Yet, a complete picture of the mechanisms contributing to cAMP compartmentation remains a mystery. Most work has focused on the role of phosphodiesterases (PDEs), which breakdown cAMP and are commonly thought to act as either functional barriers or active sinks that define different signaling domains. However, a number of studies indicate that PDE activity alone is not sufficient. The results suggest that, in addition to PDE activity, unique receptor-dependent responses can only be explained if the movement of intracellular cAMP is somehow limited by other mechanisms. Using a sophisticated new approach, we have directly measured the diffusion coefficient of cAMP in intact myocytes and found that it does indeed move dramatically slower than previously thought. Furthermore, our preliminary data have identified two factors critical to explaining this behavior. The first is buffering of cAMP by protein kinase A (PKA) immobilized by A kinase anchoring proteins (AKAPs). The second are subcellular restricted spaces. In this proposal, we will address the following specific questions: 1) Does buffering by PKA anchored to the outer membrane of mitochondria contribute to cAMP compartmentation? and 2) Does the restricted space associated with dyadic clefts contribute to cAMP compartmentation. To answer these questions, we will use a combination of molecular, biochemical, and biophysical techniques. These include raster image correlation spectroscopy (RICS) to directly measure cAMP diffusion, fluorescence resonance energy transfer (FRET)-based biosensors targeted to different subcellular locations to measure cAMP compartmentation; and patch clamp electrophysiology, Ca2+ fluorometry, and myocyte shortening to measure compartmentalized cAMP-dependent functional responses. The answers to these questions could lead to the development of novel approaches to halting the progression of cardiovascular disease and preventing the deadly consequences.