Cytokines link Toll-like receptor 4 signaling to cardiac dysfunction after global myocardial ischemia

Abstract

Background: Although Toll-like receptor 4 (TLR4) has been implicated in the myocardial injury caused by regional ischemia/reperfusion, its role in the myocardial inflammatory response and in contractile dysfunction after global ischemia/reperfusion is unclear. Cytokines, particularly tumor necrosis factor-alpha (TNF-alpha), contribute to the mechanism of myocardial dysfunction after global ischemia/reperfusion. We hypothesized that a TLR4-mediated cytokine cascade modulates myocardial contractile function after global ischemia/reperfusion. This study examined whether TLR4 regulates TNF-alpha and interleukin (IL)-1beta peptide production during global ischemia/reperfusion and whether TLR4 signaling influences postischemic cardiac function through TNF-alpha and IL-1beta.

Methods: Isolated hearts from wild-type mice, two strains of TLR4 mutants, TNF-alpha knockouts, and IL-1beta knockouts underwent global ischemia/reperfusion. Cardiac contractile function was analyzed, and myocardial nuclear factor-kappaB activity and TNF-alpha and IL-1beta levels were measured.

Results: In wild-type hearts, global ischemia/reperfusion induced nuclear factor-kappaB activation and the production of TNF-alpha and IL-1beta peptides. In TLR4-mutant hearts, these changes were significantly reduced and postischemic functional recovery was improved. Application of TNF-alpha and IL-1beta to TLR4-mutant hearts abrogated this improvement in postischemic functional recovery. Postischemic functional recovery also improved in TNF-alpha knockout and IL-1beta knockout hearts, as well as in wild-type hearts treated with TNF-binding protein or IL-1 receptor antagonist.

Conclusions: This study demonstrates that TLR4 signaling contributes to cardiac dysfunction after global ischemia/reperfusion. TLR4 signaling mediates the production of TNF-alpha and IL-1beta peptides, and these two cytokines link TLR4 signaling to postischemic cardiac dysfunction.

Fig 1

Effect of Toll-like receptor 4 (TLR4) mutation on postischemic cardiac function. Hearts from 8 TLR4-defective (TLR4-def, black squares) and 6 TLR4-deleted (TLR4-del, black circles) mice and their respective wild-type (WT) controls (WT-def, n = 8, white squares; WT-del, n = 6, white circles) underwent 20 minutes of global ischemia followed by 60 minutes of reperfusion. TLR4-mutant hearts displayed improved recovery of (A) left ventricular developed pressure (LVDP), (B) +dP/dt max, and (C) left ventricular end-diastolic pressure (LVEDP) compared with wild-type controls. Data are expressed as mean ± SEM; *p < 0.05 vs WT-def; ^#p < 0.05 vs WT-del.

Fig 2

Effect of Toll-like receptor 4 (TLR4) mutation on nuclear factor (NF)-κB phosphorylation. (A) A representative gel shows results after hearts of wild-type (WT) and TLR4-defective (TLR4-def) mice underwent either 40 minutes of perfusion (C–control), 20 minutes of ischemia (I), or 20 minutes of ischemia followed by 60 minutes of reperfusion (I/R). (B) Densitometry data of two separate experiments show that levels of phosphorylated NF-κB p65 increased after ischemia and ischemia/reperfusion in WT hearts (black bars), but not in TLR4-defective hearts (white bars).

Fig 3

Role of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in postischemic cardiac dysfunction. (A) Immunoblotting demonstrates that TNF-α knockout (TNF KO), IL-1β knockout (IL-1 KO) and wild-type (WT) hearts have comparable Toll-like receptor 4 (TLR4) levels. (B, C) Hearts isolated from TNF KO (black circles), IL-1 KO (black squares) and WT (white circles, a combined group of TNF-α WT and IL-1β WT) underwent 20 minutes of global ischemia followed by 60 minutes reperfusion. TNF KO and IL-1 KO hearts had higher left ventricular developed pressure (LVDP) and +dP/dt max after I/R than hearts from wild-type controls. Data are expressed as mean ± SEM. n = 6 in each group; *p < 0.05 vs WT; ^#p < 0.05 vs IL-1 KO. (D) Wild-type hearts underwent 20 minutes of global ischemia followed by 60 minutes reperfusion, and were treated with TNF-binding protein (TNF-BP, 1.0 μg/mL) or IL-1 receptor antagonist (IL-1 RA, 1.0 μg/mL) during reperfusion. The percentage recovery of LVDP in hearts treated with TNF-BP and IL-1 RA was greater than in untreated hearts and was similar to knockouts. Data are expressed as mean ± standard error of the mean; n = 6 in each group; *p < 0.05 vs WT.

Fig 4

Effect of tumor necrosis factor (TNF)-α and interleukin (IL)-1β on postischemic cardiac functional recovery in Toll-like receptor 4 (TLR4)-defective hearts. TLR4-defective (TLR4-def) hearts underwent 20 minutes of global ischemia followed by 60 minutes of reperfusion, and were treated with TNF-α and IL-1β (0.1 ng/mL each) during reperfusion. Postischemic (A) left ventricular developed pressure (LVDP) and (B) +dP/dt max were similar in TLR4-defective hearts treated with TNF-α and IL-1β (TLR4-def + Cytok, black circles) and wild-type (WT, white squares) hearts, and significantly lower than in untreated TLR4-defective hearts (white circles). Data are expressed as mean ± SEM; n = 6 in each group; *p < 0.05 vs WT; ^#p < 0.05 vs TLR4-def.