Abstract 11932: Distinct Patterns of Ca2+ Cycling Protein Abundance and Phosphorylation Level Facilitate Robust and Rhythmic Local Ca2+ Releases to Regulate Spontaneous Beating of Cardiac Pacemaker Cells
Spontaneous firing of sinoatrial node cells (SANC) is regulated by spontaneous submembrane local Ca2+ releases (LCR) from ryanodine receptors (RyRs). LCR activate inward Na+-Ca2+ exchange current during diastolic depolarization and thus regulate spontaneous SANC firing. In contrast to spontaneous diastolic Ca2+ sparks in ventricular myocytes (VM), which are small and stochastic, spontaneous diastolic LCR in SANC are large and rhythmic. To understand why SANC, but not VM, generate robust and rhythmic LCR under basal physiological conditions we compared expression and phosphorylation status of proteins involved in SR Ca2+ cycling in rabbit SANC and VM, using western blot or immunostaining techniques. Our data showed that phosphorylation of RyR at CaMKII-dependent Ser2815 site and PKA- and CaMKII-dependent Ser2809 site was ~10-fold and ~2-fold higher, respectively, in SANC than in VM. An increase in RyR phosphorylation facilitates RyR activation leading to generation of robust LCR in SANC. To support elevated RyR Ca2+ release the rate of Ca2+ pumping into SR in SANC should be higher compared to VM. Consistent with this idea SANC had ~40% increase in expression of SR Ca2+-ATPase (SERCA) and ~50% reduction in expression of phospholamban (PLB) proteins, than VM. Besides, phosphorylation of PLB at both PKA- (Ser16) and CaMKII-dependent (Thr17) sites was ~10-fold and ~3-fold higher, respectively, in SANC than in VM. The increased amount of SERCA protein, reduced amount of PLB protein and augmented PLB phosphorylation relieve SERCA inhibition to adjust SR Ca2+ pumping and support elevated RyR Ca2+ release in SANC. Suppression of PKA- or CaMKII-dependent phosphorylation with PKI or KN-93, respectively, markedly decreased PLB and RyR phosphorylation; reduced LCR periodicity, size and number per each spontaneous cycle (confocal microscopy, Ca2+ indicator Fluo-3); and prolonged the LCR period (time from AP-induced Ca2+ transient to the subsequent LCR), which predicted the concomitant increase in the cycle length. Thus, specific modifications in SR Ca2+ cycling protein expression and phosphorylation levels are unique mechanisms that drive intracellular Ca2+-cycling, “Ca2+ clock”, in cardiac pacemaker cells to enable normal cardiac automaticity.
Author Disclosures: T.M. Vinogradova: None. D.R. Riordon: None. D. Yang: None. S. Sirenko: None. A.E. Lyashkov: None. Y. Li: None. E.G. Lakatta: None.
- © 2014 by American Heart Association, Inc.