Abstract 17100: CaMKII Activity Modulates Basal Sarcoplasmic Reticulum Ca 2+ Cycling to Drive Normal Automaticity of Sinoatrial Node Cells

Abstract

Spontaneous beating of rabbit sinoatrial node cells (SANC) is linked to rhythmic, sarcoplasmic reticulum (SR) generated, submembrane local Ca 2+ releases (LCR) during diastolic depolarization (DD). LCRs activate inward Na + /Ca 2+ exchange current which imparts an exponential increase to the later part of DD and brings the membrane potential to threshold to fire an action potential. Although the crucial dependence of normal SANC automaticity on CaMKII signaling has previously been linked to an effect to facilitate recovery from inactivation of L-type Ca 2+ channels, there may also be direct CaMKII-dependent regulation of LCRs in SANC. Here we show that in freshly isolated rabbit SANC CaMKII inhibitor, KN-93 (0.3 μmol/L), but not its inactive analog KN-92, markedly suppresses LCRs (confocal microscopy, Ca 2+ indicator Fluo-3) reducing LCR number per each spontaneous cycle by 64% (from 1.1±0.1 to 0.4±0.1); LCR size by 32% (from 7.0±0.6 to 4.8±0.2 μm); and by markedly prolonging the LCR period (the interval between AP-induced Ca 2+ transient and subsequent LCR). Prolongation of the LCR period predicted the concomitant increase in spontaneous cycle length, indicating that CaMKII is vitally important for spontaneous SANC firing. To further delineate mechanism of CaMKII-dependent regulation of Ca 2+ cycling, avoiding interference of L-type Ca 2+ channels, we permeabilized SANC with saponin. Confocal linescan imaging of permeabilized SANC showed that KN-93, but not KN-92, decreased the LCR number by 62% (from 16.8±3.7 to 6.4±2.0 per 1sec×100μm) and LCR size by 25% (from 6.5±1.1 to 4.9±0.8 μm). The suppression of LCR parameters was associated with a ~12% reduction of the SR Ca 2+ load, assessed by a rapid application of caffeine on permeabilized SANC. The SR Ca 2+ load is controlled by phospholamban (PLB) which could be phosphorylated by CaMKII at Thr 17 site. Basal PLB phosphorylation at Thr 17 site, assessed by western blotting, is ~3 times greater in SANC than in ventricular myocytes, and KN-93, but not KN-92, markedly decreases PLB phosphorylation by ~40%. Thus, normal automaticity of SANC is regulated by basal CaMKII-dependent phosphorylation, in part, through modulation of SR Ca 2+ cycling, i.e. SR Ca 2+ load controlled by CaMKII-dependent phosphorylation of PLB.