Abstract 15542: Toll-like Receptor 7 Mediates Extracellular RNA-induced Cytokine Production in Cardiomyocytes
INTRODUCTION: We have recently reported that extracellular RNA (exRNA) released from injured cardiomyocytes induces cytokine production and may contribute to myocardial ischemia/reperfusion (I/R) injury. However, the signaling mechanism by which exRNA exhibits its pro-inflammatory effect in cardiomyocytes is unknown.
HYPOTHESIS: We hypothesize that exRNA induces inflammation through Toll-like receptors signaling.
METHODS: Mouse myocardial I/R model: WT mice were subjected to Sham procedure or coronary occlusion for 45 min followed by reperfusion. exRNA extraction and quantitation: exRNA in culture media or mouse sera was extracted using Trizol LS and quantified using Quant-iT RNA assay kit. ELISA: MIP-2 in culture media was measured by ELISA.
RESULTS: Twenty-four hours after hypoxia/serum-deprivation, RNA concentration in cardiomyocyte culture medium rose from 12 ng/ml to approx. 600 ng/ml. Freeze/thaw caused even greater RNA release (approx. 1500 ng/ml). In vivo, serum RNA level was increased from 183 ± 34 to 344 ± 68 ng/ml 24 hours after I/R. Moreover, in the presence of lipofectamine, purified exRNA induced MIP-2 production in a dose-dependent manner. exRNA-induced MIP-2 production was completely inhibited by RNase and Benzonase, but not DNase. The effect of the nucleases appeared specific as they had no effect on Pam3 (a TLR2 ligand)-induced MIP-2 production. Finally, exRNA-induced MIP-2 production in cardiomyocytes was partially (approx. 25%) and significantly inhibited by a specific TLR7 inhibitor (In), which completely blocked MIP-2 production induced by R837 (a TLR7 ligand). In contrast, TLR9-In had no effect on the exRNA-induced cytokines production. Similarly, TLR7-In, but not TLR9-In or TLR3 deletion, partially decreased exRNA-promoted MIP-2 production in macrophages.
CONCLUSIONS: These data demonstrate that 1) RNA is released from hypoxic cardiomyocytes in vitro and ischemic myocardium in vivo; 2) exRNA-induced MIP-2 production is partially through TLR7, but not TLR9 or TLR3.
Author Disclosures: Y. Feng: None. J. Cai: None. L. Zou: None. D. Li: None. W. Chao: None.
- © 2014 by American Heart Association, Inc.