Tumor necrosis factor-α does not modulate ischemia/reperfusion injury in naïve myocardium but is essential for the development of late preconditioning  Buddhadeb Dawn, Yiru Guo, Arash Rezazadeh, Ou-Li Wang, Adam B. Stein, Greg Hunt, Jai Varma, Yu-Ting Xuan, Wen-Jian Wu, Wei Tan, Xiaoping Zhu, Roberto Bolli  Journal of Molecular and Cellular Cardiology  Volume 37, Issue 1, Pages 51-61 (July 2004) DOI: 10.1016/j.yjmcc.2004.03.012

Fig. 1 Experimental protocol. Nine groups of mice were used. On day 2, mice in groups I–IV underwent a 30-min coronary occlusion followed by 24 h of reperfusion. On day 1, mice in group II (n = 10, late PC group) and group IV (n = 9, TNF-α–/– late PC group) underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles, while mice in group I (n = 15, control group) and group III (n = 10, TNF-α–/– control group) did not undergo any intervention. Mice in group V (n = 6) underwent 2 h of open-chest state (n = 3) or six cycles of 4-min coronary occlusion/4-min reperfusion (n = 3) and the hearts were harvested 2 h later for TNF-α immunolocalization. In groups VI–IX (n = 6 in each group), wild-type (groups VI and VII) and TNF-α–/– (groups VIII and IX) mice underwent six cycles of 4-min coronary occlusion/4-min reperfusion and myocardial tissue samples were harvested 30 min later for biochemical assays. Journal of Molecular and Cellular Cardiology 2004 37, 51-61DOI: (10.1016/j.yjmcc.2004.03.012)

Fig. 2 Myocardial infarct size in groups I (control group), II (PC group), III (TNF-α–/– control group), and IV (TNF-α–/– PC group). Infarct size is expressed as a percentage of the region at risk of infarction. O, Individual mice; ●, mean ± SEM; n, number of mice. Journal of Molecular and Cellular Cardiology 2004 37, 51-61DOI: (10.1016/j.yjmcc.2004.03.012)

Fig. 3 Immunolocalization of myocardial TNF-α. Representative lower magnification images (100×, panels A–C) demonstrate increased expression of TNF-α in the ischemic/reperfused myocardium. Minimal TNF-α immunoreactivity (green fluorescence) was noted in the sham-operated hearts (panel C). In contrast, extensive TNF-α expression was noted in the ischemic/reperfused zone of the hearts of preconditioned mice 30 min (panel A) and 2 h (panel B) after six cycles of occlusion/reperfusion. Higher magnification images (630×, panels D–F) demonstrate the cytoplasmic distribution of TNF-α (panel D, green fluorescence) in cardiomyocytes. Cardiomyocytes are identified by the red fluorescence of troponin T (panel E). Co-localization of TNF-α and troponin T in yellow fluorescence (panel F) confirms the cytoplasmic distribution of intracellular TNF-α in cardiomyocytes. Nuclei are identified by DAPI (panel F, blue fluorescence). Journal of Molecular and Cellular Cardiology 2004 37, 51-61DOI: (10.1016/j.yjmcc.2004.03.012)

Fig. 4 Nuclear p65 content and NF-κB DNA-binding activity. Panel A: Nuclear NF-κB p65 subunit content in the ischemic/reperfused region in groups VI–IX (n = 6/group). Upper panel, representative western immunoblots demonstrating the specific signal for p65 (signals from two mice in each group are shown). Lower panel, the densitometric data from the specific p65 signals from three separate experiments were expressed as a percentage of the wild-type control (group VI). Panel B: NF-κB DNA-binding activity in the ischemic/reperfused region as described in panel A. Upper panel, EMSAs performed in groups VI–IX (signals from two mice in each group are shown). Lower panel, densitometric quantitation of specific NF-κB signals expressed as a percentage of the wild-type control (group VI). Data are mean ± SEM. Journal of Molecular and Cellular Cardiology 2004 37, 51-61DOI: (10.1016/j.yjmcc.2004.03.012)

Fig. 5 Nuclear content of c-Jun and c-Fos and AP-1 DNA-binding activity. Panel A: Nuclear AP-1 c-Jun subunit content in the ischemic/reperfused region in groups VI–IX (n = 6/group). Upper panel, representative western immunoblots demonstrating the specific signal for c-Jun (signals from two mice in each group are shown). Lower panel, the densitometric data from the specific c-Jun signals from three separate experiments were expressed as a percentage of the wild-type control (group VI). Panel B: Nuclear AP-1 c-Fos subunit content in the ischemic/reperfused region as described in panel A. Upper panel, representative western immunoblots demonstrating the specific signal for c-Fos (signals from two mice in each group are shown). Lower panel, the densitometric data from the specific c-Fos signals from three separate experiments were expressed as a percentage of the wild-type control (group VI). Panel C: AP-1 DNA-binding activity in the ischemic/reperfused region as described in panel A. Upper panel, EMSAs performed in groups VI–IX (signals from two mice in each group are shown). Lower panel, specific densitometric quantitation of AP-1 signals expressed as a percentage of the wild-type control (group VI). Data are mean ± SEM. Journal of Molecular and Cellular Cardiology 2004 37, 51-61DOI: (10.1016/j.yjmcc.2004.03.012)