Simultaneous Adrenal and Cardiac GPCR-Gβγ Inhibition Halts Heart Failure Progression

Abstract

Objective

We propose simultaneous inhibition of Gβ γ 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 as well as 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.

Heart failure (HF) is a progressive disease with poor prognosis. A common feature of HF is elevated sympathetic nervous system activity to compensate for poor cardiac output, including excess adrenal production of the catecholamines (CA) epinephrine and norepinephrine (1–3). Chronic CA-mediated stimulation of β-adrenergic receptors (β-AR, a G-protein–coupled receptor [GPCR]), elicits pathologic upregulation of G-protein–coupled receptor kinase 2 (GRK2) that is recruited to membrane Gβγ subunits to phosphorylate β-AR, leading to their desensitization (4,5). Prior reports suggest a therapeutic role for inhibiting Gβγ–GRK2 interaction in HF to restore β-AR expression and function, utilizing a truncated Gβγ-binding GRK2 peptide that lacks the kinase domain (βARKct) (6–13), as well as a truncated Gβγ-binding phosducin (14) and a truncated phosphoinositide 3-kinase γ (PI3Kγ (15–17). Interestingly, Gβγ–GRK2 was recently found to desensitize the adrenal α₂-AR, another GPCR responsible for feedback inhibition of CA release from the adrenal gland. Importantly, adrenal α₂-AR desensitization contributes to elevated plasma CA and consequent β-AR desensitization in HF (18,19).

We recently identified and validated novel compounds that selectively inhibit Gβγ binding interactions (20), namely M119 and its highly homologous structural analog, gallein. These compounds competitively antagonize the binding of GRK2 to a specific protein– protein interaction domain of the Gβγ subunit, thus inhibiting the pathologic Gβγ–GRK2 interaction. Both compounds bind to Gβγ with equal affinity in vitro, and have equivalent efficacies in enhancing contractility in isolated cardiomyocytes as well as in acute pharmacologic and genetic mouse models of HF (21). The advantages of these compounds include their convenient route of administration (intraperitoneal injection or oral gavage) bioavailability, and cell permeability (22). Additionally, they inhibit the Gβγ–GRK2 interaction without disturbing various other aspects of basal or agonist-stimulated G-protein signaling (20). We were the first to report early proof-of-concept studies regarding possible beneficial effects of these newly identified compounds in acute pharmacological and transgenic animal models of HF (21).

In the current study, we report the therapeutic efficacy of the selective small molecule Gβγ inhibitor, gallein, when delivered after the establishment of HF in a clinically relevant surgical model. Further, we report concomitant salutary effects of gallein on the adrenal gland, including direct inhibition of adrenal CA synthesis and release both in HF animal models and in primary human adrenal pheochromocytoma tissue. We propose that simultaneous small molecule inhibition of Gβγ signaling in the heart and the adrenal gland is a novel therapeutic approach for HF, and possibly for other diseases of excess CA release, such as pheochromocytoma.

Methods

Detailed materials and methods are included in the online appendix.

Results

The small molecule Gβγ inhibitor gallein dose-dependently ameliorates cardiac dysfunction and hypertrophy in HF mice

To test the possible therapeutic effects of small molecule Gβγ inhibition in treating established HF, daily gallein administration was initiated 4 weeks after transverse aortic constriction (TAC) and continued for 8 weeks (12 weeks post-TAC). Gallein dose-dependently reduced ventricular hypertrophy (defined as the ratio of ventricular weight to tibia length) and improved cardiac function (Fig. 1 and Online fig. 1). Administration of gallein at a dose of 10 mg/kg/day was found to be the optimal therapeutic dose, and was thus utilized for subsequent studies (identified as TAC+G).

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.

Gallein improves survival and preserves cardiac contractility in pressure overload hypertrophy

After 8 weeks of daily treatments initiated 4 weeks post-TAC (i.e., at 12 weeks post-TAC), gallein significantly enhanced survival to 80%, compared with 54% survival in TAC+V (Fig. 2A). Cardiac function in TAC+V mice declined at 8 weeks post-TAC, with further deterioration at week 12. Gallein treatment initiated after establishment of HF prevented such deterioration and preserved cardiac function (Tables 1 and 2, and Fig. 2D).

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.

Table 1.

Echocardiographic Measurements of Mice at Baseline and Weeks 4, 8, and 12 Post-TAC

	Baseline		4 Weeks		8 Weeks		12 Weeks
	TAC+V	TAC+G	TAC+V	TAC+G	TAC+V	TAC+G	TAC+V	TAC+G
% EF	80.42 ± 2.01	81.97 ± 0.51	70.4 ± 5.04	74.08 ± 2.48	58.40 ± 9.29	73.82 ± 2.29	51.26 ± 9.84*	75.77 ± 3.47†
%FS	50.91 ± 0.68	49.37 ± 0.51	39.90 ± 4.04	42.26 ± 2.00	32.16 ± 6.07‡	41.94 ± 1.88	26.14 ± 5.28*	44.08 ± 2.81§
LVID; s (mm)	1.50 ± 0.07	1.54 ± 0.06	1.92 ± .25	1.92 ± 0.14	2.55 ± 0.52	1.91 ± 0.13	2.94 ± 0.71	1.80 ± 0.13§
LVID; d (mm)	3.16 ± 0.08	3.06 ± 0.09	3.44 ± 0.23	3.37 ± 0.13	4.04 ± 0.37	3.25 ± 0.12	4.41 ± 0.58‡	3.25 ± 0.12†
Volume; s (µl)	7.20 ± 0.44	6.83 ± 0.55	16.22 ± 4.56	12.31 ± 2.02	38.65 ± 15.69	11.57 ± 1.98	62.34 ± 28.75‡	11.19 ± 2.38§
Volume; d (µl)	39.76 ± 1.99	37.62 ± 2.37	50.17 ± 7.21	46.09 ± 3.97	74.64 ± 16.87	42.77 ± 3.60	98.08 ± 30.08‡	43.88 ± 3.61§

^*p < 0.01 vs. baseline TAC+V (using repeated measures analysis of variance and Bonferroni’s post-hoc analysis).

^†p < 0.05 vs. TAC+V at 12 weeks post-TAC (using repeated measures analysis of variance and Bonferroni’s post-hoc analysis).

^‡p < 0.05 vs. baseline TAC+V (using repeated measures analysis of variance and Bonferroni’s post-hoc analysis).

^§p < 0.01 vs. TAC+V at 12 weeks post-TAC (using repeated measures analysis of variance and Bonferroni’s post-hoc analysis).

EF = ejection fraction; FS = fractional shortening; LVID; d, s = left ventricular internal diameter, diastolic, systolic; TAC+G = transverse aortic constriction plus gallein; TAC+V = transverse aortic constriction plus vehicle.

Table 2.

Hemodynamic Measurements of Sham, TAC+V, and TAC+G Mice at End of Study

	Sham	TAC+V	TAC+G
LVEDP (mm Hg)	6.73 ± 1.83	23.94 ± 4.91*	8.57 ± 2.15†
+dP/dt (mm Hg/s)	8777 ± 860.3	5249 ± 913.3‡	8677 ± 743.0†
−dP/dt (mm Hg/s)	−8027 ± 926.5	−4022 ± 570.6‡	−7792 ± 824.9†
LV min (mm Hg)	4.99 ± 1.31	18.32 ± 5.03‡	5.92 ± 2.33†
LV max (mm Hg)	129.3 ± 3.98	109.9 ± 14.57	166.4 ± 13.35†

^*p < 0.01 vs. sham (using one-way analysis of variance and Bonferroni’s post-hoc analysis).

^†p < 0.05 vs. TAC+V (using one-way analysis of variance and Bonferroni’s post-hoc analysis).

^‡p < 0.05 vs. sham (using one-way analysis of variance and Bonferroni’s post-hoc analysis).

dP/dt = first derivative of left ventricular pressure; LV max = maximal left ventricular pressure; LV min = minimal left ventricular pressure; LVEDP = left ventricular end-diastolic pressure; TAC+G = transverse aortic constriction plus gallein; TAC+V = transverse aortic constriction plus vehicle.

Gallein restores β-AR density, down-regulates GRK2 expression, and inhibits GRK2 and PI3Kγ membrane recruitment

To evaluate mechanisms underlying the beneficial effects of gallein on cardiac function, we measured β-AR density, GRK2 and PI3Kγ membrane recruitment, and GRK2 gene expression. TAC mice exhibited a significant reduction in β-AR density that was normalized by gallein treatment (Fig. 2B). This was accompanied by a reduction in cardiac GRK2 gene expression and GRK2-PI3K110γ membrane translocation in TAC+G mice compared with TAC+V mice (Figs. 2C, 2E, and 2F, respectively).

Gallein attenuates cardiac remodeling and inflammation in pressure overload HF

Gallein treatment attenuated the progression of cardiac hypertrophy in TAC mice, as reflected by reduced ventricular weight to tibia length ratio (Fig. 3A) and cardiomyocyte cross-sectional area (Figs. 3B and 3C). This protective effect of gallein on cardiac hypertrophy was accompanied by reduced phosphorylation of cardiac Akt (also known as protein kinase B) (Fig. 3D) and its downstream signal, GSK-3β (Fig. 3E), and a parallel reduction in myocardial fibrosis (Figs. 4A and 4B). This may be attributed to the significantly reduced expression of the fetal genes atrial natriuretic peptide and brain natriuretic peptide (Figs. 4C and 4D), the inflammatory cytokines interleukin 1β, interleukin 6, and tumor necrosis factor α (Figs. 4E, 4F, and 4G), and the profibrotic marker α-smooth muscle actin (Fig. 4H). Moreover, we observed less myocardial apoptosis in TAC+G mice as evidenced by fewer apoptotic nuclei and reduced caspase-3 cleavage in cardiac lysates (Online fig. 2).

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.

Gallein attenuates CA production and adrenal remodeling and restores adrenal α₂-AR feedback inhibition in TAC mice

Heart failure is associated with chronically elevated plasma CA concentrations. At 12 weeks post-TAC, gallein significantly reduced plasma epinephrine and norepinephrine concentrations to 1.5-fold and 2.5-fold of baseline, respectively, whereas vehicle-treated mice showed significant elevations of 2.8-fold and 7.5-fold, respectively (Figs. 5A and 5B). Adrenal medulla hypertrophy is a common feature in HF that occurs concurrent with elevated plasma CA levels (23); this was also reduced by gallein treatment (Fig. 5C). Further, adrenal glands from TAC+G mice cultured in vitro showed significantly lower levels of basal CA secretion (Figs. 5D and 5E). Adrenal α₂-AR normally provide feedback inhibition of excess CA release, a function that is desensitized in HF. Gallein also restored α₂-AR feedback inhibitory function as compared with TAC+V mice (Figs. 5F and 5G). Further, gallein attenuated the overexpression of tyrosine hydroxylase (rate-limiting enzyme of CA production) and chromogranin A (neurokine that is synthesized, costored, and cosecreted with vesicular CA) (Fig. 5C and Online fig. 5).

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.

Gallein directly restores α₂-AR feedback inhibitory function in adrenal glands from untreated isoproterenol-pump mice

Ex vivo cultured adrenal glands from untreated mice chronically exposed to the β-AR agonist isoproterenol exhibit impaired adrenal α₂-AR function, similar to other HF models. Importantly, in vitro gallein treatment directly restored the α₂-AR feedback inhibitory function in these adrenal glands (Online fig. 3).

Gallein directly attenuates CA secretion and GRK2 protein expression and membrane translocation in cultured human pheochromocytoma

Pheochromocytoma is a tumor of the adrenal gland characterized by excessive CA production (24). Gallein treatment significantly reduced CA production in cultured pheochromocytoma slices (Figs. 6A and 6B) with attenuated GRK2 protein expression and membrane translocation (Figs. 6D and 6E, respectively). Interestingly, expression of tyrosine hydroxylase and chromogranin A was significantly downregulated by gallein treatment in cultured pheochromocytoma slices (Fig. 6C). Further, α₂-AR feedback inhibitory function trended toward improvement in gallein-treated cultured pheochromocytoma slices (Supplementary fig. 4).

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).

Discussion

Small molecule Gβγ inhibition halts HF progression post-TAC

In the present study, we demonstrate the efficacy of the small molecule Gβγ inhibitor, gallein, in ameliorating established HF through simultaneous inhibition of Gβγ in the heart and the adrenal gland. Importantly, gallein partially restores normal cardiac function and halts or reverses pathologic cardiac remodeling when administered after the establishment of chronic HF in a clinically relevant animal model. It is noteworthy that gallein treatment in sham animals mildly increases cardiac contractility as we have previously shown (21) and in online Figure 6.

We observed a dose-dependent therapeutic effect of gallein on cardiac function and hypertrophy in TAC mice in vivo, with 10mg/kg/day identified as the optimal dose. Concomitantly, gallein treatment attenuated cardiac fibrosis and apoptosis, indicating attenuated cardiac remodeling. Further, gallein treatment attenuated cardiac inflammatory cytokine expression in TAC HF mice (Figs. 4E, 4F, and 4G). Our preliminary studies have demonstrated possible systemic anti-inflammatory effects of Gβγ inhibitors in a mouse carrageenan-induced paw inflammation model (25). Prior studies have correlated inflammation and loss of cardiac function in pressure overload hypertrophy (26,27). Thus, attenuated cardiac inflammation in TAC mice by gallein may provide an additional mechanism for its therapeutic effect in pressure overload HF.

In summary, gallein increased survival, improved cardiac function, and reduced cardiac remodeling and inflammation when administered daily to mice with established HF, i.e., 4 weeks through 12 weeks post-TAC.

Small molecule Gβγ inhibition preserves cardiac membrane β-AR density by attenuating GRK2–PI3Kγ signaling

Excess sympathetic nervous system activity and outflow are salient characteristics of HF, in which reduced cardiac output triggers compensatory sympathetic nervous system overactivity (elevated local and circulating CA) that acutely rescues cardiac function. However, chronic stimulation of β-AR by CA leads to their desensitization and attenuated contractile signaling (1–3). Receptor desensitization is mainly mediated by the Gβγ subunits, which recruit receptor-desensitizing kinases such as GRK2 and PI3Kγ to agonist-stimulated β-AR, resulting in receptor phosphorylation and the recruitment of arrestins that initiate receptor internalization, desensitization, and down-regulation (5,28–30). Previous data from our laboratory and others suggests that inhibiting Gβγ signaling and its interaction with GRK2 and PI3Kγ could be of therapeutic value in HF (5,16,29,30). This has been demonstrated by large peptide inhibitors of Gβγ binding in both cell culture and animal models of HF (2,6,–17,31). In our study, small molecule Gβγ inhibition by gallein reduced GRK2 expression and GRK2–PI3K110γ membrane recruitment, thus preserving β-AR membrane expression and cardiac function.

Gallein interrupts activation of the cardiac Akt-GSK-3β hypertrophic pathway

In addition to forming a complex with GRK2 that desensitizes β-AR, PI3Kγ is of specific interest in HF because it mediates a myriad of pathological effects. PI3Kγ activates Akt/PKB after GPCR activation, leading to pathological hypertrophy (28,32,33). Conversely, loss or reduction of activity is associated with increased contractility, relaxation, and reduced hypertrophy (34). Akt is a well-known player in cardiac hypertrophy with a large number of downstream effectors. In particular, phosphorylated Akt mediates GSK-3β Ser-9 phosphorylation, thus inhibiting its antihypertrophic effects. Our results suggest that attenuated Akt activation in gallein-treated TAC mice results in elevated levels of active (nonphosphorylated) GSK-3β that attenuates cardiac hypertrophy by negatively regulating hypertrophic gene transcription and protein translation (35).

Systemic gallein treatment normalizes sympathetic tone and adrenal function in TAC mice

The adrenal glands, specifically adrenal medullary chromaffin cells, are the site of CA synthesis and secretion into the circulation. Adrenal chromaffin cell α₂-AR play a crucial role in feedback regulation of circulating levels of plasma CA (see Fig. 7). Recent reports demonstrate that HF prognosis is worse in patients with an α₂-AR deletion polymorphism that impairs feedback inhibition of CA release (36,37). Further, recent data demonstrate that in HF, adrenal α₂-AR are also desensitized by pathological Gβγ–GRK2 signaling (18,19), and that this desensitization can possibly be mitigated by adrenal βARKct (38).

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.

In our study, the salutary extracardiac effects of systemic Gβγ inhibition by gallein in chronic murine HF included reduced plasma CA levels, limited adrenal medullary hypertrophy, normalized adrenal α₂-AR function, and attenuated expression of both tyrosine hydroxylase, the ratelimiting enzyme in CA synthesis, and chromogranin A, a neurokine that is synthesized, costored, and cosecreted with CA in chromaffin cell vesicles. To investigate whether these adrenal effects resulted directly from gallein treatment or were merely the result of HF amelioration, we examined the direct effects of gallein treatment in ex vivo cultured adrenal glands isolated from vehicle-treated isoproterenol-pump HF mice, where gallein treatment directly restored adrenal α₂-AR function (Online fig. 3).

Small molecule Gβγ inhibition directly reduces CA secretion in ex vivo cultured human pheochromocytoma

To further investigate the salutary effect of gallein on CA secretion in adrenal chromaffin cells, we utilized ex vivo cultured human pheochromocytoma explants. Pheochromocytoma is a CA-secreting tumor of the adrenal medulla that is accompanied by cardiovascular complications (24). Importantly, gallein treatment reduced CA secretion and attenuated GRK2 expression and membrane translocation in ex vivo pheochromocytoma culture with attenuated protein expression of tyrosine hydroxylase and chromogranin A, suggesting a direct inhibitory effect on CA synthesis and secretion by chromaffin cells.

Study limitations

To date, pharmacological management of pheochromocytoma is poor at best, and surgical resection remains the final option. Our results suggest a possible therapeutic role for small molecule Gβγ inhibition in pheochromocytoma through its direct effect on CA secretory mechanisms. Importantly, these experiments were conducted immediately on explanted pheochromocytomas obtained from human subjects.

Conclusions

Recent reports have suggested that, if it were possible, systemic pharmacological therapy to simultaneously normalize pathological Gβγ–GRK2 signaling in both the heart and the adrenal gland could be of substantial therapeutic benefit in HF. Our current study suggests gallein to be such a systemic pharmacological therapy. Administration of gallein to animals after the establishment of HF in a clinically relevant surgical model appears to normalize dampened AR signaling in both the heart and the adrenal gland. Our data also suggest a possible therapeutic role for small molecule Gβγ inhibition in other diseases of elevated catecholamine release, such as pheochromocytoma. Future studies will seek to further establish the therapeutic efficacy of gallein in larger animal models of HF.

Abbreviations and Acronyms

AR

adrenergic receptors

βARKct

a truncated Gβγ-binding GRK2 peptide that lacks the kinase domain

CA

catecholamines

GPCR

G-protein–coupled receptor

GRK2

G-protein–coupled receptor kinase 2

HF

heart failure

PI3Kγ

phosphoinositide 3-kinase γ

TAC

transverse aortic constriction