The α1A-adrenergic receptor subtype mediates increased contraction of failing right ventricular myocardium

Abstract

Dysfunction of the right ventricle (RV) is closely related to prognosis for patients with RV failure. Therefore, strategies to improve failing RV function are significant. In a mouse RV failure model, we previously reported that α1-adrenergic receptor (α1-AR) inotropic responses are increased. The present study determined the roles of both predominant cardiac α1-AR subtypes (α1A and α1B) in upregulated inotropy in failing RV. We used the mouse model of bleomycin-induced pulmonary fibrosis, pulmonary hypertension, and RV failure. We assessed the myocardial contractile response in vitro to stimulation of the α1A-subtype (using α1A-subtype-selective agonist A61603) and α1B-subtype [using α1A-subtype knockout mice and nonsubtype selective α1-AR agonist phenylephrine (PE)]. In wild-type nonfailing RV, a negative inotropic effect of α1-AR stimulation with PE (force decreased ≈50%) was switched to a positive inotropic effect (PIE) with bleomycin-induced RV injury. Upregulated inotropy in failing RV occurred with α1A-subtype stimulation (force increased ≈200%), but not with α1B-subtype stimulation (force decreased ≈50%). Upregulated inotropy mediated by the α1A-subtype involved increased activator Ca(2+) transients and increased phosphorylation of myosin regulatory light chain (a mediator of increased myofilament Ca(2+) sensitivity). In failing RV, the PIE elicited by the α1A-subtype was appreciably less when the α1A-subtype was stimulated in combination with the α1B-subtype, suggesting functional antagonism between α1A- and α1B-subtypes. In conclusion, upregulation of α1-AR inotropy in failing RV myocardium requires the α1A-subtype and is opposed by the α1B-subtype. The α1A subtype might be a therapeutic target to improve the function of the failing RV.

Keywords: inotropic; myosin regulatory light chain; right ventricle; α1-adrenergic.

Fig. 1.

Bleomycin (Bleo) model of right ventricular (RV) failure. Echocardiographic assessment of RV and left ventricular (LV) fractional shortening before and 2 wk after bleomycin instillation was completed. A: 2 wk after bleomycin instillation, RV fractional shortening in vivo was reduced to 45% of the value before bleomycin (P = 0.002). After saline instillation, RV fractional shortening in vivo was slightly reduced to ∼80% of initial (P = 0.04). B: LV in vivo fractional shortening was not affected by bleomycin or saline instillation, demonstrating the RV-specific nature of the bleomycin model. *P < 0.05; **P < 0.01; ns, not significant.

Fig. 2.

Increased α₁-adrenergic receptor (α₁-AR) inotropy in failing RV. A: slow time-base recordings of contractions of RV myocardium from nonfailing and failing RV in response to acute α₁-AR stimulation with maximal doses of α₁A-subtype-selective agonist A61603 (100 nM) or nonsubtype-selective α₁-AR agonist phenylephrine (PE) (10 μM; (in the presence of a β-AR blocker). α₁-AR stimulation mediated negative inotropy in nonfailing RV but positive inotropy in failing RV. B: fast time-base recordings of individual contractions of RV myocardium from nonfailing or failing RV (same recordings as A). Force recordings expressed in absolute units are superimposed for contractions before addition of agonists (dotted traces) and after the inotropic responses to the agonists had developed (solid traces).

Fig. 3.

Upregulation of α₁-AR inotropy 2 wk after bleomycin instillation. A: summary of the positive inotropic (●) and negative inotropic responses (○) elicited by the α₁A-subtype agonist A61603 (A1603) for different samples of RV myocardium from different animals and for various times after a single bleomycin instillation or with biweekly bleomycin instillation (▲). Positive inotropic responses were observed 14 days after initial bleomycin administration. B: summary of inotropic responses to A61603: absolute levels of developed force before (basal) and after addition of A61603. Data from failing RV are divided into nonconverter or converter groups, depending on whether A61603 elicited a negative inotropic effect (NIE) or positive inotropic effect (PIE). **P < 0.01; n = 6–8/group.

Fig. 4.

Upregulation of α₁-AR inotropy in failing RV mediated by the α₁A-subtype. Summary of inotropic responses to maximal stimulation of the α₁A-subtype either singly using A61603 or in combination with the α₁B-subtype using PE are shown. Animals were studied >14 days after a single instillation of saline (nonfailing RV) or bleomycin (failing RV). Some animals received biweekly bleomycin instillation (triangles). Individual experimental values are shown, and the pooled means and SE indicated. A: for nonfailing RV, both A61603 and PE elicited a similar NIE. B: after bleomycin instillation, for a subset of animals (nonconverter group), A61603 and PE continued to elicit a NIE. C: in contrast, for some animals there was a conversion to a PIE mediated by A61603 (converter group). The PIE mediated by A61603 was greater than that mediated by PE (P = 0.0173). The difference between the response to A61603 minus the response to PE may reflect a NIE mediated by the α₁B-subtype. *P < 0.05.

Fig. 5.

Upregulation of α₁-AR inotropy in failing RV requires the α₁A-subtype. Slow time-base recordings of contractions of RV myocardium from knockout mice lacking the α₁A-subtype (AKO) are shown. RV myocardium was from animals with tracheal instillation of saline (nonfailing) or bleomycin (failing). Shown are acute inotropic responses to α₁-AR agonists A61603 (100 nM) or PE (10 μM) in the presence of a β-AR blocker. AKO myocardium was unresponsive to A61603. Stimulation of the remaining α₁B-subtype in AKO myocardium using PE elicited a NIE in both nonfailing RV and failing RV.

Fig. 6.

Negative inotropy mediated by the α₁B-subtype. A and B: summary of data from all AKO experiments. RV myocardium from AKO mice did not respond to A61603. For AKO mice, stimulation of the remaining α₁B-subtype using PE elicited a NIE in all experiments involving nonfailing RV or failing RV. Thus the α₁B-subtype mediates a NIE in both nonfailing and failing RV. *P < 0.05; ***P < 0.001.

Fig. 7.

Relationship between the α₁A-subtype inotropic responses versus changes in the Ca²⁺ transient. A: fast time-base recordings of Fura-2 Ca²⁺ transients (top) and simultaneously recorded contraction force (bottom) during contractions of RV myocardium from nonfailing RV (left) or failing RV (right). Records are superimposed for contractions before addition of A61603 (dotted traces) and after the inotropic responses to A61603 had developed (solid traces). A decreased Ca²⁺ transient mediated by A61603 was associated with a negative inotropic response. Conversely, an increased Ca²⁺ transient mediated by A61603 was associated with a positive inotropic response. B: changes in the amplitude of force- and Ca²⁺ transients after A61603 were expressed relative to their pre-agonist amplitude (defined as 100%). For all experiments involving myocardium from hearts at least 2 wk after bleomycin or saline instillation into the trachea, following α₁A-subtype stimulation, there was a significant positive linear relationship between changes in the amplitude of the Ca²⁺ transient and changes in the amplitude of contraction (R² = 0.787, P < 0.0001) (the relationship remained significant after excluding the most positive value). Some animals received biweekly bleomycin instillation (triangles).

Fig. 8.

α₁A-subtype effect on regulatory light chain (RLC) phosphorylation (p). Immunoblots and quantitative summary of RLC phosphorylation, with or without acute stimulation with A61603 for nonfailing RV and failing RV myocardium that had a positive inotropic response to A61603, are shown. In failing RV, RLC phosphorylation was reduced versus nonfailing RV. In failing RV, RLC phosphorylation was increased by α₁A-subtype stimulation.