G protein-coupled receptor kinase 2 contributes to impaired fatty acid metabolism in the failing heart

https://doi.org/10.1016/j.yjmcc.2018.08.025Get rights and content

Highlights

  • Elevated GRK2, as in HF, induces phosphorylation and ubiquitination of CD36.

  • GRK2 mediates CD36 downregulation and compromises FA uptake rate and energetics.

  • Knockout of GRK2 rescues CD36 phosphorylation and downregulation in HF.

  • GRK2 likely contributes to reduced CD36 and FA uptake/oxidation in HF.

  • Targeting GRK2 may restore primary contractile and energetic defects in HF.

Abstract

Increased G protein-coupled receptor kinase (GRK)2 is central to heart failure (HF) pathogenesis, via desensitization of β-adrenergic receptors and loss of contractile reserve. Since GRK2 has been shown to compromise fatty acid (FA) oxidation, this kinase may link metabolic and contractile defects in HF. The aim of this study was to investigate the mechanistic role of GRK2 in FA metabolism and bioenergetics in the heart. For that purpose, we measured FA uptake and cluster of differentiation (CD)36 expression, phosphorylation, and ubiquitination in mice with cardiac-specific overexpression of GRK2 (TgGRK2) or expression of its c-terminus (GRK2 inhibitor- TgβARKct) or in global heterozygous GRK2 knockout (GRK2+/−) mice. Cellular bioenergetics were also measured in isolated cardiomyocytes following adenoviral delivery of exogenous GRK2, βARKct, or short hairpin GRK2 (shGRK2). Additionally, CD36 expression and phosphorylation were evaluated following transverse aortic constriction (TAC) in wild type (WT) and GRK2+/− mice. Our results show a 33% ± 0.81 reduction in FA uptake rate, accompanied by 51% ± 0.17 lower CD36 protein, and 70% ± 0.23 and 69% ± 0.18 increases in CD36 phosphorylation and ubiquitination, respectively, in the TgGRK2 mice. Moreover, an in vitro kinase assay suggests that GRK2 directly phosphorylates CD36. In isolated cardiomyocytes, GRK2 overexpression induced a 26% ± 2.21 decrease in maximal respiration, which was enhanced (20% ± 4.02–5.14) with inhibition of the kinase. Importantly, in hearts with systolic dysfunction, notable reductions in CD36 mRNA and protein, as well as a significant increase in CD36 phosphorylation were normalized in the GRK2+/− mice post-TAC. Thus, we propose that GRK2 up-regulation in HF is, at least partly, responsible for reduced FA uptake and oxidation and may be a nodal link between metabolic and contractile defects.

Introduction

During heart failure (HF), sustained sympathetic drive and circulating catecholamines lead to β-adrenergic receptor (βAR) desensitization and downregulation, and, therefore, loss of contractile reserve [1]. G protein-coupled receptor (GPCR) kinase (GRK)2 is increased following cardiac injury or stress [[2], [3], [4], [5], [6]] and is responsible for phosphorylation and internalization of ligand-occupied βARs [7]. Accordingly, increased GRK2 promotes the development of HF and its inhibition improves cardiac contractile function and delays HF progression [[8], [9], [10], [11], [12], [13]]. We now also realize that GRK2 has a broader array of functions, as it has been implicated in the regulation of cardiomyocyte metabolism and mitochondrial function. In particular, following myocardial infarction, increased GRK2 phosphorylates insulin receptor substrate (IRS)1, inhibiting insulin signaling and insulin-induced glucose uptake, and contributing to insulin resistance in HF [14]. Moreover, GRK2 has been shown to localize to the mitochondria following ischemia/reperfusion where it increases oxidative stress and induces cardiomyocyte death [15,16], whereas in neonatal cardiomyocytes, it impairs β-oxidation [17]. Specific GRK2 mitochondrial substrates, however, have yet to be identified.

The contractile function of the heart is supported by a concomitant supply of energy that is tightly linked to demand. During HF this relationship becomes imbalanced, as energy demand outpaces the mitochondrial bioenergetic capacity due to both an increase in wall stress [18], as well as, compromised mitochondrial structure and function. This encompasses decreased mitochondrial content, decreased electron transport chain function, increased reactive oxygen species (ROS), abnormal morphology, and decreased respiration [19,20]. Thus, there is an overall decrease in oxidative metabolism and adenosine triphosphate (ATP) production [19,20]. These defects are further compounded by changes in myocyte FA and glucose uptake, with decreases in sarcolemmal FA transporter, cluster of differentiation/FA translocase (CD)36 [21,22]. However, the mechanisms underlying these events have not been fully elucidated, including a potential role for GRK2.

While the role of GRK2 in regulating glucose metabolism and mitochondrial function continue to be explored, the function and targets of GRK2 in FA metabolism remain uninvestigated. We hypothesized that increased GRK2 can negatively regulate cardiac FA metabolism, as observed in the injured and failing heart. Our findings demonstrate that, in the heart, increased levels of GRK2 result in an increase in the phosphorylation and ubiquitination of CD36, which is accompanied by its downregulation and decreased FA uptake rate. CD36 directs the majority of protein-mediated transport of FA, the preferred cardiac metabolic substrate, into the cardiomyocyte [23]. Our results, therefore, suggest that GRK2 may function as a molecular link between cardiac contractile and metabolic functions in the heart.

Section snippets

An increase in cardiac GRK2 compromises FA uptake

Since GRK2 is known to impair mitochondrial function [16,17] and is upregulated in the failing heart [[2], [3], [4], [5], [6]], which also exhibits reduced FA uptake and oxidation [[19], [20], [21],24,25], we hypothesized that it may regulate this process. To address this, we assessed FA uptake in cardiac-specific GRK2 transgenic (TgGRK2) mice, which exhibit 3–4 fold overexpression of GRK2 in cardiomyocytes, similar to levels found in failing human hearts [[2], [3], [4], [5], [6],26]. The

Discussion

For the last two decades, it has been clear that GRK2 up-regulation mediates the lack of inotropic reserve in the failing heart. The involvement of GRK2 in regulating metabolism, both directly and indirectly, has also been recognized. As described above, it's targeting and inhibition of IRS-1 highlights its role in the regulation of glucose metabolism and the development of insulin resistance in the failing heart [14]. This study, however, is the first to associated elevated GRK2 levels, a

Animal models

All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee of Temple University. The generation and characterization of mice with cardiac-specific overexpression of GRK2 (TgGRK2) or the expression of the βARKct (TgβARKct) as a GRK2 inhibitor peptide, or global knockout of GRK2 (GRK2+/−) are as previously described [26,43]. For all

Acknowledgments

This study is supported in part by NIH grants R37 HL061690, P01 HL108806, and P01 HL075443 (to W.J.K.). We would also like to thank Zuping Qu for maintenance of the animal colony, as well as Eric Barr and Kevin Luu for performing all of the animal genotyping. We would also like to thank Dr. J. Kurt Chuprun for support and discussions.

Funding

This work was supported by the National Institutes of Health [Grant Numbers: HL061690, HL108806, HL075443 (to W.J.K.)]. W.J.K. is the William Wikoff Smith Endowed Chair in Cardiovascular Medicine and a 2018 MERIT Awardee from the American Heart Association.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

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