Blockade of β-adrenoceptors restores the GRK2-mediated adrenal α(2) -adrenoceptor-catecholamine production axis in heart failure

Abstract

Background and purpose: Sympathetic nervous system (SNS) hyperactivity is characteristic of chronic heart failure (HF) and significantly worsens prognosis. The success of β-adrenoceptor antagonist (β-blockers) therapy in HF is primarily attributed to protection of the heart from the noxious effects of augmented catecholamine levels. β-Blockers have been shown to reduce SNS hyperactivity in HF, but the underlying molecular mechanisms are not understood. The GPCR kinase-2 (GRK2)-α(2) adrenoceptor-catecholamine production axis is up-regulated in the adrenal medulla during HF causing α(2) -adrenoceptor dysfunction and elevated catecholamine levels. Here, we sought to investigate if β-blocker treatment in HF could lower SNS activation by directly altering adrenal GRK2 levels.

Experimental approach: Four weeks after myocardial infarction-induced HF, adult rats were randomized to 10-week treatment with vehicle (HF/C) or bisoprolol (HF/B). Cardiac function and dimensions were measured. In heart and adrenal gland, GRK2 levels were assessed by RT-PCR and Western blotting and adrenoceptors studied with radioligand binding. Catecholamines and α(2) adrenoceptors in adrenal medulla chromaffin cell cultures were also measured.

Key results: Bisoprolol treatment ameliorated HF-related adverse cardiac remodelling and reduced plasma catecholamine levels, compared with HF/C rats. Bisoprolol also attenuated adrenal GRK2 overexpression as observed in HF/C rats and increased α(2) adrenoceptor density. In cultures of adrenal medulla chromaffin cells from all study groups, bisoprolol reversed HF-related α(2) adrenoceptor dysfunction. This effect was reversed by GRK2 overexpression.

Conclusion and implications: Blockade of β-adrenoceptors normalized the adrenal α(2) adrenoceptor-catecholamine production axis by reducing GRK2 levels. This effect may contribute significantly to the decrease of HF-related sympathetic overdrive by β-blockers.

Figure 1

Ejection fraction (EF, as %) measured by echocardiography 4 weeks post MI (before treatments start) (A) and at the end of the study period, 10 weeks after bisoprolol or placebo treatments (B). (C) Percentage change in ejection fraction after the 10 weeks of treatment. LV internal diameter at diastole (LVIDd) measured by echocardiography before (D) and after treatment (E). (F) Heart rate (HR) at 14 weeks after MI. Sham, n= 12; HF/C, n= 18; HF/B, n= 20. Data are presented as mean ± SEM. *P < 0.05 versus sham; #P < 0.05 versus HF/C; & P < 0.05 versus sham and HF/C. anova analysis and Bonferroni test among all groups.

Figure 2

mRNA levels for (A) BNP, (B) collagen type I (Col1), (C) TGFβ1 in hearts from all experimental groups at the end of the study period (Sham, n= 10; HF/C, n= 12; HF/B, n= 12). All values were standardized to amplified 28S rRNA. *P < 0.05 versus Sham or HF/B groups. anova analysis and Bonferroni test among all groups. Data are presented as mean ± SEM and plotted as fold over sham values.

Figure 3

Plasma catecholamine (CA) levels in the three experimental groups at the end of the study. Data for noradrenaline and adrenaline are presented separately as means ± SEM. *P < 0.05 versus Sham or HF/B groups (n= 10 for each group); ^#P < 0.05 versus Sham or HF/C. anova analysis and Bonferroni test among all groups.

Figure 4

(A) GRK2 expression in adrenal homogenates purified from all three experimental groups at the end of the study period (n= 7 for each group). Representative Western blots (upper panel) and average densitometric quantitative analysis from blots showing the ratio of GRK2 to GAPDH (lower panel). += positive control. (B) Total α₂-adrenoceptor (α₂AR) density in plasma membranes purified from the adrenal glands of all three experimental groups at the end of the study (n= 6 for each group). *P < 0.05 versus Sham or HF/B; #P < 0.05 versus HF/C. Data are presented as mean ± SEM. anova analysis and Bonferroni test among all groups.

Figure 5

Secretion of adrenaline from cultures of chromaffin cells, isolated from the adrenals of sham, HF/C and HF/B rats, in response to nicotine (20 µmol·L⁻¹⁾, following pretreatment with vehicle (Nicotine) or with UK14304 (10 µmol·L⁻¹; UK + Nicotine). *P < 0.05, versus HF/C-UK + Nicotine, n= 6. Data are presented as mean ± SEM. anova analysis and Bonferroni test among all groups.

Figure 6

Representative Western blot showing expression of transgenes in cultures of chromaffin cells (upper panel). Secretion of adrenaline from chromaffin cells isolated from the adrenals of HF/B rats infected in vivo with AdGFP or AdGRK2 in response to nicotine treatment with or without pretreatment with UK14304 (UK + Nicotine) (lower panel). *P < 0.05,versus AdGRK2-UK + Nicotine, n= 6. Data are presented as mean ± SEM. anova analysis and Bonferroni test among all groups.