Elsevier

Cellular Signalling

Volume 26, Issue 6, June 2014, Pages 1226-1234
Cellular Signalling

The eIF2B-interacting domain of RGS2 protects against GPCR agonist-induced hypertrophy in neonatal rat cardiomyocytes

https://doi.org/10.1016/j.cellsig.2014.02.006Get rights and content

Highlights

  • RGS2 eIF2B-interacting domain (RGS2eb) inhibits cardiomyocyte hypertrophy.

  • RGS2eb has no effect on GPCR-dependent signaling.

  • RGS2 knockdown potentiates GPCR-mediated cardiomyocyte hypertrophy.

  • RGS2 regulation of protein synthesis may contribute to its antihypertrophic effects.

Abstract

The protective effect of Regulator of G protein Signaling 2 (RGS2) in cardiac hypertrophy is thought to occur through its ability to inhibit the chronic GPCR signaling that promotes pathogenic growth both in vivo and in cultured cardiomyocytes. However, RGS2 is known to have additional functions beyond its activity as a GTPase accelerating protein, such as the ability to bind to eukaryotic initiation factor, eIF2B, and inhibit protein synthesis. The RGS2 eIF2B-interacting domain (RGS2eb) was examined for its ability to regulate hypertrophy in neonatal ventricular myocytes. Both full-length RGS2 and RGS2eb were able to inhibit agonist-induced cardiomyocyte hypertrophy, but RGS2eb had no effect on receptor-mediated inositol phosphate production, cAMP production, or ERK 1/2 activation. These results suggest that the protective effects of RGS2 in cardiac hypertrophy may derive at least in part from its ability to govern protein synthesis.

Introduction

Pathological cardiac hypertrophy is an enlargement of the heart accompanying many forms of heart disease, and is an independent risk factor for cardiovascular morbidity and mortality [1]. In response to a variety of stimuli including chronic hypertension and acute myocardial infarction, the myocardium increases in size in an attempt to normalize wall stress and maintain cardiac function [1], [2]. Although initially believed to be a compensatory mechanism, prolonged hypertrophic stimuli can tip the balance from an adaptive towards a maladaptive response, leading to abnormal metabolic, structural, and functional changes that over time can result in cardiac remodeling, increased cardiac fibrosis, dilation and ultimately heart failure [1], [2]. As cardiac hypertrophy progresses, cardiomyocytes undergo increased protein synthesis and become larger [3], and also exhibit characteristic genetic changes [4] and signs of ER stress [5].

G-protein coupled receptor (GPCR) signaling regulates essential functions in the cardiovascular system such as heart rate and contractility; however, sustained stimulation of certain G protein-coupled receptors promotes cardiomyocyte hypertrophy and thus plays a pivotal role in the development of human heart failure. These include angiotensin II, endothelin, and α1-adrenergic receptors, which couple primarily to Gq, and also β-adrenergic receptors that primarily activate Gs [6], [7]. GPCRs and G proteins are themselves under the control of another family of proteins, the Regulator of G protein Signaling (RGS) proteins [8], [9]. RGS proteins are negative modulators of cellular signaling that function by acting as GTPase accelerating proteins (GAP) for members of the Gαi and/or Gαq subfamilies of heterotrimeric G proteins [8], [9], [10]. In addition, RGS proteins have also been shown to block signal transduction by interfering with G protein–effector interactions [8], [9], [10].

RGS2 is unique among RGS proteins in being Gαq/11-selective [11], [12], which results from its low affinity for Gαi [13]. Although it has no observable effect on the GTPase activity of Gαs [11], [14], RGS2 is able to inhibit Gαs-stimulated adenylyl cyclase activity [15]. Apart from its effects on G protein-mediated signaling, RGS2 can also regulate tubulin polymerization [16], TRPV6 cation channels [17], and the initiation of mRNA translation [18]. RGS2 inhibits translation via its effects on the initiation factor eIF2B, which results in a reduction in global protein synthesis [18]. This function maps to a 37 amino acid residue domain that overlaps extensively with the RGS domain of RGS2 (RGS2 eIF2B-interacting domain), and a corresponding peptide is able to inhibit in vitro translation in a dose-dependent manner [18].

RGS2 is upregulated in response to, and can inhibit both Gq- and Gs-mediated signals [15], [19], [20], [21], [22]. Additionally, RGS2 is upregulated in many cells due to various forms of stress and may contribute to the cellular stress response [23]. RGS2 can impede Gq- and Gs-associated hypertrophic growth in cardiomyocytes, and its loss contributes to the development of hypertrophy [19], [20], [22], [24], [25]. Notably, mice lacking RGS2 that undergo transverse aortic constriction exhibit a greater degree of cardiac enlargement and develop heart failure more rapidly than their wild type counterparts [25]. The observed protective effects of RGS2 against hypertrophy are generally assumed to reflect its ability to limit GPCR signaling, however the possible contributions of its other functions have not been explicitly considered. Specifically, the ability of RGS2 to limit global protein synthesis would be expected to limit cellular growth, and in the present study we test the hypothesis that the 37 amino acid eIF2B-interacting domain of RGS2 (RGS2eb) might decrease GPCR-induced cardiomyocyte hypertrophy.

The primary factor controlling the physical growth associated with myocardial hypertrophy is regulation of protein synthesis in cardiomyocytes [26], and it has been shown that β-adrenergic receptor-induced hypertrophy is mediated through eIF2Bε [27]. Here we report that both RGS2 and RGS2eb are able to inhibit α- and β-adrenergic receptor-induced hypertrophy in rat neonatal cardiomyocytes. Unlike the full length protein, RGS2eb did not inhibit G protein-mediated effects on second messenger levels or ERK phosphorylation, suggesting that its antihypertrophic effects are due to the direct inhibition of mRNA translation. It follows that the antihypertrophic effect of full length RGS2 may arise at least in part from its ability to curtail protein synthesis.

Section snippets

Recombinant adenoviruses

Replication-defective adenoviruses encoding GFP (Ad-GFP), RGS2 (Ad-RGS2), and the 37 amino acid residue eIF2Bε binding domain of RGS2 (Ad-RGS2eb) were generated with the AdMax adenovirus vector creation kit according to the manufacturer's protocol (Microbix Biosystems, Inc., Toronto, Canada). Empty adenovirus (Ad-Ctr) lacking a gene insert was also used as a control. AdEasy Viral Titer kit (Agilent Technologies) was used to determine adenoviral titers following the manufacturer's procedure.

Isolation and primary cell culture of neonatal rat ventricular myocytes

RGS2eb expression attenuates the development of cardiomyocyte hypertrophy in response to phenylephrine and isoproterenol

Our previous work showed that full length RGS2 as well as the 37 amino acid residue eIF2B-interacting domain (RGS2eb) can inhibit de novo protein synthesis in vitro as well as in multiple cell types. The ability of RGS2eb to impede protein synthesis is sequence specific, as a control scrambled 37-mer peptide (same residues as RGS2eb but in randomized order) did not exhibit any inhibitory effects on in vitro translation (data not shown). These results likely reflect the sequence and structural

Discussion

The key finding of the present study is that cellular expression of the eIF2B-interacting domain of RGS2 is sufficient to impede the development of hypertrophy triggered by α1- and β-adrenergic agonists in neonatal cardiomyocytes, notwithstanding its lack of any measureable effect on second messenger production or ERK activation. Overexpression of full length RGS2, in contrast, inhibited both Gq- and Gs-mediated receptor signals as well as agonist-induced cardiomyocyte hypertrophy, while RGS2

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by operating grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Ontario. PC was supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario. RG is supported by a New Investigator Award from the Heart and Stroke Foundation of Canada.

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