A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation

Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states.Author Summary: Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain how this one signaling molecule produces unique receptor-dependent functional responses. But, how exactly compartmentation occurs, is unknown. This is because there has been no way to measure the regulation and movement of cAMP in cells with intact subcellular structures. In this study, we applied novel computational approaches to predict whether PDE activity alone or in conjunction with restricted diffusion is sufficient to produce cAMP gradients in submicroscopic signaling domains. We also used the models to test the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Our simulations suggest that PDE activity alone is not sufficient to explain compartmentation, but if diffusion of cAMP is limited by potential factors such as molecular crowding, PKA buffering, and anatomical barriers, then compartmentation is predicted to occur.