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.

Keywords: L-type Ca2+ channels; calyculin A; endogenous phosphatase inhibitors; local Ca2+ releases; numerical model; okadaic acid; phospholamban; phosphoprotein phosphatase; ryanodine receptors; sinoatrial node cells.

Figure 1

Schematic diagram of the coupled-clock system that includes four major sub-systems or functional modules: the sarcoplasmic reticulum, the Ca²⁺ clock; sarcolemmal ion channels and transporters, the membrane clock; biochemical drivers; and the autonomic modulation of the system components. The Ca²⁺ clock cycles Ca²⁺ via SR Ca²⁺ pump (SERCA) and Ca²⁺ release channels (RyRs). The membrane clock generates APs and interacts with the Ca²⁺ clock via multiple Ca²⁺-dependent mechanism, including NCX current that accelerates the diastolic depolarization. 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 autonomic nervous system modulates the clock system via G protein-coupled receptor signaling. 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 PDEs. Abbreviations: I_K,_Ach, acetylcholine-activated K⁺ current; NCX, Na⁺/Ca²⁺ exchanger; I_CaL, L-type Ca²⁺ current; K⁺ channels, potassium channels; I_f, hyperpolarization-activated current; Ca²⁺, calcium ions; LCRs, local submembrane Ca²⁺ releases; RyR, ryanodine receptors; SR, sarcoplasmic reticulum; SERCA, SR Ca²⁺ ATPase; CaMKII, calcium-calmodulin-dependent protein kinase II; PLM, phospholemman; PLB, phospholamban; P, phosphorylation; PKA, protein kinase A; cAMP, cyclic adenosine 3′,5′-monophosphate; AC, adenylyl cyclase; G_gsα, G-protein coupled receptors stimulatory alpha subunit; G_iα, G-protein coupled receptors inhibitory alpha subunit; G_iαβγ, G-protein coupled receptors inhibitory alpha, betta, gamma subunits; G_βγ, G-protein coupled receptors beta, gamma subunits; PPs, phosphoprotein phosphatases; PPIs, phosphoprotein phosphatase inhibitors; PDEs, phosphodiesterases.

Figure 2

mRNA expression (A,B) and protein abundance (C,D) (Mean+/−SEM) patterns of PP1, PP2A, and PP1 endogenous inhibitors (I-1, KEPI and DARPP-32) normalized to β-tubulin (mRNA) or α-actinin in LVC and SANC (n = 6 independent hearts analyzed per group); * p < 0.05, ** p < 0.01 compared to LVC. For alternative transcript names see Table S1. For Primers and probes used for RT-QPCR see Table S2.

Figure 3

Average effects of CyA on the magnitude of SANC PLB phosphorylation at PKA-dependent Ser¹⁶ and CaMKII-dependent Thr¹⁷ sites. (A,B) Concentration dependence (30 min CyA treatment, n = 4). (C,D) Rates of phosphorylation in response to 100 nmol/L CyA. Representative Western blots of phosphorylated PLB and total PLB are presented in the upper part of each panel. Number of samples is shown in each column. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Figure 4

(A,B) Representative confocal line-scan images and (C) a fast Fourier transform (FFT) of Ca²⁺ oscillations in a permeabilized SANC bathed in 150 nmol/L [Ca²⁺] in control conditions and during superfusion with 500 nmol/L CyA. (D–H) Average changes in local Ca²⁺ release characteristics (LCRs) in control and during superfusion with 500 nmol/L CyA (n = 6): (D) Number of LCR events per 100 µm of the line-scan image and during a 1-sec time interval; (E) LCR size (as FWHM, the full width at half-maximum amplitude); (F) LCR duration (as FDHM, the full duration at half-maximum amplitude); (G) Ca²⁺ signals of Individual LCRs (µmxmsxΔCa²⁺ nmol/L); (H) Ca²⁺ signals of the LCR ensemble (the integrated Ca²⁺ signal of all LCRs), * p < 0.05, ** p < 0.01 paired t-test vs control, n = 6. Representative confocal images (I,J) and the average amplitude of the SR Ca²⁺ release (K) induced by a rapid spritz application of 20 mM caffeine in permeabilized SANC during 500 nmol/L CyA superfusion (n = 12) and in control (n = 7). * p < 0.05, 2-tailed t-test.

Figure 5

Representative confocal line-scan images of LCRs in permeabilized SANC bathed in 150 nmol/L [Ca²⁺] in control conditions and during incubation with purified PP1 (5 U mL⁻¹) (A) or PP2A (5 U mL⁻¹) catalytic subunits (F). Changes in local Ca²⁺ release (LCRs) characteristics before and during exposure to PP1 (n = 4) (B–E) or PP2A (n = 6) (G–J); (B,G) Number of LCR events per 100 µm of the line-scan image and during a 1-sec time interval; (C,H) LCR amplitude (F/F₀); (D,I) LCR size (as FWHM, the full width at half-maximum amplitude); (E,J) Average ensemble of LCR Ca²⁺ signals (as log of the integrated Ca²⁺ signal produced by each LCR; #-represents number of LCR events). * p < 0.05, the paired t-test vs. control (B–E); one way ANOVA (G–J).

Figure 6

Effects of suppression of PP activity by CyA or OKA on spontaneous beating of isolated rabbit SANC. (A) Representative example of APs recorded in SANC before and during superfusion with 100 nmol/L CyA and following washout of CyA. (B) Representative example of SANC APs recorded prior to and during superfusion with 100 nmol/L OKA and after OKA washout. (C) The average increase in the spontaneous AP firing rate did not significantly differ in response to 100 (n = 10), 300 (n = 5) or 500 nmol/L (n = 6) of CyA. (D) Average spontaneous AP firing rate responses to OKA (n = 6). * p < 0.05 vs. control.

Figure 7

PP inhibition by CyA increases the LCR number and size of LCRs in SANC during spontaneous AP firing. (A) Confocal line-scan image of a representative SANC prior to and during exposure to 100 nmol/L CyA; CyA-induced changes in: (B) LCR period; (C) LCR number per cycle; (D) LCR size (n = 12). (E) PP inhibition-induced changes in the LCR periods predict the concurrent changes in the AP cycle lengths (n = 12). * p < 0.05 vs. control.

Figure 8

PP inhibition-induced potentiation of L-type Ca²⁺ current amplitude. (A) The voltage clamp protocol and representative original L-type Ca²⁺ current recordings prior to and during PP inhibition by CyA. Average effects of CyA on the amplitude (B) and current-voltage relationship (C) of I_Ca in SANC (n = 9). L-type Ca²⁺ current amplitudes were normalized to membrane capacitance. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 9

Numerical model simulations of the experimentally defined effects of protein phosphatase inhibition by CyA on spontaneous AP firing rate of rabbit SANC. (A) A numerical solution in which the maximum SR Ca²⁺ pumping rate, P_up was increased to 24.25 mM/s, reproduced the experimentally observed increase in AP firing rate by 25% in the presence of 100 nM of CyA. (B) Simulations of the membrane potential (V_m), NCX current (I_NCX), L-type Ca²⁺ current (I_CaL), and Ca²⁺ release flux (j_SRCarel) before (dashed line) and during CyA application (CyA, solid line).