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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.


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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).
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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).

Mentions: To find the dominant pacemaker mechanisms that mediate autonomic regulation crosstalk via cAMP- PKA signaling, we performed numerical simulations where some specific SR or membrane proteins were made PKA-phosphorylation insensitive (i.e., their degree of phosphorylation does not change under adrenergic or cholinergic stimulation and is maintained at the basal level). Figure 6 shows the results of the model simulations when varying the level of ISO and CCh treatment. PKA-dependent phosphorylation of L-type channels was predicted to play a relatively minor role in mediating the decrease in AP firing rate under low activation of ChR, although it was observed to gain relative importance as the concentration of CCh was increased. For CCh = 100 nM, the reduction in the AP firing rate was 22.7% when disabling ICaL modulation by PKA (purple curve in Figure 6B) in comparison to 25.9% with all the mechanisms active (thus a difference of 3.2%). Interestingly, under β-AR stimulation, “clamping” the phosphorylation of ICaL resulted in a 4.4% increase in AP firing rate with respect to when the phosphorylation modulation of ICaL was activated (yellow curve in Figure 6A).


The Autonomic Nervous System Regulates the Heart Rate through cAMP-PKA Dependent and Independent Coupled-Clock Pacemaker Cell Mechanisms
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).
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Related In: Results  -  Collection

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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).
Mentions: To find the dominant pacemaker mechanisms that mediate autonomic regulation crosstalk via cAMP- PKA signaling, we performed numerical simulations where some specific SR or membrane proteins were made PKA-phosphorylation insensitive (i.e., their degree of phosphorylation does not change under adrenergic or cholinergic stimulation and is maintained at the basal level). Figure 6 shows the results of the model simulations when varying the level of ISO and CCh treatment. PKA-dependent phosphorylation of L-type channels was predicted to play a relatively minor role in mediating the decrease in AP firing rate under low activation of ChR, although it was observed to gain relative importance as the concentration of CCh was increased. For CCh = 100 nM, the reduction in the AP firing rate was 22.7% when disabling ICaL modulation by PKA (purple curve in Figure 6B) in comparison to 25.9% with all the mechanisms active (thus a difference of 3.2%). Interestingly, under β-AR stimulation, “clamping” the phosphorylation of ICaL resulted in a 4.4% increase in AP firing rate with respect to when the phosphorylation modulation of ICaL was activated (yellow curve in Figure 6A).

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.


Related in: MedlinePlus