RESUMO
Dengue virus (DENV) infection elevates intracellular Ca2+ concentration ([Ca2+]i), but it is unknown whether Ca2+ and calmodulin (CaM) are involved in DENV infection. We conducted immunofluorescence and western blot experiments and measured [Ca2+]i examining the effects of DENV infection and drugs that alter Ca2+/CaM functions on CaM translocation, DENV2 infection, protein expression, virus-inducible STAT2 protein abundance, and CREB phosphorylation in H9c2 cells. DENV infection increased CaM expression, its nuclear translocation and NS3 and E viral proteins expression and colocalization in a manner that could be blocked by the ryanodine receptor antagonist dantrolene. DENV infection also increased CREB phosphorylation, an effect inhibited by either dantrolene or the CaM inhibitor W7. Dantrolene substantially hindered infection as assessed by focus assays in Vero cells. These results suggest that Ca2+ and CaM play an important role in DENV infection of cardiac cells and that dantrolene may protect against severe DENV cardiac morbidity.
Assuntos
Calmodulina/metabolismo , Núcleo Celular/metabolismo , Dantroleno/farmacologia , Vírus da Dengue/fisiologia , Mioblastos Cardíacos/virologia , Transporte Ativo do Núcleo Celular , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citosol/metabolismo , Vírus da Dengue/efeitos dos fármacos , Mioblastos Cardíacos/efeitos dos fármacos , Mioblastos Cardíacos/metabolismo , Fosforilação , Poli I-C/farmacologia , Ratos , Fator de Transcrição STAT2/metabolismo , Regulação para Cima , Proteínas Virais/metabolismoRESUMO
Voltage-dependent Ca2+ channels and store-operated Ca2+ channels (SOCs) are the major routes of Ca2+ entry into mammalian cells. Previously, we reported that pharmacological preconditioning (PPC) leads to a decrease in the amplitude of L-type calcium channel current in the heart. In this study, we examined PPC-associated changes in SOC function. We measured adult cardiomyocyte membrane currents using the whole-cell patch-clamp technique, and we evaluated reactive oxygen species (ROS) production and intracellular Ca2+ levels in cardiomyocytes using fluorescent probes. Diazoxide (Dzx) and thapsigargin (Tg) were used to induce PPC and to deplete internal stores of Ca2+, respectively. Ca2+ store depletion generated inward currents with strong rectification, which were suppressed by the SOC blocker GSK-7975-A. These currents were completely abolished by PPC, an effect that could be countered with 5-hydroxydecanoate (5-HD; a selective mitochondrial ATP-sensitive K+ channel blocker), an intracellular mitochondrial energizing solution, or Ni2+ [a blocker of sodium-calcium exchanger (NCX)]. Buffering of ROS and intracellular Ca2+ also prevented PPC effects on SOC currents. Refilling of intracellular stores was largely suppressed by PPC, as determined by measuring intracellular Ca2+ with a fluorescent Ca2+ indicator. These results indicate that influx of Ca2+ through SOCs is inhibited by their ROS and Ca2+-dependent inactivation during PPC and that NCX is a likely source of PPC-inactivating Ca2+. We further showed that NCX associates with Orai1. Down-regulation of SOCs by PPC may play a role in cardioprotection following ischemia-reperfusion.
RESUMO
Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection.
Assuntos
Vírus da Dengue/fisiologia , Miócitos Cardíacos/virologia , Animais , Linhagem Celular , Vírus da Dengue/crescimento & desenvolvimento , Modelos Biológicos , RatosRESUMO
The role of mitochondrial K(ATP) (mitoK(ATP)) channels on muscle fatigue was assessed in adult mouse skeletal muscle bundles. Muscle fatigue was produced by eliciting short repetitive tetani. Isometric tension and the rate of production of reactive oxygen species (ROS) were measured at room temperature (20-22 degrees C) using a force transducer and the fluorescent indicator CM-H(2)DCFDA. We found that opening mitoK(ATP) channels with diazoxide (100 microM) significantly reduced muscle fatigue. Fatigue tension was 34% higher in diazoxide-treated fibers relative to controls. This effect was blocked by the mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD), by the protein kinase C (PKC) inhibitor chelerythrine, and by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) but was not accompanied by a change in the rate of ROS production during fatigue. A physiological role of mitoK(ATP) channels on muscle fatigue is proposed.