Cytokines Link Toll-Like Receptor 4 Signaling to Cardiac Dysfunction After Global Myocardial Ischemia John Cha, MD, Zhiping Wang, MD, Lihua Ao, BS, Ning.

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Cytokines Link Toll-Like Receptor 4 Signaling to Cardiac Dysfunction After Global Myocardial Ischemia John Cha, MD, Zhiping Wang, MD, Lihua Ao, BS, Ning Zou, MD, PhD, Charles A. Dinarello, MD, Anirban Banerjee, PhD, David A. Fullerton, MD, Xianzhong Meng, MD, PhD The Annals of Thoracic Surgery Volume 85, Issue 5, Pages 1678-1685 (May 2008) DOI: 10.1016/j.athoracsur.2008.01.043 Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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. The Annals of Thoracic Surgery 2008 85, 1678-1685DOI: (10.1016/j.athoracsur.2008.01.043) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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). The Annals of Thoracic Surgery 2008 85, 1678-1685DOI: (10.1016/j.athoracsur.2008.01.043) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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. The Annals of Thoracic Surgery 2008 85, 1678-1685DOI: (10.1016/j.athoracsur.2008.01.043) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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. The Annals of Thoracic Surgery 2008 85, 1678-1685DOI: (10.1016/j.athoracsur.2008.01.043) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions