title: RGS2 overexpression or G(i) inhibition rescues the impaired PKA signaling and slow AP firing of cultured adult rabbit pacemaker cells.
abstract: Freshly isolated adult rabbit sinoatrial node cells (f-SANC) are an excellent model for studies of autonomic signaling, but are not amenable to genetic manipulation. We have developed and characterized a stable cultured rabbit SANC (c-SANC) model that is suitable for genetic manipulation to probe mechanisms of spontaneous action potential (AP) firing. After 48 h in culture, c-SANC generate stable, rhythmic APs at 34±0.5°C, at a rate that is 50% less than f-SANC. In c- vs. f-SANC: AP duration is prolonged; phosphorylation of phospholamban at Ser(16) and type2 ryanodine receptor (RyR2) at Ser(2809) are reduced; and the level of type2 regulator of G-protein signaling (RGS2), that facilitates adenylyl cyclases/cAMP/protein kinase A (PKA) via G(i) inhibition, is substantially reduced. Consistent with the interpretation that cAMP/PKA signaling becomes impaired in c-SANC, acute β-adrenergic receptor stimulation increases phospholamban and RyR2 phosphorylation, enhances RGS2-labeling density, and accelerates the AP firing rate to the similar maximum in c- and f-SANC. Specific PKA inhibition completely inhibits all β-adrenergic receptor effects. Adv-RGS2 infection, or pertussis toxin treatment to disable G(i)-signaling, each partially rescues the c-SANC spontaneous AP firing rate. Thus, a G(i)-dependent reduction in PKA-dependent protein phosphorylation, including that of Ca(2+) cycling proteins, reduces the spontaneous AP firing rate of c-SANC, and can be reversed by genetic or pharmacologic manipulation of PKA signaling.
authors.affiliation: Laboratory of Cardiovascular Science, IRP, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6825, USA.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Yang
authors.forename: Alexey E
authors.initials: AE
authors.lastname: Lyashkov
authors.forename: Yue
authors.initials: Y
authors.lastname: Li
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Ziman
authors.forename: Edward G
authors.initials: EG
authors.lastname: Lakatta
mesh_terms: D000200:Action Potentials; D000256:Adenoviridae / Q000235:genetics; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D048430:Cell Shape; D002478:Cells, Cultured; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D005822:Genetic Vectors; D007545:Isoproterenol / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D015290:Second Messenger Systems; D012849:Sinoatrial Node / Q000166:cytology*; D014162:Transfection; D000099211:Phospholamban
chemical_list: D000318:Adrenergic beta-Agonists; D002135:Calcium-Binding Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D007545:Isoproterenol; D037342:Pertussis Toxin; D047428:Protein Kinase Inhibitors; D020710:RGS Proteins; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D000099211:Phospholamban