RESUMEN
A high global prevalence of diabetes and its implications on the heart in vivo and in vitro tools have been pursued to alleviate the complications of high glucose. This chapter oulines the methods used for maintaining H9C2 cardiomyoblasts in vitro and for stimulating hyperglycemic situation. In addition, we present a method to assess cellular GLUT-4 expression using qRT-PCR. This cellular model also allows us to examine the therapeutic approach of an antioxidant, Trolox, for upregulating GLUT-4 and uptake of glucose under hyperglycemic condition.
Asunto(s)
Antioxidantes , Diabetes Mellitus , Miocardio , Reacción en Cadena de la Polimerasa , Antioxidantes/farmacología , Cromanos/farmacología , Glucosa , Transportador de Glucosa de Tipo 4/análisis , Humanos , Miocardio/citología , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Type-2 diabetes, characterized by hyperglycemia causing various symptoms of metabolic disorders in the heart, kidneys, and brain, has many underlying molecular mechanisms leading to functional insufficiency of these organs. We describe protocols wherein we have optimized conditions for maintenance of hyperglycemic H9c2 cell lines and design to assess the effect of a water-soluble vitamin, Trolox, on the apoptotic pathway. Primarily, the design provides researchers to analyze apoptosis by flow cytometry.
Asunto(s)
Apoptosis , Citometría de Flujo , Hiperglucemia , Miocitos Cardíacos , Línea Celular , Humanos , Miocardio/citologíaRESUMEN
Redox imbalance due to hyperglycemia is a causative factor for an increased generation of reactive oxygen species (ROS) that leads to mitochondrial dysfunction and the release of cytochrome-c. The aim of the present study is to elucidate the functional role of oxidative stress (OS) in the induction of apoptosis in H9c2 cells in the hyperglycemic state through glucose transporter-4 (GLUT-4) regulation and antioxidant status. H9c2 cells were incubated with 15, 24, and 33 mM glucose for 24, 48, and 72 hr to induce hyperglycemic stress. Hyperglycemic episodes have significantly influenced GLUT-4 mRNA regulation, depleted glutathione (GSH) and its associated enzymes, reduced cellular antioxidant enzymes (AOEs), caused nuclear condensation, and induced apoptosis by activating caspase-9 and 3 and annexin V binding in a concentration and duration-dependent manner. Trolox pretreatment significantly enhanced the GLUT-4 mRNA and antioxidant defense mechanism, suppressed nuclear condensation, and prevented cytochrome-c release, thereby reducing mitochondrial-dependent apoptosis. The present study shows that the toxic effect of high glucose is significantly regulated and that OS induction can be prevented through a water-soluble vitamin E analog "Trolox" treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , Cromanos/farmacología , Transportador de Glucosa de Tipo 4/metabolismo , Hiperglucemia/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Apoptosis/genética , Apoptosis/fisiología , Citocromos c/metabolismo , Enzimas/metabolismo , Glucosa/efectos adversos , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/genética , Glutatión/metabolismo , Hiperglucemia/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Estrés Oxidativo/efectos de los fármacos , RatasRESUMEN
Oxidative stress (OS) has been implicated in a variety of pathological conditions, including diabetes mellitus, characterized by hyperglycemia. In the present study, OS induced by hyperglycemia and the effect of trolox, a vitamin E analog, were studied in cardiomyocytes and H9c2 cells exposed to 15 to 33 mM glucose (HG) for 24 to 72 hours in Dulbecco modified Eagle medium. Cells treated wirh 24 or 33 mM glucose for 24 hours or above showed decreased viability and adenosine triphosphate (ATP) content with a concomitant increase in radicals of oxygen species, calcium (Ca2+ ), mitochondrial permeability transition, and oxidative markers, confirming that the cells were under stress. However, upon exposure to 15 mM glucose for 24 hours, H9c2 cells maintained homeostasis and ATP generation. Pretreatment of cells with trolox reduced HG-induced OS to control levels. Here, we report that the toxic effect of HG is highly regulated and that OS induction can be prevented with Trolox, a potential inhibitor of membrane damage.