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Cardiovascular Research 2002 55(3):672-680; doi:10.1016/S0008-6363(02)00325-5
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Copyright © 2002, European Society of Cardiology

Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts

Adrian T Saurina,*, Daniel J Penningtonb, Nicolaas J.H Raatc, David S Latchmand, Michael J Owenb and Michael S Marbera

aDepartment of Cardiology, KCL, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, UK
bImperial Cancer Research Fund, London, UK
cAcademic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
dInstitute of Child Health, Great Ormond Street Hospital, University College London, London WC1N 1EH, UK

* Corresponding author. Tel.: +44-171-922-8191; fax: +44-171-960-5659 adrian.saurin{at}kcl.ac.uk

Received 30 October 2001; accepted 20 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Activation of protein kinase C (PKC) isoforms is associated with the cardioprotective effect of early ischaemic preconditioning (IP). PKC consists of at least 10 different isoforms, encoded by separate genes, which mediate distinct physiological functions. Although the PKC-{varepsilon} isoform has been implicated in preconditioning, uncertainty remains. We investigated whether preconditioning still occurs in a mouse line lacking cardiac PKC-{varepsilon} protein due to a targeted disruption within the pkc-{varepsilon} allele. Methods: The isolated buffer-perfused hearts from knockout mice lacking PKC-{varepsilon} (–/–) and sibling heterozygous mice (+/–), with a normal PKC-{varepsilon} complement, were preconditioned by 4x4 min ischaemia/6 min reperfusion. Hearts then underwent 45 min of global ischaemia followed by 1.5 h of reperfusion. Results: In PKC-{varepsilon} (+/–) hearts ischaemic preconditioning reduced infarction volume as a percentage of myocardial volume (24.3±4.5 vs. 41.3±4.7%, P<0.001). In contrast, in PKC-{varepsilon} (–/–) hearts preconditioning failed to diminish infarction (36.4±2.9 vs. 38.8±4.5%). Surprisingly however, although preconditioning did not reduce infarct size, it did enhance contractile recovery in PKC-{varepsilon} (–/–) mice (43.1±3.9 vs. 24.9±5.1%, P<0.05), similar to the level seen in PKC-{varepsilon} (+/–) hearts (35.2±3.9 vs. 20.9±5.0%, P<0.05). Conclusions: These data suggest that PKC-{varepsilon} is essential for the reduction in infarction that follows early ischaemic preconditioning, but is not associated with the improvement in functional recovery.

KEYWORDS Preconditioning; Protein kinases; Ischemia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Ischaemic preconditioning (IP) describes the reduction in susceptibility to myocardial infarction that follows brief periods of sublethal ischaemia [1]. This protection, which can result in a 4-fold reduction in infarct size, is secondary to a delay in the onset and rate of necrosis during the subsequent lethal ischaemia [2,3]. Although the protection afforded by brief periods of ischaemia is short-lived [4] and cannot be sustained [5], manipulation of the underlying signalling mechanisms may enable persistent protection without the risk of an ischaemic trigger [6].

The first intracellular kinase to be implicated in preconditioning was protein kinase C (PKC) [7]. PKC consists of at least 10 different isoforms, encoded by separate genes, which mediate distinct physiological functions [8]. Although protection can be blocked or mimicked by PKC inhibitors or activators [9–13], it is uncertain which isoform(s) is/are responsible. Understanding the role of individual PKC isoforms may provide new therapeutic targets with high specificity.

It has been suggested that PKC-{varepsilon} activation is crucial to protection since isoform-specific inhibitory peptides are able to abolish protection in response to IP [14,15]. The peptides function by preventing the binding of specific PKC isoforms to their respective receptors for activated C-kinase (RACKs). The rationale is that they prevent a specific PKC isoform from localizing with its substrate(s), thus causing a loss of function. The difficulty in measuring isoform activation makes evaluation of the selectivity and efficacy of these peptides complex. Moreover selective activation of PKC-{delta}, a closely related isoform to PKC-{varepsilon}, is able to protect both isolated myocytes [16] and the whole heart against ischaemia [17]. There is therefore a need to further clarify the role of individual isoforms during ischaemic preconditioning. We used mice with a targeted disruption of the pkc-{varepsilon} gene to investigate the role of this isoform during preconditioning in isolated buffer-perfused hearts.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
All experiments were performed in accordance with the United Kingdom Home Office Guidance on the Operation of Animals (Scientific Procedures) Act 1986, published by Her Majesty's Stationary Office, London.

2.1 Generation of PKC-{varepsilon}-deficient mice
A detailed description of the generation and morphology of PKC-{varepsilon} (–/–) mice can be found elsewhere [18]. In brief, a 9.3-kb EcoRI fragment containing the first exon of the pkc-{varepsilon} gene was isolated from a murine 129/Sv genomic DNA library (gift from T. Rabbits, LMB, Cambridge). To generate a targeting vector, a positive selectable cassette was introduced into the PstI site of exon 1. This cassette contains stop codons in all three frames, an independent ribosomal entry site (IRES) followed by the LacZ gene with an SV40 polyadenylation sequence, and a neomycin phosphotransferase gene (MC1Neo poly(A); gift from A. Smith, Center for Genome Research, Edinburgh). Correctly targeted GK129 ES cells were selected and then injected into C57BL/6 blastocysts. Chimeric mice were bred to C57BL/6 mice in specific pathogen free (SPF) conditions and offspring crossed to generate pkc-{varepsilon} homozygous mutant animals. PKC-{varepsilon} deficient mice were shown to be negative for PKC-{varepsilon} protein by Western blotting.

2.2 Heart isolation
All male animals were anaesthetized with a ketamine/xylazine mixture (150 and 24 mg/kg with 100 IU heparin, i.p.), hearts rapidly excised from the thorax and placed in ice-cold modified Krebs–Henseleit (K–H) buffer. After removal of excess thymic and fatty tissue, the aorta was cannulated with a 23-G blunt and grooved stainless steel needle. The heart was retrogradely perfused with K–H buffer containing (in mmol/l): NaCl 118.5, NaHCO3 25.0, KCl 4.75, KH2PO4 1.18, MgSO4 1.19, D-glucose 11.0, and CaCl2 1.41 at a constant pressure of 80 mmHg (1080 mm H2O). The K–H buffer was pre-filtered using a 0.8-µM micro-filter (Whatman, UK) and constantly gassed with 95% O2/5% CO2.

2.3 Buffer perfusion
Following isolation the heart temperature was monitored continuously by a K-Type thermocouple, passed retrogradely through the pulmonary valve into the right ventricle, and attached to a C9001 Thermometer (Comark, UK). The temperature was maintained at 37.0±0.1 °C by immersing the heart and cannula in K–H buffer kept at 37.0 °C in a waterjacketed chamber. Contractile function was monitored by a fluid-filled balloon inserted into the left ventricle. The balloon was attached to a pressure transducer, which was coupled to a 4S Powerlab (AD Instruments, UK). The frequency response characteristics of the isovolumic fluid-filled balloons, coupling tubing and transducer were flat to at least 30 Hz. The balloon was gradually inflated until the end-diastolic pressure reached between 2 and 5 mmHg. Hearts were then paced at 580 bpm by a 0.075-mm silver wire (Advent, UK), placed through the right ventricular wall into the right ventricular apex. The wire and aortic cannula were attached to an SD9 stimulator (Grass Instruments, USA) delivering square wave pulses of 5 mS duration at 1 V amplitude.

2.4 Experimental protocol
After retrograde perfusion commenced, the hearts were stabilized for 30 min. For inclusion in the study all hearts had to fulfill the following criteria: coronary flow between 1.5 and 4.0 ml/min; heart rate >300 bpm (unpaced); left ventricular developed pressure (LVDP)>60 mmHg; time from thoracotomy to aortic cannulation <3 min; no persistent dysrhythmia (during 30 min stabilization). Hearts were then randomly assigned to two groups; (1) control (no ischaemic preconditioning (continued perfusion for 40 min after stabilization)); (2) ischaemic preconditioning (IP) (4x4 min ischaemia/6 min reperfusion). All hearts then underwent 45 min global ischaemia, by clamping the aortic inflow tubing, followed by 1.5 h reperfusion. Electrical pacing was stopped when contraction ceased during ischaemia and restarted 30 min into reperfusion or when spontaneous contraction returned.

2.5 Infarct size assessment
Following 1.5 h reperfusion, hearts were perfused for 1 min with 5 ml 1% (w/v) triphenyltetrazolium chloride (TTC) in phosphate buffer (Na2HPO4 45.1 mmol/l, NaH2PO4 3.3 mmol/l, pH 7.8). Hearts were then removed from the cannula and placed in an identical TTC solution at 37 °C for 10 min. The atria were then removed, and the hearts blotted dry, weighed and stored at –20 °C for up to 1 week. Hearts were then thawed, placed in 2.5% glutaraldehyde for 1 min and then set in 5% agarose solution. The agarose heart blocks were then sectioned from apex to base in 0.7-mm slices using a vibratome (Agar Scientific, UK). Following sectioning, slices were placed overnight in 10% formaldehyde at room temperature, before transferring into phosphate-buffered saline for a further 2 days at 4 °C. Sections were then compressed between Perspex plates (0.57 mm apart), and imaged using a TK-1280E digital camera (JVC). After magnification (x25), planimetry was carried out using image analysis software (NIH Image v1.61) and surface area transformed to volume by multiplication with tissue depth.

2.6 Subcellular protein fractionation
Hearts were homogenised in lysis buffer (20 mM Tris–HCl (pH 7.4), 100 mM NaF, 20 µM leupeptin, 120 µM pepstatin A, 200 µM PMSF, 10 µM E-64) (10 ml of buffer/g wet wt of tissue), using a Polytron homogeniser. Following brief centrifugation at 13 000 rpm to yield the cytosolic fraction, the pellet was resuspended in lysis buffer containing 0.1% Triton X-100. After incubation for 15 min at 4 °C, the suspension was spun again for 10 min at 13 000 rpm to yield a triton-extractable membrane fraction.

2.7 Western blot analysis
Samples were obtained from the whole mouse heart by homogenization in electrophoresis sample buffer (250 mM Tris–HCl, 4% SDS, 10% glycerol, and 2% β-mercaptoethanol, pH 6.8). Western blotting was then carried out, as described previously [19]. All total PKC antibodies were murine monoclonal antibodies (Transduction Laboratories, UK), which were detected using a peroxidase-conjugated rabbit anti-mouse IgG secondary antibody (Dako, Denmark).

2.8 Statistical analysis
Data for functional recovery at the end of reperfusion were analyzed by one-way ANOVA, followed where appropriate by the Tukey test for pair-wise comparisons. Linear regression was carried out using SigmaStat statistical package. Infarct size comparisons were by analysis of covariance (ANCOVA) with respect to total myocardial volume using an Excel plug-in (Ferris State University). A probability value of less than 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Preliminary studies
Preconditioning has been proposed to delay the onset and rate of necrosis during index ischaemia [3]. We chose 45 min as the duration of lethal ischaemia because in a standard inbred strain (T/O mice, B&K Universal, UK) infarction was significantly larger than after 30 min (26.3±3.7 vs. 8.0±2.7%, P<0.05). Since we were able to detect this difference, and preconditioning is thought to delay infarction by 10–15 min, we reasoned that preconditioning prior to 45 min ischaemia should cause a significant reduction in infarct size.

Yellon's group has previously shown that four cycles of 5 min ischaemia/5 min reperfusion preconditions the isolated mouse heart [20]. Initial experiments in our model showed that 5 min ischaemia caused a slight elevation in end diastolic pressure and predisposed hearts to dysrhythmias during subsequent reperfusions (results not shown). We therefore used 4x4 min ischaemia/6 min reperfusion, which caused no observable detrimental effects. Moreover, extending reperfusion to 2.5 h after this IP stimulus caused no perturbation in LVDP compared to aerobically perfused controls and infarction was undetectable by TTC staining (results not shown).

TTC, which is reduced from a yellow to a red pigment in living cells, was used to demarcate the infarct zone. Insufficient washout of dehydrogenases, NADH and other co-factors from the necrotic tissue may cause false-positive staining of dead cells [21]. In keeping with previous studies [22], pilot experiments showed that diminishing the duration of reperfusion to 1 h had no significant effect on TTC-determined infarct size (results not shown). Moreover, functional recovery plateaued between 1 and 1.5 h reperfusion, therefore 1.5 h was used as the duration of reperfusion in all subsequent studies.

3.2 Preconditioning studies in T/O mice
A total of 33 male T/O mice were randomized to either control (no preconditioning) or IP (4x4 min ischaemia/6 min reperfusion) groups. Four hearts were excluded during the stabilization period due to high aortic flows (two), low developed pressure (one) or persistent dysrhythmia (one). There were no significant differences in baseline parameters between groups (Table 1).


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  		Table 1 Morphometric characteristics and baseline parameters of isolated buffer-perfused hearts from T/O mice after the 30 min stabilization perioda

 
Preconditioning prior to the index ischaemia reduced infarct size (Fig. 1a) and improved functional recovery during subsequent reperfusion (recovery LVDP: 53.8±2.5 vs. 38.9±2.1%, P<0.001). Although the reduction in mean infarct size seen following preconditioning was significant, there was a large intragroup variability in individual infarct size (Fig. 1a). Fig. 1b shows that this variability can be explained by a variation in myocardial volume (IP group: r = 0.96, P<0.001). When analyzed (using analysis of covariance (ANCOVA)) with respect to total myocardial volume, the reduction in infarction in preconditioned hearts reached a higher level of significance (Fig. 1b, P<0.001).


Figure 1
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  		Fig. 1 Effect of ischaemic preconditioning (IP) on infarct size in buffer perfused hearts from T/O mice. (A) Mean data for infarct volume as a percentage of total heart volume in control (n = 15) and IP (n = 14) groups (*P<0.05). In the control group infarction was 26.3±3.7%, whereas infarction in IP group is 12.2±1.7%. (B) Infarct volume in control (bullet) and IP ({circ}) hearts expressed with respect to total heart volume (**P<0.001, by ANCOVA). Linear regression for control (r = 0.96) and IP (r = 0.92) groups both reached statistical significance (P<0.001).

 
3.3 Characterization of the genetically altered PKC-{varepsilon} mouse line
The genotypes of the genetically altered PKC-{varepsilon} mice were determined by PCR (as shown in Fig. 2B). Western blot analysis with mouse monoclonal antibodies (Transduction Laboratories) raised against the N-terminal portion of PKC was performed to confirm the absence of PKC-{varepsilon} protein in mice homozygous for a disruption within the PKC-{varepsilon} allele (Fig. 2A). Identical Western blots were obtained using a polyclonal antibody (Santa Cruz Biotech.) raised against a C-terminal peptide fragment of PKC-{varepsilon} (results not shown).


Figure 2
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  		Fig. 2 Analysis of protein kinase C (PKC) isoform expression in hearts from mice wild type (+/+), heterozygous (+/–), and homozygous (–/–) for a disruption within the pkc-{varepsilon} allele. (A) Hearts from (–/–) mice have no detectable expression of PKC-{varepsilon} protein, whereas hearts from (+/+) and (+/–) mice show similar levels of PKC-{varepsilon} protein expression. PKC-{varepsilon} genotype has no significant effect on expression of PKC-{delta} or PKC-{alpha}, two other PKC isoforms implicated in IP. Equal protein loading was confirmed by Coomassie staining of identically loaded gels. There was no difference in actin levels between groups. (B) Mouse genotypes were determined by PCR. (+/+) mice yield a fragment encoding 350 bp, whereas (–/–) mice generate a larger fragment 700 bp, and (+/–) mice produce both fragments.

 
We examined the expression of PKC-{alpha} and PKC-{delta} in genetically altered PKC-{varepsilon} mice, to determine whether other PKC isoforms were upregulated in the absence of PKC-{varepsilon}. Fig. 2 shows that PKC-{alpha} and PKC-{delta} levels were unaffected by genotype. It is possible that PKC translocation following preconditioning is altered in genetically altered PKC-{varepsilon} mice, since other PKC isoforms have a low affinity for the PKC-{varepsilon} RACK (RACK2), thus in the absence of PKC-{varepsilon} these isoforms may behave in a ‘PKC-{varepsilon}-like’ manner by binding to RACK2. In our model, however, the localisation of PKC-{alpha} and PKC-{delta} is unaltered following preconditioning in all PKC-{varepsilon} groups, as assessed by subcellular fractionation of soluble and triton-soluble proteins (results not shown).

3.4 Preconditioning studies in PKC-{varepsilon} knockout mice
The offspring of heterozygous (+/–)xknockout (–/–) breeding pairs were randomized to either control or IP groups. A total of 48 male mice entered the study, but five hearts were excluded during stabilization due to high aortic flows (two), low developed pressure (one) or persistent dysrhythmia (2). The genotypes were determined by PCR and confirmed by Western blot analysis (Fig. 2) after all contractility and infarction data had been recorded. There were no significant differences in baseline parameters between groups (Table 2). Moreover, the myocardial contractility was not significantly different between groups, either before, during, or following 45 min global no-flow ischaemia (results not shown).


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  		Table 2 Morphometric characteristics and baseline parameters of isolated buffer-perfused hearts from heterozygote (+/–) and PKC-{varepsilon} knockout (–/–) mice after the 30 min stabilization perioda

 
In heterozygous (+/–) mice, which contain the full complement of PKC-{varepsilon} protein (Fig. 2), preconditioning resulted in a significant reduction in infarct size (Fig. 3a, P<0.05), although again this reached higher significance when expressed against heart size (Fig. 3b, P<0.001). In knockout (–/–) hearts that lack the pkc-{varepsilon} gene and PKC-{varepsilon} protein (Fig. 2), there was no statistical difference between IP and controls hearts, even with respect to total myocardial volume (Fig. 4). The control level of infarction in heterozygous (+/–) and knockout (–/–) hearts was identical, as was the relationship between myocardial volume and infarct size in all hearts except preconditioned heterozygotes. Paradoxically, however, although there was no benefit of IP on infarct size in hearts from (–/–) mice, contractile recovery (i.e., % baseline LVDP) was preserved in this group to a level similar to that seen in preconditioned (+/–) hearts (Fig. 5).


Figure 3
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  		Fig. 3 The effect of ischaemic preconditioning (IP) in isolated buffer-perfused hearts from mice heterozygous (+/–) for a disruption within the pkc-{varepsilon} allele. (A) Mean data for infarct volume as a percentage of total heart volume in control (bullet, n = 10) and IP ({circ}, n = 12) groups from heterozygous mice (*P<0.05). In the control group infarction was 41.3±4.7%, whereas infarction in IP group is 24.3±4.5%. (B) Infarct volume in control (bullet) and IP ({circ}) hearts from (+/–) mice expressed against total heart volume. Preconditioning prior to ischaemia significantly reduced infarct volume compared to controls (**P<0.001 (ANCOVA)).

 

Figure 4
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  		Fig. 4 The effect of ischaemic preconditioning (IP) in isolated buffer-perfused hearts from mice homozygous (–/–) for a disruption within the pkc-{varepsilon} allele. (A) Mean data for infarct volume as a percentage of total heart volume in control (bullet, n = 8) and IP ({circ}, n = 13) groups from heterozygous mice. In the control group infarction was 38.8±4.5%, which did not differ significantly from the IP group whose infarction averaged 36.4±2.9%. (B) Infarct volume in control ({blacktriangleup}) and IP ({bigtriangleup}) hearts from (–/–) mice expressed against total heart volume. There was no statistical difference in infarct size between groups (P = 0.23).

 

Figure 5
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  		Fig. 5 The effect of ischaemic preconditioning on contractile recovery. Mean data for recovery in LVDP (% baseline) in all groups. IP significantly improved contractile recovery in hearts from both (+/–) and (–/–) mice (*P<0.05).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1 Ischaemic preconditioning in isolated buffer-perfused mouse hearts
To our knowledge, there have been only two previous reports of preconditioning in the isolated mouse heart [20,23]. We therefore characterized our own protocol for preconditioning with four cycles of 4 min ischaemia with 6 min of intervening reperfusions before 45 min global ischaemia. The endpoints used to assess myocardial injury were contractile recovery and infarct size, which were enhanced and attenuated, respectively, by IP. This is unlike the findings of Xi et al. who, in a similar model, showed that whilst preconditioning reduced infarct size there was no effect on post-ischaemic ventricular function [23].

In this model, infarction volume is related to heart volume and weight. Thus when heart volume is less than 80 mm3 (110 mg) we found little or no infarction after 45 min global ischaemia. Superficially, this seems surprising and counter-intuitive. However identical relationships between volume at risk of infarction and final infarction volume are documented in better characterized small animal models of myocardial infarction [24].

4.2 The role of PKC isoforms in preconditioning
The difficulty in measuring the activity of individual PKC isoforms has led to contradictory reports implicating different isoform(s) in IP. Using translocation as a surrogate for activation, it has been suggested that {alpha}, {delta}, {varepsilon} and {eta} are all activated following a preconditioning stimulus [25,26]. Until recently this was simply associative data, but the design of isoform specific peptides that inhibit PKC translocation has provided the strongest evidence in favor of PKC-{varepsilon} in early ischaemic preconditioning. These studies, in isolated neonatal cardiac myocytes [14] and adult cardiomyocytes [15], show that preconditioning requires the translocation of PKC-{varepsilon} for protection against simulated ischaemia. Moreover, peptides that function by promoting the translocation of PKC-{varepsilon} have also been shown to protect both neonatal cardiac myocytes and the mouse heart against ischaemia [27]. Furthermore, Ping et al. have showed that overexpression of pkc-{varepsilon} protected adult cardiomyocytes against simulated ischaemia [28]. The primary concern in all these studies is the inability to accurately assess isoform activation. This is because isoform-specific substrates are currently unknown and differing antibody affinities, isotype abundance and in vitro activation complicates the interpretation of PKC activity after immunoprecipitation of individual isoforms. The requirement for some measure of activity is highlighted in the activator/inhibitor peptide studies, since these selective strategies rely on the premise that PKC translocation is synonymous with activation. This premise may not be correct with the possibility of non-translocation dependent activation [29], and even translocation dependent inhibition [30]. Furthermore, other uncertainties exist since we [16] and others [17] have suggested that specific activation of PKC-{delta} in rat myocytes or whole heart protects against simulated and true ischaemia, respectively. In fact, the downstream signaling pathways activated by PKC-{delta} and -{varepsilon}, which are thought to lead to protection, have many similarities [19,31]. For these reasons we sought to examine IP in hearts from mice deficient in pkc-{varepsilon}.

4.3 Ischaemic preconditioning in isolated hearts from pkc-{varepsilon} knockout mice
For controls, we chose to use mice heterozygous (+/–) for a disrupted pkc-{varepsilon} allele, rather than wild type (+/+) mice. This decision was reached after ensuring that mice with these genotypes had a similar complement of PKC-{varepsilon} within their hearts (Fig. 2). We also chose to use male mice throughout the study since female hormones such as oestrogen have been shown to confound data interpretations by altering sensitivity to ischaemia in the mouse [32]. Thus, all male offspring of (+/–) to (–/–) matings were randomized before genotyping, which reduced colony maintenance costs and eliminated bias since genotypes were not determined until all physiological data had been collected. A further advantage of this approach is that mice share the same genetic background, thus reducing the effect of modifier genes and thereby increasing the probability that the effects observed are due to the gene of interest. Moreover, hearts from mice heterozygous for a disruption within the pkc-{varepsilon} allele retain the ability to precondition (Fig. 3). We have shown that protection is present in heterozygous (+/–) mice, whereas the knockout (–/–) littermates lacking PKC-{varepsilon} show no reduction in infarct size following IP. This demonstrates that preconditioning, at least in the mouse, protects via a signaling pathway that involves the specific activation of PKC-{varepsilon}.

A major concern of knockout technologies is the possibility that closely related genes may be developmentally upregulated to counteract the knockout phenotype. In this respect, although no significant changes in PKC{alpha} or PKC{delta} protein levels could be detected, it is still possible that these isoforms may counteract the knockout effect by binding to and activating PKC-{varepsilon}-specific substrates. However, the inability to precondition knockout mice argues against this hypothesis. Furthermore subcellular fractionation on heart samples (into soluble and triton-soluble protein fractions) following preconditioning in heterozygous and control mice demonstrate that the localisation of PKC-{alpha} and PKC-{delta} is unaffected by genotype (results not shown).

4.4 The dichotomy of contractile recovery and infarct size
In hearts lacking PKC-{varepsilon}, although infarct size is unaltered by IP, contractile recovery is still preserved. We are not able to provide a definite explanation for this dichotomy. However, one can speculate that it is related to the mutually exclusive processes of stunning and infarction. For example, antioxidants given at the onset of reperfusion have been shown to improve contractile recovery with no effect of necrosis. Puett et al. [33] showed in dogs that oxypurinol, which inhibits xanthine oxidase and decreases free radical production, improved regional ventricular function when given during reperfusion following 60 min ischaemia, but failed to reduce infarct size. The authors suggested oxygen radicals contribute to stunning of reversibly damaged myocardium but not to the final extent of necrosis. Ischaemic preconditioning has also been shown to reduce oxygen radical production during reperfusion following ischaemia [34], it is therefore possible that this reduction in oxidant stress and consequent stunning is independent of PKC-{varepsilon} activation in the mouse heart. This would explain the improvement in functional recovery, but not infarction, in PKC-{varepsilon} deficient hearts following IP. This would also agree with recent work from Downey's laboratory which demonstrates that the preservation of myocardial function in the rat is likely to result from a decreased purine release following preconditioning which diminishes free radical generation by xanthine oxidase [35]. These data therefore reinforce the importance of using multiple endpoints in studies designed to infer the mechanism(s) of early ischaemic preconditioning.

4.5 How does PKC-{varepsilon} protect?
Although PKC-{varepsilon} is critical for protection in the mouse heart, it is unknown whether this is because PKC-{varepsilon} is the only isoform activated by preconditioning or whether PKC-{varepsilon} is the only isoform capable of protecting. Future studies designed to assess whether the non-isoform selective PKC activator PMA can protect PKC-{varepsilon} (–/–) hearts will help to address this question. A proteomic approach has already been adopted in an attempt to discern candidate substrates using hearts from mice expressing constitutively active PKC-{varepsilon} [36]. The PKC-{varepsilon} knockout model described within these studies, and the inhibitory and activating RACK peptides described by others [37], should provide valuable tools for future proteomic studies designed to reveal the downstream substrate(s) of PKC-{varepsilon} following ischaemic preconditioning.

Time for primary review 28 days.


   Acknowledgements
 
This work was supported by the Wellcome Trust project grant no. 005696 and the British Heart Foundation.


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

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S. J. Clarke, I. Khaliulin, M. Das, J. E. Parker, K. J. Heesom, and A. P. Halestrap
Inhibition of Mitochondrial Permeability Transition Pore Opening by Ischemic Preconditioning Is Probably Mediated by Reduction of Oxidative Stress Rather Than Mitochondrial Protein Phosphorylation
Circ. Res., May 9, 2008; 102(9): 1082 - 1090.
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