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.

Ca ²⁺ and V _m transitions occurring throughout AP cycles in sinoatrial nodal (SAN) cells are cues that: (1) not only regulate activation states of molecules operating within criticality (Ca ²⁺ domain) and limit-cycle (V _m domain) mechanisms of a coupled-clock system that underlies SAN cell automaticity; (2) but are also regulated by the activation states of the clock molecules they regulate. In other terms, these cues are both causes and effects of clock molecular activation (recursion). Recently, we demonstrated that Ca ²⁺ and V _m transitions during AP cycles in single SAN cells isolated from mice, guinea pigs, rabbits and humans are self-similar (obey a power law) and are also self-similar to trans-species AP firing intervals of these cells in vitro , to heart rate in vivo , and to body mass. Neurotransmitter stimulation of β adrenergic receptor or cholinergic receptor initiated signaling in SAN cells modulates their AP firing rate and rhythm by impacting on the degree to which SAN clocks couple to each other, creating the broad physiologic range of SAN cell mean AP firing intervals and firing interval variabilities. Here we show that Ca ²⁺ and V _m domain kinetic transitions (time to AP ignition in diastole and 90% AP recovery) occurring within given AP, the mean AP firing intervals, and AP firing interval variabilities within time-series of APs in 230 individual SAN cells are self-similar (obey power laws). In other terms, these long-range correlations inform on self-similar distributions of order among SAN cells across the entire broad physiologic range of SAN AP firing intervals, regardless of whether autonomic receptors of these cells are stimulated or not, and regardless of the type (adrenergic or cholinergic) of autonomic receptor stimulation. These long-range correlations among distributions of Ca ²⁺ and V _m kinetic functions that regulate SAN cell clock coupling during each AP cycle in different individual , isolated SAN cells not in contact with each other. Our numerical model simulations further extended our perspectives to the molecular scale and demonstrated that many ion currents also behave self-similar across autonomic states. Thus, to ensure rapid flexibility of AP firing rates in response to different types and degrees of autonomic input, nature “did not reinvent molecular wheels within the coupled-clock system of pacemaker cells”, but differentially engaged or scaled the kinetics of gears that regulate the rate and rhythm at which the “wheels spin” in a given autonomic input context.