Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion

L. Breivik 0 E. Helgeland 0 E. K. Aarnes 0 J. Mrdalj 0 A. K. Jonassen 0 0 L. Breivik (&) E. Helgeland E. K. Aarnes J. Mrdalj A. K. Jonassen Department of Biomedicine, Faculty of Medicine and Dentistry, University of Bergen , Bergen, Norway Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by C55% versus control hearts (P 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P \ 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P \ 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) \ 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion. - Impaired coronary circulation caused by a myocardial ischemic episode will lead to compromised hemodynamic function and ultimately cell death. Rapid restoration of coronary blood flow to the infarct-related coronary artery by thrombolysis or primary coronary angioplasty is effective interventions for limiting myocardial infarct size and improving clinical outcomes [20, 39]. Although early reperfusion is essential to limit infarct size, it may paradoxically induce cell death (lethal reperfusion injury) [2]. Two ways of protecting the heart against reperfusioninduced injury are ischemic preconditioning (IPC) [22] and ischemic postconditioning (IPost) [40], applying transient non-lethal episodes of myocardial ischemia and reperfusion either before or after the index ischemic event, respectively. Another strategy for achieving cardioprotection is remote ischemic preconditioning (RIPC), whereby brief ischemia in one distant region or organ protects the heart from a sustained episode of ischemia [26]. Remote IPost (RIPost) is a more clinically relevant cardioprotective strategy, and has been shown to be effective in experimental studies when the remote ischemic conditioning was applied after the onset of myocardial ischemia [15]. There is strong evidence that one or more factors released in response to brief episodes of ischemia during IPC can interact with receptors, locally or at a distance, triggering a cytoprotective response. Dickson et al. [6] found that IPC-induced cardioprotection could be transferred between individuals by transfusion of whole blood from preconditioned to non-preconditioned rabbits and that effluent from preconditioned isolated ex vivo rabbit hearts offered protection to non-preconditioned hearts [5]. Furthermore, Serejo et al. [28] recently showed that cardioprotective factors released into coronary IPC effluent from ex vivo rat hearts are thermolabile hydrophobic substances with molecular weights higher than 3.5 kDa offering cytoprotection via PKC activation in the recipient heart. In addition, Lang et al. [17] suggested that mediator(s) released during IPC in rat hearts are smaller than 8 kDa. IPC and IPost reduce infarct size when studied in a wide variety of experimental models including rat, rabbit, dog, pig and primates by sharing similar mechanisms and exerting most of their cytoprotection at reperfusion [9, 12, 26, 37]. IPC activates PI3-kinase which facilitates phosphorylation and activation of the protein kinase Akt through its mediator PDK-1 [1]. Akt is part of the reperfusion injury salvage kinases (RISK) that confer cardioprotection when activated at reperfusion [9]. IPost activates similar signaling pathways to IPC [9] and is thereby also dependent on RISK activation to exert its protection [35], although the involvement of RISK has lately been proven to be species dependent [27, 30]. The underlying mechanism of RIPC and RIPost remains unclear, but many of the signaling pathways underlying myocardial preconditioning and postconditioning have been implicated in RIPC [11]. Numerous studies have focused on characterizing the signaling mechanisms involved in IPC. However, the precise adaptive response triggered by agents released during the short reperfusion episodes of IPC remains unresolved. Identification of such mediators would be of great importance for the development of pharmacological therapies to enhance myocardial tolerance to ischemiareperfusioninduced stress in patients with acute myocardial infarction (AMI) [38]. The aims of this study were therefore to (1) investigate the protective capabilities of the IPC effluent when delivered at ischemic reperfusion and its potential to induce remote postconditioning (RIPost); (2) determine if coronary IPC effluent mediates its protection through a PI3K/Akt-dependent cytoprotective signaling pathway; (3) characterize the protective mediator(s) by fractionation of effluent based on different properties such as hydrophobicity and size. Langendorff perfusion procedure All experiments were approved by the Norwegian State Commission for Laboratory Animals, and carried out in accordance with the European Communities Council Directive of 1986 (86/609/EEC). Male Wistar rats (250350 g, n = 183) fed a standard diet were heparinized (200 IU) and anesthetized with sodium pentobarbital (50 mg/kg) i.p. The hearts were excised, placed in ice-cold KrebsHeinseleit buffer (KHB) and rapidly mounted onto a Langendorff perfusion system. KHB (pH 7.4, oxygenated with 95% O2/5% CO2) contained in mM: 118 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 KH2 PO4, 1.2 MgSO4, 25.2 NaHCO3, 11.0 glucose. Perfusion pres sure was maintained at 100 cmH2O, and the myocardial temperature kept at 37.5 C. A water-filled latex balloon, connected to a hydrostatic pressure transducer (SP84 (...truncated)