Toll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury

Abstract

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3(-/-)) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3(-/-) and WT mice were subjected to myocardial ischemia (45min) followed by reperfusion for up to 3days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3(-/-) mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3(-/-) mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1β) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.

Keywords: Apoptosis; Inflammatory cytokine; Myocardial I/R; NF-κB; TLR.

Figure 1. TLR3 deficiency attenuates cardiac dysfunction following myocardial infarction or I/R injury and decreases myocardial infarct size following myocardial I/R injury

TLR3^−/− and age-matched WT mice were subjected to myocardial infarction (MI) by permanent ligation of LAD artery (A) or myocardial ischemia (45 min) followed by reperfusion for up to 3 days (B). Cardiac function was measured by echocardiography before (baseline) and after ischemia/reperfusion. (C) TLR3 deficiency decreases myocardial infarct size. TLR3^−/− and age-matched WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion (4 hrs). Infarct size was determined by TTC staining. Blue color shows non-ischemic areas, red color indicates ischemic areas. Pale (white) indicates necrotic tissues. Ratios of risk area vs. left ventricle area (RA/LV) and infarct area vs. risk area (IA/RA) were calculated and are presented in the graphs. Photographs of representative heart sections are shown above. There were 8 mice in each group. *p < 0.05 compared with indicated groups. ^#p < 0.05 compared with respective baseline.

Figure 2. TLR3 deficiency attenuates I/R-induced myocardial apoptosis

TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion (24 h). Sham surgical operation served as sham control. Myocardial apoptosis was examined by the TUNEL assay in the heart sections. (A) DAPI stained nuclei are blue color and TUNEL positive cells show green fluorescence. The bar graph shows the percent of apoptotic cells. n=3 in each group. (B) TLR3 deficiency prevents I/R-induced activity of caspase-3/7 and caspase-8 in the myocardium. There were 6 mice in each group. *p < 0.05 compared with indicated groups.

Figure 3. TLR3 deficiency attenuates I/R-increased FasL, Fas, FADD, Bak, and Bax expression, and prevents I/R-induced decreases in myocardial Bcl-2 levels

TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion (24 h). Sham surgical operation serve as sham control. TLR3 deficiency attenuates I/R-increased the levels of Fas (A), FasL (B), FADD (C), Bak (D), and Bax (E) in the myocardium. (F) TLR3 deficiency increases the levels of Bcl2 in the myocardium following myocardial I/R. There were 6 mice in each group. *p < 0.05 compared with indicated groups.

Figure 4. TLR3-deficiency prevents I/R-induced myocardial NF-κB binding activity and pro-inflammatory cytokine production in the circulation

TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion (24 h). Sham surgical operation serve as sham control. Hears were harvested for the preparation of the nuclear and cytoplasmic proteins. TLR3 deficiency prevents I/R-induced NF-κB binding activity (A), TNF-α (B), and IL-1β (C) production. There were 6 mice in each group. *p < 0.05 compared with indicated groups.

Figure 5. TLR3 deficiency attenuates I/R-induced infiltration of neutrophils and macrophages into the myocardium

TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion for indicated time. Sham surgical operation served as sham control. (A) TLR3 deficiency decreases the infiltration of neutrophils (A) and macrophages (B) into the myocardium. The pink color indicates positive neutrophils in the myocardium (A). The dark brown color indicates positive macrophages (B). There were 3 mice in each group. TLR3 deficiency prevents I/R-induced increases in the expression of VCAM-1 (C) and ICAM-1 (D) in the myocardium. The dark brown color indicates positive staining of VCAM-1 or ICAM-1 in the myocardium. There were 6 mice in each group. *p < 0.05 compared with indicated groups.

Figure 5. TLR3 deficiency attenuates I/R-induced infiltration of neutrophils and macrophages into the myocardium

TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion for indicated time. Sham surgical operation served as sham control. (A) TLR3 deficiency decreases the infiltration of neutrophils (A) and macrophages (B) into the myocardium. The pink color indicates positive neutrophils in the myocardium (A). The dark brown color indicates positive macrophages (B). There were 3 mice in each group. TLR3 deficiency prevents I/R-induced increases in the expression of VCAM-1 (C) and ICAM-1 (D) in the myocardium. The dark brown color indicates positive staining of VCAM-1 or ICAM-1 in the myocardium. There were 6 mice in each group. *p < 0.05 compared with indicated groups.