The Autonomic Nervous System Regulates the Heart Rate through cAMP-PKA Dependent and Independent Coupled-Clock Pacemaker Cell Mechanisms

Abstract

Sinoatrial nodal cells (SANCs) generate spontaneous action potentials (APs) that control the cardiac rate. The brain modulates SANC automaticity, via the autonomic nervous system, by stimulating membrane receptors that activate (adrenergic) or inactivate (cholinergic) adenylyl cyclase (AC). However, these opposing afferents are not simply additive. We showed that activation of adrenergic signaling increases AC-cAMP/PKA signaling, which mediates the increase in the SANC AP firing rate (i.e., positive chronotropic modulation). However, there is a limited understanding of the underlying internal pacemaker mechanisms involved in the crosstalk between cholinergic receptors and the decrease in the SANC AP firing rate (i.e., negative chronotropic modulation). We hypothesize that changes in AC-cAMP/PKA activity are crucial for mediating either decrease or increase in the AP firing rate and that the change in rate is due to both internal and membrane mechanisms. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, PKA activity and AP firing rate were tightly linked in response to either adrenergic receptor stimulation (by isoproterenol, ISO) or cholinergic stimulation (by carbachol, CCh). To identify the main molecular targets that mediate between PKA signaling and pacemaker function, we developed a mechanistic computational model. The model includes a description of autonomic-nervous receptors, post- translation signaling cascades, membrane molecules, and internal pacemaker mechanisms. Yielding results similar to those of the experiments, the model simulations faithfully reproduce the changes in AP firing rate in response to CCh or ISO or a combination of both (i.e., accentuated antagonism). Eliminating AC-cAMP-PKA signaling abolished the core effect of autonomic receptor stimulation on the AP firing rate. Specifically, disabling the phospholamban modulation of the SERCA activity resulted in a significantly reduced effect of CCh and a failure to increase the AP firing rate under ISO stimulation. Directly activating internal pacemaker mechanisms led to a similar extent of changes in the AP firing rate with respect to brain receptor stimulation. Thus, Ca²⁺ and cAMP/PKA-dependent phosphorylation limits the rate and magnitude of chronotropic changes in the spontaneous AP firing rate.

Keywords: autonomic regulation; cAMP-PKA signaling; coupled clock system; mathematical model; pacemaker cells; phospholamban.

Figure 1

Schematic diagram of sinoatrial node mechanisms. Autonomic regulation via adenylyl cyclase-cyclic adenosine monophosphate-Protein kinase A (AC-cAMP-PKA) signaling: the internal pacemaker mechanisms are tightly coupled with cAMP/PKA signaling through the stimulation of G-protein-coupled receptors that activate (adrenergic receptors, β-AR) or inactivate (cholinergic receptors, ChR) AC as well as Ca²⁺-calmodulin activated AC. Only the main ion channels modulated in our model by AC-cAMP-PKA signaling are represented in the figure. SERCA, sarcoplasmic reticulum Ca²⁺ ATPase; PLB, phospholamban; RyR, ryanodine; PDE, phosphodiesterase; PPT, protein phosphatase; P, phosphate.

Figure 2

Schematic illustration of the AC-AC-cAMP-PKA signaling cascade. Adenylyl cyclase (AC) is activated by adrenergic receptors (β-AR) and calmodulin, and deactivated by cholinergic receptor (ChR) stimulation. Activated AC converts adenosine triphosphate (ATP) into cAMP, which itself is transformed into protein kinase A (PKA). PKA phosphorylates a number of targets, including phospholamban (PLB) proteins, whose phosphorylation level will regulate the activation of SERCA and thus the speed at which Ca²⁺ enters the SR. The model includes two restraining mechanisms that act like brakes: protein phosphatase (PPT), which removes phosphate groups from proteins, and phosphodiesterase (PDE), which breaks the phosphodiester bond in cAMP and degrades its level.

Figure 3

Experimental measurements of PKA activity and action potential (AP) firing rate as a function of sympathetic stimulation (via adrenergic receptors, A,B) using isoproterenol (ISO) and parasympathetic stimulation (via cholinergic receptors, C,D) using carbachol (CCh) in rabbit pacemaker cell. (A,C) Show representative examples, (B,D) present average data for n = 5 and n = 4 pacemaker cells (from 5 and 4 rabbits, respectively), respectively, with the vertical bars representing the standard errors. (E) Shows the relationship between the AP firing rate and PKA activity, as obtained from the ISO and CCh measurements. Equation for the curve fitting of the AP-PKA relationship: 26.4434+90.3024·x^16.9029/(0.6713^16.9029+x^16.9029).

Figure 4

Model simulations in response to sympathetic and parasympathetic stimulation. Membrane voltage, main membrane currents, AC-cAMP-PKA signaling, and Ca²⁺ cycling (flux and concentration) in the sarcoplasmic reticulum (SR) in the basal state or in response to sympathetic stimulation (via adrenergic receptors, red curve) using isoproterenol (ISO) or parasympathetic stimulation (via cholinergic receptors, green curve) using carbachol (CCh). The panels show: (A) The membrane voltage (V_m); (B) cAMP level; (C) PKA activity level; (D) the flux of Ca²⁺ exiting the sarcoplasmic reticulum (j_SRCarel); (E) Ca²⁺ concentration in the junctional sarcoplasmic reticulum compartment (Ca_jSR); (F) the Na⁺-Ca²⁺ exchanger current (I_NCX); (G) funny-current (I_f); (H) L-type current (I_CaL); and (I) muscarinic activated current (I_KACh).

Figure 5

Relationship between action potential (AP) firing rate and cAMP level. The relationship between action potential (AP) firing rate and cAMP, as predicted by the model (continuous line) in comparison to experimental results (crosses) from Yaniv et al. (2013d). The model predictions are obtained for varying quantities of ISO (orange curve) and CCh (blue curve). The concentrations of drugs used for the experiments were: isoproterenol (ISO, 1 μM), calyculin A (1 μM), blebbistatin (10 μM), caesium chloride (CsCl, 2 mM), cyclopiazonic acid (CPA, 5 μM; Yaniv et al., 2011).

Figure 6

Analysis of coupled-clock mechanisms. Action potential firing rate (% basal) change under the effect of adrenergic receptor (β-AR) stimulation by ISO (A) or cholinergic receptor (ChR) stimulation by CCh (B). In order to highlight the relative contribution of different system components, some mechanisms were virtually deactivated by disabling their modulation by PKA/cAMP. Of note, the RyR modulation by PKA is very minor in the current formulation of the model and thus is left intact for all the runs. SERCA: sarcoplasmic reticulum Ca²⁺ ATPase, PLB: phospholamban. (C) Representative western blots of PLB phosphorylated at serine¹⁶ site and total PLB in rabbit SANC in the basal state and following milrinone (50 μM), phosphodiesters inhibitor (IBMX, 100 μM), β-AR stimulation [0.1 μM (ISO1) or 1 μM (ISO2) isoproterenol], and PKA inhibitor (PKI, 10 μM; reproduce from Vinogradova et al., 2010). (D) Representative western blots of PLB phosphorylated at serine¹⁶ site and total PLB in rabbit SANC in the basal state and following graded concentrations of CCh (Lyashkov et al., 2009).

Figure 7

Positive chronotropic response of SANCs to flash photolysis of caged ISO or caged cAMP. (A) Change in action potential (AP) firing rate, cAMP, and PKA activity after release of the caged cAMP following the flash at t = 50 s. During the same cycle, the AP firing rate instantaneously increased upon flash photolysis of caged-cAMP or with flash photolysis of caged ISO. (B) Action potential and dV_m/dt before, during, and after release of the caged cAMP or caged ISO for a 2 s segment. Notice the higher number of cycles and higher maximum dV_m/dt during cAMP release or ISO treatment (in red).

Figure 8

Caged cAMP and ISO experiments. Effect of caged cAMP (A) and ISO (B) on cellular currents: main membrane currents (a–c), AC-cAMP-PKA signaling (d,e), and Ca²⁺ cycling (flux and concentration) in the sarcoplasmic reticulum before and during cAMP release from the cage (f,g). Notice the increase in the Ca²⁺ released from the RyR (j_SRCarel, f) and of the Ca²⁺-Na⁺ exchanger (I_NCX, c) after cAMP release from the cage. I_CaL, L-type current; I_f, funny-current; I_NCX, the Na⁺-Ca²⁺ exchanger current; Ca_jSR, the junctional sarcoplasmic reticulum Ca²⁺ concentration.

Figure 9

Sympathovagal compensation. Compensatory effects of cholinergic receptor (ChR) stimulation by CCh to adrenergic receptor (β-AR) stimulation by ISO. (A) Trend of the sympathovagal compensation curve, i.e., the concentrations of ISO and CCh that compensate for each other's effects on the action potential (AP) firing rate; (B) An example of β-AR stimulation by ISO starting at t = 900 s (10 nM) followed by ChR stimulation by CCh (88 nM) starting at t = 950 s. The AP firing rate returns to its basal rate after the addition of CCh.

Figure 10

Non additivity of adrenergic and cholinergic stimulation. (A) Simulating the response to 20 nM ISO. The AP firing rate increases by 13%. (B) Simulating the response to 80 nM of CCh. The AP firing rate decreases by 16%. (C) Simulating the response to 80 nM of CCh followed by the addition of 20 nM of ISO. The AP firing rate increases by 6%. (D) Simulating the response to 20 nM of ISO followed by 80 nM of CCh. The AP firing rate increases by 6%. These set of simulations show that the response to treatment by ISO and CCh is not equal to the simple addition of the individual ISO and CCh effect on the AP firing rate (13–16% = −3% ≠ 6%).

Figure 11

Phase dependency of the vagal effects. The phasic changes in pacemaker cycle length depend on the timing of the vagal stimulation during the AP cycle. To simulate this effect, flash release of 120 nM of caged CCh is simulated at different points of the action potential cycle [indicated by the arrows and markers S1–S4 in (A)]. Simulations when the release of caged CCh is performed at: S1, peak action potential (PAP); S2, the end of the repolarization (RP); S3, early diastolic depolarization (DD); S4, late DD. (A) Action potential variation as a function of caged CCh release upon flash. (B) Variation in cycle length for four cycles, starting one cycle before caged release of CCh. Flash lasts for 50 ms and, C_b = 120 nM, k_{CCh, off} = 0.11 0.3 ms⁻¹, k_{CCh, on} = 10e−7 mM⁻¹ ms⁻¹.