The interaction between myocardial depressant factors in endotoxemic cardiac dysfunction: role of TNF-alpha in TLR4-mediated ICAM-1 expression

Abstract

Multiple pro-inflammatory mediators contribute to cardiac dysfunction caused by bacterial lipopolysaccharide (LPS). The rapid TNF-alpha response is likely involved in the induction of down-stream myocardial depressant factors. Studies by our laboratory and others indicate an important role for ICAM-1 in endotoxemic cardiac dysfunction through leukocyte-independent mechanisms. The purpose of this study was to determine: whether ICAM-1 knockout improves cardiac function during endotoxemia and whether TLR4 and TNF-alpha regulate LPS-induced myocardial ICAM-1 expression.

Methods and results: Mice were treated with Escherichia coli LPS (0.5mg/kg iv). Myocardial ICAM-1 levels were analyzed by immunoblotting and left ventricular developed pressure (LVDP) was assessed by the Langendorff technique. In wild-type mice, peak ICAM-1 levels were observed at 4h when myocardial contractility was depressed. Myocardial contractility was improved following LPS in mice lacking functional TLR4, TNF-alpha or ICAM-1. TLR4 mutation abolished ICAM-1 expression with abrogation of precedent TNF-alpha response. Similarly, TNF-alpha knockout reduced myocardial ICAM-1 level following LPS treatment.

Conclusions: ICAM-1 contributes to the mechanism of endotoxemic cardiac dysfunction. TNF-alpha is involved in the regulation of myocardial ICAM-1 expression by TLR4.

Figure 1. Time course of myocardial ICAM-1 expression

Myocardial samples were collected from wild-type (C57BL/6) mice at 0, 1, 2 and 4 h following administration of LPS or saline for analysis of ICAM-1 levels by immunoblotting. A. A representative gel shows that ICAM-1 level increased at 1, 2 and 4 h following LPS treatment in wild-type mice, and ICAM-1 was not detectable at 4 h after LPS treatment in ICAM-1 KO mice. B. Densitometric data show peak ICAM-1 level at 4 h after LPS treatment. n = 4 in each group.

Figure 2. Cardiac dysfunction during endotoxemia

Wild-type mice were treated with E. coli LPS (serotype 055:B5, 0.5 mg/kg ip) or normal saline (0.2 ml ip). Hearts were isolated at 1, 2 and 4 h after treatment, and left ventricular developed pressure (LVDP, A) and +dP/dt (B) were assessed by Langendorff. Cardiac Dysfunction occurs at 4 h after LPS treatment. Data are expressed as mean ± SE. n = 6 in each group; *P<0.05 vs. saline control.

Figure 3. The roles of ICAM-1, TLR4 and TNF-α in endotoxemic cardiac dysfunction

Hearts were isolated at 4 h after LPS or normal saline treatment from wild-type mice and mutant mice, and left ventricular developed pressure (LVDP, A) and +dP/dt (B) were assessed by Langendorff. In comparison to that in wild-type mice, myocardial contractile function was improved following LPS treatment in mice lacking ICAM-1, functional TLR4 or TNF-α. Data are expressed as mean ± SE. n = 6 in each group; * P<0.05 vs. LPS-treated wild-type group.

Figure 4. The effect of TLR4 mutation on myocardial ICAM-1 levels

Myocardial samples were collected from wild-type (BALB/cJ) mice and TLR4 defective mutant mice at 0, 2 and 4 h following LPS treatment for analysis of ICAM-1 levels. A. A representative gel of 2 separate experiments shows that TLR4 mutation abrogates the induction of myocardial ICAM-1 by LPS. B. Densitometric data confirm the absence of ICAM-1 induction at both 2 and 4 h after LPS treatment in TLR4 defect mice.

Figure 5. The effect of TNF-α knockout on myocardial ICAM-1 levels

Myocardial samples were collected from wild-type (B6.129) mice and TNF-α knockoutmice at 0, 2 and 4 h following LPS treatment for analysis of ICAM-1 levels. A. A representative gel of 2 separate experiments shows that TNF-α KO attenuates the increase in myocardial ICAM-1. B. Densitometric analysis shows slightly increased, but markedly reduced in comparison to wild-type, myocardial ICAM-1 levels in TNF-α knockoutmice following LPS treatment.