Study Of Extracellular RNA And TLR3-Trif Signaling In Myocardial Ischemia-reperfusion Injury

Objectives:Recent studies have suggested a role of TLR3as a sensor for endogenous RNA in tissue inflammatory injury. However, the impact of TLR3and its adaptor, Trif, mediated signaling in myocardial ischemia-reperfusion (I/R) injury, which involves significant innate immune response, is unknown. Here, we tested the hypotheses that1) TLR3-Trif signaling is involved in myocardial infarction (MI) and LV dysfunction after I/R.2) TLR3-Trif signaling contributes to myocardial I/R injury by mediating myocardial apoptosis.3) Extracellular RNA (exRNA) is responsible for myocardial I/R injury.Methods and Results:C57BL/6wild type (WT), Trif-/-and TLR3-/-mice were subjected to ligation of the left anterior descending coronary artery (LAD) for45min followed by reperfusion for4h or24h. At the end of reperfusion, the hearts were harvested and examined for area-at-risk (AAR) and MI sizes. Compared to WT, TLR3-/-and Trif-/-,but not IFNAR1-/-, mice had significantly smaller MI24h after I/R. Echocardiography demonstrated better-preserved LV function in TLR3-/-and Trif-/-than in WT mice24h after I/R. Myocardial apoptosis was significantly attenuated in Trif-/-compared with WT mice subjected to45min of ischemia and4h of reperfusion as demonstrated by fluorescent annexin-V imaging, DNA fragementation assay, and caspase-3activity. Surprisingly, Trif and TLR3deficiency had no impact on myocardial inflammation after I/R. Moreover, hypoxia and I/R led to release of RNA including small noncoding microRNAs from cardiomyocytes and cardiac tissue, respectively. Finally, RNase treatment inhibited necrotic cell-induced cytokine responses in cardiomyocytes in vitro. In vivo study, compared with the normal saline control group, RNase treatment group had significantly reduced myocardial cytokines production, caspase-3activity, and infarction size following ischemic insult.Conclusion:TLR3-Trif signaling represents an injurious pathway during I/R. Extracellular RNA released during I/R is a contributor to ischemic myocardial injury and thus may serve as a potential therapeutic target in the management of myocardial I/R injury.