Protein phosphatase1 regulates normal automaticity of heart pacemaker node cells

National Institute On Aging

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Abstract

Spontaneous AP firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for generation of spontaneous, diastolic local Ca2+ releases (LCRs). However, the expression pattern of phosphoprotein phosphatases (PP) and phosphatase inhibitors is unknown in SANC, and the impact of phosphatase inhibition on the spontaneous local Ca2+ releases (LCRs) and other players of the oscillatory coupled-clock system is also unknown. Our paper shows that rabbit SANC express both PP1 and PP2A, and PP Inhibitor-1 (I-1), Dopamine and cAMP-regulated phosphoprotein (DARPP-32), and these exceeded kinase C-enhanced PP1 inhibitor (KEPI). Furthermore, the PP1 inhibitor, Calyculin A, (CyA) applied to intact, freshly isolated single SANC, significantly increased phospholamban (PLB) phosphorylation (by 2-3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner, and increased RyR phosphorylation at the Ser2809 site. CyA substantially increased sarcoplasmic reticulum Ca2+ load, augmented L-type Ca2+ current amplitude, and augmented LCRs characteristics in intact and permeabilized SANC, decreased LCR period, and increased the spontaneous basal AP firing rate. The selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters or PLB phosphorylation. In permeabilized SANC, purified PP1 suppressed LCRs, whereas purified PP2A had no effect on LCR characteristics. A numerical model quantitatively predicted changes in SANC AP firing rate in response to PP inhibition. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.