CaMKII-Dependent Phosphorylation Modulates Ca2+ Cycling in Sinoatrial Node Cells to Regulate Cardiac Pacemaker Function

Abstract

Spontaneous beating of rabbit sinoatrial node cells (SANC) is linked to rhythmic, submembrane, sarcoplasmic reticulum (SR) generated local Ca2+releases (LCR), which activate inward Na+/Ca2+exchange current to impart exponential increase of the late diastolic depolarization rate and fire an action potential (AP). Rabbit SANC have high basal PKA-dependent phosphorylation, but basal CaMKII-dependent phosphorylation has never been ascertained. Here we show that SANC maintain high basal PLB phosphorylation at Thr17 site (western blotting), employed as a marker of CaMKII-dependent protein phosphorylation, that is markedly suppressed by CaMKII inhibitor, KN-93, but not its inactive analog KN-92. Considering that CaMKII activity is critically dependent on intracellular [Ca2+]i we employed BAPTA-AM to chelate [Ca2+]i. Both KN-93 and BAPTA-AM markedly decrease the LCR size (confocal line-scan imaging), LCR number per each spontaneous cycle and prolong the LCR period (time between AP-induced Ca2+ transient and subsequent LCR). The increase in the LCR period predicts concurrent increase in the spontaneous cycle length. To delineate mechanisms of CaMKII-dependent regulation of Ca2+ cycling, avoiding interference of L-type Ca2+channels, which are also regulated by CaMKII, we permeabilized SANC with saponin. Elevation of cytosolic [Ca2+] from 50 to 150 nmol/L in permeabilized SANC significantly increases the LCR size and amplitude due, in part, to a marked increase of PLB phosphorylation at Thr17site and resultant increase in the SR Ca2+ content and, in part, to RyR phosphorylation at PKA- and CaMKII-dependent Ser2809 site (immunostaining). CaMKII-inhibitor AIP suppresses LCR characteristics in permeabilized SANC, decreases phosphorylation of RyR, PLB and reduces the SR Ca2+ load. Thus, normal automaticity of SANC is regulated by basal CaMKII-dependent phosphorylation, partly via modulation of intrinsic SR Ca2+ cycling, i.e. SR Ca2+ pumping and release attained through phosphorylation of PLB and RyR.