RESUMO
A vast body of literature has established GRK2 as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function post injury in numerous animal models. In recent years, discovery of several non-canonical GRK2 targets has expanded our view of this kinase. Here, we describe the novel and exciting finding that cardiac GRK2 activity can regulate whole body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (TgßARKct) display an enhanced obesogenic phenotype when fed a high fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD fed TgßARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics we found that cardiac GRK2 signaling altered myocardial BCAA and endocannabinoid metabolism and modulated circulating BCAA and endocannabinoid metabolite profiles on a HFD, and one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. Taken together, these results suggest that metabolic changes in the heart due to GRK2 signaling on a HFD control whole body metabolism.
Assuntos
Tecido Adiposo Branco/metabolismo , Adiposidade/fisiologia , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Miocárdio/metabolismo , Obesidade/metabolismo , Adipócitos/fisiologia , Tecido Adiposo Branco/citologia , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Diferenciação Celular/fisiologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Endocanabinoides/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/genética , Humanos , Masculino , Metabolômica , Camundongos , Camundongos Transgênicos , Obesidade/etiologia , Transdução de Sinais/fisiologia , Aumento de Peso/fisiologiaRESUMO
Increased abundance of GRK2 [G protein-coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser670 is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.
Assuntos
Apoptose , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Glucose/química , Insuficiência Cardíaca/prevenção & controle , Isquemia/fisiopatologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Animais , Quinase 2 de Receptor Acoplado a Proteína G/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Masculino , Camundongos , Mitocôndrias/patologia , Miócitos Cardíacos/patologia , Oxirredução , Consumo de Oxigênio , Fosforilação , Mutação Puntual , Serina/química , Serina/genética , Serina/metabolismo , Transdução de SinaisRESUMO
RATIONALE: G protein-coupled receptor kinase 2 (GRK2) is an important molecule upregulated after myocardial injury and during heart failure. Myocyte-specific GRK2 loss before and after myocardial ischemic injury improves cardiac function and remodeling. The cardiac fibroblast plays an important role in the repair and remodeling events after cardiac ischemia; the importance of GRK2 in these events has not been investigated. OBJECTIVE: The aim of this study is to elucidate the in vivo implications of deleting GRK2 in the cardiac fibroblast after ischemia/reperfusion injury. METHODS AND RESULTS: We demonstrate, using Tamoxifen inducible, fibroblast-specific GRK2 knockout mice, that GRK2 loss confers a protective advantage over control mice after myocardial ischemia/reperfusion injury. Fibroblast GRK2 knockout mice presented with decreased infarct size and preserved cardiac function 24 hours post ischemia/reperfusion as demonstrated by increased ejection fraction (59.1±1.8% versus 48.7±1.2% in controls; P<0.01). GRK2 fibroblast knockout mice also had decreased fibrosis and fibrotic gene expression. Importantly, these protective effects correlated with decreased infiltration of neutrophils to the ischemia site and decreased levels of tumor necrosis factor-α expression and secretion in GRK2 fibroblast knockout mice. CONCLUSIONS: These novel data showing the benefits of inhibiting GRK2 in the cardiac fibroblast adds to previously published data showing the advantage of GRK2 ablation and reinforces the therapeutic potential of GRK2 inhibition in the heart after myocardial ischemia.
Assuntos
Fibroblastos/enzimologia , Quinase 2 de Receptor Acoplado a Proteína G/deficiência , Coração/fisiopatologia , Contração Miocárdica/fisiologia , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Animais , Animais Recém-Nascidos , AMP Cíclico/metabolismo , Fibrose , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Isquemia Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/genética , Miocárdio/patologia , NF-kappa B/metabolismo , Infiltração de Neutrófilos , RNA Interferente Pequeno/genética , Ratos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Volume Sistólico , Transdução Genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a ß2-adrenergic receptor (ß2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as ß2AR-induced hypertrophy.
Assuntos
Clembuterol/efeitos adversos , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/enzimologia , Doenças Musculares/enzimologia , Transdução de Sinais/efeitos dos fármacos , Animais , Clembuterol/farmacocinética , Quinase 2 de Receptor Acoplado a Proteína G/genética , Hipertrofia/induzido quimicamente , Hipertrofia/enzimologia , Hipertrofia/genética , Hipertrofia/patologia , Camundongos , Camundongos Knockout , Contração Muscular/genética , Músculo Esquelético/patologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/genética , Doenças Musculares/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Transdução de Sinais/genéticaRESUMO
Heart failure (HF) causes a tremendous burden on the worldwide healthcare system, affecting >23 million people. There are many cardiovascular disorders that contribute to the development of HF and multiple risk factors that accelerate its occurrence, but regardless of its underlying cause, HF is characterized by a marked decrease in myocardial contractility and loss of pump function. One biomarker molecule consistently shown to be upregulated in human HF and several animal models is G protein-coupled receptor kinase-2 (GRK2), a kinase originally discovered to be involved in G protein-coupled receptor desensitization, especially ß-adrenergic receptors. Higher levels of GRK2 can impair ß-adrenergic receptor-mediated inotropic reserve and its inhibition, or molecular reduction has shown to improve pump function in several animal models including a preclinical pig model of HF. Recently, nonclassical roles for GRK2 in cardiovascular disease have been described, including negative regulation of insulin signaling, a role in myocyte cell survival and apoptotic signaling, and it has been shown to be localized in/on mitochondria. These new roles of GRK2 suggest that GRK2 may be a nodal link in the myocyte, influencing both cardiac contractile function and cell metabolism and survival and contributing to HF independent of its canonical role in G protein-coupled receptor desensitization. In this review, classical and nonclassical roles for GRK2 will be discussed, focusing on recently discovered roles for GRK2 in cardiomyocyte metabolism and the effects that these roles may have on myocardial contractile function and HF development.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/fisiopatologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/enzimologia , Animais , Biomarcadores/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Insuficiência Cardíaca/patologia , Humanos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologiaRESUMO
Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. ß-adenergic receptors (ßAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous ßAR in primary neonatal rat cardiac fibroblasts (NRCF). Catecholamine stimulation of the cells induced a negative DMR deflection resulting in a concentration-dependent pharmacological response that was competitively blocked by ßAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Pharmacological profiling of subtype-selective ßAR agonists and antagonists revealed a dominant role of ß2AR in mediating the DMR responses, consistent with the relative expression levels of ß2AR and ß1AR in NRCF. Additionally, ßAR-mediated cAMP generation was assessed via a fluorescence biosensor, revealing similar kinetics between DMR responses and cAMP generation. As such, ßAR-dependent DMR responses were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, we assessed G protein-independent ßAR signaling through epidermal growth factor receptor (EGFR). While inhibition of EGFR reduced the DMR response to ßAR stimulation, our results demonstrate that G protein-dependent signaling produces a majority of the biological response to ßAR stimulation in NRCF. Altogether, measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous ßAR signaling via both G protein-dependent and -independent pathways.