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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.


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·x16.9029/(0.671316.9029+x16.9029).
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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·x16.9029/(0.671316.9029+x16.9029).

Mentions: Equation (3) models the AC activated by internal Ca2+ (denoted ) or by GPCR (denoted [AC]GPCR), which sense the nervous stimulations outside the cell and activate intracellular AC. Equation (4) breaks down the GPCR activation of the primary effector protein AC into its two components: positive activation caused by the stimulation of adrenergic receptors and inactivation caused by the stimulation of cholinergic receptors. Equation (5) expresses the positive activation and inactivation as a function of ISO, CCh, and the level of ATP. Finally, Equations (6, 7) give the rate of cAMP change as a function of the ATP that is converted into cAMP via Ca2+-AC or via GPCR, and the cAMP that is degraded through the production of PKA or by PDE. Brackets represent the concentration of a substance (e.g., [cAMP], [ATP]) or the level of activation of a substance (e.g., [PKA] ∈ [0 − 1], [PLBp] ∈ [0 − 1]). The terms for k1 − k5 are adapted from the YL model (Yaniv et al., 2013d, 2015a). The functions kiso and kcch are evaluated using the experimental relationships between the AP firing rate and cAMP (AP-cAMP) and cAMP and ATP (cAMP-ATP) (Yaniv et al., 2015a). In addition, to estimate kiso, experimental measurements from Yaniv et al. (2015a) (republished here in Figures 3A,B) are used to evaluate the relationship between the AP firing rate and the ISO level (AP-ISO). For kcch we used our new experimental measurements to evaluate the AP-CCh relationship (see Figures 3C,D). Because the data for the ISO and CCh experiments were obtained on cultured cells, which typically have a reduced basal AP firing rate with respect to fresh cells (Yang et al., 2012), the basal AP firing rate is assumed to be obtained for ISO = 1nM (13). The relationship between cAMP and PKA was adapted from the Saucerman et al. model (Saucerman et al., 2003). More details are available in the Supplementary Material.


The Autonomic Nervous System Regulates the Heart Rate through cAMP-PKA Dependent and Independent Coupled-Clock Pacemaker Cell Mechanisms
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·x16.9029/(0.671316.9029+x16.9029).
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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·x16.9029/(0.671316.9029+x16.9029).
Mentions: Equation (3) models the AC activated by internal Ca2+ (denoted ) or by GPCR (denoted [AC]GPCR), which sense the nervous stimulations outside the cell and activate intracellular AC. Equation (4) breaks down the GPCR activation of the primary effector protein AC into its two components: positive activation caused by the stimulation of adrenergic receptors and inactivation caused by the stimulation of cholinergic receptors. Equation (5) expresses the positive activation and inactivation as a function of ISO, CCh, and the level of ATP. Finally, Equations (6, 7) give the rate of cAMP change as a function of the ATP that is converted into cAMP via Ca2+-AC or via GPCR, and the cAMP that is degraded through the production of PKA or by PDE. Brackets represent the concentration of a substance (e.g., [cAMP], [ATP]) or the level of activation of a substance (e.g., [PKA] ∈ [0 − 1], [PLBp] ∈ [0 − 1]). The terms for k1 − k5 are adapted from the YL model (Yaniv et al., 2013d, 2015a). The functions kiso and kcch are evaluated using the experimental relationships between the AP firing rate and cAMP (AP-cAMP) and cAMP and ATP (cAMP-ATP) (Yaniv et al., 2015a). In addition, to estimate kiso, experimental measurements from Yaniv et al. (2015a) (republished here in Figures 3A,B) are used to evaluate the relationship between the AP firing rate and the ISO level (AP-ISO). For kcch we used our new experimental measurements to evaluate the AP-CCh relationship (see Figures 3C,D). Because the data for the ISO and CCh experiments were obtained on cultured cells, which typically have a reduced basal AP firing rate with respect to fresh cells (Yang et al., 2012), the basal AP firing rate is assumed to be obtained for ISO = 1nM (13). The relationship between cAMP and PKA was adapted from the Saucerman et al. model (Saucerman et al., 2003). More details are available in the Supplementary Material.

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.