Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2+ cycling proteins of sinoatrial node cells (SANC) is higher than in left ventricular myocytes (LVC) and is crucial for SANC basal normal automaticity. We hypothesized that the activity of Ser/Thr phosphoprotein phosphatases (PP) PP1 and PP2A or endogenous phosphatase inhibitors limits the basal action potential (AP) firing rate. SANC transcript abundance (RT-qPCR) and protein expression (western blot) for PP1 equaled to these of PP2A. Inhibitor-1 (I-1), and Dopamine- and cAMP-regulated phosphoprotein (DARPP-32) RNA expression and protein levels in SANC were similar and exceeded those of kinase Cenhanced PP1 inhibitor (KEPI). Inhibition of both PP1 and PP2A activities by Calyculin A (CyA) in intact freshly isolated SANC significantly increased phospholamban (PLB) phosphorylation (by 2-3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites and increased RyR phosphorylation at Ser2809 site. In single rabbit SANC, CyA (100 nmol/L) decreased the spontaneous SANC cycle length and increased the average spontaneous SANC firing rate (perforated patch-clamp technique) by 30%. CyA substantially increased diastolic local Ca2+ release (LCR) number and size (confocal line-scan imaging) and reduced the LCR period (time from the action potential induced Ca2+ transient to subsequent LCR). The CyA-induced increase in LCR parameters was accompanied by an increase in RyR phosphorylation at Ser2809 site, concurrent augmentation of L-type Ca2+ current amplitude and elevation of sarcoplasmic reticulum Ca2+ load. The selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on SANC spontaneous AP firing, LCR parameters or PLB phosphorylation. In permeabilized SANC Cy A markedly increased LCR number and size and purified PPI suppressed LCRs, while neither okadaic acid nor purified PP2A affected LCR characteristics. A numerical model, fine-tuned by experimentally observed changes in ICaL, PLB phosphorylation and changes in SR Ca2+ pumping produced by PP inhibition in SANC quantitatively predicted changes in SANC AP firing rate in response to PP inhibition. Thus, basal PP activity in SANC is regulated by PP1 which modulates the basal spontaneous firing rate of SANC via suppression of SR Ca2+ cycling protein phosphorylation, SR-generated LCRs and L-type Ca2+ current.
| Sirenko, Syevda; Maltsev, Victor A; Maltseva, Larissa A et al. (2014) Sarcoplasmic reticulum Ca2+ cycling protein phosphorylation in a physiologic Ca2+ milieu unleashes a high-power, rhythmic Ca2+ clock in ventricular myocytes: relevance to arrhythmias and bio-pacemaker design. J Mol Cell Cardiol 66:106-15 |
