Abstract 18768: Role of Tlr4 in Modulating Isoproterenol Induced-Autophagic Genes in the Brain: An in vivo and in vitro Study
Exaggerated sympathetic activity is the hallmark of cardiovascular disease. It is increasingly evident that inflammatory molecules play an important role in the pathogenesis of heart failure (HF). Increased beta-adrenergic stimulation might cause damage to proteins that might contribute to increased autophagic flux and inflammation. Since studies from our lab have shown that TLR4 within the brain contribute to sympathoexcitatory process, we hypothesized that TLR4 mediated autophagic flux in the hypothalamus may be critical in isoproterenol (ISO) induced sympathoexcitation. To test our hypothesis, we used both in vitro and in vivo studies to demonstrate the role of TLR4 in modulating ISO induced autophagy in the murine neuroblastoma cells and in the hypothalamus. For in vitro studies, we pretreated neuroblastoma cells with specific TLR4-inhibitor peptide (VIPER), and knocked down TLR4 using a SiRNA-TLR4, followed by challenge with isoproterenol (100uM) for 24h. Cells were harvested post-treatment and processed for the expression of mRNA and proteins levels of autophagy related proteins. ISO treatment significantly increased autophagic protein (specifically ATG5, ATG16 and LC3 show 3.5-, 3.9-, 4.6- fold increase respectively, p < 0.01). These increased were significantly reduced by TLR4 inhibition in murine neuroblastoma cells in response to challenge with ISO. To determine the effect of ISO in vivo, WT and TLR4-/- mice were infused s/c with ISO (85mg/kg/day) for 2 days. Cardiac function were measured at baseline and at 48h post ISO treatment. Subsequently, mice were euthanized, plasma samples collected for norepinephrine and the hypothalamus for quantification of mRNA levels of autophagy related gene and proteins. In the WT+ISO treatment showed increased mRNA expression levels of autophagic related genes ATG5, ATG16 and LC3, by 2.7-, 2.5- & 5- fold respectively (p < 0.01) as compared to WT, whereas from TLR4-/-+ISO group showed decreased expression of ATG5, ATG16 and LC3 by 1.7-,1.5- and 2.19- fold respectively, p < 0.01). These findings suggest an important role for TLR4 in regulating autophagic genes in the brain and contributing to sympathoexcitatory process in HF.
Author Disclosures: P. Bagam: None. F.A. Rosa: None. R. Dharmakumar: None. S. Batra: None. J. Francis: None.
- © 2016 by American Heart Association, Inc.