RESUMEN
Trans fats are not a homogeneous group of molecules and less is known about the cellular effects of individual members of the group. Vaccenic acid (VA) and elaidic acid (EA) are the predominant trans monoenes in ruminant fats and vegetable oil, respectively. Here, we investigated the mechanism of cell death induced by VA and EA on primary rat ventricular myofibroblasts (rVF). The MTT assay demonstrated that both VA and EA (200µM, 0-72 h) reduced cell viability in rVF (P<0.001). The FACS assay confirmed that both VA and EA induced apoptosis in rVF, and this was concomitant with elevation in cleaved caspase-9, -3 and -7, but not caspase-8. VA and EA decreased the expression ratio of Bcl2:Bax, induced Bax translocation to mitochondria and decrease in mitochondrial membrane potential (Δψ). BAX and BAX/BAK silencing in mouse embryonic fibroblasts (MEF) inhibited VA and EA-induced cell death compared to the corresponding wild type cells. Transmission electron microscopy revealed that VA and EA also induced macroautophagosome formation in rVF, and immunoblot analysis confirmed the induction of several autophagy markers: LC3-ß lipidation, Atg5-12 accumulation, and increased beclin-1. Finally, deletion of autophagy genes, ATG3 and ATG5 significantly inhibited VA and EA-induced cell death (P<0.001). Our findings show for the first time that trans fat acid (TFA) induces simultaneous apoptosis and autophagy in rVF. Furthermore, TFA-induced autophagy is required for this pro-apoptotic effect. Further studies to address the effect of TFA on the heart may reveal significant translational value for prevention of TFA-linked heart disease.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia , Miofibroblastos/efectos de los fármacos , Miofibroblastos/patología , Ácidos Grasos trans/farmacología , Animales , Western Blotting , Células Cultivadas , Citometría de Flujo , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Cardiac myofibroblasts are key players in chronic remodeling of the cardiac extracellular matrix, which is mediated in part by elevated transforming growth factor-ß1 (TGF-ß1). The c-Ski proto-oncoprotein has been shown to modify TGF-ß1 post-receptor signaling through receptor-activated Smads (R-Smads); however, little is known about how c-Ski regulates fibroblast phenotype and function. We sought to elucidate the function of c-Ski in primary cardiac myofibroblasts using a c-Ski overexpression system. Cardiac myofibroblasts expressed three forms of c-Ski with the predominant band at 105 kDa, and adenoviral c-Ski treatment resulted in overexpression of 95-kDa c-Ski in cellular nuclei. Exogenous c-Ski led to significant inhibition of type I collagen secretion and myofibroblast contractility using two-dimensional semifloating gel contraction assay in both basal and with TGF-ß1 (10 ng/ml for 24 h) stimulation. Overexpressed c-Ski did not inhibit nuclear translocation of phosphorylated R-Smad2, despite their binding, as demonstrated by immunoprecipitation. Acute treatment of primary myofibroblasts with TGF-ß1 in vitro revealed a marked nuclear shuttling of c-Ski at 24 and 48 h following stimulation. Remarkably, overexpression of c-Ski led to a stepwise reduction of the myofibroblast marker α-smooth muscle actin with increasing multiplicity of infection, and these results indicate that 95-kDa c-Ski overexpression may effect a loss of the myofibroblastic phenotype. Furthermore, adenovirus (Ad) for hemagglutinin-tagged c-Ski infection led to a reduction in the number of myofibroblasts versus Ad-LacZ-infected and uninfected controls, due to induction of apoptosis. Finally, we observed a significant increase in 105-kDa c-Ski in the cytosolic fraction of cells of the infarct scar and adjacent remnant myocardium vs. noninfarcted controls.
Asunto(s)
Fibrosis/metabolismo , Contracción Miocárdica/fisiología , Miocardio/citología , Miofibroblastos/citología , Proteínas Proto-Oncogénicas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Apoptosis/fisiología , Diferenciación Celular , Supervivencia Celular , Regulación de la Expresión Génica/fisiología , Miofibroblastos/clasificación , Proteínas Proto-Oncogénicas/genética , Ratas , Proteína Smad2/genética , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
In fibrosing hearts, myofibroblasts are associated with cardiac extracellular matrix remodeling. Expression of key genes in the transition of cardiac fibroblast to myofibroblast phenotype in post-myocardial infarction heart and in vitro has not been well addressed. Contractile, focal adhesion-associated, receptor proteins, fibroblast growth factor-2 (FGF-2) expression, and motility were compared to assess phenotype in adult and neonatal rat cardiac fibroblasts and myofibroblasts. Neonatal and adult fibroblasts undergo phenotypic transition to myofibroblastic cells, marked by increased alpha-smooth muscle actin (alphaSMA), smooth muscle myosin heavy chain (SMemb), extra domain-A (ED-A) fibronectin, paxillin, tensin, FGF-2, and TbetaRII receptor. Elevated ED-A fibronectin confirmed fibroblast to supermature myofibroblastic phenotype transition. Presence of myofibroblasts in vivo was noted in sections of healed infarct scar after myocardial infarction, and their expression is similar to that in culture. Thus, cultured neonatal and adult cardiac fibroblasts transition to myofibroblasts in vitro and share expression profiles of cardiac myofibroblasts in vivo. Reduced motility with in vitro passage reflects enhanced production of focal adhesions.