Abstract

Chronic heart failure (HF) is characterized by sympathetic overactivity and enhanced circulating catecholamines (CAs), which significantly increase HF morbidity and mortality. We recently reported that adrenal G protein-coupled receptor kinase 2 (GRK2) is up-regulated in chronic HF, leading to enhanced CA release via desensitization/down-regulation of the chromaffin cell alpha(2)-adrenergic receptors that normally inhibit CA secretion. We also showed that adrenal GRK2 inhibition decreases circulating CAs and improves cardiac inotropic reserve and function. Herein, we hypothesized that adrenal-targeted GRK2 gene deletion before the onset of HF might be beneficial by reducing sympathetic activation. To specifically delete GRK2 in the chromaffin cells of the adrenal gland, we crossed PNMTCre mice, expressing Cre recombinase under the chromaffin cell-specific phenylethanolamine N-methyltransferase (PNMT) gene promoter, with floxedGRK2 mice. After confirming a significant ( approximately 50%) reduction of adrenal GRK2 mRNA and protein levels, the PNMT-driven GRK2 knock-out (KO) offspring underwent myocardial infarction (MI) to induce HF. At 4 weeks post-MI, plasma levels of both norepinephrine and epinephrine were reduced in PNMT-driven GRK2 KO, compared with control mice, suggesting markedly reduced post-MI sympathetic activation. This translated in PNMT-driven GRK2 KO mice into improved cardiac function and dimensions as well as amelioration of abnormal cardiac beta-adrenergic receptor signaling at 4 weeks post-MI. Thus, adrenal-targeted GRK2 gene KO decreases circulating CAs, leading to improved cardiac function and beta-adrenergic reserve in post-MI HF. GRK2 inhibition in the adrenal gland might represent a novel sympatholytic strategy that can aid in blocking HF progression.

Highlights

  • ␣2ARs play a crucial role in autocrine feedback inhibition of CA release from cardiac sympathetic nerve terminals and from the adrenal medulla

  • G protein-coupled receptor kinase 2 (GRK2) gene deletion induced by adrenal chromaffin cell expression of Cre recombinase led to a significant (53.1 Ϯ 0.5%) reduction of total adrenal GRK2 mRNA levels in the phenylethanolamine N-methyltransferase (PNMT)-driven GRK2 KO mice compared with control wild-type (WT) floxedGRK2ϩ/ϩ mice (Fig. 1B)

  • We documented a crucial role for adrenal GRK2 in regulation of cardiac sympathetic stimulation, both under normal conditions and in the context of chronic heart failure (HF) (17, 24; reviewed in Ref. 11)

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Summary

Chromaffin Cell Culture and in

Vitro CA Secretion Assays—Chromaffin cells were isolated from adrenal glands excised from the mice and cultured as described previously [17]. ␣2AR Density Measurements— Plasma membranes from excised adrenal glands were prepared as described [17], and saturation bindficial by reducing sympathetic activation. PNMTCre mice, expressing Cre recombinase under the Cardiac ␤AR Density and cAMP Measurements—␤AR dengene promoter of the chromaffin cell-specific enzyme phe- sity was measured in isolated cardiac plasma membranes using nylethanolamine N-methyl transferase (PNMT) [20], with 125I-CYP (iodocyanopindolol), as described before [23]. To induce HF, resultant mice diac cAMP levels were measured with the BIOMOL Cyclic homozygous for the floxed allele (i.e. PNMTCreϩ/Ϫ/ floxedGRK2ϩ/ϩ) and control mice with endogenous GRK2 expression underwent myocardial infarction (MI) and were studied at 4 weeks post-MI.

EXPERIMENTAL PROCEDURES
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