Compartmentation of cAMP signaling in cardiac myocytes: a computational study

Abstract

Receptor-mediated changes in cAMP production play an essential role in sympathetic and parasympathetic regulation of the electrical, mechanical, and metabolic activity of cardiac myocytes. However, responses to receptor activation cannot be easily ascribed to a uniform increase or decrease in cAMP activity throughout the entire cell. In this study, we used a computational approach to test the hypothesis that in cardiac ventricular myocytes the effects of beta(1)-adrenergic receptor (beta(1)AR) and M(2) muscarinic receptor (M(2)R) activation involve compartmentation of cAMP. A model consisting of two submembrane (caveolar and extracaveolar) microdomains and one bulk cytosolic domain was created using published information on the location of beta(1)ARs and M(2)Rs, as well as the location of stimulatory (G(s)) and inhibitory (G(i)) G-proteins, adenylyl cyclase isoforms inhibited (AC5/6) and stimulated (AC4/7) by G(i), and multiple phosphodiesterase isoforms (PDE2, PDE3, and PDE4). Results obtained with the model indicate that: 1), bulk basal cAMP can be high ( approximately 1 microM) and only modestly stimulated by beta(1)AR activation ( approximately 2 microM), but caveolar cAMP varies in a range more appropriate for regulation of protein kinase A ( approximately 100 nM to approximately 2 microM); 2), M(2)R activation strongly reduces the beta(1)AR-induced increases in caveolar cAMP, with less effect on bulk cAMP; and 3), during weak beta(1)AR stimulation, M(2)R activation not only reduces caveolar cAMP, but also produces a rebound increase in caveolar cAMP following termination of M(2)R activity. We conclude that compartmentation of cAMP can provide a quantitative explanation for several aspects of cardiac signaling.

FIGURE 1

Compartmentation of cAMP signaling pathways in a cardiac ventricular myocyte. β₁-adrenergic receptor (β₁); M₂ muscarinic receptor (M₂); stimulatory (G_s) and inhibitory G-proteins (G_i); adenylyl cyclase type 5 or 6 (AC5/6) and 4 or 7 (AC4/7); phosphodiesterase type 2 (PDE2), 3 (PDE3), and 4 (PDE4); cAMP flux between caveolar and extracaveolar (J_Cav/Ecav), extracaveolar and bulk cytosolic (J_Ecav/Cyt), and caveolar and bulk cytosolic (J_Cav/Cyt) compartments.

FIGURE 2

Effect of β₁-adrenergic receptor activation on cAMP kinetics. Effect of submaximally (3 nM) and maximally (100 nM) stimulating concentrations of isoproterenol on the time course of changes in cAMP concentration. (A) Average concentration of cAMP in all compartments (total cAMP, simulation). (B) Concentration of cAMP in the caveolar compartment (caveolar cAMP, simulation). (C) Dependence of total and caveolar cAMP (simulations) on concentration of isoproterenol used to stimulate β₁-adrenergic receptors.

FIGURE 3

Simulation of β₁-adrenergic responses. (A) In vivo activation of type II protein kinase A (PKA) by submaximally (0.3 nM) and maximally (1 μM) stimulating concentrations of isoproterenol (experimental). Increase in PKA activity measured as change in FRET response (ΔR/R₀) of PKA-based biosensor expressed in an adult ventricular myocyte. Data from Warrier et al. (47). (B) cAMP sensitivity of PKA-based biosensor in vitro. Data from Mongillo et al. (4). (C) Predicted response of PKA-based biosensor in a ventricular myocyte exposed to submaximally and maximally stimulating concentrations of isoproterenol (simulation). (D) Predicted isoproterenol sensitivity of PKA-based biosensor in vivo (simulation).

FIGURE 4

Simulation of muscarinic inhibition of β₁-adrenergic response. Effect of acetylcholine ((ACh) 10 μM) on the response to a maximally stimulating concentration (200 nM) of isoproterenol (Iso). (A) In vivo PKA activity measured as change in FRET response (ΔR/R₀) of PKA-based biosensor expressed in an adult ventricular myocyte (experimental). Data from Warrier et al. (47). (B) Predicted response of PKA-based biosensor in a ventricular myocyte (simulation). (C) Average concentration of cAMP in all compartments (total cAMP, simulation). (D) Concentration of cAMP in the caveolar compartment (caveolar cAMP, simulation).

FIGURE 5

Simulation of muscarinic stimulatory response. Rebound stimulatory effect observed following transient exposure to acetylcholine ((ACh) 10 μM) in the presence of a submaximally stimulating concentration (0.1 nM) of isoproterenol (Iso). (A) In vivo PKA activity measured as change in FRET response (ΔR/R₀) of PKA-based biosensor expressed in an adult ventricular myocyte (experimental). Data from Warrier et al. (47). (B) Predicted response of PKA-based biosensor in a ventricular myocyte (simulation). (Dashed line) Predicted response if biosensor were able to detect decreases in cAMP activity below basal levels (see Appendix II).

FIGURE 6

Kinetics of changes in cAMP activity associated with muscarinic stimulatory response. Changes in caveolar and extracaveolar cAMP production and concentration caused by transient exposure to acetylcholine ((ACh) 10 μM) in the presence of a submaximally stimulating concentration (0.1 nM) of isoproterenol (Iso) (simulations). (A) Concentration of cAMP in the caveolar compartment. (B) Rate of cAMP concentration change in the caveolar compartment due to the activity of AC5/6. (C) Rate of cAMP concentration change in the caveolar compartment (Cav) due to the flux from the extracaveolar compartment (Ecav). (D) Concentration of cAMP in the extracaveolar compartment. (E) Rate of cAMP concentration change in the extracaveolar compartment due to the activity of AC4/7.

FIGURE 7

Simulation of M₂ muscarinic responses in the absence of β₁-adrenergic stimulation. (A) Effect of a maximally stimulating concentration of acetylcholine on the activity of the PKA-based biosensor expressed in an adult ventricular myocyte (experimental). Data from Warrier et al. (47). (B) Predicted response of PKA-based biosensor in a ventricular myocyte exposed to a maximally stimulating concentration of acetylcholine (simulation). (Dashed line) Predicted response if biosensor were able to detect decreases in cAMP activity below basal levels. (C) Change in total cAMP concentration in response to a maximally stimulating concentration of acetylcholine (simulation). (D) Change in caveolar cAMP concentration in response to a maximally stimulating concentration of acetylcholine (simulation).