Real-time relationship between PKA biochemical signal network dynamics and action potential firing rate in heart pacemaker cells

National Institute On Aging

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Abstract

cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. Although PKA-dependent mechanisms are linked to action potential (AP) firing rate, real-time measurements of PKA signaling in the context of heart pacemaker function are lacking. Specifically, it has never been demonstrated that the kinetics and stoichiometry of increases in PKA activity and protein phosphorylation in individual pacemaker cell are similar to and sufficient to account for changes in the kinetics and stoichiometry of increases in AP firing rate that occur in response to physiological (-AR stimulation) or phosphodiesterase (PDE) inhibition. In cultured adult rabbit pacemaker cells infected with an adenovirous expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of -AR stimulation (by Isoproterenol), the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.40.1nM vs. 0.60.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to -AR stimulation or PDE inhibition were tightly linked. We characterized the system rate-limiting biochemical reactions by integrating these experimentally derived data into a mechanistic-computational model. Model simulations predicted that phospholamban phosphorylation is a potent target of the increase in PKA activity that links to increase in spontaneous AP firing rate. In summary, the kinetics and stoichiometry of increases in PKA activity in response to a physiological (-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca2+-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate. In cultured adult rabbit pacemaker cells infected with Ad-CMV-Ateam expressing ATP-FRET sensor, the cytosolic ATP level keeps stable in beating or non-beating cells with / without the incubation of PDE inhibitor IBMX (20 micro Mole). So no correlation was detected between the cytosolic APT level and the spontaneous beating rate,