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Cardiovascular Research 2002 55(3):553-560; doi:10.1016/S0008-6363(02)00283-3
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Copyright © 2002, European Society of Cardiology

Classic ischemic but not pharmacologic preconditioning is abrogated following genetic ablation of the TNF{alpha} gene

Robert M Smith, Naushaad Suleman, Joy McCarthy and Michael N Sack*

Hatter Institute for Cardiology Research, MRC Inter-University Cape Heart Group, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa

* Corresponding author. Tel.: +27-21-406-6350; fax: +27-21-447-8789 sack{at}capeheart.uct.ac.za

Received 19 November 2001; accepted 1 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Tumor necrosis factor alpha (TNF{alpha}) is known to mimic ischemic preconditioning (IP). However, it is not known whether TNF{alpha}-preconditioning is mediated by ‘established’ preconditioning signaling or via novel signaling cascades. Moreover, whether TNF{alpha} is required to induce the ischemic preconditioning phenotype has not been determined. Methods: To evaluate the role of TNF{alpha}, we determined the infarct-sparing effect of IP comparing TNF{alpha} null (TNF{alpha}–/–) and wild-type mice. The IP protocol included 4x5 min ischemia/reperfusion (I/R) prior to the index 35 min of global ischemia followed by 45 min of reperfusion in isolated perfused murine hearts. Infarct size was measured as a percentage of cardiac volume. To evoke particular signaling pathways numerous pharmacologic studies were performed. Results: Following IP, infarct size was significantly reduced by 43% in wild-type mice. In contrast, infarct size was not attenuated by IP in the TNF{alpha}–/– group versus I/R controls (Infarct size—36±3%). Interestingly, pharmacologic preconditioning with adenosine (100 µM) and diazoxide (30 µM) mimicked IP in both the wild-type (infarct size—11±4% and 18±2%) and in TNF{alpha}–/– mice (infarct size—15±4% and 23±3%) versus respective I/R controls. Recombinant TNF{alpha} (0.5 ng/ml) administered for 7 min followed by a 10-min washout mimicked IP in wild-type mice but not in the TNF{alpha} deficient mouse hearts. The cardioprotective effects of IP, adenosine and TNF{alpha} were abolished by the co-administration of the putative mitochondrial KATP blocker 5-hydroxydecanoate. Conclusions: We demonstrate that cardiac TNF{alpha} production is required for ischemic preconditioning-induced cardioprotection but not necessary in pharmacologic preconditioning with adenosine or diazoxide in TNF{alpha}–/– mice. Moreover, TNF{alpha} administration is sufficient to activate preconditioning in wild-type mice. Finally, as 5-hydroxydecanoate abrogates ischemic, adenosine and TNF{alpha} induced preconditioning, this suggests that diverse signaling pathways converge at the level of mitochondrial KATP channel activation to mediate this cardioprotection.

KEYWORDS Adenosine; Immunology; Ischemia; K-ATP channel; Preconditioning


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Brief periods of sub-lethal ischemia that precede sustained ischemia result in a marked limitation of infarct size and are described as ischemic preconditioning [1–4]. Understanding this innate biologic phenomenon could enable us to develop newer strategies to enhance myocardial tolerance against ischemic injury. Until recently, the role of the innate immune system in preconditioning has not been extensively explored. Interestingly, the pleiotropic cytokine TNF{alpha} which is known to be an apical regulator of innate immunity has been shown to be modestly elevated in the serum of rabbits following ischemic preconditioning [5]. Moreover, pharmacologic administration and endogenous production of TNF{alpha} has induced preconditioning-like cardioprotection in both rats and rabbits. This cardioprotection is thought to be due, in part to activation of the free radical scavenger manganese superoxide dismutase (MnSOD) [6–8]. To establish the requirement of TNF{alpha} signaling in innate cardioprotection, Mann and colleagues carried out a series of acute coronary artery occlusion studies in mice lacking one or both of the known cognate TNF{alpha} cell surface receptors [9]. The genetic ablation of either receptor alone did not alter myocardial infarct size compared to wild-type control mice. Conversely, the genetic ablation of both cognate TNF{alpha} cell surface receptors in mice resulted in a marked augmentation of cardiac infarction in response to coronary artery occlusion [9]. Collectively these data suggest that TNF{alpha} signaling could be an important regulator in preconditioning and may play an important role in promoting innate cytoprotection against cardiac ischemic injury.

Preconditioning, which in essence unmasks an innate cytoprotective program is classically thought to be activated predominantly via GI-protein-coupled receptor mediated signaling [10]. The ischemic preconditioning induced ligands that activate Gi-receptor coupled signaling and are proposed to activate the preconditioning cytoprotective programs include adenosine, bradykinin and opioids [2]. To date, these GI-protein-coupled receptor mediated signaling events have been shown to activate the mitochondrial ATP sensitive potassium channel (mKATP) [11]. In turn, activation of the mKATP channel is thought to promote tolerance against ischemia, via mechanisms that have not been completely elucidated [12–14].

In contrast, TNF{alpha} acts via type II membrane receptors and directs a multitude of diverse downstream signaling events that are thought to be both distinct from and potentially overlapping with Gi-receptor associated signaling [15,16]. Questions that arise include whether: TNF{alpha} mediated signaling is required in classical ischemic preconditioning; whether TNF{alpha} signaling is necessary to confer preconditioning-like cardioprotection and whether TNF{alpha}-induced preconditioning acts via mKATP channel activation?

The present study was undertaken to address these questions and to further characterize the interplay between Gi-receptor coupled signaling, TNF{alpha} signaling and mitochondrial KATP channel activation in the context of classical preconditioning in the heart. Of note, numerous investigators have demonstrated that the operational procedures, including sham-procedures and cardiac ischemia–reperfusion studies in-vivo result in the acute and sustained activation of numerous cytokines, including TNF{alpha}, IL-1β and IL-6 [17–20]. To bypass this we have used a reductionist approach, i.e. the isolated perfused mouse heart preparation, to study the relative role of TNF{alpha} in preconditioning. TNF{alpha} null mice (TNF{alpha}–/–) were compared to wild-type control mice in response to ischemic and pharmacologic preconditioning comparing infarct size between the groups using an isolated perfused heart preparation [21,22].

In the present study, we found that ischemic preconditioning results in endogenous biosynthesis of TNF{alpha} by the murine heart. We also demonstrate that in ischemic preconditioning TNF{alpha} is required to attain a tolerant phenotype against ischemia/reperfusion-induced infarction. Furthermore, we demonstrate that TNF{alpha} is not necessary to mediate pharmacologic preconditioning when adenosine or the mKATP channel activator diazoxide [23] were administered as preconditioning ‘triggers’ in the TNF{alpha} deficient mouse hearts. We also confirm that recombinant TNF{alpha} administration is sufficient to mimic ischemic preconditioning in the wild-type mice. Finally, the pharmacologic antagonist studies suggest a convergence of signaling at the mitochondrial level via both Gi-receptor coupled and TNF{alpha} activated cardioprotection.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academic Press, Washington, DC, 1996), and all procedures were approved by the University of Cape Town Medical School Research Ethics Committee.

2.1 TNF{alpha} deficient mice
Homozygous TNF-{alpha} deficient mice (TNF{alpha}–/–) and wild-type control mice were used in this study and were a generous gift from Bernhard Ryffel, Department of Immunology, University of Cape Town Medical School. Targeted deletion of the TNF{alpha} gene has been described and genotyping by PCR is performed routinely in our laboratory [24].

2.2 Mouse isolated heart perfusion protocol
Male mice (18–25 g) were anesthetized (pentabarbitone sodium, 60 mg kg–1 i.p.) and heparinized (25 IU i.p.). Once an adequate level of anesthesia had been achieved the chest was opened excising the sternum and attached costal cartilages to give adequate access to the mediastinum. The heart was rapidly removed and placed in ice cold (4 °C) Krebs–Henseleit buffer and the aorta cannulated. Hearts were then perfused with a modified Krebs–Henseleit buffer (NaCl 118.0 mM; NaHCO3 24.0 mM; KCl 4.0 mM; NaH2PO4 1.0 mM; CaCl2 2.5 mM; MgCl2 1.2 mM; di-sodium EDTA 0.5 mM; glucose 10 mM; gassed with 95% O2/5%CO2 at 37 °C) in a retrograde fashion with a constant pressure of 110 cmH2O. Temperature was measured by the placement of a fine thermocouple wire (Physitemp, NJ, USA) and monitored on a Digitron 2600T (Torquay, UK). Hearts were fastened, via a rigid lightweight lexan coupling rod, to a force displacement transducer (Grass FT03C, MA, USA) by means of a 4-0 silk (on a 20-mm curved atraumatic needle) placed through the apex of the heart. Diastolic tension was adjusted to 2 g and hearts paced at 600 bpm. Developed tension was recorded on a chart recorder (Lectromed Multitrace-2, Letchworth, UK). The coronary flow rate was measured by timed collection.

Following 20 min of stabilization, hearts were exposed to the protocols shown in Fig. 1. At the end of the experimental protocol, infarct size was assessed by TTC staining as described previously [21,22,25]. A minimum of six hearts were used in all pharmacologic agonist studies and five hearts in antagonist studies. Hearts were excluded from the study if coronary flow was >5 ml/min or <1.5 ml/min and/or if the heart rate could not be paced at 600 beats/min at the end of the 20-min stabilization period. Following the protocols hearts were frozen and sectioned into 1.5-mm slices. The slices were then laid out and compressed between thin glass plates 0.5 mm apart. The sections were then scanned, enlarged, and infarct size assessed using computerized planimetry (Planimetry+, Boreal Software, Norway) by a researcher blinded to the groups.


Figure 1
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  		Fig. 1 Schematic representation of preconditioning protocols in the isolated perfused murine hearts. In all experiments, the index ischemia lasted for 35 min and the final reperfusion time for 45 min. Ischemic preconditioning was conferred by four times 5 min of ischemia and reperfusion prior to the index ischemic insult. The pharmacologic preconditioning mimetic agents were administered for 7 min followed by a 10-min washout prior to the index ischemia. 5-HD bracketed both ischemic and pharmacologic preconditioning by administration for 3 min prior to and extending for 5 min after the preconditioning triggers.

 
2.3 Immunoprecipitation and Western blot analysis
Hearts were perfused and freeze clamped using Wallenberger tongs precooled in liquid nitrogen. The tissue was stored at –80 °C until protein extraction was performed. Cell lysates were prepared in ice cold radioimmunoprecipitation assay (RIPA) buffer (1xPBS; 1% Igepal; 0.5% sodium deoxycholate; 0.1% SDS) supplemented with protease inhibitors (100 µg/ml PMSF; 40 µg/ml aprotinin and 1 mM sodium orthovanadate, pH 7.0). Cellular debris was removed by centrifugation at 5000xg for 5 min, and protein concentrations were determined in triplicate by the Lowry method. For all samples, 500 µg of total protein were immunoprecipitated using rabbit polyclonal TNF{alpha} antibody (Santa Cruz Biotech, CA, USA) and agarose conjugated Protein A/G (Santa Cruz Biotech, CA, USA). Washed proteins (4x in PBS, pH 7.4) were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and were transferred to PVDF membranes by electrophoretic transfer. Non-specific binding sites were blocked by incubation in Tris-buffered saline containing 0.1% Tween 20 and 5% (wt/vol) skimmed milk powder. Immunoblotting analyses were carried out and immunocomplexes were visualized with the appropriate horseradish peroxidase-conjugated immunoglobulin G (IgG; Santa Cruz Biotech, CA, USA) and chemiluminescent reagents (Amersham Pharmacia Biotech, Amersham, UK). Relative peptide levels were measured using densitometric analysis with UVIband (UVI Tech, Cambridge, UK) software on a PC.

2.4 Statistical analysis
Results are expressed as mean values±S.E.M. and were analyzed by one-way ANOVA with Dunn's post test, using GraphPad InStat version 3.01 (GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant at values of P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Cardiac biosynthesis of TNF{alpha} is induced in response to ischemic preconditioning
Lethal ischemia is well known to result in endogenous cardiac TNF{alpha} production [17,26–28]. However, we wanted to establish whether the non-lethal ischemia and reperfusion that activates the preconditioning cardioprotective phenotype could result in endogenous TNF{alpha} biosynthesis in the isolated perfused murine heart. Steady-state TNF{alpha} peptide levels were measured using Western blot analysis comparing normoxic controls versus ischemic preconditioned heart tissue. The tissue was extracted immediately prior to the index ischemia (Fig. 2). Here we demonstrate that the four by 5-min ischemic preconditioning ‘triggers’ results in a twofold induction of TNF{alpha} in the ischemic preconditioned heart versus the temporally matched perfused isolated control hearts (Fig. 2).


Figure 2
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  		Fig. 2 TNF{alpha} levels in response to ischemic preconditioning. Panel A: Schematic of time point of protein extraction to assess endogenous TNF{alpha} biosynthesis by the heart (depicted by vertical arrow). Hatched boxes represent 4x5 min of ischemia, horizontal lines represent normoxic perfusion. Panel B: Bar graphs represent the mean±S.E.M. densitometric values in arbitrary units (AU) of whole heart steady-state levels of TNF{alpha} in the control hearts versus hearts exposed to the preconditioning ‘trigger’. The AU are normalized to control=100. The inset is a representative Western blot showing the steady-state levels of TNF{alpha}.

 
3.2 TNF{alpha} is required in ischemic preconditioning induced cardioprotection against myocardial infarction
To determine whether TNF{alpha} is a required component of ischemic preconditioning mediated cardioprotection, we compared infarct size in the wild-type versus TNF{alpha}–/– mice following the four times 5-min preconditioning protocol (Fig. 1). Infarct size in the ischemic preconditioned wild-type mice was reduced by 42.8±9.0% compared to the wild-type ischemic control mice (Fig. 3). The TNF{alpha}–/– mice were resistant to ischemic preconditioning and the mean infarct size was similar when comparing the TNF{alpha}–/– ischemic control and the ischemic preconditioned TNF{alpha}–/– mice. Of note, there was no difference in infarct size when comparing the infarct size in the ischemic control wild-type and the ischemic control TNF{alpha}–/– mice. The putative mitochondrial KATP channel blocker—5-hydroxydecanoate that is known to inhibit ischemic preconditioning in all other species investigated was also shown to abrogate ischemic preconditioning-induced cardioprotection against infarct size in the isolated perfused wild-type mice used in this study.


Figure 3
Figure 3
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  		Fig. 3 (A) Infarct size is presented as a percentage of ventricular volume in isolated mouse hearts subjected to 35 min of global (index) ischemia and 45 min of reperfusion. Wild-type hearts exposed to four cycles of 5 min ischemia and 5 min reperfusion (IPC) prior to the index ischemia have significantly smaller infarcts than those in control hearts (P<0.05). The protection afforded by IPC in the wild-type hearts could be abrogated by the putative mitochondrial KATP channel blocker 5-hydroxydecanoate (100 µM). The TNF{alpha}–/– mice could not be protected by IPC. The open circles represent individual experimental values; Closed circles represent the group mean±S.E.M. (B) Representative sections of hearts stained with TTC following the ischemia and reperfusion protocols (see Methods and Fig. 1 for details).

 
3.3 Pharmacologic preconditioning with adenosine and diazoxide restores the cardioprotective phenotype in TNF{alpha} null mice
As TNF{alpha} orchestrates signaling via distinctive and previously unrecognized pathways in preconditioning, we then went on to evaluate whether a classic Gi-receptor coupled signaling pathway (adenosine) or whether direct activation of the mitochondrial KATP channel with diazoxide could rescue this ischemic preconditioning resistant phenotype in the TNF{alpha}–/– mice. Firstly we demonstrate that these two agents can mimic ischemic preconditioning in the wild-type mice with a reduction in infarct size by 68.0±10.8% by adenosine and by 46.7±5.2% in the diazoxide pretreated wild-type mice compared to wild-type ischemic controls (Fig. 4). Interestingly, the preconditioning phenotype could be rescued by using both of these agents as preconditioning mimetics in the TNF{alpha}–/– mice. The cardioprotection afforded by these agents was similar in the TNF{alpha}–/– mice (62.0±10.5% and 39.4±7.3% for adenosine and diazoxide, respectively) and in the wild-type mice pretreated with the same agents (Fig. 4). In this murine preparation we demonstrate that 5-hydroxydecanoate abrogates the preconditioning effect of adenosine.


Figure 4
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  		Fig. 4 Wild-type and TNF{alpha}–/– hearts pretreated with adenosine (100 µM) or diazoxide (30 µM) as preconditioning-mimetics prior to the index ischemia had significantly smaller infarcts than control hearts (P<0.01). The protection afforded by adenosine could be reversed in the presence of the mitochondrial KATP channel blocker 5-hydroxydecanoate (100 µM). Symbols as in Fig. 3 legend.

 
3.4 Recombinant TNF{alpha} can mimic ischemic preconditioning and can rescue the cardioprotective phenotype in TNF{alpha} null mice when administered using a delayed preconditioning protocol
As TNF{alpha} has been shown to mimic preconditioning, especially delayed preconditioning we evaluated whether administration of TNF{alpha} could mimic preconditioning in mice. In the wild-type mice hearts, recombinant TNF{alpha} mimicked ischemic, adenosine and diazoxide preconditioning with a reduction in infarct size by 69.6±4.3% compared to ischemic wild-type controls (Fig. 5). Here, the mitochondrial KATP channel blocker 5-hydroxydecanoate completely abolished this cardioprotective phenotype. When TNF{alpha} was administered as a classical preconditioning mimetic (protocol—Fig. 1) the cardioprotective phenotype could not be obtained in the TNF{alpha}–/– mice (Fig. 5).


Figure 5
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  		Fig. 5 TNF{alpha} is sufficient to confer preconditioning like cardioprotection in wild-type mice as evidenced by the reduction in infarct size as a percentage of ventricular volume in these mice. The TNF{alpha}–/– mice could not be preconditioned with recombinant murine TNF{alpha}. When using the priming protocol schematized (panel A) and described in the results, recombinant TNF{alpha} could confer acute protection against infarction as shown in panel B.

 
The inability to precondition the TNF{alpha}–/– mice with recombinant TNF{alpha} begs the question as to whether the downstream TNF{alpha} signaling pathway is latent or downregulated in these mice. To begin to answer this question we attempted to activate this signaling pathway by priming the TNF{alpha}–/– mice with intravenous TNF{alpha} 24 h prior to the TNF{alpha} preconditioning experiments. When TNF{alpha} was administered 24 h prior to the preconditioning experiments, a second dose of TNF{alpha} administered as a preconditioning mimetic rescued the TNF{alpha}–/– cardioprotective phenotype. Here the infarct size was diminished by 59.4±12.3% compared to the TNF{alpha}–/– ischemic controls (Fig. 5).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study, we demonstrate that ischemic preconditioning stimulates endogenous cardiac TNF{alpha} production and that this ligand is required in ischemic preconditioning-induced cardioprotection against myocardial infarction in mice. Conversely we demonstrate that TNF{alpha} is not necessary for pharmacologic preconditioning when either adenosine or diazoxide are used to ‘trigger’ this cardioprotective program. Moreover, TNF{alpha} administration is sufficient to activate preconditioning in wild-type mice. Finally, the fact that 5-hydroxydecanoate abrogates ischemic, adenosine and TNF{alpha} induced preconditioning in mice suggests that distinct signaling pathways converge at the level of mitochondrial KATP channel activation to mediate this cardioprotective regulatory program.

The pleiotropic cytokine TNF{alpha} is postulated to have a multitude of adverse effects in cardiac pathologies such as in heart failure [29,30], in atherosclerosis [31], in post-ischemic reperfusion injury [32] and in post-transplant rejection [33]. In contrast recent scientific evidence suggests that TNF{alpha} may have adaptive effects directing innate cardiac protection against ischemia [9], against viral cardiac pathogens [34] and in promoting adaptive cardiac hypertrophic growth [24]. These divergent effects have been postulated to result from differential temporal and dose effects of this autacoid on the heart [16,35]. As preconditioning is known to unmask cellular programs that promote innate cytoprotection, the data from our studies strongly support a role for TNF{alpha} in the induction of cardioprotective cellular events against ischemic injury.

The cell surface ligands that have been shown to induce preconditioning have classically acted via Gi-protein coupled receptors and have been postulated to act predominantly via similar intracellular signaling events [2]. Interestingly, recent data are beginning to question the commonality of cell surface receptor mediated signaling in preconditioning [36]. Our data add to this knowledge base and identifies a possible Gi-protein-independent signaling cascade that may also induce the preconditioning mediated cytoprotective program. The mitochondrial KATP pharmacologic antagonist data does however implicate a common role for mitochondrial biology as a pivotal intracellular regulator of these programs.

The temporal effects of regulatory peptides and events in the preconditioning program is becoming more apparent [37,38]. This temporal effect has also been elegantly illustrated where reactive oxygen species (ROS) have been identified as ‘triggers’ in the preconditioning program [3,39–41] and concurrently where preconditioning results in an attenuation of ROS release during the post-ischemic reperfusion [42]. We postulate that a similar temporal effect of TNF{alpha} signaling is involved in the preconditioning program. We base this hypothesis on data from Heusch and colleagues [5] and from our results in this study that show that TNF{alpha} is produced in response to ischemic preconditioning and that it is required to activate this cardioprotective program. Moreover, as demonstrated by Meldrum et al. [43], ischemic and adenosine-induced preconditioning result in a decrease in post-ischemic TNF{alpha} production at reperfusion in comparison to non-preconditioned rat hearts. The identification of the negative regulatory feedback loops concerning ROS and TNF{alpha} production in preconditioning may shed light on the underlying cellular mechanism driving this cell survival program.

The fact that pre-administration of TNF{alpha} 24 h prior to TNF{alpha}-preconditioning rescues the cardioprotected phenotype suggests that the signaling effector system downstream of TNF{alpha} may be downregulated in TNF{alpha} deficient mice. However, these data are preliminary in that TNF{alpha} receptor density and receptor–ligand interaction studies have not been performed. Hence further functional studies are required to evaluate the validity of this observation.

The isolated perfused murine heart preparation was used in this study as this enabled us to dissect out both different signaling cascades and the distinct requirement of TNF{alpha} in ischemic preconditioning. This isolated heart system probably limits the known activation of the cytokine cascade following surgical stress in the in-vivo preparation [19]. However, the perfused murine heart preparation model does have limitations in that post-ischemic reperfusion can only be maintained for a short time period [22]. This ex-vivo time frame does not enable the heart to completely evolve the post-infarct remodeled heart. However, our reductionist approach does identify a putatively novel signaling pathway in preconditioning. This work does need to be coupled to further in-vivo work to more comprehensively evaluate the TNF{alpha} signaling system in preconditioning and its interaction with the full complement of cytokines known to be induced in response to ischemia and reperfusion [17].

In conclusion, in this study, TNF{alpha} is required to induce classical ischemic preconditioning in the murine heart and that in parallel to GI-coupled receptor signaling seems to confer this cardioprotection via mKATP channel activation. Moreover, this study in conjunction with other findings in studies described in this manuscript identifies TNF{alpha} signaling as a novel system to study in order to facilitate our understanding of the molecular and cellular mechanisms underlying the cardio-protected phenotype induced by preconditioning. Finally, the data presented support the emerging concept that the innate immune system may play an important role in adaptive cyto-protective effects in the heart [44].

Time for primary review 24 days.


   Acknowledgements
 
This study was supported by a Wellcome Trust Equipment Grant, UK (MNS), and in part by the South African MRC, The Hatter Foundation, UK and by non-restrictive start-up funds to The Hatter Institute (Cape Town) from Roche Pharmaceuticals and Servier Laboratories.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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N. Suleman, S. Somers, R. Smith, L. H. Opie, and S. C. Lecour
Dual activation of STAT-3 and Akt is required during the trigger phase of ischaemic preconditioning
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