CaMKII-dependent phosphorylation regulates basal cardiac pacemaker function via modulation of local Ca2+ releases

Abstract

Spontaneous beating of the heart pacemaker, the sinoatrial node, is generated by sinoatrial node cells (SANC) due to gradual change of the membrane potential called diastolic depolarization (DD). Spontaneous, submembrane local Ca(2+) releases (LCR) from ryanodine receptors (RyR) occur during late DD and activate an inward Na(+)/Ca(2+)exchange current to boost the DD rate and fire an action potential (AP). Here we studied the extent of basal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation and the role of basal CaMKII-dependent protein phosphorylation in generation of LCRs and regulation of normal automaticity of intact rabbit SANC. The basal level of activated (autophosphorylated) CaMKII in rabbit SANC surpassed that in ventricular myocytes (VM) by approximately twofold, and this was accompanied by high basal level of protein phosphorylation. Specifically, phosphorylation of phospholamban (PLB) at the CaMKII-dependent Thr(17) site was approximately threefold greater in SANC compared with VM, and RyR phosphorylation at CaMKII-dependent Ser(2815) site was ∼10-fold greater in the SA node, compared with that in ventricle. CaMKII inhibition reduced phosphorylation of PLB and RyR, decreased LCR size, increased LCR periods (time from AP-induced Ca(2+) transient to subsequent LCR), and suppressed spontaneous SANC firing. Graded changes in CaMKII-dependent phosphorylation (indexed by PLB phosphorylation at the Thr(17)site) produced by CaMKII inhibition, β-AR stimulation or phosphodiesterase inhibition were highly correlated with changes in SR Ca(2+) replenishment times and LCR periods and concomitant changes in spontaneous SANC cycle lengths (R(2) = 0.96). Thus high basal CaMKII activation modifies the phosphorylation state of Ca(2+) cycling proteins PLB, RyR, L-type Ca(2+) channels (and likely others), adjusting LCR period and characteristics, and ultimately regulates both normal and reserve cardiac pacemaker function.