Dataset Information

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 ?-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 ?-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.

SUBMITTER: Yang D

PROVIDER: S-EPMC3472119 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

RGS2 overexpression or G(i) inhibition rescues the impaired PKA signaling and slow AP firing of cultured adult rabbit pacemaker cells.

Yang Dongmei D Lyashkov Alexey E AE Li Yue Y Ziman Bruce D BD Lakatta Edward G EG

Journal of molecular and cellular cardiology 20120819 5

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 ...[more]

PMID: 22921807

Similar Datasets

Project description:Pacemaker cells residing in the sinoatrial node generate the regular heartbeat. Ca2+ signaling controls the heartbeat rate-directly, through the effect on membrane molecules (NCX exchange, K+ channel), and indirectly, through activation of calmodulin-AC-cAMP-PKA signaling. Thus, the physiological role of signaling in pacemaker cells can only be assessed if the Ca2+ dynamics are in the physiological range. Cultured cells that can be genetically manipulated and/or virally infected with probes are required for this purpose. Because rabbit pacemaker cells in culture experience a decrease in their spontaneous action potential (AP) firing rate below the physiological range, Ca2+ dynamics are expected to be affected. However, Ca2+ dynamics in cultured pacemaker cells have not been reported before. We aim to a develop a modified culture method that sustains the global and local Ca2+ kinetics along with the AP firing rate of rabbit pacemaker cells. We used experimental and computational tools to test the viability of rabbit pacemaker cells in culture under various conditions. We tested the effect of culture dish coating, pH, phosphorylation, and energy balance on cultured rabbit pacemaker cells function. The cells were maintained in culture for 48 h in two types of culture media: one without the addition of a contraction uncoupler and one enriched with either 10 mM BDM (2,3-Butanedione 2-monoxime) or 25 μM blebbistatin. The uncoupler was washed out from the medium prior to the experiments. Cells were successfully infected with a GFP adenovirus cultured with either BDM or blebbistatin. Using either uncoupler during culture led to the cell surface area being maintained at the same level as fresh cells. Moreover, the phospholamban and ryanodine receptor densities and their phosphorylation level remained intact in culture when either blebbistatin or BDM were present. Spontaneous AP firing rate, spontaneous Ca2+ kinetics, and spontaneous local Ca2+ release parameters were similar in the cultured cells with blebbistatin as in fresh cells. However, BDM affects these parameters. Using experimental and a computational model, we showed that by eliminating contraction, phosphorylation activity is preserved and energy is reduced. However, the side-effects of BDM render it less effective than blebbistatin.

Dataset Information

RGS2 overexpression or G(i) inhibition rescues the impaired PKA signaling and slow AP firing of cultured adult rabbit pacemaker cells.

Publications

RGS2 overexpression or G(i) inhibition rescues the impaired PKA signaling and slow AP firing of cultured adult rabbit pacemaker cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets