Abstract
Spontaneous AP (action potential) 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 the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) 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; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. 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.
Highlights
IntroductionExchange current (INCX ), accelerating the rate of diastolic depolarization and leading to an increase in the spontaneous action potential (AP) firing rate (Figure 1)
PP2A transcript abundance in left ventricle cells (LVC) exceeded that in sinoatrial nodal cells (SANC) four-fold (Figure 2A)
The main functional effects of phosphorylation on both M and Ca2+ clock proteins is an increase in the size and an earlier occurrence of spontaneous ryanodine receptor (RyR)-generated local Ca2+ releases (LCRs)
Summary
Exchange current (INCX ), accelerating the rate of diastolic depolarization and leading to an increase in the spontaneous AP firing rate (Figure 1). Ca2+ cycling proteins and L-type Ca2+ channels by PKA and CaMKII in SANC is markedly higher than in ventricular myocytes (VM) and both (CaMKII and PKA) are required for the generation of basal rhythmic LCRs and spontaneous AP firing of SANC [5]. The system’s biochemical driver is cAMP, which is generated by Ca2+ -activated AC1and AC8 and leads to activation of PKA. PKA and CaMKII increase phosphorylation of clock proteins (black arrows). The cAMP level is kept in check by PDEs. The focus of the present study is to determine whether PPs, by keeping clock protein phosphorylation levels in check, form the double braking system with
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