RESUMO
RATIONALE: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator-activated receptor (PPAR)α and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown. OBJECTIVE: To determine the role of Krüppel-like factor 5 (KLF5) in transcriptional regulation of Ppara. METHODS AND RESULTS: We discovered that KLF5 activates Ppara gene expression via direct promoter binding. This is blocked in hearts of septic mice by c-Jun, which binds an overlapping site on the Ppara promoter and reduces transcription. We generated cardiac myocyte-specific Klf5 knockout mice that showed reduced expression of cardiac Ppara and its downstream fatty acid metabolism-related targets. These changes were associated with reduced cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation, and cardiac dysfunction. Diabetic mice showed parallel changes in cardiac Klf5 and Ppara expression levels. CONCLUSIONS: Cardiac myocyte KLF5 is a transcriptional regulator of Ppara and cardiac energetics.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Diabetes Mellitus Experimental/metabolismo , Metabolismo Energético , Fatores de Transcrição Kruppel-Like/metabolismo , Miócitos Cardíacos/metabolismo , PPAR alfa/metabolismo , Sepse/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/fisiopatologia , Linhagem Celular , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Ácidos Graxos/metabolismo , Genótipo , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , PPAR alfa/genética , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sepse/genética , Sepse/fisiopatologia , Transdução de Sinais , Transportador 2 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Triglicerídeos/metabolismo , Regulação para CimaRESUMO
While immunotherapy has become standard-of-care for cutaneous melanoma patients, primary and acquired resistance prevent long-term benefits for about half of the late-stage patients. Pre-clinical models are essential to increase our understanding of the resistance mechanisms of melanomas, aiming to improve the efficacy of immunotherapy. Here, we present two novel syngeneic transplantable murine melanoma cell lines derived from the same primary tumor induced on BrafV600E Pten-/- mice: MeVa2.1 and MeVa2.2. Derivatives of these cell lines expressing the foreign antigen ovalbumin (dOVA) showed contrasting immune-mediated tumor control. MeVa2.2.dOVA melanomas were initially controlled in immune-competent hosts until variants grew out that had lost their antigens. By contrast, MeVa2.1.dOVA tumors were not controlled despite presenting the strong OVA antigen, as well as infiltration of tumor-reactive CD8+ T cells. MeVa2.1.dOVA displayed reduced sensitivity to T cell-mediated killing and growth inhibition in vitro by both IFN-γ and TNF-α. MeVa2.1.dOVA tumors were transiently controlled in vivo by either targeted therapy, adoptive T cell transfer, regulatory T cell depletion, or immune checkpoint blockade. MeVa2.1.dOVA could thus become a valuable melanoma model to evaluate novel immunotherapy combinations aiming to overcome immune resistance mechanisms.
Assuntos
Melanoma , Neoplasias Cutâneas , Camundongos , Animais , Melanoma/patologia , Neoplasias Cutâneas/genética , Imunoterapia , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , AntígenosRESUMO
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
Cardiomyopathy frequently complicates sepsis and is associated with increased mortality. Increased cardiac oxidative stress and mitochondrial dysfunction have been observed during sepsis, but the mechanisms responsible for these abnormalities have not been determined. We hypothesized that NADPH oxidase 2 (NOX2) activation could be responsible for sepsis-induced oxidative stress and cardiomyopathy. Treatment of isolated adult mouse cardiomyocytes with low concentrations of the endotoxin lipopolysaccharide (LPS) increased total cellular reactive oxygen species (ROS) and mitochondrial superoxide. Elevated mitochondrial superoxide was accompanied by depolarization of the mitochondrial inner membrane potential, an indication of mitochondrial dysfunction, and mitochondrial calcium overload. NOX2 inhibition decreased LPS-induced superoxide and prevented mitochondrial dysfunction. Further, cardiomyocytes from mice with genetic ablation of NOX2 did not have LPS-induced superoxide or mitochondrial dysfunction. LPS decreased contractility and calcium transient amplitude in isolated cardiomyocytes, and these abnormalities were prevented by inhibition of NOX2. LPS decreased systolic function in mice, measured by echocardiography. NOX2 inhibition was cardioprotective in 2 mouse models of sepsis, preserving systolic function after LPS injection or cecal ligation and puncture (CLP). These data show that inhibition of NOX2 decreases oxidative stress, preserves intracellular calcium handling and mitochondrial function, and alleviates sepsis-induced systolic dysfunction in vivo. Thus, NOX2 is a potential target for pharmacotherapy of sepsis-induced cardiomyopathy.
Assuntos
Cálcio/metabolismo , Cardiomiopatias/prevenção & controle , Mitocôndrias Cardíacas/metabolismo , NADPH Oxidase 2/antagonistas & inibidores , Sepse/complicações , Animais , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/etiologia , Modelos Animais de Doenças , Ecocardiografia , Lipopolissacarídeos/farmacologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , NADPH Oxidase 2/genética , Fosforilação Oxidativa , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The relative role of acute myocardial ischemia and infarction in ventricular arrhythmogenesis is incompletely understood. We compared the arrhythmia pattern after ischemia/infarction to that observed after direct myocardial necrosis without preceding ischemia in rats. METHODS: Coagulation necrosis was induced in Wistar rats (n=20, 280±3 g) by radiofrequency current application (for 15 s) from a 4-mm-tip ablation catheter. Myocardial infarction was induced by coronary artery ligation with (n=10) or without (n=10) reperfusion. Using 24-h telemetry recording, we examined ventricular arrhythmias, voluntary motor activity and indices of sympathetic activation. RESULTS: The coagulation-necrosis volume was 24.4%±0.6%, comparable to the infarct size in the absence of reperfusion. Acute left ventricular failure and sympathetic activation were similar in the three groups. Coagulation necrosis induced ventricular fibrillation immediately, followed by a second peak after â¼1 h. Reperfusion decreased ventricular arrhythmias, whereas a second arrhythmogenic period (between the third and the eight hour) was noted in non-reperfused infarcts (mainly monomorphic ventricular tachycardia). CONCLUSIONS: Distinct arrhythmia patterns occur after myocardial infarction (with or without reperfusion) and after direct necrosis. They are not produced by differences in sympathetic activation and are likely related to the evolution of myocardial injury. The necrosis rat model may be useful in studies of arrhythmogenesis.
Assuntos
Arritmias Cardíacas/patologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Animais , Arritmias Cardíacas/etiologia , Modelos Animais de Doenças , Eletrocardiografia , Frequência Cardíaca/fisiologia , Masculino , Infarto do Miocárdio/complicações , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/patologia , Necrose , Ratos Wistar , Índice de Gravidade de Doença , Disfunção Ventricular Esquerda/complicações , Disfunção Ventricular Esquerda/patologiaRESUMO
The antiarrhythmic potential of postconditioning in in vivo models remains poorly defined. We compared the effects of pre- and postconditioning on ventricular arrhythmogenesis against controls with and without reperfusion. Wistar rats (n = 40, 269 ± 3 g) subjected to ischemia (30 minutes)--reperfusion (24 hours) were assigned to the following groups: (1) preconditioning (2 cycles), (2) postconditioning (6 cycles), or (3) no intervention and were compared with (4) nonreperfused infarcts and (5) sham-operated animals. Infarct size was measured, and arrhythmogenesis was evaluated with continuous telemetric electrocardiographic recording, heart rate variability indices, and monophasic action potentials (MAPs). During a 24-hour observation period, no differences in mortality were observed. Reperfusion decreased infarct size and ameliorated sympathetic activation during the late reperfusion phase. Preconditioning decreased infarct size by a further 35% (P = .0017), but only a marginal decrease (by 18%, P = .075) was noted after postconditioning. Preconditioning decreased arrhythmias during ischemia and early reperfusion, whereas postconditioning almost abolished them during the entire reperfusion period. No differences were noted in MAPs or in the magnitude of sympathetic activation between the 2 interventions. Compared to postconditioning, preconditioning affords more powerful cytoprotection, but both interventions exert antiarrhythmic actions. In the latter, these are mainly evident during the ischemic phase and continue during early reperfusion. Postconditioning markedly decreases reperfusion arrhythmias during a prolonged observation period. The mechanisms underlying the antiarrhythmic effects of pre- and postconditioning are likely different but remain elusive.