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 language | English (US) |
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Pages (from-to) | 687-694 |
Number of pages | 8 |
Journal | Journal of Molecular and Cellular Cardiology |
Volume | 53 |
Issue number | 5 |
DOIs | |
State | Published - Nov 2012 |
Externally published | Yes |
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