Abstract 1537: Basal Phosphorylation Of Ca2+ Cycling Proteins By Both PKA And CAMKII is Required For Robust Generation Of Local Subsarcolemmal Ca2+ Releases To Drive Sinoatrial Node Cell Automaticity
Spontaneous beating of rabbit sinoatrial node cells (SANC) is controlled by cAMP-mediated PKA-dependent local subsarcolemmal Ca releases (LCRs) from sarcoplasmic reticulum (SR). LCRs activate inward Na-Ca exchange current during late diastolic depolarization and, as a result, regulate spontaneous SANC firing. To understand why SANC, but not ventricular myocytes (VM), generate LCR under basal physiological conditions, we compared SR Ca loading and release in permeabilized SANC and VM. SR Ca load, assessed by rapid application of caffeine (Fluo-4, line-scan confocal imaging) was similar or larger in VM than in SANC over a wide range of free [Ca]: 50 – 250 nmol/L. When free [Ca] was elevated above 150 nmol/L, SANC, compared to VM, spontaneously released 2–3 fold more Ca (estimated as a total Ca signal mass of all spontaneous SR Ca releases per 100 μm over 400 ms), suggesting that at a given SR Ca load, SR Ca cycling in ‘skinned’ SANC is more robust than in ‘skinned’ VM. To further characterize mechanisms that allowed SANC to generate vigorous Ca release at physiological [Ca], we studied effects of free [Ca] on phosphorylation of Ca cycling proteins in skinned cells, using phospholamban (PLB) phosphorylation as an index protein. Elevation of free [Ca] from 0 to 150 nmol/L increased PLB phosphorylation ~80% at PKA-dependent site (Ser16, western blot) and ~37% at CaMKII-dependent site (Thr17, immunostaning) in SANC, but not in VM. To compare the contributions of PKA and CaMKII-dependent phosphorylation to Ca cycling in ‘skinned’ SANC we used either specific PKA-inhibitor peptide, PKI, or CaMKII inhibitor, KN-93. PKI (15 μmol/L) decreased PLB phosphorylation at Ser16 site and suppressed number of LCRs and SR Ca load in SANC. KN-93 (3 μmol/L) decreased PLB phosphorylation at Thr17 site and the number of LCRs in ‘skinned’ SANC, but the SR Ca load was unchanged. Thus, Ca-dependent phosphorylation of Ca cycling proteins, including PLB phosphorylation at both PKA and CaMKII dependent sites, underlies robust basal Ca cycling in SANC. Our results also suggest that PKA and CaMKII dependent phosphorylation may have distinct roles in the modulation of basal SR Ca cycling that drives robust automaticity of cardiac pacemaker cells.