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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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 (jSRCarel, f) and of the Ca2+-Na+ exchanger (INCX, c) after cAMP release from the cage. ICaL, L-type current; If, funny-current; INCX, the Na+-Ca2+ exchanger current; CajSR, the junctional sarcoplasmic reticulum Ca2+ concentration.
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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 (jSRCarel, f) and of the Ca2+-Na+ exchanger (INCX, c) after cAMP release from the cage. ICaL, L-type current; If, funny-current; INCX, the Na+-Ca2+ exchanger current; CajSR, the junctional sarcoplasmic reticulum Ca2+ concentration.

Mentions: We next tested whether, similar to autonomic modulation, direct alteration of intracellular AC-cAMP-PKA signaling resulted in changes in the magnitude and kinetics of the AP firing rate. Specifically, we wished to compare the kinetic responses of rapid changes in either neural stimulator of AC and Ca2+-calmodulin activated AC. To this end, we compared the changes in the dynamics of internal pacemaker mechanisms in response to internal stimulation by release of caged cAMP (50 μM) or to autonomic receptor stimulation by release of caged-ISO (3 μM). In contrast to perfusion with ISO, the release of cAMP from caged molecules produced an immediate response. Our model simulations were compared to the only reported experimental results on the effect of caged-cAMP or ISO on the AP firing rate (Tanaka et al., 1996). Figure 7A, shows the effect before, during, and after caged cAMP release, as well as before, during, and after application of ISO. During the same cycle, the AP firing rate instantaneously increased by 19% upon flash photolysis of caged-cAMP and by up to 19% with flash photolysis of caged ISO. Figure 7B shows a snapshot of action potential membrane voltage (Vm) and dVm/dt before and after flash release of caged cAMP as well as during caged-ISO release; in particular it can be observed that (dVm/dt)max increased after flash photolysis, thus showing that early DD was initiated more quickly after cAMP release. The same observation can be made for (dVm/dt)max during caged-ISO release. Figure 8 shows the main membrane currents (A-C), AC-cAMP-PKA signaling (D-E), and Ca2+ cycling (flux and concentration) in the sarcoplasmic reticulum (F-G) before and during cAMP release (Figure 8A) or ISO release (Figure 8B) from the caged molecule. In particular, there were important increases in the amount of Ca2+ released from RyR (jSRCarel, Figure 8f) and in the Ca2+-Na+ exchanger current (INCX, Figure 8c) after cAMP release from the cage. Shortly after the flash photolysis of caged cAMP, the different fluxes, currents and ion concentration revert to their basal values.


The Autonomic Nervous System Regulates the Heart Rate through cAMP-PKA Dependent and Independent Coupled-Clock Pacemaker Cell Mechanisms
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 (jSRCarel, f) and of the Ca2+-Na+ exchanger (INCX, c) after cAMP release from the cage. ICaL, L-type current; If, funny-current; INCX, the Na+-Ca2+ exchanger current; CajSR, the junctional sarcoplasmic reticulum Ca2+ concentration.
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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 (jSRCarel, f) and of the Ca2+-Na+ exchanger (INCX, c) after cAMP release from the cage. ICaL, L-type current; If, funny-current; INCX, the Na+-Ca2+ exchanger current; CajSR, the junctional sarcoplasmic reticulum Ca2+ concentration.
Mentions: We next tested whether, similar to autonomic modulation, direct alteration of intracellular AC-cAMP-PKA signaling resulted in changes in the magnitude and kinetics of the AP firing rate. Specifically, we wished to compare the kinetic responses of rapid changes in either neural stimulator of AC and Ca2+-calmodulin activated AC. To this end, we compared the changes in the dynamics of internal pacemaker mechanisms in response to internal stimulation by release of caged cAMP (50 μM) or to autonomic receptor stimulation by release of caged-ISO (3 μM). In contrast to perfusion with ISO, the release of cAMP from caged molecules produced an immediate response. Our model simulations were compared to the only reported experimental results on the effect of caged-cAMP or ISO on the AP firing rate (Tanaka et al., 1996). Figure 7A, shows the effect before, during, and after caged cAMP release, as well as before, during, and after application of ISO. During the same cycle, the AP firing rate instantaneously increased by 19% upon flash photolysis of caged-cAMP and by up to 19% with flash photolysis of caged ISO. Figure 7B shows a snapshot of action potential membrane voltage (Vm) and dVm/dt before and after flash release of caged cAMP as well as during caged-ISO release; in particular it can be observed that (dVm/dt)max increased after flash photolysis, thus showing that early DD was initiated more quickly after cAMP release. The same observation can be made for (dVm/dt)max during caged-ISO release. Figure 8 shows the main membrane currents (A-C), AC-cAMP-PKA signaling (D-E), and Ca2+ cycling (flux and concentration) in the sarcoplasmic reticulum (F-G) before and during cAMP release (Figure 8A) or ISO release (Figure 8B) from the caged molecule. In particular, there were important increases in the amount of Ca2+ released from RyR (jSRCarel, Figure 8f) and in the Ca2+-Na+ exchanger current (INCX, Figure 8c) after cAMP release from the cage. Shortly after the flash photolysis of caged cAMP, the different fluxes, currents and ion concentration revert to their basal values.

View Article: PubMed Central - PubMed

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.

No MeSH data available.