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Cardiovascular Research 2007 73(1):153-163; doi:10.1016/j.cardiores.2006.10.013
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Copyright © 2006, European Society of Cardiology

Reperfusion kinase phosphorylation is essential but not sufficient in the mediation of pharmacological preconditioning: Characterisation in the bi-phasic profile of early and late protection

Robert M. Bell, James E. Clark, David J. Hearse and Michael J. Shattock*

Cardiac Physiology, King's College London, Cardiovascular Division, The Rayne Institute, St. Thomas' Hospital, London, SE1 7EH, UK

* Corresponding author. Tel.: +44 20 7188 0945; fax: +44 20 7188 3902. Email address: michael.shattock{at}kcl.ac.uk

Received 17 March 2006; revised 11 October 2006; accepted 18 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Pharmacological preconditioning (PPC) triggers early (ePPC) and delayed protection (dPPC), occurring within 1 h or after 24 h following the preconditioning stimulus, respectively, through recruitment of protein kinase signalling. Angiotensin II (ATII) is a recognised trigger of PPC, recruiting kinases and transcription factors known to be involved in both phases of protection. Our objectives were to determine whether ATII is capable of triggering dPPC and whether recruitment of pro-survival kinases, Akt and extracellular signal-regulated kinase (ERK), following the injurious ischaemic insult is essential for the mediation of PPC.

Methods: In a mouse Langendorff model of ischaemia/reperfusion injury, we undertook to determine whether ATII triggers both ePPC and dPPC. Western blot analysis was used to determine kinase phosphorylation at reperfusion, and kinase inhibitors wortmannin and PD98059 were used to ascertain the significance of kinase regulation.

Results: We demonstrated that ATII triggered PPC with attenuation of infarction at 1 and 24 h (19±4% and 25±4% versus control, 35±4% of risk zone, p<0.05), consistent with the ePPC and dPPC time-course. This bi-phasic protection was associated with significant post-ischaemic phosphorylation of both Akt and ERK within the first 5 min of reperfusion. Akt and ERK phosphorylation was increased following ePPC by 4.5±0.5 and 1.9±0.6 fold, respectively (p<0.001), and dPPC by 24±2.0 and 2.1±0.1 fold, respectively (p<0.001). Both wortmannin and PD98059 administered during reperfusion ameliorated the phosphorylation of Akt and ERK and abrogated the resistance to infarction resulting from both ePPC and dPPC (33±3% and 35±4%, respectively, versus controls 33±4% and 33±5%, p=NS). There was no evidence of augmented phosphorylation of either p38 kinase or JNK at either time point.

Conclusion: We demonstrate that PPC results in a clearly delineated time-course of bi-phasic protection against injurious ischemic injury that is correlated with reperfusion kinase phosphorylation of both Akt and ERK. These data indicate a novel mechanism of early and particularly delayed preconditioning.

KEYWORDS Agiotensin II; Preconditioning; Ischaemia; Infarct; Necrosis; Akt; ERK; p38; JNK; MAPK; Reperfusion


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Preconditioning, with either transient ischaemia or with a pharmacological ‘preconditioning mimetic’, results in significant cardioprotection against a subsequent lethal ischemic insult. Interestingly, the protection appears in two separate phases or ‘windows’ of protection. Early preconditioning results in significant protection over 2–3 h following a preconditioning stimulus [1]. This phase of preconditioning is largely acknowledged to be mediated by protein kinase activity, including isoforms of PKC, tyrosine kinase [2] and more recently, the serine/threonine kinase, Akt [3]. Following the resolution of this ‘first window of protection’, there is a delay until the second window of protection appears 24 h after the preconditioning stimulus, resulting in protection which then persists for a period of some days [4]. In contrast to early preconditioning, the second window of protection is attributable to the significant de-novo protein synthesis of a variety of cardioprotective proteins [5]. A number of candidate proteins have been identified, including inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 [6], as well as the expression of specific heat shock proteins [7]. The regulation of expression of these cardioprotective proteins is mediated through the activation of gene transcription factors (including nuclear factor (NF)-{kappa}B [8,9] and activator protein (AP)-1 [10,11]), recruited through the increased activity of a range of mitogen activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK) [12], p38 kinase [13] and JNK [12] by the initiating preconditioning stimulus [9].

However, the relative significance of kinase phosphorylation in context of cardioprotection remains unclear, as evidence from Heusch's laboratory has suggested; the magnitude of phosphorylation detected by quantitative Western blotting for the various MAPKs appeared to be poorly correlated with the observed cardioprotection [14]. Therefore, whilst MAPK activity may be essential for triggering protection it is unlikely to represent the whole story of myocardial resistance to necrotic injury. Another role for MAPK activity has been alluded to in recent work by Hausenloy et al. [15], where kinase activation (including PI3K, ERK and p70S6K) following the injurious ischaemic insult was found to play a pivotal role in the protection against infarction resulting from an ischaemic preconditioning stimulus. However, this study concentrated upon the early ischaemic preconditioning time frame; how kinase activation at reperfusion is modified in second window of protection and the period between phases of protection is not currently known.

Angiotensin II (ATII) is a recognised trigger of preconditioning-like protection in myocardium [16], a trigger thought to be related to the generation of reactive oxygen species (ROS) via the activity of NADPH oxidase [3,17]. Characterised as an early-phase preconditioning-mimetic, ATII has been shown to recruit various MAPKs, including ERK [18], p38 [18,19], JNK [18], as well as the serine/threonine kinase, Akt [20,21]. Moreover, ATII has been linked to insulin-like growth factor-1 receptor (IGF-1R) expression up-regulation through recruitment of the gene transcription factor, NF-{kappa}B [22]. Thus, ATII has been linked to the same groups of kinases that characterise the signalling cascades of both early and delayed preconditioning. ATII also regulates the same gene-transcription factors involved in the mediation of the second window of protection, and through regulation of IGF-1R, is possibly also linked to mechanisms recently implicated in Reperfusion Salvage [23,24]. It is therefore attractive to hypothesise that ATII not only triggers early preconditioning, but also a second window of protection. Given the identity of the kinases and processes involved in ATII signalling, it is also possible that the protection observed following both phases of protection is through the activation of pro-survival kinases such as Akt and ERK during reperfusion.

In this study, we investigate the ATII triggered time-course of bi-phasic preconditioning protection against infarction and whether there is contemporaneous post-ischaemic kinase activation/phosphorylation upon reperfusion that correlates with the preconditioning-protection observed.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
All work was conducted in accordance with the Guidelines on the Operation of the Animals (Scientific Procedures) Act 1986, published by The Stationery Office (London, UK), conforming with National Institute of Health Guidelines for the Care and Use of Laboratory Animals [25]. Wortmannin and PD98059 were purchased from Sigma (Poole, UK). Dimethyl Sulfoxide (DMSO, BDH laboratory supplies, Poole, UK) was used as the solvent for wortmannin and PD98059 and used at a final concentration in the perfusion buffer of not more than 0.01%.

2.1. Langendorff perfusion
Male C57BL6 mice (3–4 months of age, 20–30 g weight) were used, and randomly assigned to either control or treatment groups. Heart isolation and Langendorff perfusion (with modified Krebs–Henseleit buffer consisting of NaCl 118.5 mmol/L, NaHCO3 25 mmol/L, glucose 11 mmol/L, KCl 4.7 mmol/L, KH2PO4 1.2 mmol/L, MgSO4 1.2 mmol/L, CaCl2 1.4 mmol/L) were performed as previously described. In brief, mice were anaesthetised with an intra-peritoneal injection of 60 mg/kg pentobarbitone. Hearts were then harvested via a para-medial thoracotomy and rapidly transferred to a dissection dish filled with ice cold Krebs–Henseleit buffer. The aorta was cannulated with a 21-gauge cannula and the heart retrograde perfused on a murine Langendorff perfusion rig at 80 mmHg pressure, maintained using a STH pump controller (AD Instruments, Australia) that enables real-time coronary flow monitoring. Hearts were paced throughout the stabilisation period and during the final 20 min of reperfusion (600 beats/min) following the injurious ischaemia. Each Langendorff perfused mouse heart was randomly attributed to one of each of the experimental protocols (summarised in Fig. 1 and described in further detail below). Hearts harvested for Western blot analysis were snap frozen at the end of the perfusion protocol in liquid nitrogen and stored at –80 °C prior to protein extraction.


Figure 1
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  		Fig. 1 Experimental protocols. A: Isoflurane anaesthetised mice were stabilised for 10 min before the injection of 0.5–500 µg/kg angiotensin II (ATII), thereafter, haemodynamic parameters were monitored for 30 min. For all subsequent protocols, mice were pre-treated with vehicle (water for injection) or ATII prior to harvesting the heart and Langendorff perfusion. B: ATII dose response. Animals were pre-treated 1 h prior to harvesting the heart, with ATII at doses ranging from 0.5 to 500 µg/kg i.p. The hearts were stabilised for 20 min before being subjected to 35 min global, normothermic ischaemia and 30 min reperfusion prior to determination of infarct size. This infarct protocol was used in all subsequent infarct size studies. C: ATII time-course. Using the optimal dose of ATII, a 96 h post-injection cardioprotection time-course was determined, using infarct size as the primary end-point. D: Reperfusion kinase phosphorylation. Hearts were pre-treated 1, 6 or 24 h prior to subjecting the hearts to 20 min stabilisation, 35 min ischaemia and 5 min reperfusion prior to snap-freezing the heart for later Western blot analysis. E: Importance of Akt and ERK phosphorylation upon reperfusion. Hearts were pre-treated using ATII as in protocol D, 1, 6 and 24 h prior to subjecting the hearts to ischaemia/reperfusion. During reperfusion, the first 15 min were in the presence of vehicle, DMSO (<0.01%), wortmannin (100 nmol/l) or PD98059 (10 µmol/l). Hearts were reperfused for 5 min (and snap frozen) for Western blot analysis, or 30 min for infarct size assessment.

 
2.2. Preconditioning and perfusion protocols
The experiments were divided into two phases (Fig. 1). All Langendorff-perfused hearts for infarct analysis were stabilised for 20 min prior to being subjected to 35 min global, normothermic ischaemia, prior to 30 min reperfusion and infarct analysis by TTC staining, as has been previously characterised in the C57BL6 mouse model [26].

Phase 1 was designed to characterise the angiotensin II (ATII) preconditioning regimen. To ascertain the impact of various concentrations of ATII upon systemic blood pressure, Isoflurane-anaesthetised mice were injected, following a 10 min stabilisation period, with 0.5–500 µg/kg ATII via intra-peritoneal (i.p.) bolus injection and systemic blood pressure measured over a time-course of 30 min (Fig. 1A). Then, to determine the optimal dosing regimen of ATII, mice were injected with the same range of ATII doses, and then after one hour, the hearts were excised and subjected to ischaemia/reperfusion (Fig. 1B). To determine whether angiotensin resulted in the expected bi-phasic preconditioning predicted in other animal models of ischaemic and pharmacological preconditioning, mice were injected with ATII 1–96 h prior to harvesting and subjecting the heart to ischaemia/reperfusion (Fig. 1C).

Phase 2 was designed to determine kinase phosphorylation upon reperfusion, following 35 min of global, normothermic ischaemia (Fig. 1D). After 5 min of reperfusion following injurious ischaemia, the hearts were snap-frozen prior to protein extraction and Western blot analysis. To ascertain the importance of phosphorylation of Akt and ERK, kinase inhibitors wortmannin (100 nmol/l) and PD98059 (10 µmol/l) or vehicle, DMSO (0.01%) were added to the Krebs–Henseleit buffer from the onset of reperfusion for the first 15 min of reperfusion. The hearts were either perfused for 30 min prior to infarct size analysis or perfused in presence of inhibitor/vehicle for 5 min and snap-frozen for protein extraction and Western blot analysis (Fig. 1E).

2.3. Systemic blood pressure measurement
Measurement of blood pressure following i.p. injection of angiotensin was performed on male C57BL6 mice. Anaesthesia was induced (4%) and maintained (1.5%) with inhaled isoflurane (Abbott Laboratories Ltd, Queensborough, UK), with an inspired O2 concentration of 100%. With the mice spontaneously ventilating, the right common carotid artery was exposed and a microtip volume/pressure conductance catheter (Millar Instruments Inc., Houston, USA) inserted and advanced into the arch of the aorta. Following instrumentation, animal core temperature was maintained at 37 °C using a STH heating mat and the animal was stabilised for 10 min prior to injection of angiotensin. Thereafter, heart rate, systolic, diastolic and mean arterial pressure were recorded using PowerLab (AD Instruments, Australia) bioamplifiers and software on a PC for 30 min, by which time haemodynamic parameters returned to pre-injection stabilisation levels.

2.4. Infarct size analysis
Infarct size was determined by triphenyl tetrazolium chloride (TTC) staining, as previously described [26]. In brief, hearts at the end of the experimental protocol were infused with TTC in phosphate buffered solution (pH 7.4), prior to incubation of the whole heart at 37 °C for 10 min in TTC solution. The tetrazolium dye turns viable tissue a dark-red colour, whilst non-viable, necrotic myocardium appears pale. Hearts are then frozen at –80 °C and then sliced perpendicular to the long-axis of the heart at 1 mm intervals, providing between 8–10 slices per heart. Fixing the tissue slices in 10% v/v formaldehyde for 12 h augments the colour contrast between viable and necrotic tissue. The stained and fixed heart slices were then digitally photographed for planimetry using NIH Image 1.62 on an Apple iMac computer. Infarct size is expressed as an infarct to risk zone ratio (where the risk zone in the global ischemic heart is the whole ventricular volume).

2.5. Western blot analysis
Frozen tissue samples were extracted into 100 mg heart wt/ml 50 mmol/l TRIS (pH 6.8)-based extraction buffer containing 5 mmol/l NaF, 2 mmol/l NaVO4, 2 mmol/l EDTA plus Roche Complete EDTA-free protease inhibitor, and homogenised. In brief, Western blot analysis were performed on 12% polyacrylamide gels, loaded at 10 µl/lane and transferred onto Hybond-P membrane (Amersham Biosciences, Chalfont, England). Loading confirmed both by Coumassie staining of the membranes after and by quantification of the relevant non-phosphorylated protein (Akt, ERK, p38-MAPK or JNK) by Western blot analysis. The membranes were probed with rabbit polyclonal antibodies for Akt, phospho-(serine 473) Akt, ERK, phospho-ERK, p38, phospho-p38 and JNK and phospho-JNK, phospho GSK-3β (serine 9), phospho Bad (serine 136) and phospho eNOS (serine 1177) antibodies purchased from Cell Signalling, and used in accordance to the manufacturer's instructions. Membranes were then incubated in Anti-rabbit IgG-HRP secondary antibodies (Amersham Biosciences, Chalfont, England) and quantification achieved using the ECL chemoluminescence technique, with exposure to photographic film. The developed films were scanned using a flatbed document scanner and the relative densitometry assessed using the NIH Shareware program, NIH Image 1.62 on an Apple iMac PC.

2.6. Statistical analysis
All values are expressed as mean±standard error of the mean. Differences in continuously distributed variables between predetermined experimental groups were analysed using one-way ANOVA followed by Fisher's protected test of least significant difference. P values of 0.05 were considered to be at the limit of statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Angiotensin II characterisation: haemodynamics and cardioprotection
Angiotensin II (ATII) is a well-characterised pressor agent upon systemic blood pressure, but in the context of acute intra-peritoneal (i.p.) injection, the haemodynamic consequence of the drug's administration and the time-course of action is unknown. To characterise this, we undertook measurements of systemic blood pressure in isoflurane anaesthetised animals. Using a range of ATII concentrations from 0.5 to 500 µg/kg, we demonstrated a dose-dependent effect of ATII upon systemic blood pressure (Fig. 2A and representative traces, Fig. 2B). There was no significant difference between groups of baseline mean arterial pressure (MAP, Fig. 2A). MAP was not significantly increased over baseline or vehicle (water for injection) control until an ATII dose of 50 µg/kg or more was employed (MAP 97±3 (p<0.05) and 131±9 mmHg (p<0.001) for 50 and 500 µg/kg respectively, versus vehicle control 81±3 mmHg). The lower 0.5 and 5 µg/kg doses of ATII had no significant impact upon the MAP in these anaesthetised animals (84±2 and 88±2 mmHg respectively). The haemodynamic impact of ATII in the isoflurane-treated animals corresponded with observation of animal discomfort in conscious mice (as determined by scoring of social behaviour, exploratory activity, posture and piloerrection) used for the later infarct-limiting dose-ranging study. Lower ATII doses caused almost no alteration of animal behaviour in contrast to that seen at pressor doses of ATII (50–500 µg/kg).


Figure 2
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  		Fig. 2 Angiotensin II (ATII) dose-response. To determine the optimal concentration of ATII to be used in the study, ATII was administered i.p. at doses ranging from 0.5 to 500 µg/kg and compared to vehicle (water for injection). A: Mean arterial pressure (MAP) measurements. The white bars show baseline MAP, as recorded prior to administration of ATII. Peak ATII induced increase of MAP is shown by the gray bars. There were no significant differences in baseline MAP between groups, whereas ATII at doses greater than 5 µg/kg resulted in significant increases in MAP (*p<0.05 and **p<0.001 versus baseline and vehicle controls, n=4 per group). B: Representative systolic blood pressure traces (n=4 per group) are shown. The higher doses of ATII result in a greater increase in blood pressure, and the effect tended to be of longer duration. C: Administering ATII 1 h prior to subjecting the heart to ischaemia/reperfusion resulted in a bell-shaped curve, with optimal concentration observed at 5 µg/kg – a dose used in all subsequent ATII studies. (*p<0.05 and **p<0.001 versus vehicle control group, n=6 per group).

 
To determine the optimal cardioprotective concentration of ATII we undertook a second dose-response curve, this time using infarct size as the primary end-point. ATII triggered protection in the isolated mouse heart 1 h after i.p. injection in a dose-dependent manner with an optimal dose of 5 µg/kg (Fig. 2C), with an infarct to risk zone ratio of 19±4% versus control, 35±4% (p<0.01). The dose-response curve was found to be bell-shaped, where at higher doses of ATII, protection is lost. The optimal protective dose of ATII was found to be sub-pressor, and therefore all further experiments using ATII were performed using a dose of 5 µg/kg i.p. – coincidentally, a dose similar to that used by Kimura et al. in rat pharmacological preconditioning [3].

3.2. Angiotensin II characterisation: does ATII trigger bi-phasic preconditioning?
To determine whether ATII triggers bi-phasic preconditioning and its time-course, hearts were pre-treated with 5 µg/kg ATII 1 to 96 h prior to the hearts being isolated and subjected to ischaemia and reperfusion and infarct size determined (protocol: Fig. 1C). ATII resulted in significant protection against lethal ischaemia/reperfusion injury (Fig. 3) at 1 and 2 h post preconditioning (19±3% versus control, 36±2%, p<0.01), but this protection was lost by 6 h (32±2%). Protection re-emerged by 24 h (25±4%, p<0.05 versus control), and significant protection against necrosis was maintained through to 72 h (25±1%, p<0.05 versus control) before waning at 96 h (31±2%) post ATII injection. Thus the time-course of mouse preconditioning with ATII would appear identical to ischemic and pharmacological preconditioning previously described in pig [27] and rabbit [4,28], for both classical preconditioning and the second window of protection.


Figure 3
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  		Fig. 3 Angiotensin II preconditioning time-course. 5 µg/kg ATII injected 1–96 h prior to subjecting the hearts to ischaemia/reperfusion. Pharmacological preconditioning demonstrated the typical bi-phasic pattern of protection, with early protection appearing within an hour, lasting at least 2 h before disappearing by 6 h post injection. 24 h following the preconditioning stimulus, protection re-appeared and persisted until waning by 96 h following the i.p. bolus injection of ATII. (*p<0.05 versus vehicle control, n=5–6 per group).

 
3.3. Does ATII preconditioning augment MAPK phosphorylation at reperfusion?
To determine whether pharmacological preconditioning results in phosphorylation of pro-survival kinases after injurious ischaemia, in both windows of protection, we performed Western blot analysis on protein extracted from hearts reperfused for 5 min after injurious ischaemia (protocol: Fig. 1D).

We determined phosphorylation levels of Akt, ERK, p38 and JNK at three major time points, corresponding to early preconditioning (1 h post ATII injection), the window of no protection (6 h post AT injection) and the second window of protection (24 h post ATII injection). ATII pre-treatment resulted in significant increases in phosphorylation of both Akt and ERK at 1-hour following the pharmacological preconditioning injection of ATII (41±2 AU versus control, 1±1 AU p<0.05, and 585±97 AU versus control, 276±60 AU p<0.001 respectively, Fig. 4A and B). This was mirrored by similar increases in phosphorylation of both Akt and ERK (242±12 AU and 487±33 AU, p<0.001 versus respective controls, respectively) at 24 h. However, there was no apparent regulation of either p38-MAPK or JNK following pharmacological preconditioning (Fig. 4C and D), although an upward trend was observed in p38-MAPK phosphorylation, it did not achieve significance. Unexpectedly, there was an increase of observed phosphorylation of both Akt and ERK at 6 h following the pharmacological preconditioning stimulus, in the absence of protection. This raised the question of whether kinase phosphorylation upon reperfusion is merely an epiphenomenon of pharmacological preconditioning that is coincidentally observed in a similar time frame to that of protection.


Figure 4
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  		Fig. 4 Kinase-phosphorylation upon reperfusion. 1, 6 and 24 h following bolus injection, Akt (A) and ERK (B) were found to be significantly phosphorylated 5 min into reperfusion following injurious ischaemia. In contrast to kinase phosphorylation, Akt and ERK were phosphorylated corresponding to protection at 1 h. No evidence of p38-MAPK (C) or JNK (D) was observed. (*p<0.05 versus respective control group, n=4 per group; representative Western blots shown).

 
3.4. Is Akt and ERK phosphorylation upon reperfusion essential for early and late preconditioning?
To determine whether Akt and ERK phosphorylation are essential for the protection associated with pharmacological preconditioning or merely an epiphenomenon, we examined the effect of inhibiting PI3K and MEK1 with wortmannin and PD98059 respectively, administered for the first 15 min of reperfusion (protocol described in Fig. 1E). The DMSO vehicle itself had no impact upon infarct size when compared to KHB perfused hearts (33±2% versus 35±4% respectively, p=NS, Fig. 5A, B). ATII resulted in significant early protection at 1 h (24±3%, p<0.05), which resolved by 6 h (30±3%, p=NS) and was re-instituted by 24 h (24±5%, p<0.05). Inhibiting PI3K with wortmannin however, completely attenuated preconditioning in both the early (34±2%, p=NS) and late phases (35±4%, p=NS) (Fig. 5C). Similarly, inhibition of MEK with PD98059 also abrogated early (28±3%, p=NS) and delayed protection (33±3%, p=NS, Fig. 5D). In both cases, neither wortmannin nor PD98059 had any impact upon infarct size in control hearts (33±5% and 33±2% respectively), nor upon the infarct size at 6 h post-preconditioning stimulus (33±1% and 31±3% versus vehicle control, 30±3% and KHB control, 32±2%, p=NS respectively).


Figure 5
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  		Fig. 5 Impact of PI3 kinase and MEK inhibition at reperfusion upon ATII-triggered bi-phasic preconditioning. Relative to control groups (A), drug vehicle, DMSO (0.01%, B) had no impact upon the protection in early or delayed phases of pharmacological preconditioning related to ATII. In contrast, both wortmannin (C) and PD98059 (D) (PI3 kinase and MEK inhibitors respectively) abrogated the protection associated with pharmacological preconditioning at both 1 and 24 h following ATII bolus injection. (*p<0.05 versus respective control, n=6 per group).

 
To determine whether the inhibitors used were effective in abrogating the phosphorylation of Akt and ERK, we performed Western blot analysis on hearts treated with ATII (Fig. 6). Both wortmannin and PD98059 had the expected effect, attenuating Akt and ERK phosphorylation respectively in both the early and delayed preconditioning time frames. Interestingly, both inhibitors impacted upon phosphorylation of the alternate pathway, with wortmannin attenuating ERK phosphorylation, and PD98059 attenuating Akt phosphorylation. This observation would appear to suggest a cross talk between the two kinases, although the mechanism of how this may be occurring was not investigated in this study.


Figure 6
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  		Fig. 6 Impact of PI3 kinase and MEK inhibition at reperfusion upon Akt and ERK phosphorylation. Representative Western blots are shown. Wortmannin completely abrogates the phosphorylation of Akt in early reperfusion associated with pharmacological preconditioning. A similar diminution of ERK phosphorylation is also seen. Similarly, PD98059 is found to attenuate the Akt phosphorylation signal, as well as abrogating ERK phosphorylation following pharmacological preconditioning. Thus there appears to be evidence of cross-talk between the MEK and PI3 kinase pathways in this model (*p<0.05 versus respective control, n=4 per group).

 
3.5. Are downstream kinase pathways intact during the phase of no-protection?
The data suggest that phosphorylation of both Akt and ERK at reperfusion may be pivotal to the protection observed in both early and delayed preconditioning. However, the period of no protection between these two phases of protection would seem to represent a curious dichotomy. Despite significant phosphorylation of Akt and ERK at reperfusion 6 h after ATII administration, it is apparent that these kinases, although important for preconditioning-protection, are insufficient to ensure amelioration of ischaemia/reperfusion injury. We hypothesised that during the phase of no protection at 6 h following ATII administration, the kinase cascade may be inhibited downstream of Akt and ERK, and thus unable to recruit protective end-effector mechanisms. To ascertain whether this is the case, we looked at the phosphorylation of a number of downstream targets of the Akt signalling cascade that have been highlighted as potential mediators of myocardial protection: GSK-3β [29], Bad [30] and eNOS [31].

Concomitant with increase of phosphorylation of both Akt and ERK (Fig. 7), GSK-3β demonstrated increased phosphorylation at 1, 6 and 24 h following ATII administration (2.6±0.2, 2.5±0.5 and 4.0±0.9 fold increases respectively over control samples, and significantly increased over contemporaneous time-matched controls, p<0.05). Similar increases were also seen with Bad (3.3±0.5, 2.3±0.01 and 2.9±0.1 fold increase, p<0.05 versus time matched controls) and eNOS (1.8±0.4, 1.6±0.1 and 2.6±0.1 fold increase, p<0.05 versus time matched controls). Therefore, it would seem that there is no inhibition of signalling to these downstream kinases during the period of no protection, rather, perhaps, the potential target of these signalling pathways is insensitive to their activity.


Figure 7
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  		Fig. 7 Downstream kinase phosphorylation. Concomitant with the augmentation of Akt and ERK phosphorylation at 1, 6 and 24 h following ATII administration, similar increases of phosphorylation were seen in three disparate, putative downstream mediators of myocardial protection, GSK-3β (serine 9), Bad (serine 136) and eNOS (serine 1177). Therefore the loss of protection observed at 6 h would appear associated with insensitivity of a downstream target of these signalling pathways. (*p<0.05 versus respective time-matched control group, n=4 per group; representative Western blots shown).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study we demonstrate that pharmacological preconditioning, using angiotensin II (ATII) as a trigger, results in bi-phasic preconditioning in mouse heart, inducing both ‘classical’ early preconditioning and a second window of protection 24 h after the preconditioning stimulus. The time-course of protection against infarction appears identical to that seen in other mammalian species including pig [27] and rabbit [4,28], and represents the first full characterisation of the time-course of preconditioning protection against myocardial infarction in the mouse model, albeit pertaining to a single pharmacological trigger. In addition, we show that bi-phasic preconditioning is critically dependent upon the phosphorylation of ‘pro-survival’ kinases, Akt and ERK, during the post-ischaemic reperfusion phase in both phases of protection. This is, therefore, the first demonstration of the pivotal role of reperfusion kinase activation for the protection observed in the second window of protection.

ATII is a well established trigger of a preconditioning-like protection in myocardium [16,32,33]. The downstream signalling cascade is thought to include the generation of reactive oxygen species (ROS) in the myocardium via the activity of NADPH oxidase [3,17], leading to recruitment of protein kinases including ERK [12], p38 kinase [13] and JNK [12], as well as Akt [20,21]. In the present study, we have undertaken to investigate bi-phasic preconditioning, the second phase of which has not previously been studied in regard to ATII administration. Delayed pharmacological preconditioning, due to the prolonged period of time between preconditioning stimulus and the start of the experiment, negates the possibility of administrating ATII in vitro, in contrast to earlier ATII studies of early preconditioning which were universally based in the Langendorff system [16,32,33]. Out of necessity therefore, ATII was administered systemically (as it was in the study by Kimura et al. [3]), which introduces the potential confounding impact of ATII upon systemic blood pressure. The haemodynamic impact of low-dose acute bolus i.p. injection is unknown, so we undertook to characterise the pressor effect of ATII over a dose range of 0.5–500 µg/kg. We found that ATII at cardioprotective concentrations (0.5 and 5 µg/kg) were sub-pressor, in contrast to the higher doses studied (50–500 µg/kg), which coincided with dose-dependent loss of protection against infarction. Therefore, it would appear that the protection observed with the ATII dose employed in this study was not confounded by hypertension secondary to systemic vasoconstriction. That the ATII infarct limitation curve was bell-shaped with loss of protection at higher doses is interesting; the mechanism by which cardiac protection is obscured in this instance is unknown, but could potentially be related to the pressor response to high-dose ATII.

That ATII should trigger both phases of protection is perhaps not surprising. As previously mentioned, ATII up-regulates a similar pattern of kinases to that seen following other stimuli of preconditioning [34], including the recruitment of gene-transcription factors thought to be essential to the recruitment of cytoprotective proteins required for the development of the second window of protection. However, we wanted to determine whether, as has recently been found following early ischaemic preconditioning [15], kinase recruitment in reperfusion following the injurious ischaemic insult is essential for the protection seen during the second window. One mechanism by which this could be realised is through the increased expression of insulin-like growth factor (IGF)-1 receptors (IGF-1R), shown to be triggered by administration of ATII via the recruitment of the transcription factor, NF-{kappa}B. [22] Signalling via the IGF-1 receptor appears to play an important role in ameliorating ischaemia/reperfusion injury. Previous studies have shown that administration of IGF-1R following injurious ischaemia attenuates ischaemia/reperfusion injury [35,36]. Moreover, over-expression of IGF-1R in a transgenic model demonstrates increased basal Akt activity [23,37], an important ‘pro-survival’ kinase in the context of ischaemia/reperfusion injury, which leads to attenuated cell death in models of ischaemia/reperfusion [23]. Therefore, it is plausible that ATII triggered delayed protection could be mediated through the recruitment/phosphorylation of Akt and ERK in early reperfusion. Interestingly, this is exactly what we found: as in early preconditioning resulting from ATII, delayed protection was also characterised by a marked increase of phosphorylation of both Akt and ERK. Moreover, inhibition of PI3K and MEK upon reperfusion by wortmannin and PD98059 completely abrogated the protection associated with both phases of protection. Thus it appears that both PI3K and MEK activity is essential for both phases of protection arising as a consequence of ATII preconditioning. How these kinases interact with cardioprotective proteins such as iNOS, COX and heat shock proteins remains unclear; it is conceivable that post-transcriptional modifications of either constitutive or induced proteins is required in order for the heart to demonstrate a cardioprotective trait.

Our data however does throw up an unexpected anomaly. As shown in Fig. 3, 6 h after ATII administration there is no observable protection, despite the clearly augmented phosphorylation of Akt and ERK. Given the pharmacological evidence that PI3K and MEK activities are essential for the observed protection, it seems unlikely that the observed phosphorylation of Akt and ERK in reperfusion is an epiphenomenon. But the data does indicate that phosphorylation of Akt and ERK is not sufficient to imbue protection upon the reperfused heart. That neither wortmannin nor PD98059 exacerbated the necrotic injury suggests that the loss of protection between windows of preconditioning is not due to a coincident deleterious process, but rather, it would seem, an absence of a down-stream effector mechanism, the identity of which remains to be ascertained. Given that phosphorylation of three down-stream targets of the proposed cytoprotective pathway, GSK-3β, Bad and eNOS remain unhindered, it would appear that the kinase signalling cascade remains intact – perhaps indicating end-target insensitivity. That GSK-3β [29], Bad [38] and eNOS [26,39] are all known or thought to interact with the mitochondria in the context of myocardial ischaemic/reperfusion injury, and with mitochondria gaining much interest as a potentially crucial target in this model [40,41], it is tempting to postulate that it is the mitochondria themselves that are responsible for the loss of protection between the two phases of protection. Clearly this is a hypothesis that requires further study, and given the important role that mitochondria play in the initiation of apoptotic signalling, another potential hypothesis to consider concerns caspase activation and recruitment of apoptotic cell death cascades. All three kinase targets studied – GSK-3β, Bad and eNOS – are thought to be associated with the down-regulation of pro-apoptotic pathways, be that through attenuation of permeability transition pore open probability by GSK-3β [42], and therefore amelioration of cytochrome-c release and caspase-9 activation – a mechanism that may also be linked with phosphorylation of Bad and its sequestration by 14-3-3 [43], or direct inhibition of caspase-3 by nitrosylation by nitric oxide synthesised by eNOS [44]. We have not measured caspase activity nor apoptotic cell death as part of this investigation – however we would reasonably anticipate that caspase-9 and caspase-3 activities would be attenuated by the Akt and ERK signalling cascades, but whether these caspases are inhibited or indeed whether alternate injurious mitochondria-independent apoptotic cascades are recruited during the ‘window of no protection’ is an intriguing possibility. However as caspase activity has been linked to necrotic injury [45], and given the observation that inhibition of the Akt and ERK signalling pathways failed to increase necrotic injury over control hearts, the possibility of competing injurious caspase activity appears an unlikely, but whether there is a bi-phasic attenuation of caspase activity concomitant with the bi-phasic protection observed – with loss of caspase inhibition during the ‘window of no protection’ – is an interesting possibility deserving future study.

Thus we present a number of novel observations. (a) This is, to our knowledge, the first time that the full time-course of preconditioning has been mapped in the mouse. (b) Our data suggest that MAPK activation is crucial in pharmacological preconditioning in the early reperfusion phase in both early and delayed pharmacological preconditioning. (c) Our data also highlights the conundrum of why protection is lost between the early phase and the second window of protection: whereas potential pro-survival kinases are demonstrably activated, there appears to be a down-stream inhibition or deficiency of the signalling pathway that has yet to be delineated.


   Notes
 
Time for primary review 17 days


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

  1. Murry C.E., Jennings R.B., Reimer K.A. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation (1986) 74:1124–1136.[Abstract/Free Full Text]
  2. Baines C., Wang L., Cohen M., Downey J. Protein tyrosine kinase is downstream of protein kinase C for ischemic preconditioning's anti-infarct effect in the rabbit heart. J Mol Cell Cardiol (1998) 30:383–392.[CrossRef][Web of Science][Medline]
  3. Kimura S., Zhang G.X., Nishiyama A., Shokoji T., Yao L., Fan Y.Y., et al. Role of NAD(P)H oxidase-and mitochondria-derived reactive oxygen species in cardioprotection of ischemic reperfusion injury by angiotensin II. Hypertension (2005) 45:860–866.[Abstract/Free Full Text]
  4. Baxter G., Goma F., Yellon D. Characterisation of the infarct-limiting effect of delayed preconditioning: time-course and dose-dependency studies in rabbit myocardium. Basic Res Cardiol (1997) 92:159–167.[CrossRef][Web of Science][Medline]
  5. Rizvi A., Tang X.L., Qiu Y., Xuan Y.T., Takano H., Jadoon A.K., et al. Increased protein synthesis is necessary for the development of late preconditioning against myocardial stunning. Am J Physiol (1999) 277:H874–H884.[Web of Science][Medline]
  6. Dawn B., Xuan Y.T., Guo Y., Rezazadeh A., Stein A.B., Hunt G., et al. IL-6 plays an obligatory role in late preconditioning via JAK-STAT signaling and upregulation of iNOS and COX-2. Cardiovasc Res (2004) 64:61–71.[Abstract/Free Full Text]
  7. Marber M.S., Latchman D.S., Walker J.M., Yellon D.M. Cardiac stress protein elevation 24 h after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation (1993) 88:1264–1272.[Abstract/Free Full Text]
  8. Xuan Y., Tang X., Banerjee S., Takano H., Li R., Han H., et al. Nuclear factor-kappaB plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res (1999) 84:1095–1109.[Abstract/Free Full Text]
  9. Zhao T.C., Taher M.M., Valerie K.C., Kukreja R.C. p38 Triggers late preconditioning elicited by anisomycin in heart: involvement of NF-kappaB and iNOS. Circ Res (2001) 89:915–922.[Abstract/Free Full Text]
  10. Dawn B., Guo Y., Rezazadeh A., Wang O.L., Stein A.B., Hunt G., et al. Tumor necrosis factor-alpha does not modulate ischemia/reperfusion injury in naive myocardium but is essential for the development of late preconditioning. J Mol Cell Cardiol (2004) 37:51–61.[CrossRef][Web of Science][Medline]
  11. Xi L., Taher M., Yin C., Salloum F., Kukreja R.C. Cobalt chloride induces delayed cardiac preconditioning in mice through selective activation of HIF-1alpha and AP-1 and iNOS signaling. Am J Physiol Heart Circ Physiol (2004) 287:H2369–H2375.[Abstract/Free Full Text]
  12. Li R.C., Ping P., Zhang J., Wead W.B., Cao X., Gao J., et al. PKCepsilon modulates NF-kappaB and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes. Am J Physiol Heart Circ Physiol (2000) 279:H1679–H1689.[Abstract/Free Full Text]
  13. Maulik N., Sato M., Price B.D., Das D.K. An essential role of NFkappaB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett (1998) 429:365–369.[CrossRef][Web of Science][Medline]
  14. Behrends M., Schulz R., Post H., Alexandrov A., Belosjorow S., Michel M.C., et al. Inconsistent relation of MAPK activation to infarct size reduction by ischemic preconditioning in pigs. Am J Physiol Heart Circ Physiol (2000) 279:H1111–H1119.[Abstract/Free Full Text]
  15. Hausenloy D.J., Tsang A., Mocanu M.M., Yellon D.M. Ischemic preconditioning protects by activating prosurvival kinases at reperfusion. Am J Physiol Heart Circ Physiol (2005) 288:H971–H976.[Abstract/Free Full Text]
  16. Liu Y., Tsuchida A., Cohen M.V., Downey J.M. Pretreatment with angiotensin II activates protein kinase C and limits myocardial infarction in isolated rabbit hearts. J Mol Cell Cardiol (1995) 27:883–892.[CrossRef][Web of Science][Medline]
  17. Das S., Engelman R.M., Maulik N., Das D.K. Angiotensin preconditioning of the heart: evidence for redox signaling. Cell Biochem Biophys (2006) 44:103–110.[CrossRef][Web of Science][Medline]
  18. El Bekay R., Alvarez M., Monteseirin J., Alba G., Chacon P., Vega A., et al. Oxidative stress is a critical mediator of the angiotensin II signal in human neutrophils: involvement of mitogen-activated protein kinase, calcineurin, and the transcription factor NF-kappaB. Blood (2003) 102:662–671.[Abstract/Free Full Text]
  19. Ushio-Fukai M., Alexander R.W., Akers M., Griendling K.K. p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy. J Biol Chem (1998) 273:15022–15029.[Abstract/Free Full Text]
  20. Ushio-Fukai M., Alexander R.W., Akers M., Yin Q., Fujio Y., Walsh K., et al. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem (1999) 274:22699–22704.[Abstract/Free Full Text]
  21. Takahashi T., Taniguchi T., Konishi H., Kikkawa U., Ishikawa Y., Yokoyama M. Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells. Am J Physiol (1999) 276:H1927–H1934.[Web of Science][Medline]
  22. Ma Y., Zhang L., Peng T., Cheng J., Taneja S., Zhang J., et al. Angiotensin II stimulates transcription of insulin-like growth factor I receptor in vascular smooth muscle cells: role of nuclear factor-kappaB. Endocrinology (2006) 147:1256–1263.[Abstract/Free Full Text]
  23. Yamashita K., Kajstura J., Discher D.J., Wasserlauf B.J., Bishopric N.H., Anversa P., et al. Reperfusion-activated Akt kinase prevents apoptosis in transgenic mouse hearts overexpressing insulin-like growth factor-1. Circ Res (2001) 88:609–614.[Abstract/Free Full Text]
  24. Hausenloy D.J., Yellon D.M. New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res (2004) 61:448–460.[Abstract/Free Full Text]
  25. Council N.R. Guide for the care and use of laboratory animals. (1996) Washington, DC: National Academy Press.
  26. Bell R.M., Maddock H.L., Yellon D.M. The cardioprotective and mitochondrial depolarising properties of exogenous nitric oxide in mouse heart. Cardiovasc Res (2002) 57:405–415.[CrossRef][Web of Science]
  27. Tang X.-L., Qiu Y., Sun J.-Z., Park S.-W., Kalya A., Bolli R. Time-course of late preconditioning against myocardial stunning in conscious pigs. Circ Res (1996) 79:424–434.[Abstract/Free Full Text]
  28. Baxter G.F., Yellon D.M. Time-course of delayed myocardial protection after transient adenosine A1 receptor activation in the rabbit. J Cardiovasc Pharmacol (1997) 29:631–638.[CrossRef][Web of Science][Medline]
  29. Juhaszova M., Zorov D.B., Kim S.H., Pepe S., Fu Q., Fishbein K.W., et al. Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest (2004) 113:1535–1549.[CrossRef][Web of Science][Medline]
  30. Das S., Otani H., Maulik N., Das D.K. Redox regulation of angiotensin II preconditioning of the myocardium requires MAP kinase signaling. J Mol Cell Cardiol (2006) 41(2):223–225.[CrossRef][Web of Science][Medline]
  31. Tsang A., Hausenloy D.J., Mocanu M.M., Yellon D.M. Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ Res (2004) 95:230–232.[Abstract/Free Full Text]
  32. Sharma A., Singh M. Role of angiotensin in cardioprotective effect of ischemic preconditioning. J Cardiovasc Pharmacol (1999) 33:772–778.[CrossRef][Web of Science][Medline]
  33. Sharma A., Singh M. Possible mechanism of cardioprotective effect of angiotensin preconditioning in isolated rat heart. Eur J Pharmacol (2000) 406:85–92.[CrossRef][Web of Science][Medline]
  34. Das D.K., Maulik N., Engelman R.M. Redox regulation of angiotensin II signaling in the heart. J Cell Mol Med (2004) 8:144–152.[Web of Science][Medline]
  35. Otani H., Yamamura T., Nakao Y., Hattori R., Kawaguchi H., Osako M., et al. Insulin-like growth factor-I improves recovery of cardiac performance during reperfusion in isolated rat heart by a wortmannin-sensitive mechanism. J Cardiovasc Pharmacol (2000) 35:275–281.[CrossRef][Web of Science][Medline]
  36. Davani E.Y., Brumme Z., Singhera G.K., Cote H.C., Harrigan P.R., Dorscheid D.R. Insulin-like growth factor-1 protects ischemic murine myocardium from ischemia/reperfusion associated injury. Crit Care (2003) 7:R176–R183.[CrossRef][Web of Science][Medline]
  37. McMullen J.R., Shioi T., Huang W.Y., Zhang L., Tarnavski O., Bisping E., et al. The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase(p110alpha) pathway. J Biol Chem (2004) 279:4782–4793.[Abstract/Free Full Text]
  38. Uchiyama T., Engelman R.M., Maulik N., Das D.K. Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning. Circulation (2004) 109:3042–3049.[Abstract/Free Full Text]
  39. Loke K.E., McConnell P.I., Tuzman J.M., Shesely E.G., Smith C.J., Stackpole C.J., et al. Endogenous endothelial nitric oxide synthase-derived nitric oxide is a physiological regulator of myocardial oxygen consumption. Circ Res (1999) 84:840–845.[Abstract/Free Full Text]
  40. Halestrap A.P., Clarke S.J., Javadov S.A. Mitochondrial permeability transition pore opening during myocardial reperfusion-a target for cardioprotection. Cardiovasc Res (2004) 61:372–385.[Abstract/Free Full Text]
  41. Honda H.M., Korge P., Weiss J.N. Mitochondria and ischemia/reperfusion injury. Ann NY Acad Sci (2005) 1047:248–258.[CrossRef][Web of Science][Medline]
  42. Juhaszova M., Zorov D.B., Kim S.H., Pepe S., Fu Q., Fishbein K.W., et al. Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest (2004) 113:1535–1549.[CrossRef][Web of Science][Medline]
  43. Murriel C.L., Churchill E., Inagaki K., Szweda L.I., Mochly-Rosen D. Protein kinase Cdelta activation induces apoptosis in response to cardiac ischemia and reperfusion damage: a mechanism involving BAD and the mitochondria. J Biol Chem (2004) 279:47985–47991.[Abstract/Free Full Text]
  44. Rossig L., Fichtlscherer B., Breitschopf K., Haendeler J., Zeiher A.M., Mulsch A., et al. Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. J Biol Chem (1999) 274:6823–6826.[Abstract/Free Full Text]
  45. Mocanu M.M., Baxter G.F., Yellon D.M. Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury. Br J Pharmacol (2000) 130:197–200.[CrossRef][Web of Science][Medline]

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