RESUMEN
The activation of signaling pathways involving protein tyrosine kinases (PTKs) has been demonstrated during Trypanosoma cruzi invasion. Herein, we describe the participation of FAK/Src in the invasion of cardiomyocytes by T. cruzi. The treatment of cardiomyocytes with genistein, a PTK inhibitor, significantly reduced T. cruzi invasion. Also, PP1, a potent Src-family protein inhibitor, and PF573228, a specific FAK inhibitor, also inhibited T. cruzi entry; maximal inhibition was achieved at concentrations of 25µM PP1 (53% inhibition) and 40µM PF573228 (50% inhibition). The suppression of FAK expression in siRNA-treated cells and tetracycline-uninduced Tet-FAK(WT)-46 cells significantly reduced T. cruzi invasion. The entry of T. cruzi is accompanied by changes in FAK and c-Src expression and phosphorylation. An enhancement of FAK activation occurs during the initial stages of T. cruzi-cardiomyocyte interaction (30 and 60min), with a concomitant increase in the level of c-Src expression and phosphorylation, suggesting that FAK/Src act as an integrated signaling pathway that coordinates parasite entry. These data provide novel insights into the signaling pathways that are involved in cardiomyocyte invasion by T. cruzi. A better understanding of the signal transduction networks involved in T. cruzi invasion may contribute to the development of more effective therapies for the treatment of Chagas' disease.
Asunto(s)
Quinasa 1 de Adhesión Focal/fisiología , Miocitos Cardíacos/parasitología , Transducción de Señal/fisiología , Trypanosoma cruzi/fisiología , Familia-src Quinasas/fisiología , Animales , Proteína Tirosina Quinasa CSK , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Quinasa 1 de Adhesión Focal/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Fosforilación , Pirazoles/farmacología , Pirimidinas/farmacología , Quinolonas/farmacología , ARN Interferente Pequeño/fisiología , Sulfonas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismoRESUMEN
Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.
Asunto(s)
Endotoxemia/enzimología , Quinasa 1 de Adhesión Focal/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas Musculares/biosíntesis , Miocardio/enzimología , ARN Interferente Pequeño/farmacología , Animales , Colágeno/metabolismo , Endotoxemia/inducido químicamente , Endotoxemia/patología , Activación Enzimática/efectos de los fármacos , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Silenciador del Gen/efectos de los fármacos , Humanos , Lipopolisacáridos/toxicidad , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocardio/patología , Fosforilación/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Hypertrophy is a critical event in the onset of failure in chronically overloaded hearts. Focal adhesion kinase (FAK) has attracted particular attention as a mediator of hypertrophy induced by increased load. Here, we demonstrate increased expression and phosphorylation of FAK in the hypertrophic left ventricles (LVs) of aortic-banded mice. We used an RNA interference strategy to examine whether FAK signaling plays a role in the pathophysiology of load-induced LV hypertrophy and failure. Intrajugular delivery of specific small interfering RNA induced prolonged FAK silencing ( approximately 70%) in both normal and hypertrophic LVs. Myocardial FAK silencing was accompanied by prevention, as well as reversal, of load-induced left ventricular hypertrophy. The function of LVs was preserved and the survival rate was higher in banded mice treated with small interfering RNA targeted to FAK, despite the persistent pressure overload. Studies in cardiac myocytes and fibroblasts harvested from LVs confirmed the ability of the systemically administered specific small interfering RNA to silence FAK in both cell types. Further analysis indicated attenuation of cardiac myocyte hypertrophic growth and of the rise in the expression of beta-myosin heavy chain in overloaded LVs. Moreover, FAK silencing was demonstrated to attenuate the rise in the fibrosis, collagen content, and activity of matrix metalloproteinase-2 in overloaded LVs, as well as the rise of matrix metalloproteinase-2 protein expression in fibroblasts harvested from overloaded LVs. This study provides novel evidence that FAK may be involved in multiple aspects of the pathophysiology of cardiac hypertrophy and failure induced by pressure overload.