RGS2 overexpression or Gi inhibition rescues the impaired PKA signaling and slow AP firing of cultured adult rabbit pacemaker cells

Dongmei Yang, Alexey E. Lyashkov, Yue Li, Bruce D. Ziman, Edward G. Lakatta

Research output: Contribution to journalArticlepeer-review

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 48h 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 Ser16 and type2 ryanodine receptor (RyR2) at Ser2809 are reduced; and the level of type2 regulator of G-protein signaling (RGS2), that facilitates adenylyl cyclases/cAMP/protein kinase A (PKA) via Gi 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 Gi-signaling, each partially rescues the c-SANC spontaneous AP firing rate.Thus, a Gi-dependent reduction in PKA-dependent protein phosphorylation, including that of Ca2+ cycling proteins, reduces the spontaneous AP firing rate of c-SANC, and can be reversed by genetic or pharmacologic manipulation of PKA signaling.

Original languageEnglish (US)
Pages (from-to)687-694
Number of pages8
JournalJournal of Molecular and Cellular Cardiology
Volume53
Issue number5
DOIs
StatePublished - Nov 2012
Externally publishedYes

Keywords

  • Action potential firing rate
  • Cultured adult rabbit sinoatrial node cells
  • PKA-dependent protein phosphorylation
  • Type2 regulator of G-protein signaling

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

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