Adenosine reduces sinoatrial node cell action potential firing rate by uncoupling its membrane and calcium clocks

Abstract

The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca²⁺ and membrane ion current clocks driven by Ca²⁺-calmodulin-activated adenylyl cyclase-protein kinase-A signaling. The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors (A₁, A₃) that couple to G_i protein-coupled receptors, reducing spontaneous AP firing rate via G_βγ signaling that activates I_KAch,Ado. Adenosine also inhibits adenylyl cyclase activity via G_αi signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to I_KAch,Ado activation, adenosine impacts also Ca²⁺ via G_αi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca²⁺ and membrane clock coupling. To this end, we measured Ca²⁺ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL (on average by 43%, n = 10) and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (n = 10) measured via membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (n = 40) measured via Ca²⁺ (assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca²⁺releases (LCRs) (Ca²⁺-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100 µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate G_βγ and I_KACh,Ado and to activate G_αi, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.

Keywords: adenosine; calcium release; cardiac arrhythmia; coupled-clock pacemaker system; sarcoplasmic reticulum (SR); sick sinus syndrome; sinoatrial node (SAN); sinus node arrest.

FIGURE 1

(A) Action potentials and AP-Induced Ca²⁺ transients are highly correlated in SANC in which both Ca²⁺ and V_m were simultaneously measured (17 cycles measured simultaneously). (B), example of a simultaneous V_m and Ca²⁺ recording. (C,D) Illustrate V_m and Ca²⁺ parameters measured in this study.

FIGURE 2

Adenosine slows the firing rate of rhythmically firing SANC in a dose-response manner. The IC₅₀ for our dose response curve was 31 µM adenosine. (n = 4 for 100 nm and 1 µM adenosine, n = 5 for 10 µM adenosine, and n = 7 for 100 µM adenosine). The data of firing rate response were normalized to be between 0% and 100% of baseline and fitted to Variable slope model using GraphPad program https://www.graphpad.com/guides/prism/latest/curve-fitting/reg_dr_inhibit_normalized_variable.htm. The equation for this fitting function was: Y = 100/(1 + 10^((LogIC50-X)*HillSlope)].

FIGURE 3

(A) an example of a cell that increased APCL from 577 ms at baseline to 1009 ms with adenosine and recovered to 597 ms with washout (washout not shown). (B) Statistical analysis of adenosine effect: SANC action potential cycle length (APCL) increased with adenosine and recovered with washout in SANC (n = 14). (C) APCL and coefficient of variation (CV) increased with adenosine. *p < 0.05 via one-way repeated measures ANOVA.

FIGURE 4

Time controls of Ca²⁺ and electrophysiology measurements show that SANC action potential cycle length (APCL) and coefficient of variation (CV) do not change with time. Repeated measures ANOVAs of Ca²⁺ and V_m measurements showed there was no significant time effect on SANC firing rate and CV within twenty minutes. Based on this data, all experiments were performed within twenty minutes.

FIGURE 5

(A) an example of a cell that increased APCL from 480 ms at baseline to 888 ms with adenosine and recovered to 648 ms with adenosine and recovered to 648 ms with washout (washout not shown). (B) SANC AP-induced Ca²⁺ transient cycle length increased with adenosine and recovered partially with washout. (n = 46). (C) SANC AP-Induced Ca²⁺ transient cycle length and coefficient of variation (CV) increased with adenosine; *p < 0.05 via one-way repeated measures ANOVA.

FIGURE 6

Histograms of LCR size (A), LCR duration (B), and mean LCR period (C) percentage distributions in all rhythmic SANC before and in the presence of adenosine (n = 34). Mean LCR period was the averaged LCR period of 3–7 cycles at baseline and with adenosine for each SANC. Mean LCR period also increased in response to adenosine; *p < 0.05 via one-way repeated measures ANOVA.

FIGURE 7

(A) an example of a rhythmically firing SANC that decreases in rate of LCR ensemble growth in response to adenosine. There is an overall decrease in global cytosolic Ca²⁺ with adenosine compared to baseline. Panel (B) depicts the average LCR ensemble growth rate of 5 cycles at baseline and with ado. The average LCR ensemble growth rate decreased to 42% of baseline with adenosine. This decrease reflects the changes in intracellular Ca²⁺ availability, manifesting in changes to LCR parameters. This results in an extended time period of Ca²⁺ cycling between AP-induced Ca²⁺ transients. Panel (C), the correlation between mean LCR Period (3–7 beats) and AP-induced Ca²⁺ transient cycle length is reduced at longer cycle lengths. At baseline, there is a strong correlation between APCL and LCR period, indicating robust clock coupling (n = 35). Adenosine, in the same population of cells, the mean APCL and LCR period increased for many cells. For a subset of cells, mean APCL increased more than mean LCR period, indicating reduced fidelity of clock coupling indicated by the orange circles that digress up and leftward from the linear trendline (dashed line). The linear trendline includes baseline and ado values. Panel (D), an example of a SANC with ado where LCR ensemble growth propagation was insufficient and failed to generate an AP, resulting in clock uncoupling and a “failed AP attempt”.

FIGURE 8

(A) simultaneous V_m and Ca²⁺ recordings of a rhythmically firing SANC at baseline that fires with decreased rhythmicity in response to 10 µM Ado. (B) the rate of LCR ensemble growth is steeper at baseline (blue line) and decreases in response to adenosine (magenta line). The asterisk indicates the baseline and ado cycle lengths measured in Figure 8A.

FIGURE 9

Relationships between key parameters of the coupled-clock system in control (baseline) and in the presence of adenosine in SANC in which Ca²⁺ and V_m were measured simultaneously. The LCR period informs on the APCL because the LCR period informs on the time-to-AP-ignition onset. Both Time-to-AP-ignition onset (A) and LCR period (B,C) and increase with adenosine.

FIGURE 10

(A) Phase V_m-Ca²⁺ relationship. (A) one AP cycle (* in Figure 5) prior to (baseline) and during adenosine (Ado) superfusion in which V_m and Ca²⁺ were measured simultaneously. (B) phase V_m-Ca²⁺ diagram depicting the relationship between V_m and global cytosolic Ca²⁺ during several AP cycles (electrochemical gradient oscillations) at baseline (blue) and with adenosine (orange); 4 consecutive cycles from Figure 5 are shown. Numbers 1–5 indicate the times during the AP cycles in Panel (A).

FIGURE 11

(A) Simplified schematic of the coupled system of membrane ion current oscillators and Ca²⁺ oscillators (Coupled-clock system) operative within sinoatrial nodal cells (SANC). The system provides robust and flexible AP firing rates. Constitutively active Ca²⁺-AC-cAMP-PKA signaling intrinsic to SANC, that is activated by Ca²⁺ oscillations, couples to an ensemble of electrogenic surface membrane molecules (current oscillators). AC, adenylyl cyclase; cAMP, cyclic AMP; PKA, protein kinase A; A₁R, adenosine A₁ receptor; G_αi, Gi protein alpha subunit; G_βγ, G_i protein beta gamma subunit; RyR, ryanodine receptor; SERCA, sarco-endoplasmic reticulum Ca²⁺-ATPase; PLB, phospholamban; SR, Sarcoplasmic reticulum; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; I_K, delayed rectifier K⁺ current; I_KACh,Ado, Acetylcholine/Adenosine-activated K⁺ current; I_f, HCN currents; I_CaL, L-type Ca²⁺ current; I_NCX, Na⁺/Ca²⁺ exchanger currents; and I_NaKATPase, Na⁺/K⁺ pump. (B) A schematic for SANC clock coupling/uncoupling. Clocks are coupled in the awake basal state, in response to physiologic stress (beta-adrenergic stimulation) coupling increases and beat-to-beat variability is reduced. In physiologic responses to vagal stimulation or adenosine, the clocks become partially uncoupled and the APCL becomes prolonged. These effects become more exaggerated as G_i coupled stimulation increases at higher drug concentrations.

FIGURE 12

Synergism of different component mechanisms in SANC rate slowing by ACh. Results of numerical model predictions for spontaneous AP rate reduction at various moderate ACh concentrations (up to 100 nM) for different mechanisms (labels). Since ACh and ado act via the same signaling mechanism, a similar result is expected for ado. Modified from (Maltsev and Lakatta, 2010).