A full range of mouse sinoatrial node AP firing rates requires protein kinase A-dependent calcium signaling

Abstract

Recent perspectives on sinoatrial nodal cell (SANC)(*) function indicate that spontaneous sarcoplasmic reticulum (SR) Ca(2+) cycling, i.e. an intracellular "Ca(2+) clock," driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules ("surface membrane clock") to drive SANC normal automaticity. The role of AC-cAMP-PKA-Ca(2+) signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca(2+) cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca(2+) releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca(2+)]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by β-adrenergic receptor (β-AR) stimulation requires normal intracellular Ca(2+) cycling, because the β-AR chronotropic effect is markedly blunted when SR Ca(2+) cycling is disrupted. Thus, AC-cAMP-PKA-Ca(2+) signaling cascade is a major mechanism of normal automaticity in mouse SANC.

Figure 1

Molecular phenotype of Ca²⁺ cycling proteins in mouse SAN/SANC. A. Abundant expressions of RyR2, SERCA2 and NCX1 protein are found in both SAN region and neighboring atrium (scale=50 μm); B. Western blots of SAN, atrial and ventricular tissue for RyR2, SERCA2a and NCX1 confirm abundant quantities are expressed in SAN compared to contracting myocardium. Note that SAN strips pooled from mice are required for western blots.

Figure 2

Isolated mouse SANC with positive immuno-labeling of SERCA2 (A), NCX1 (B), RyR2 and HCN4 (C), Cav-3 (D) with their specific patterns. Note SANC are negative for Cx43 (A) and HCN4 (green) and RyR2 (red) co-localize in SANC (C) sub-sarcolemmal area (scale=20 μm).

Figure 3

SAN pacemaking function depends on intact SR Ca²⁺ function. Change of spontaneous beating SAN rate under: removal of calcium by Ca²⁺-free Tyrode solution (A); depleting SR Ca²⁺ content by 20 mM caffeine (B); inhibition of Ca²⁺ releasing from SR by ryanodine (C); inhibition of Ca²⁺ pumping into SR by CPA (D).

Figure 4

AC-cAMP-PKA dependent PLB phosphorylation in mouse SANC. A: Immuno-reactivity change of PLB-PS16 (green) and PLN total (red) in untreated SANC (basal, A), PKA inhibition (PKI, B and H89, C), AC inhibition (MDL, D), PDE inhibition (IBMX, E) and β-AR stimulation (ISO, F), scale = 20 μm.

Figure 5

A. The average change in PS-16/total PLB ratio is shown in the bar graph. B. The relationship of changes in beating rate to changes in PLB16 phosphorylation in response to β-AR stimulation, PDE inhibition or PKA inhibition comparing to the basal level.

Figure 6

Basal cAMP-PKA signaling drives rhythmic spontaneous basal local SR Ca²⁺ releases in permeabilized mouse SANC. A. Confocal linescan images of a representative skinned mouse SANC bathed in 100 nM free Ca²⁺ before (top) and during superfusion with 15 μM PKI (bottom). B. Power spectrum of a continuous 4 second recording of LCRs (the average signal in A between two arrows) in panel A. C. Autocorrelation function (right panels) and comparison of average LCR characteristics in control conditions and during application of 15 μM PKI and 1 μM of cAMP in the presence of 15 μM PKI: number (normalized per 1 second and 100 μm); amplitude (F/F0); size (FWHM), measured as full width at half maximum amplitude; total integrated LCR signal mass (product of the LCR size, amplitude, duration, (see online supplement for details), rhythmicity index (RI) (estimated from the amplitude of the third peak of the autocorrelation function). Bars are mean±SEM, *P<0.05 PKI vs. control.

Figure 7

PDE inhibition accelerates rhythmic spontaneous basal local SR Ca²⁺ releases in permeabilized mouse SANC. A, Confocal linescan images of a representative skinned mouse SANC bathed in 100 nM free Ca²⁺ before (top) and during exposure to 5 μM IBMX (bottom). B, Power spectrum of a continuous 4-s-long recording of LCRs (for the average signal between two arrows in panel A. C, Autocorrelation function and comparison of average LCR characteristics in control conditions and after 5 μM IBMX: A crude index to estimate normalized number of activated RyR within an LCR (see the supplementary method); size (FWHM), measured as full width at half maximum amplitude; duration (FDHM) measured as the full duration at half-maximum amplitude; total integrated LCR signal mass (normalized sum of size, amplitude, duration, see the method part), rhythmicity index (RI) (estimated from the amplitude of the third peak of the autocorrelation function). Bars are mean±SEM, *P<0.05.

Figure 8

Augmentation of beating rate in response to an increase in cAMP/PKA activation requires intact SR function. Beating rate increase in response to IBMX (A) and is significantly blunted in the presence of inhibition of SR Ca²⁺ pumping by CPA (B and C). The average responses in beating rate to IBMX or ISO in the presence or absence of CPA or ryanodine (D).