Postconditioning with a CpG containing Oligodeoxynucleotide ameliorates myocardial infarction in a murine closed-chest model Se-Chan Kim a, ⁎ ,1 , Shuijing Wu a,c,1 , Xiangming Fang c , Jens Neumann a , Lars Eichhorn a , Grigorij Schleifer a , Olaf Boehm a , Rainer Meyer b , Stilla Frede a , Andreas Hoeft a , Georg Baumgarten a , Pascal Knuefermann a a Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Sigmund-Freud-Str. 25, D-53115 Bonn, Germany b Institute of Physiology, University of Bonn, Nussallee 11, D-53115 Bonn, Germany c Department of Anesthesiology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China abstract article info Article history: Received 2 January 2014 Accepted 29 September 2014 Available online 29 October 2014 Keywords: postconditioning Toll-like receptor myocardial infarction innate immunity cytokines Aims: Toll-like receptor (TLR)9 ligand CpG-oligodeoxynucleotide (CpG-ODN) exerts preconditioning in myocar- dial ischemia/reperfusion. We hypothesized a postconditioning effect of CpG-ODN in a murine closed-chest model of myocardial infarction. Materials and Methods: C57BL/6 (12 weeks, male, WT) mice were instrumented at the left anterior descending artery, then allowed 5d of recovery before 30 min ischemia. Treatments comprised: 1) PBS: 250 μl phosphate buffer solution intraperitoneally 5 min before reperfusion and 2) IPC (ischemic postconditioning): 3 twenty- second reperfusion and occlusion episodes at the end of ischemia 3) CpG-ODN: 1668 thioate 0.2 μmol/kg BW intraperitoneally 5 min before reperfusion. Infarct size was assessed via triphenyltetrazolium chloride (TTC) staining after 2 and 24 h reperfusion. Myocardial mRNA-expression of cytokines was measured using real-time PCR after 2 h reperfusion. Phosphatidylinositol-3 kinase (PI3K)-inhibitor wortmannin was injected intraperito- neally in WT 15 min before postconditioning and PBS in each group. Cardiac function in WT was assessed with a left-ventricular pressure-volume catheter at 24 h reperfusion. Key findings: Following 30 min ischemia and 2 h reperfusion, infarct size was diminished by 90% in WT postconditioned with CpG-ODN (2.4 ± 1.55 IS/AAR%) and IPC (1.98 ± 1.03 IS/AAR%) compared to PBS mice (23.2 ± 3.97 IS/AAR%). Infarct size increased following 24 h reperfusion but the differences remained robust. Expression of TNF-α and IL-10 was increased in CpG-ODN. Wortmannin abolished the postconditioning effect of CpG-ODN and IPC. Ejection fraction and preload-recruitable stroke work were significantly greater in CpG-ODN mice. Significance: CpG-ODN confers postconditioning via activation of TLR9. Cardiac function is preserved following CpG-ODN postconditioning. The PI3K -inhibitor wortmannin attenuates CpG-ODN postconditioning. © 2014 Elsevier Inc. All rights reserved. Introduction Ischemic postconditioning by repetitive cycles of reperfusion and oc- clusion following ischemia has been shown to ameliorate myocardial infarction in animal studies and clinical trials [38,44]. Pharmacological postconditioning using autacoids such as adenosine, opioids, bradykinin and cytokines is a potential therapeutic adjunct to primary percutane- ous coronary intervention (PCI) in patients with acute myocardial infarction (AMI) [29]. The activation of survival kinases, including phosphoinositide 3-kinase (PI3K)/Akt, inhibits the opening of mito- chondrial permeability transition pore by inhibition of glycogen syn- thase kinase (GSK)3β. This so-called reperfusion injury salvage kinase (RISK) pathway has been widely propagated as a critical underlying mechanism of ischemic postconditioning [10]. Alternatively, activation of the Janus kinase(JAK)-Signal Transducer and Activator of Transcrip- tion (STAT) pathway by cytokines has recently been discussed as a po- tential mechanism of postconditioning [24]. Cardioprotection following activation of Toll-like receptors (TLR) 2, 4 and 9 has been demonstrated in several animal ischemia/reperfusion models [12,27]. Downstream of TLR2 and 4, activation of PI3K/Akt has been proposed. Furthermore, stimulation of TLR9 could reduce ac- tivity of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2 modulating mitochondrial ATP synthesis [36,37]. Recent studies also suggest that application of synthetic cytosine-phospho-guanine oligodeoxynucleotide (CpG-ODN) exerts a preconditioning effect and improves hemodynamic function [25,26]. CpG-ODN containing unmethylated CpG motifs mimic the immunostimulatory effects of genomic bacterial motifs [20]. The activation of the innate immune Life Sciences 119 (2014) 1–8 ⁎ Corresponding author. Tel.: +49 228 287 14110; fax: +49 228 287 14125. E-mail address: Se-Chan.Kim@ukb.uni-bonn.de (S.-C. Kim). 1 Se-Chan Kim and Shuijing Wu contributed equally to this work. http://dx.doi.org/10.1016/j.lfs.2014.09.029 0024-3205/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie