Fig 2: Myocardial infarction (MI) increased TLR3 expression and its physical association with Trif in mouse myocardium. Heart samples were taken from infarct area at 4 weeks after MI. (A) and (B) show mRNA and protein levels of TLR3 in sham and MI hearts. n = 4 mice/group. Data are means ± S.D. A P < 0.01 versus sham. (C) Representative immunohistochemistry images of heart sections stained for TLR3 (brown colour). An isotype IgG control was performed to verify the specificity of TLR3 reactivity. (D) Lysates of heart tissue were immunoprecipitated with anti-TLR3 antibodies (IP: TLR3), followed by SDS–PAGE and immunoblotting (IB) with indicated antibodies. IP with isotype IgG (IP: IgG) was performed as a control to exclude the non-specific binding of antibodies to cellular proteins. Green arrows indicate non-specific bands. The association between TLR3 and Trif, but not MyD88, was detectable in sham myocardium and was increased in infarct myocardium.

Fig 3: A TLR3 agonist polyinosinic-polycytidylic acid (poly(I:C)) induced autophagy in cultured cardiomyocytes through a TRIF-dependent pathway. (A) Poly(I:C) increased autophagy markers in cultured H9c2 rat ventricular cells. (B) Poly(I:C) stimulated autophagosome formation but did not affect autophagic flux. Primary cultured neonatal rat ventricular myocytes (NRVMs) were transfected with a tandem mRFP-GFP-LC3 adenovirus for 24 hrs, followed by treatment with poly(I:C) (100 µg/ml, 4 hrs). Autophagosomes and autolysosomes were, respectively, visualized as yellow- and red-only punctas under a confocal microscope. (C) An autophagic flux inhibitor chloroquine (CQ) induced accumulations of LC3-II and p62/SQSTM1 proteins in H9c2 myocytes receiving poly(I:C) (100 µg/ml, 4 hrs). CQ was applied at 10 µM, immediately prior to poly(I:C). (D) Effects of indicated siRNA on poly(I:C)-induced changes in autophagy markers in NRVMs. All the siRNAs were transfected at 50 nM for 48 hrs, and poly(I:C) was added at 100 µg/ml for 4 hrs before cell harvest. Negative control (NC) siRNA served as control. RNAiMAX transfection reagent was used in all the siRNA experiments. The upper panel shows the knockdown effects of siRNAs, and the lower panel shows representative Western blot images (presented from four independent experiments) and densitometry quantitative data (normalized into ‘fold of vehicle group’). All quantitative data are expressed as means ± S.D. a P < 0.05, A P < 0.01 versus vehicle; b P < 0.05, B P < 0.01 versus poly(I:C).