Simultaneous adrenal and cardiac g-protein-coupled receptor-gβγ inhibition halts heart failure progression

Abstract

Objectives: The authors propose simultaneous inhibition of Gβγ signaling in the heart and the adrenal gland as a novel therapeutic approach for heart failure (HF).

Background: Elevated sympathetic nervous system activity is a salient characteristic of HF progression. It causes pathologic desensitization of β-adrenergic receptors (β-AR), facilitated predominantly through Gβγ-mediated signaling. The adrenal glands are key contributors to the chronically elevated plasma catecholamine levels observed in HF, where adrenal α2-AR feedback inhibitory function is impaired also through Gβγ-mediated signaling.

Methods: We investigated the efficacy of a small molecule Gβγ inhibitor, gallein, in a clinically relevant, pressure-overload model of HF.

Results: Daily gallein treatment (10 mg/kg/day), initiated 4 weeks after transverse aortic constriction, improved survival and cardiac function and attenuated cardiac remodeling. Mechanistically, gallein restored β-AR membrane density in cardiomyocytes, attenuated Gβγ-mediated G-protein-coupled receptor kinase 2-phosphoinositide 3-kinase γ membrane recruitment, and reduced Akt (protein kinase B) and glycogen synthase kinase 3β phosphorylation. Gallein also reduced circulating plasma catecholamine levels and catecholamine production in isolated mouse adrenal glands by restoring adrenal α2-AR feedback inhibition. In human adrenal endocrine tumors (pheochromocytoma), gallein attenuated catecholamine secretion, as well as G-protein-coupled receptor kinase 2 expression and membrane translocation.

Conclusions: These data suggest small molecule Gβγ inhibition as a systemic pharmacologic therapy for HF by simultaneously normalizing pathologic adrenergic/Gβγ signaling in both the heart and the adrenal gland. Our data also suggest important endocrine/cardiovascular interactions and a possible role for small molecule Gβγ inhibition in treating endocrine tumors such as pheochromocytoma, in addition to HF.

Keywords: catecholamines; fibrosis; heart failure; hypertrophy; sympathetic nervous system.

Figure 1. Dose-Response Efficacy of Gallein After Transverse Aortic Constriction

(A) A schematic representation of the experimental time line showing initiation of gallein treatment after the establishment of heart failure, i.e., 4 weeks after transverse aortic constriction (TAC). A dose-dependent cardioprotective effect of daily intraperitoneal (i.p.) gallein (G) was observed by both (B) cardiac morphometry (ventricular weight to tibia length, VW/TL) and (C) cardiac function (echocardiography, % fractional shortening). Optimal therapeutic dose was 10 mg/kg/day. *p < 0.001 vs. sham; †p < 0.01 and ‡p < 0.05 vs. TAC+V (using one-way analysis of variance and Bonferroni’s post-hoc analysis); §p < 0.05 vs. baseline TAC+V; ‖p < 0.01 vs. all groups at baseline; and ¶p < 0.05 vs. 12 weeks TAC+V (using repeated measures analysis of variance with Bonferroni’s post-hoc analysis). TAC+G =transverse aortic constriction plus gallein in varying doses; TAC+V =transverse aortic constriction plus vehicle.

Figure 2. Salutary Effect of Gallein Post-Transverse Aortic Constriction

(A) Gallein (G; 10 mg/kg/day) -treated mice showed enhanced survival (80%; 8 of 10), whereas mice receiving vehicle injection (V) showed lower survival rate (54.55%; 6 of 11) relative to 100% survival in the sham group. (B) Cardiac β-adrenergic receptor (β-AR) density was significantly reduced in transverse aortic constriction (TAC) mice and was recovered to almost normal levels by gallein treatment. (C) G-protein–coupled receptor kinase 2 (GRK2) gene expression was elevated in TAC+V mice and was reduced by gallein treatment. (D) M-mode echocardiographic images showing impaired contractile function in TAC+V group and recovered function in gallein-treated animals. This likely resulted from gallein-mediated recovery of β-AR function due to attenuation of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ) membrane recruitment (E and F). *p < 0.05 vs. sham; †p < 0.001, ‡p < 0.01, and §p < 0.05 vs. TAC+V (using one-way analysis of variance with Bonferroni’s post-hoc analysis). Nonparametric analysis of β-AR binding utilizing Kruskal Wallis test yielded p < 0.05 for sham vs. TAC+V. GAPDH =glyceraldehyde 3-phosphate dehydrogenase.

Figure 3. Gallein Reduces Ventricular Hypertrophy and Akt Phosphorylation

(A) Hypertrophy (ventricular weight to tibia length, VW/TL) was attenuated in gallein-treated (G) post-transverse aortic constriction (TAC) animals. (B) Reduced cardiomyocyte cross-sectional area (CM CA) in gallein-treated mice as a quantification of (C) wheat germ agglutinin staining (WGA, green; nuclear 4′,6-diamidino-2-phenylindole, blue; scale bar =50 µm). (D) Reduced cytosolic Ser473-Akt phosphorylation as compared with total Akt protein expression, and (E) Ser9-GSK-3β phosphorylation relative to total GSK-3β protein expression in gallein-treated mice (densitometric analysis and fold change), in parallel with VW/TL and CM CA data. *p < 0.001, †p < 0.01, and ‡p < 0.05 vs. sham; §p < 0.05, ‖p < 0.01, and ¶p < 0.001 vs. TAC+V (using one-way analysis of variance with Bonferroni’s post-hoc analysis). Nonparametric analysis of pGSK/GSK utilizing Kruskal-Wallis test yielded p < 0.05 for sham and p < 0.01 for TAC+G vs. TAC+V.

Figure 4. Reduced Cardiac Fibrosis and Inflammatory Markers in Gallein-Treated Mice Post-Transverse Aortic Constriction

(A) Picrosirius red and (B) Masson’s trichrome staining shows less cardiac fibrosis in gallein-treated mice after transverse aortic constriction (TAC+G) than in vehicle-treated mice (TAC+V). (C–H) Real time polymerase chain reaction analysis of inflammatory and profibrotic gene expression (normalized to glyceraldehyde 3-phosphate dehydrogenase [GAPDH] as housekeeping gene) in cardiac RNA extracts show attenuated gene expression of these markers by gallein treatment. *p < 0.001, †p < 0.01, and ‡p < 0.05 vs. sham; §p < 0.001 and ‖p < 0.05 vs. TAC+V (using one-way analysis of variance with Bonferroni’s post-hoc analysis). Nonparametric analysis of Nppb and Il6 utilizing Kruskal-Wallis test yielded p < 0.05 for sham vs. TAC+V and p < 0.01 for TAC+G vs. TAC+V, respectively. Acta2 =actin α2; Il1β =interleukin 1b; Il6 =interleukin 6; Nppa =atrial natriuretic peptide; Nppb =brain natriuretic peptide; TNFa =tumor necrosis factor α.

Figure 5. Gallein Reduces Plasma Catecholamines and Adrenal Hypertrophy, and Restores Adrenal α₂-AR Feedback Inhibition of Catecholamine Release

(A, B) Gallein treatment in post-transverse aortic constriction mice (TAC+G) reduces circulating plasma catecholamines (norepinephrine [NEpi], A; epinephrine [Epi], B) compared with vehicle-treated mice (TAC+V). (C) Gallein attenuates adrenal medullae hypertrophy post-TAC (hematoxylin-eosin staining of paraffin-fixed adrenal sections; scale bar =25 µm) and attenuates tyrosine hydroxylase (TH) as well as chromogranin A (CgA) protein expression in adrenal chromaffin cells (immunofluorescent staining; scale bar =50 µm). (D, E) Gallein reduces chronically elevated catecholamine secretion in ex vivo cultures of post-TAC adrenal medullae. (F, G) Adrenal α₂-adrenergic receptor (α₂-AR) feedback inhibitory function is recovered by gallein treatment in TAC mice. *p < 0.0001, †p < 0.001, ‡p < 0.01, and §p < 0.05 vs. sham; ‖p < 0.01 and ¶p < 0.05 vs. TAC+V (using one-way analysis of variance with Bonferroni’s post-hoc analysis). Nonparametric analysis of plasma epinephrine utilizing Kruskal-Wallis test yielded p < 0.001 for sham vs. TAC+V.

Figure 6. Gallein Reduces Catecholamine Secretion and Normalizes α₂-AR Feedback Inhibition in Diseased Human Adrenal Medullae

Ex vivo cultured human adrenal pheochromocytoma slices were treated with gallein (G; 10 µmol/l) or vehicle (V) for 48 h. (A, B) Gallein treatment attenuates catecholamine secretion (norepinephrine [NEpi], A; epinephrine [Epi], B). (C) Gallein attenuates tyrosine hydroxylase (TH) and chromogranin A (CgA) protein expression levels and (D, E) attenuates G-protein–coupled receptor kinase 2 (GRK2) protein expression and membrane translocation (densitometric analysis and fold change; normalized to glyceraldehyde 3-phosphate dehydrogenase [GAPDH]). *p < 0.0001, †p < 0.001, ‡p < 0.01 vs. group V (using Student t test and nonparametric utilizing Mann-Whitney U test).

Figure 7. Model for Dual Efficacy of Small Molecule Gβγ Inhibition in Heart and Adrenal Gland During HF

(A) In cardiomyocytes, Gβγ–G-protein–coupled receptor kinase 2 (GRK2) interaction, triggered by elevated sympathetic nervous system (SNS) activity in heart failure (HF), signals β-adrenergic receptor (β-AR) desensitization. (B) In adrenal chromaffin cells, the site of catecholamine (CA) production, 1) central nicotinic stimulation triggers 2) synthesis and 3) secretion of CA into plasma. High levels of plasma CA 4) stimulate α₂-AR-mediated 5) feedback inhibition of CA synthesis and secretion. (C) In HF, continuous CA stimulation of α₂-AR triggers its Gβγ-GRK2–mediated desensitization and the loss of feedback inhibition of CA release, contributing to SNS overactivity in HF. (D) Small molecule Gβγ inhibitors may provide a novel therapeutic approach for HF by inhibiting Gβγ–GRK2 signaling simultaneously in the heart and the adrenal gland, thus breaking this vicious cycle.