Nuclear Factor-kB Plays an Essential Role in the Late Phase of Ischemic Preconditioning in Conscious Rabbits

Although it is recognized that late preconditioning (PC) results from upregulation of cardioprotective genes, the specific transcription factor(s) that govern this genetic adaptation remains unknown. The aim of this study was to test the hypothesis that the development of late PC is mediated by nuclear factor-kB (NF-kB) and to elucidate the mechanisms that control the activation of NF-kB after an ischemic stimulus in vivo. A total of 152 chronically instrumented, conscious rabbits were used. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles, which elicits late PC, induced rapid activation of NF-kB, as evidenced by a marked increase in p65 content (1164%; Western immunoblotting) and NF-kB DNA binding activity (1306%; electrophoretic mobility shift assay) in nuclear extracts isolated 30 minutes after the last reperfusion. These changes were attenuated 2 hours after ischemic PC and resolved by 4 hours. Competition and supershift assays confirmed the specificity of the NF-kB DNA complex signals. The mobility of the NF-kB DNA complex was shifted by anti-p65 and anti-p50 antibodies but not by anti–c-Rel antibodies, indicating that the subunits of NF-kB involved in gene activation after ischemic PC consist of p65-p50 heterodimers. Pretreatment with the NF-kB inhibitor diethyldithiocarbamate (DDTC; 150 mg/kg IP 15 minutes before ischemic PC) completely blocked the nuclear translocation and increased DNA binding activity of NF-kB. The same dose of DDTC completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that NF-kB activation is essential for the development of this phenomenon in vivo. The ischemic PC-induced activation of NF-kB was also blocked by pretreatment with N-nitro-L-arginine (L-NA), a nitric oxide synthase (NOS) inhibitor, N-2-mercaptopropionyl glycine (MPG), a reactive oxygen species (ROS) scavenger, chelerythrine, a protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor (all given at doses previously shown to block late PC), indicating that ischemic PC activates NF-kB via formation of NO and ROS and activation of PKCand tyrosine kinase–dependent signaling pathways. A subcellular redistribution and increased DNA binding activity of NF-kB quantitatively similar to those induced by ischemic PC could be reproduced pharmacologically by giving the NO donor diethylenetriamine/NO (DETA/NO) (at a dose previously shown to elicit late PC), demonstrating that NO in itself can activate NF-kB in the heart. Taken together, these results provide direct evidence that activation of NF-kB is a critical step in the signal transduction pathway that underlies the development of the late phase of ischemic PC in conscious rabbits. The finding that four different pharmacological manipulations (L-NA, MPG, chelerythrine, and lavendustin A) produced similar inhibition of NF-kB suggests that this transcription factor is a common downstream pathway through which multiple signals elicited by ischemic stress (NO, ROS, PKC, tyrosine kinases) act to induce gene expression. To our knowledge, this is the first demonstration that NO can promote NF-kB activation in the heart, a finding that identifies a new biological function of NO and may have important implications for various pathophysiological conditions in which NO is involved and for nitrate therapy. (Circ Res. 1999;84:1095-1109.)