Abstract 21546: Protein Phosphatase 1 Regulates Normal Automaticity of the Heart's Pacemaker Node Cells by Site-specific Modulation of Phospholamban Phosphorylation that Regulates Spontaneous Subsarcolemmal Local Ca2+ Releases
The heart's rhythm is driven by spontaneous action potential (AP) firing of sinoatrial node cells (SANC), in which cAMP-mediated, PKA-dependent local subsarcolemmal Ca2+ releases (LCRs) play a crucial role. SANC sarcomplasmic reticulum (SR) has been referred to as an intracellular ”Ca2+ clock” because it generates spontaneous periodic submembrane Ca2+ oscillations controlled by phosphorylation of phospholamban (PLB). Protein kinase A (PKA) and the Ca2+/calmodulin-dependent protein kinase II (CAMKII) regulate PLB by phosphorylation at Ser16 and Thr17 sites. We aimed to determine how protein phosphatase (PP) activity is involved in regulation of PLB phosphorylation, LCR generation and spontaneous rhythmic AP firing of SANC. In freshly isolated rabbit SANC, the PP1/2A inhibitor calyculin A (CyA), 100–500 nM, increased the spontaneous SANC beating rate, recorded using perforated patch, by 24–30%, from 179.4±13.1 to 230.6±7.45 beat/min (n=21). Confocal Ca2+ imaging of isolated SANC demonstrated that PP inhibition significantly increased LCR size from 5.5±0.5 to 8.1±1.3 um (n=12), and decreased the LCR period from 304.8±15.9 to 242.3±13.2 ms (n=12). The decrease in the LCR period predicted the reduction in the spontaneous SANC cycle length (CL) from 390.3±17.1 to 314.4±14.5 ms, R2=84 (n=12). Assessment of PLB phosphorylation at PKA-dependent and CaMKII-dependent sites using phosphorylation site-specific polyclonal antibodies indicated that basal phosphorylation at Ser16 and Thr17 was increased by 1.65- and 3.34-fold after incubation with 100 nM CyA for 10 min (n=5, 4). Okadaic acid, at 1–100 nM, a concentration specific for the inhibition of PP2A, did not have a significant effect on the spontaneous AP, CL, LCRs parameters and PLB phosphorylation indicating that PP1 rather than PP2A is involved in regulation of spontaneous rhythmic beating in SANC. Biochemical measurement showed a high level of PP activity which was inhibited by CyA. Thus basal PLB phosphorylation in SANC is determined by the balanced activity of phosphatases and kinases. We show, for the first time, that basal PP1 activity regulates spontaneous SANC AP firing rate and modulates SR generated LCRs via its effect on PLB phosphorylation, while PP2A is not involved in this regulation.
- © 2010 by American Heart Association, Inc.