New evidence for coupled clock regulation of the normal automaticity of sinoatrial nodal pacemaker cells: bradycardic effects of ivabradine are linked to suppression of intracellular Ca²⁺ cycling

Abstract

Beneficial clinical bradycardic effects of ivabradine (IVA) have been interpreted solely on the basis of If inhibition, because IVA specifically inhibits If in sinoatrial nodal pacemaker cells (SANC). However, it has been recently hypothesized that SANC normal automaticity is regulated by crosstalk between an "M clock," the ensemble of surface membrane ion channels, and a "Ca(2+) clock," the sarcoplasmic reticulum (SR). We tested the hypothesis that crosstalk between the two clocks regulates SANC automaticity, and that indirect suppression of the Ca(2+) clock further contributes to IVA-induced bradycardia. IVA (3 μM) not only reduced If amplitude by 45 ± 6% in isolated rabbit SANC, but the IVA-induced slowing of the action potential (AP) firing rate was accompanied by reduced SR Ca(2+) load, slowed intracellular Ca(2+) cycling kinetics, and prolonged the period of spontaneous local Ca(2+) releases (LCRs) occurring during diastolic depolarization. Direct and specific inhibition of SERCA2 by cyclopiazonic acid (CPA) had effects similar to IVA on LCR period and AP cycle length. Specifically, the LCR period and AP cycle length shift toward longer times almost equally by either direct perturbations of the M clock (IVA) or the Ca(2+) clock (CPA), indicating that the LCR period reports the crosstalk between the clocks. Our numerical model simulations predict that entrainment between the two clocks that involves a reduction in INCX during diastolic depolarization is required to explain the experimentally AP firing rate reduction by IVA. In summary, our study provides new evidence that a coupled-clock system regulates normal cardiac pacemaker cell automaticity. Thus, IVA-induced bradycardia includes a suppression of both clocks within this system.

Keywords: 50% decay time of intracellular Ca(2+); 90% decay time of intracellular Ca(2+); AC; AP; Action potential; Adenylyl-cyclases; CPA; Ca(2+) cycling; Cyclopiazonic acid; IVA; Ion channels; Ivabradine; LCR; Local Ca(2+) release; M; MDP; Maximum diastolic depolarization; Membrane; PKA; PLB; Phospholamban; Physiology; Protein kinase A; SANC; SR; Sarcoplasmic reticulum; Sinoatrial nodal pacemaker cells; Sinoatrial-node cells; T-50(c); T-90(c).

Figure 1. Schematic illustrations of the coupled clock system

The interplay of Ca²⁺-calmodulin adenylyl-cyclases (AC), PDE activity and cAMP-mediated, protein kinase A (PKA)-dependent and Ca²⁺/calmodulin protein kinase II (CaMKII) signaling to the cardiac pacmaker sarcoplasmic reticulum Ca²⁺ cycling proteins, surface membrane ion channels and mitochondria.

Figure 2. Effect of IVA on I_f amplitude and activation kinetics

(A) Representative I_f traces in SANC recorded before and 5 min after application of IVA and (B) average peak I_f amplitude-voltage relationship (inset depicts I_f measured over the physiological voltage range), (C) representative I_f tail traces recorded in SANC before and after application of IVA and (D) I_f steady-state activation curve under control conditions, and in the presence of IVA (n=9). I_f tail current density at each membrane potential in control and with IVA is expressed relative to its maximal value at −120 mV.

Figure 3. Effect of IVA on spontaneous AP firing rate and spatiotemporal characteristics of LCRs

(A) Representative AP recordings and (B) average changes in the rate of AP firing in the presence of IVA (n=9). (C) Confocal line scan images of Ca²⁺ in a representative SANC before and following exposure to IVA. LCRs are indicated by arrowheads. The LCR period is defined as the time from the peak of the prior AP-induced Ca²⁺ transient to an LCR onset. (D) LCR size (full width at half-maximum amplitude) (n=12 cells, 123 LCR) and (E) A linear function describes the relationship of the 90% decay time of the AP-induced Ca²⁺ transient (T-90c) to the LCR period in control and in the presence of IVA. The line is the best least squares linear-regression fit to the data. *p<0.05 vs. drug.

Figure 4. SR load estimation from rapid caffeine application

Effects of a rapid application of caffeine (indicated by the arrow) onto a SANC in (A) control, or in the presence of 3 μM IVA. (B) Average effects of IVA on the amplitude of caffeine-induced cytosolic Ca²⁺ transient (n=12 in control and in response to IVA). Effects of a rapid application of caffeine onto a SANC in (C) control, or in the presence of CPA. (D) Average effects of CPA on the amplitude of the caffeine-induced cytosolic Ca²⁺ transient (n=9 for control and CPA). (Note that the caffeine response can be usually measured only once in a given SANC, because following caffeine application a prolonged period is required for AP firing rate to return to the control AP firing rate. Therefore, the effects of caffeine before (i.e., control) and following application of IVA are measured in different cells.) ^*p<0.05 vs. control.

Figure 5. Effect of CPA on spontaneous AP firing rate and spatiotemporal characteristics of LCRs

(A) Ca²⁺ confocal line scan images of a representative SANC before and following exposure to CPA, (B) LCR size in control (n=12 cells, 117 LCRs) and the presence of CPA (n=12 cells, 86 LCRs). (C) A linear function describes the relationship of the 90% decay time of the AP-induced Ca²⁺ transient (T-90c) to the LCR period in control and in the presence of CPA. The line is the best least squares linear-regression fit to the data. (D) Representative AP recordings and (E) average changes in the rate of AP firing in the presence of CPA (n=9). *p<0.05 vs. drug.

Figure 6. Effect of IVA and CPA on LCR period

The LCR period in control or in the presence of IVA (panel A) or CPA (panel B) predicts the concurrent AP cycle length. (C) Combined data in A and B for IVA and CPA conform to a least square fit of the data in A and B for IVA or CPA alone, respectively. Inset illustrates the average data points for control, IVA and CPA. (D) Data in panel C for IVA or CPA are expressed as % change from control prior to drug application in each cell. The IVA and CPA prolongation of the LCR period predict the concurrent prolongation of AP cycle length. The line is the best least squares linear-regression fit to the data. The dash line is the line of identity.

Figure 7. Effect of IVA on spontaneous LCR characteristics in saponin permeabilized SANC

(A) Ca²⁺ confocal line scan images of a representative SANC before and following exposure to IVA. Average effects of IVA on the (B) LCR duration, (C) LCR size and (D) LCR amplitude.

Figure 8. Coupled clock numerical model

Simulations of the effects of IVA on (A) I_f, (B) AP, (C) I_Ca.L, (D) cytosolic Ca²⁺, (E) junctional, (F) network SR Ca²⁺ and (G) sarolemmal Na⁺-Ca²⁺ exchanger current. Blue traces are model simulations prior to drug. Red traces are model simulations when feed-forward entrainment and with only partial feed-back between the M and Ca²⁺ clock. Green traces are model simulations when both feed-forward and full feed-back entrainments exist between the clocks. Note the dashed line represent the diastolic depolarization phase.

Figure 9

Numerical simulations of the relationships between membrane potential, I_f and sodium calcium exchange current during (A) and (C) one complete AP cycle; (B) and (D) during diastolic depolarization only. Note, that the ordinate scale in A and C differs from that in B and D.