Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells

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.

High cardiomyocyte diversity in human early prenatal heart development

“…Such adaptations might prevent SAN CMs from becoming contracting fast-conducting atrial CMs. 38 , 42 In addition, expression of PPP1R1A, gene that encodes a cytoplasmic protein phosphatase inhibitor that modulates a clock mechanism that impacts on intrinsic automaticity of the SAN pacemaker cells, 43 was higher (p < 0.0001) in the SAN than in cluster 5.

Figure 2 Atrial CM subtype properties (A) Two-dimensional UMAP of atrial trabecular, conduit, and SAN CMs and heatmaps of relevant genes.

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Chinese natural compound decreases pacemaking of rabbit cardiac sinoatrial cells by targeting second messenger regulation of f-channels

“…cAMP synthesis is operated by the Ca 2+ -sensitive and Ca 2+ -insensitive adenylyl cyclase (AC1/8 and AC5/6, respectively), while cAMP conversion to AMP is catalyzed by the action of the PDE. AC and PDE are therefore central elements of a regulatory pathway that controls cell cAMP dynamics at rest and during autonomic stimulation ( Mika and Fischmeister, 2021 ; Robinson et al, 2021 ; Sirenko et al, 2021 ; Yaniv et al, 2015 ). According to St Clair et al, 2017 , PDE4 is particularly relevant in basal conditions, while PDE3 activity is important during β-adrenergic stimulation.…”

Compartmentalized cAMP signalling and control of cardiac rhythm

“…CaN also has been shown to interact with multiple AKAPs in the cardiac myocyte, including the plasmalemma anchored AKAP5 [ 95 ], the Z-line localized AKAP Cypher/Zasp [ 96 ], the mitochondrial AKAP1 [ 97 ] and mAKAP at the nuclear envelope [ 98 ]. Currently, limited information is available on the specific role of phosphatases in the regulation of β-AR signalling in SAN cells, although both PP1 [ 99 ] and PP2A [ 100 ] have been implicated. A significant basal phosphatase activity has been proposed to act in concert with PDEs activity to maintain a low phosphorylation level of clock molecules (see below) in the resting state, thus enabling a rapid, robust response when the β-ARs are activated.…”