26. SUBCELLULAR MODELING OF PKA ACTIVATION AND CAMP DIFFUSION IN LOCALIZED MICRODOMAINS OF ADULT CARDIAC MYOCYTES

Department: Bioengineering
Faculty Advisor(s): Andrew McCulloch

Primary Student
Name: Britton Warren Boras
Email: bboras@ucsd.edu
Phone: 858-534-5796
Grad Year: 2015

Abstract
The beta adrenergic pathway in cardiomyocytes activates protein kinase A (PKA) to phosphoregulate several Ca2+ handling proteins, including the L-type Ca channel, ryanodine receptor, and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (via phospholamban), resulting in inotropic, lusitropic and chronotropic responses. However, based on PKA kinetics measured in vitro, almost all PKA should be activated by basal concentrations of cAMP. Several recent studies have postulated a role for local degradation of cAMP by phosphodiesterases (PDE) in maintaining microdomains with lower cAMP concentrations. However, the degradation rate of cAMP is slow compared with the rate of cytosolic diffusion, suggesting that local clustering of PDE is insufficient to maintain the cAMP microdomains alone. In this study, Virtual Cell, a finite volume solver, was used to create 3D diffusional models that were then validated with in vitro FRET experiments to probe other potential mechanisms of PKA?s compartmentalized response. We examined the effects of structural obstruction, cAMP buffering by exchange protein directly activated by cAMP (EPAC), PKA isoform localization, ion gradients, and physical coupling between PKA and PDE, mediated by A-kinase anchoring protein (AKAP). Our results suggest that structural obstructions, obtained from cryo-TEM images, are insufficient to decrease the rate of diffusion of cAMP. However, PKA isoform localization around the SERCA pumps is essential in maintaining phosphoregulation. Furthermore, AKAP complexes and basal ion concentrations also contribute to localized control of PKA. These in vitro and in silico experiments help us understand how microdomains in adult cardiomyocytes are maintained.

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