RGS2 inhibits β-adrenergic receptor-induced cardiomyocyte hypertrophy

doi:10.1016/j.cellsig.2010.03.015

Cellular Signalling

Volume 22, Issue 8, August 2010, Pages 1231-1239

https://doi.org/10.1016/j.cellsig.2010.03.015 Get rights and content

Abstract

The chronic stimulation of certain G protein-coupled receptors promotes cardiomyocyte hypertrophy and thus plays a pivotal role in the development of human heart failure. The beta-adrenergic receptors (β-AR) are unique among these in that they signal via Gs, whereas others, such as the alpha1-adrenergic (α1-AR) and endothelin-1 (ET-1) receptors, predominantly act through Gq. In this study, we investigated the potential role of regulator of G protein signalling 2 (RGS2) in modulating the hypertrophic effects of the β-AR agonist isoproterenol (ISO) in rat neonatal ventricular cardiomyocytes. We found that ISO-induced hypertrophy in rat neonatal ventricular myocytes was accompanied by the selective upregulation of RGS2 mRNA, with little or no change in RGS1, RGS3, RGS4 or RGS5. The adenylyl cyclase activator forskolin had a similar effect suggesting that it was mediated through cAMP production. To study the role of RGS2 upregulation in β-AR-dependent hypertrophy, cardiomyocytes were infected with adenovirus encoding RGS2 and assayed for cell growth, markers of hypertrophy, and β-AR signalling. ISO-induced increases in cell surface area were virtually eliminated by the overexpression of RGS2, as were increases in α-skeletal actin and atrial natriuretic peptide. RGS2 overexpression also significantly attenuated ISO-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt activation, which may account for, or contribute to, its observed antihypertrophic effects. In contrast, RGS2 overexpression significantly activated JNK MAP kinase, while decreasing the potency but not the maximal effect of ISO on cAMP accumulation. In conclusion, the present results suggest that RGS2 negatively regulates hypertrophy induced by β-AR activation and thus may play a protective role in cardiac hypertrophy.

Introduction

RGS (regulator of G protein signalling) proteins are negative regulators of G protein signalling. They achieve this by accelerating the hydrolysis of GTP to GDP on the activated G protein, thereby converting it to its inactive state [1]. In addition to promoting G protein deactivation, RGS proteins may also negatively regulate signalling by blocking effector activation. All RGS proteins promote GTP hydrolysis by members of the Gαi subfamily of G proteins, and a subset also produces such effects on Gαq proteins [1].

RGS2 is a member of the B/R4 subfamily of RGS proteins which are relatively small and simple in structure. RGS2 is unique among the RGS proteins, since it binds to Gαi proteins with relatively low affinity and thus selectively attenuates Gq-mediated signals [2]. RGS2 inhibits Gq-dependent phospholipase Cβ activation both by promoting GTP hydrolysis and also apparently by physically disrupting the G protein-effector interaction [1]. In addition to Gq, RGS2 can inhibit Gs-stimulated adenylyl cyclase activity [3], [4], albeit through a mechanism that does not appear to involve changes in the rate at which this G protein hydrolyzes GTP [4], [5].

RGS2 is one of the predominant RGS proteins expressed in human heart [6]. Previously, we have shown that RGS2 expression in isolated neonatal rat cardiomyocytes is upregulated by Gq activation [7]. Furthermore, RGS2 overexpression attenuates cardiomyocyte hypertrophy induced by the activation of Gq-coupled receptors, whereas gene silencing of RGS2 by RNAi exacerbates Gq-mediated hypertrophy [9]. Mende et al. observed similar changes in adult ventricular myocytes [8], but also found that in vivo RGS2 ultimately may be downregulated subsequent to prolonged Gq activation [9]. Importantly, recent animal studies have substantiated the causal relationship between deficiency of RGS2 and cardiac hypertrophy. Takimoto et al. demonstrated that mice lacking RGS2 had a normal basal cardiac phenotype, yet responded rapidly to pressure overload, with increased myocardial Gq signalling, marked cardiac hypertrophy and failure, and early mortality [10]. Collectively, these observations suggest that RGS2 can protect against Gq-associated hypertrophic growth in cardiomyocytes, and that its loss contributes to the development of hypertrophy.

Signalling though the Gs-coupled β-AR is also able to stimulate cardiomyocyte hypertrophy, and RGS2 in other cells is upregulated by, and also can attenuate, signalling via Gs coupled receptors. Therefore, we hypothesized that signalling though the β-AR may regulate RGS2 expression in cardiomyocytes and that, in turn, RGS2 may regulate β-AR-induced cardiomyocyte hypertrophy. To test this, the effects of the β-AR agonist isoproterenol (ISO) on RGS2 expression, and the effects of overexpression of RGS2 on ISO-induced cardiomyocyte hypertrophy were examined in cultured rat cardiomyocytes. Notably, ISO treatment selectively induced RGS2 expression in primary rat cardiomyocytes. Furthermore, overexpression of RGS2 not only significantly attenuated ISO-induced cardiomyocyte hypertrophy, but also significantly blocked ISO-induced hypertrophic gene expression and attenuated ISO-induced ERK1/2 and Akt activation. These data indicate that RGS2 is important in regulating Gs-coupled hypertrophic responses in cardiomyocytes.

Section snippets

Cell culture

Primary rat ventricular myocytes were isolated from 2-day-old Sprague–Dawley rats following previously described procedures [7]. The isolated myocytes were cultured in plating medium (supplementary material) for 2–3 days prior to all experiments. All studies using animals were approved by the University of Western Ontario Animal Care and Use Committee and complied with guidelines of the Canadian Council on Animal Care.

Recombinant adenovirus

RGS2 adenovirus was generated as described previously [7]. Myocytes were

ISO induces RGS2 expression in neonatal ventricular myocytes

We investigated the ability of the β-AR agonist isoproterenol (ISO) to induce expression of endogenous RGS proteins in neonatal rat ventricular myocytes. Cells were treated with ISO (10 μM) for up to 24 h and mRNA levels of RGS1, 2, 3, 4 and 5 were determined using quantitative RT-PCR. Of the five RGS proteins examined, only the RGS2 mRNA transcript was significantly enhanced by ISO treatment (Fig. 1A). The stimulation of RGS2 expression by ISO reached a maximum after 1 h and tapered off

Discussion

Previous studies examining RGS2 function in cardiac tissue and cultured cardiomyocytes have focused primarily on signalling through Gq coupled receptors. The aim of the present study was to determine the role of RGS2 in regulating Gs coupled hypertrophic stimuli in rat neonatal ventricular cardiomyocytes. Our data demonstrate that RGS2 mRNA and protein are upregulated in cardiomyocytes, not only by Gq coupled signalling, but also by ISO which acts through the Gs coupled β-adrenergic (β-AR)

Acknowledgements

This work was supported by operating grants from the Heart and Stroke Foundation of Ontario (PC) and the Canadian Institutes of Health Research (PC, LAK). CN was supported by a Postdoctoral Fellowship from the Canadian Institutes of Health Research. AJS holds a Master's Studentship from the Heart and Stroke Foundation of Ontario. PC holds a Career Investigator Award from the Heart and Stroke Foundation of Ontario. We are grateful to Dr Morris Karmazyn for the helpful discussions and for sharing

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¹: These authors contributed equally to this work.

²: Current address: Hospital for Special Surgery. 535 East 70th Street, New York, NY 10021, United States.

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RGS2 inhibits β-adrenergic receptor-induced cardiomyocyte hypertrophy

Abstract

Introduction

Section snippets

Cell culture

Recombinant adenovirus

ISO induces RGS2 expression in neonatal ventricular myocytes

Discussion

Acknowledgements

Cell. Signal.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell. Signal.

J. Mol. Cell. Cardiol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Mol. Cell. Cardiol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell Cardiol.

Cell Signal.

Brain Res. Mol. Brain Res.

Nature

Cardiovasc. Res.

Invest

Endocrinology

Annu. Rev. Physiol.