Negative impact of β-arrestin-1 on post-myocardial infarction heart failure via cardiac and adrenal-dependent neurohormonal mechanisms

Abstract

β-Arrestin (βarr)-1 and β-arrestin-2 (βarrs) are universal G-protein-coupled receptor adapter proteins that negatively regulate cardiac β-adrenergic receptor (βAR) function via βAR desensitization and downregulation. In addition, they mediate G-protein-independent βAR signaling, which might be beneficial, for example, antiapoptotic, for the heart. However, the specific role(s) of each βarr isoform in cardiac βAR dysfunction, the molecular hallmark of chronic heart failure (HF), remains unknown. Furthermore, adrenal βarr1 exacerbates HF by chronically enhancing adrenal production and hence circulating levels of aldosterone and catecholamines. Herein, we sought to delineate specific roles of βarr1 in post-myocardial infarction (MI) HF by testing the effects of βarr1 genetic deletion on normal and post-MI cardiac function and morphology. We studied βarr1 knockout (βarr1KO) mice alongside wild-type controls under normal conditions and after surgical MI. Normal (sham-operated) βarr1KO mice display enhanced βAR-dependent contractility and post-MI βarr1KO mice enhanced overall cardiac function (and βAR-dependent contractility) compared with wild type. Post-MI βarr1KO mice also show increased survival and decreased cardiac infarct size, apoptosis, and adverse remodeling, as well as circulating catecholamines and aldosterone, compared with post-MI wild type. The underlying mechanisms, on one hand, improved cardiac βAR signaling and function, as evidenced by increased βAR density and procontractile signaling, via reduced cardiac βAR desensitization because of cardiac βarr1 absence, and, on the other hand, decreased production leading to lower circulating levels of catecholamines and aldosterone because of adrenal βarr1 absence. Thus, βarr1, via both cardiac and adrenal effects, is detrimental for cardiac structure and function and significantly exacerbates post-MI HF.

Keywords: aldosterone; catecholamines; knockout mice; β-arrestin-1.

Figure 1

(A) Ejection fraction (EF) % of sham-operated (sham) or of 4-week post-MI (MI) βarr1KO and WT mice. *, p<0.05, vs. either Sham; **, p<0.05, vs. WT MI; n=7 mice/group. (B) Basal and maximal dose (333 ng/kg) of isoproterenol (Max. Iso)-stimulated +dP/dt_max responses of these mice. *, p<0.05, vs. WT Sham-Max. Iso; ^#, p<0.05, vs. either Sham; **, p<0.05, vs. WT MI-Max. Iso; ^, p<0.05, vs. WT MI-Basal; n=5 mice/group.

Figure 2

(A) Kaplan-Meier survival curves of the 4 groups of the study: sham-operated (Sham) and post-MI (MI) βarr1KO and WT mice. p=0.012 between MI WT and MI βarr1KO; n=15 mice/group for sham, 37 mice/group for MI mice. (B) Plasma circulating NE and Epi levels in sham-operated (Sham) or in 4-week post-MI (MI) βarr1KO (KO) and WT mice. *, p<0.05, vs. Sham-either genotype; ^, p<0.05, vs. WT MI; n=6 mice/group. (C) Serum aldosterone levels in these mice. *, p<0.05, vs. all other groups; n=6 mice/group.

Figure 3

(A) Apoptotic cell death at 24 hrs post-MI in βarr1KO (KO) and WT mice, as measured by TUNEL performed in the border zone of the infarct. Representative images of TUNEL-positive nuclei are shown (left), along with their quantitation (right). No difference in rate of apoptosis in the remote zone was found (data not shown). *, p<0.05, vs. either Sham; ^#, p<0.05, vs. WT MI; n=6 hearts/group. (B) Left Ventricular End Diastolic Diameter (LVEDD) of these mice at 4 weeks post-MI or post-sham operation. ^#, p<0.05, vs. either Sham; *, p<0.05, vs. WT MI; n=7 mice/group. (C–D) Infarct size at 4 weeks post-MI. Representative triphenyltetrazolium chloride (TTC)–stained cardiac cross-sections are shown in (C), and average left ventricular (LV) infarct size, expressed as % LV infarction and measured from 6 mice/group, is shown in (D). **, p<0.05, vs. WT MI.

Figure 4

(A–C) Levels of pro-inflammatory cytokines TNFα (A), IL-6 (B), and IL-1β (C), measured in serum of intra-cardiac blood from βarr1KO (KO) and WT mice at 4 weeks post-MI (MI) or post-sham operation (Sham). *, p<0.05, vs. all other groups; **, p<0.05, vs. WT Sham; n=5 mice/group. (D–E) Cardiac fibrosis staining in the 4-week post-MI mice. Blue denotes collagen fibers, red denotes muscle fibers, and black represents cell nuclei. Representative images are shown in (D) and quantification of the % fibrotic area in (E). *, p<0.05; n=5 hearts/group. (F–G) Heart mRNA levels of Collagen-1α1 (Col-1α1) (F) and of brain natriuretic peptide (BNP) (G) in these mice. ^#, p<0.05, vs. either Sham; *, p<0.05, vs. WT MI; n=6 hearts/group.

Figure 5

(A) βAR density in cardiac plasma membranes of sham-operated (Sham) or of 4-week post-MI (MI) βarr1KO (KO) and WT mice. *, p<0.05, vs. all other groups; n=5 hearts/group. (B) Steady-state total cAMP levels in cardiac homogenates purified from these mice. *, p<0.05 vs. all other groups; n=5 hearts/group. (C) Cardiac SERCA activity measured in membrane homogenates from the hearts of these mice. *, p<0.05, vs. WT Sham; **, p<0.05, vs. WT MI; n=5 hearts/group. (D–E) Cardiac EGFR transactivation in these mice. Representative western blots in total cardiac protein extracts for phospho-Tyr845-EGFR or total EGFR (loading control) are shown in (D) and their densitometric quantitation in (E). Relative EGFR transactivation was calculated by comparing the ratios of phospho-EGFR to total EGFR for each sample. *, p<0.05, vs. either WT; **, p<0.05, vs. KO Sham; n=5 hearts/group.

Figure 6

Schematic illustration of the cardiac (left) and adrenal (right) signaling mechanisms underlying the effects of βarr1 on cardiovascular function. G_s: stimulatory G protein; P: Phosphorylation; α₂AR: alpha2-adrenergic receptor; AT₁R: Angiotensin II type 1 receptor; CA: Catecholamine. See text for details and all other molecular acronym descriptions.