beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current

Abstract

beta3-adrenergic receptor (beta3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of beta3-AR in the human atrium. Unexpectedly, beta3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (I Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I(Ca,L) and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective beta3-AR agonists, SR58611 and BRL37344, and a beta3-AR partial agonist, CGP12177, stimulated I(Ca,L) in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The beta3-AR agonists also increased contractility but with a much lower efficacy (approximately 10%) than isoprenaline. The beta3-AR antagonist L-748,337, beta1-/beta2-AR antagonist nadolol, and beta1-/beta2-/beta3-AR antagonist bupranolol were used to confirm the involvement of beta3-ARs (and not beta1-/beta2-ARs) in these effects. The beta3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I(Ca,L) stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, beta3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.

Figure 1. β₃-AR stimulation of I_Ca,L in HAMs.

Each experiment shows the time course of I_Ca,L amplitude recorded in 2 HAMs that were simultaneously exposed to increasing concentrations of SR58611 (A), BRL37344 (B), and CGP12177 (C). Each symbol indicates a net amplitude of I_Ca,L measured every 8 seconds at 0 mV membrane potential. The individual current traces were obtained in each experimental condition at the times indicated by the corresponding letters in the main graph. (D) Concentration-response curve for the effects of the 3 agonists on I_Ca,L. The points show the mean ± SEM of 6 (SR58611, filled square), 10 (BRL37344, filled circle), or 8 (CGP12177, open square) cells.

Figure 2. Effects of β₃-AR and β₁-/β₂-AR antagonists on β₃-AR stimulation of I_Ca,L in HAMs.

In each experiment, I_Ca,L was recorded in 1 (B and C) or 2 (A) HAMs, which were simultaneously exposed to BRL37344 (1 μM, A; 0.1 μM, B) or CGP12177 (1 μM, C) and then to the β₁-/β₂-AR antagonist nadolol (1 μM, A) or to the β₃-AR antagonist L-748,337 (1 μM, B and C). The dotted lines in A indicates spontaneous rundown.

Figure 3. NO pathway is not involved in the stimulation of I_Ca,L by β₃-AR agonists.

In each experiment, I_Ca,L was recorded in a single HAM exposed to CGP12177 in the presence of either 1 mM L-NMMA in intracellular and extracellular solutions (A) or serotonin (100 nM, B). The dotted line in B indicates spontaneous rundown.

Figure 4. PKA mediates the stimulation of I_Ca,L by β₃-AR agonists.

I_Ca,L was recorded in a single HAM exposed to either 1 μM BRL3744 (A) or 1 μM CGP12177 (B). When the current was at its maximal stimulation, the PKA inhibitor H89 (1 μM) was added to the extracellular solutions in the presence of the β₃-AR agonists, and the current returned to basal levels.

Figure 5. Effect of β₃-AR ligands on contractility in human atrial trabeculae.

CGP12177 (A), SR58611 (B), or BRL37344 (C) were applied to human atrial preparations at the concentrations indicated, after 15 minutes of perfusion with 200 nM nadolol followed by the continuous presence of nadolol. The individual contractile traces were obtained in each experimental condition at the times indicated by the corresponding arrows. All 3 compounds significantly (P < 0.05) increased force of contraction.

Figure 6. PDE inhibition potentiates the effects of β₃-AR agonists on contractility in human atrial trabeculae.

BRL37344 (A) or CGP12177 (B) were applied to human atrial preparations at the concentrations indicated, in the presence of 200 nM nadolol and in the absence (A) or presence (A and B) of the PDE inhibitor IBMX (10 μM). (C) Summary of the effects of the β₃-AR agonists on contractility. The effects of SR58611 (100 nM), BRL37344 (10 μM), and CGP12177 (1 μM), with or without IBMX (10 μM), are compared with that of ISO (1 μM). The bars show the mean ± SEM of the number of experiments indicated above the bars. *P < 0.05; ***P < 0.005 versus control. ANOVA followed by post-hoc Bonferroni’s test was used to compare the effects of the β₃-AR agonists in the absence or presence of IBMX. ^#P < 0.05.