Modeling the effects of β1-adrenergic receptor blockers and polymorphisms on cardiac myocyte Ca2+ handling

Abstract

β-Adrenergic receptor blockers (β-blockers) are commonly used to treat heart failure, but the biologic mechanisms governing their efficacy are still poorly understood. The complexity of β-adrenergic signaling coupled with the influence of receptor polymorphisms makes it difficult to intuit the effect of β-blockers on cardiac physiology. While some studies indicate that β-blockers are efficacious by inhibiting β-adrenergic signaling, other studies suggest that they work by maintaining β-adrenergic responsiveness. Here, we use a systems pharmacology approach to test the hypothesis that in ventricular myocytes, these two apparently conflicting mechanisms for β-blocker efficacy can occur concurrently. We extended a computational model of the β(1)-adrenergic pathway and excitation-contraction coupling to include detailed receptor interactions for 19 ligands. Model predictions, validated with Ca(2+) and Förster resonance energy transfer imaging of adult rat ventricular myocytes, surprisingly suggest that β-blockers can both inhibit and maintain signaling depending on the magnitude of receptor stimulation. The balance of inhibition and maintenance of β(1)-adrenergic signaling is predicted to depend on the specific β-blocker (with greater responsiveness for metoprolol than carvedilol) and β(1)-adrenergic receptor Arg389Gly polymorphisms.

Fig. 1.

Extended ternary complex model of the β₁-adrenergic receptor, coupled with the β₁-adrenergic pathway and ventricular myocyte excitation-contraction coupling. K_L, equilibrium dissociation constant of the agonist receptor complex; K_R, propensity for switching between active and inactive receptor states; K_G, dissociation constant for binding of G-protein to the receptor; α, differential affinity of the ligand for the inactive receptor; γ, differential affinity of the ligand-receptor complex for G-protein.

Fig. 2.

Experimental validation of coupled β₁-adrenergic signaling and excitation-contraction coupling model. (A) Model reproduces shift in agonist binding affinity in the presence of guanosine 5′-[β,γ-imido] triphosphate (GPP), which displaces G_s from the receptor. (B) Kinetics of [cAMP] in response to 10 nM isoproterenol (ISO) stimulation. (C) cAMP dose response to ISO. (D) PKA activity measured by FRET reporter AKAR3. (E) Phospholamban phosphorylation in response to ISO. (F) Ca²⁺ dose response to ISO. Results in (A)–(C), (E), and (F) show direct comparison with published experimental data (Mason et al., 1999), (Vila Petroff et al., 2001), (De Arcangelis et al., 2010), (Vittone et al., 1998), and (Collins and Rodrigo, 2010), whereas data in (D) and (F) were acquired in the current study.

Fig. 3.

Propranolol both inhibits and maintains β₁-adrenergic-mediated regulation of Ca²⁺ transients. (A) Model-predicted individual Ca²⁺ transients in response to increasing concentration of isoproterenol (ISO). (B) Ca²⁺ concentration increased in response to 0.1 µM ISO, with no further response to subsequent stimulation with 10 µM ISO. (C) The model predicted that propranolol (PRO) inhibits response to 0.1 μM ISO, but the responsiveness to 10 μM ISO is maintained (large sensitivity). (D) Individual Ca²⁺ transients as measured by fluo-4 from rat ventricular myocytes exposed to increasing [ISO]; scale bar 20 µm. (E) Similar to model predictions, myocytes were not responsive to further stimulation with 10 µM ISO. (F) PRO inhibited response to 0.1 μM ISO, but myocytes were responsive to further stimulation with 10 µM ISO. Sensitivity was quantified as the increase in Ca²⁺ transient magnitude when increasing from 0.1 µM ISO (analogous to chronically elevated catecholamines in heart failure) to 10 µM ISO (analogous to exercise).

Fig. 4.

Propranolol both inhibits and maintains the β₁-adrenergic-mediated Ca²⁺ and PKA response after 24-hour isoproterenol (ISO) pretreatment. (A) Expression and cytosolic distribution of PKA activity biosensor AKAR3 in rat adult ventricular myocytes (YFP emission); scale bar 40 µM. Following 24-hour pretreatment with both 0.1 µM ISO and 0.1 µM propranolol (PRO), both (B) PKA activity measured by AKAR3 and (C) Ca²⁺ response as measured by fluo-4 were still sensitive to a subsequent increase to 10 µM ISO.

Fig. 5.

Ligand-binding affinity and inverse agonism were both predicted to influence ligand cAMP sensitivity. (A) In silico screen of 19 β₁-adrenergic ligands predicts differential cAMP sensitivity. (B) Effect of ligand dissociation constant (K_L) on predicted cAMP sensitivity. (C) Effect of ligand inverse agonism (α) on predicted cAMP sensitivity. Propranolol (PRO), metoprolol, and carvedilol (highlighted in red) were predicted to have both distinct effects on cAMP sensitivity with distinct combinations of ligand dissociation constant and inverse agonism.

Fig. 6.

Metoprolol and carvedilol differentially influence β₁-adrenergic responsiveness. (A) In adult ventricular myocytes, metoprolol (MET) blocked response to 0.1 μM isoproterenol (ISO), but the responsiveness to 10 μM ISO was maintained. (B) Carvedilol (CAR) blocked the response to both 0.1 μM ISO and 10 μM ISO. (C) Summary of model-predicted Ca²⁺ responses to 0.1 µM and 10 µM ISO in the presence of β-blockers. MET and propranolol (PRO) were both predicted to substantially enhance cAMP sensitivity to 10 µM ISO, but CAR was not. (D) Summary of experimental validations from adult ventricular myocytes for PRO, MET, and CAR.

Fig. 7.

β₁AR-Arg389 polymorphism responds differently to β-blockers. (A) Model reproduces the shift in agonist binding affinity in the presence of guanosine 5′-[β,γ-imido] triphosphate for Arg389. (B) Concentration dependence of adenylyl cyclase (AC) activity to isoproterenol for Gly389 and Arg389. (C) Arg389 is predicted to have higher cAMP sensitivity and Ca²⁺ response versus Gly 389 in cardiac myocytes. (D) In silico screen of 19 β₁-adrenergic ligands against Arg389. (E) Differential cAMP sensitivity between Arg389 and Gly389 for different β₁-adrenergic ligands predicted for cardiac myocytes. Experimental data in panels (A) and (B) are from (Mason et al., 1999; Mialet Perez et al., 2003).