Abstract
Compartmentation and dynamics of cAMP and PKA signaling are important
determinants of specificity among cAMP's myriad cellular roles. Both
cardiac inotropy and the progression of heart disease are affected
by spatiotemporal variations in cAMP/PKA signaling, yet the dynamic
patterns of PKA-mediated phosphorylation that influence differential
responses to agonists have not been characterized. We performed live-cell
imaging and systems modeling of PKA-mediated phosphorylation in neonatal
cardiac myocytes in response to G-protein coupled receptor stimuli
and UV photolysis of "caged" cAMP. cAMP accumulation was rate-limiting
in PKA-mediated phosphorylation downstream of the beta-adrenergic
receptor. Prostaglandin E1 stimulated higher PKA activity in the
cytosol than at the sarcolemma, whereas isoproterenol triggered faster
sarcolemmal responses than cytosolic, likely due to restricted cAMP
diffusion from submembrane compartments. Localized UV photolysis
of caged cAMP triggered gradients of PKA-mediated phosphorylation,
enhanced by phosphodiesterase activity and PKA-mediated buffering
of cAMP. These findings indicate that combining live-cell FRET imaging
and mechanistic computational models can provide quantitative understanding
of spatiotemporal signaling.
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