title: Real-time relationship between PKA biochemical signal network dynamics and increased action potential firing rate in heart pacemaker cells: Kinetics of PKA activation in heart pacemaker cells.
abstract: cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. These functions are predicted to be involved in regulation of spontaneous action potential (AP) generation of these cells. Here we investigate if the kinetics and stoichiometry of increase in PKA activity match the increase in AP firing rate in response to β-adrenergic receptor (β-AR) stimulation or phosphodiesterase (PDE) inhibition, that alters the AP firing rate of heart sinoatrial pacemaker cells. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of β-AR stimulation (by Isoproterenol) the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.4±0.1nM vs. 0.6±0.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to β-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 (β-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca(2+)-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate.
authors.affiliation: Biomedical Engineering Faculty, Technion-IIT, Haifa, Israel. Electronic address: yaely@bm.technion.ac.il.
authors.forename: Yael
authors.initials: Y
authors.lastname: Yaniv
authors.affiliation: Department of Biomedical Engineering, The Johns Hopkins University of Medicine, Baltimore, MD, USA.
authors.forename: Ambhighainath
authors.initials: A
authors.lastname: Ganesan
authors.affiliation: Laboratory of Cardiovascular Science, Biomedical Research Center Intramural Research Program, National Institute on Aging, NIH, Baltimore, MD, USA.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Yang
authors.affiliation: Laboratory of Cardiovascular Science, Biomedical Research Center Intramural Research Program, National Institute on Aging, NIH, Baltimore, MD, USA.
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Ziman
authors.affiliation: Laboratory of Cardiovascular Science, Biomedical Research Center Intramural Research Program, National Institute on Aging, NIH, Baltimore, MD, USA.
authors.forename: Alexey E
authors.initials: AE
authors.lastname: Lyashkov
authors.affiliation: System Biology Institute, Yale University, New Haven, CT, USA.
authors.forename: Andre
authors.initials: A
authors.lastname: Levchenko
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; The Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Laboratory of Cardiovascular Science, Biomedical Research Center Intramural Research Program, National Institute on Aging, NIH, Baltimore, MD, USA. Electronic address: lakattae@grc.nia.nih.gov.
authors.forename: Edward G
authors.initials: EG
authors.lastname: Lakatta
mesh_terms: D000200:Action Potentials / Q000187:drug effects*; D000818:Animals; D020013:Calcium Signaling; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010726:Phosphodiesterase Inhibitors / Q000008:administration & dosage; D010766:Phosphorylation / Q000187:drug effects; D011817:Rabbits; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D012849:Sinoatrial Node / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
chemical_list: D010726:Phosphodiesterase Inhibitors; D011943:Receptors, Adrenergic, beta; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Computational modeling; Coupled-clock system; Pacemaker; Phosphorylation