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1.
Circ Res ; 119(10): 1116-1127, 2016 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-27601479

RESUMO

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/metabolismo
2.
J Biol Chem ; 291(42): 21913-21924, 2016 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-27566547

RESUMO

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/genética
3.
Circ Res ; 117(12): 1001-12, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26515328

RESUMO

RATIONALE: G protein-coupled receptor kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is upregulated in heart failure. Although GRK5 is a critical regulator of cardiac G protein-coupled receptor signaling, recent data has uncovered noncanonical activity of GRK5 within nuclei that plays a key role in pathological hypertrophy. Targeted cardiac elevation of GRK5 in mice leads to exaggerated hypertrophy and early heart failure after transverse aortic constriction (TAC) because of GRK5 nuclear accumulation. OBJECTIVE: In this study, we investigated the role of GRK5 in physiological, swimming-induced hypertrophy (SIH). METHODS AND RESULTS: Cardiac-specific GRK5 transgenic mice and nontransgenic littermate control mice were subjected to a 21-day high-intensity swim protocol (or no swim sham controls). SIH and specific molecular and genetic indices of physiological hypertrophy were assessed, including nuclear localization of GRK5, and compared with TAC. Unlike after TAC, swim-trained transgenic GRK5 and nontransgenic littermate control mice exhibited similar increases in cardiac growth. Mechanistically, SIH did not lead to GRK5 nuclear accumulation, which was confirmed in vitro as insulin-like growth factor-1, a known mediator of physiological hypertrophy, was unable to induce GRK5 nuclear translocation in myocytes. We found specific patterns of altered gene expression between TAC and SIH with GRK5 overexpression. Further, SIH in post-TAC transgenic GRK5 mice was able to preserve cardiac function. CONCLUSIONS: These data suggest that although nuclear-localized GRK5 is a pathological mediator after stress, this noncanonical nuclear activity of GRK5 is not induced during physiological hypertrophy.


Assuntos
Cardiomegalia/metabolismo , Cardiomegalia/patologia , Quinase 5 de Receptor Acoplado a Proteína G/fisiologia , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Cardiomegalia/genética , Células Cultivadas , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/patologia , Ratos
4.
Circ Res ; 114(10): 1661-70, 2014 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-24812353

RESUMO

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/fisiologia
6.
bioRxiv ; 2023 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-36824711

RESUMO

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.

7.
Nat Commun ; 14(1): 5031, 2023 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-37596294

RESUMO

Mitochondrial quality control is critical for cardiac homeostasis as these organelles are responsible for generating most of the energy needed to sustain contraction. Dysfunctional mitochondria are normally degraded via intracellular degradation pathways that converge on the lysosome. Here, we identified an alternative mechanism to eliminate mitochondria when lysosomal function is compromised. We show that lysosomal inhibition leads to increased secretion of mitochondria in large extracellular vesicles (EVs). The EVs are produced in multivesicular bodies, and their release is independent of autophagy. Deletion of the small GTPase Rab7 in cells or adult mouse heart leads to increased secretion of EVs containing ubiquitinated cargos, including intact mitochondria. The secreted EVs are captured by macrophages without activating inflammation. Hearts from aged mice or Danon disease patients have increased levels of secreted EVs containing mitochondria indicating activation of vesicular release during cardiac pathophysiology. Overall, these findings establish that mitochondria are eliminated in large EVs through the endosomal pathway when lysosomal degradation is inhibited.


Assuntos
Vesículas Extracelulares , Lisossomos , Animais , Camundongos , Mitocôndrias , Transporte Biológico , Corpos Multivesiculares
8.
JCI Insight ; 52019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30946029

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/fisiologia
9.
JCI Insight ; 52019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30990467

RESUMO

The E3 ubiquitin ligase Parkin plays an important role in regulating clearance of dysfunctional or unwanted mitochondria in tissues, including the heart. However, whether Parkin also functions to prevent cardiac aging by maintaining a healthy population of mitochondria is still unclear. Here, we have examined the role of Parkin in the context of mtDNA damage and myocardial aging using a mouse model carrying a proofreading defective mitochondrial DNA polymerase gamma (POLG). We observed both decreased Parkin protein levels and development of cardiac hypertrophy in POLG hearts with age; however, cardiac hypertrophy in POLG mice was neither rescued, nor worsened by cardiac specific overexpression or global deletion of Parkin, respectively. Unexpectedly, mitochondrial fitness did not substantially decline with age in POLG mice when compared to WT. We found that baseline mitophagy receptor-mediated mitochondrial turnover and biogenesis were enhanced in aged POLG hearts. We also observed the presence of megamitochondria in aged POLG hearts. Thus, these processes may limit the accumulation of dysfunctional mitochondria as well as the degree of cardiac functional impairment in the aging POLG heart. Overall, our results demonstrate that Parkin is dispensable for constitutive mitochondrial quality control in a mtDNA mutation model of cardiac aging.


Assuntos
Envelhecimento/patologia , Cardiomegalia/patologia , Mitocôndrias/patologia , Miocárdio/patologia , Ubiquitina-Proteína Ligases/metabolismo , Envelhecimento/genética , Animais , Cardiomegalia/diagnóstico , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Células Cultivadas , DNA Polimerase gama/genética , DNA Polimerase gama/metabolismo , DNA Mitocondrial/genética , Modelos Animais de Doenças , Ecocardiografia , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mitofagia/genética , Mutação , Miocárdio/citologia , Miócitos Cardíacos , Cultura Primária de Células , Ubiquitina-Proteína Ligases/genética
10.
J Biol Chem ; 283(4): 2335-43, 2008 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-18024432

RESUMO

Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the alpha2beta1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the alpha2 I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of alpha2beta1. Microvascular endothelial cells from alpha2beta1(-/-) mice, but not those isolated from either wild-type or alpha1beta1(-/-) mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in alpha2beta1(-/-) mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin alpha2beta1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the alpha2beta1 integrin receptor.


Assuntos
Proteínas Angiostáticas/genética , Endotélio Vascular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Integrina alfa2beta1/metabolismo , Neovascularização Patológica/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Angiostáticas/metabolismo , Proteínas Angiostáticas/farmacologia , Animais , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Endotélio Vascular/patologia , Feminino , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/farmacologia , Humanos , Integrina alfa1beta1/genética , Integrina alfa1beta1/metabolismo , Integrina alfa2beta1/genética , Camundongos , Camundongos Knockout , Transplante de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Transplante Heterólogo
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