INTRODUCTION

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Ischemic preconditioning describes the resistance to myocardial infarction that follows short, sublethal episodes of ischemia (1). The protection associated with ischemic preconditioning is profound but, unfortunately, short lived (2) and prone to tachyphylaxis (3). These features have cast doubts on the eventual clinical utility of this powerful protective phenomenon (4). For this and other reasons intense research activities and interests have focused upon the trigger(s), mediator(s), and final effector(s) of this phenomenon. The eventual aim is specific manipulation of the signaling pathway to achieve durable protection without an ischemic trigger (5).

The most popular hypothesis first proposed by Cohen and Downey (5) views the pathway leading to preconditioning as follows. The brief ischemic trigger causes the release of endogenous mediators such as adenosine that bind in a retaliatory fashion to cognate heptahelical transmembrane receptors (6, 7). When bound to agonist, these receptors activate phospholipase C() through a G_qGTP protein-mediated mechanism (6). Phospholipase C, in turn, mediates the hydrolysis of phosphatidylinositol 4,5-biphosphate producing the dual second messengers of diacylglycerol and inositol 1,4,5-trisphosphate (5, 6). The diacylglycerol binds to the regulatory domain of protein kinase C causing activation (6). Protection then occurs by protein kinase C-mediated phosphorylation of serine/threonine residues within a specific amino acid consensus sequence on a target protein (6). Evidence suggests that this target protein may be the ATP-sensitive potassium channel K_ATP (8, 9).

Although very attractive, the Downey hypothesis has come under attack. There is general agreement that adenosine, or a similar heptahelical transmembrane receptor agonist, is the trigger for preconditioning. However, the role of PKC¹ as the mediator of preconditioning is far more controversial (4, 9-11). This is in part related to the complexities of PKC biology and pharmacology.

Protein kinase C describes an ever expanding family of enzymes of related structure and function (6). These proteins have a carboxyl-terminal catalytic domain linked through a hinged region to an amino-terminal regulatory domain. Within the regulatory domain lies a pseudosubstrate subdomain that binds to the catalytic site preventing substrate phosphorylation by steric interference (6, 12). On activation a conformational change occurs within the enzyme to expose the catalytic site and allow substrate binding.

Despite increasing knowledge of how the functional domains interact to cause and regulate PKC activity, the measurement and pharmacological manipulation of this family of enzymes remain complex with multiple pitfalls (6, 13). Activators may have a bimodal effect first activating and then down-regulating PKC and its activity, whereas inhibitors may interfere with other kinase pathways. Unfortunately, the most specific inhibitors, peptide fragments of pseudosubstrate domains, are difficult to use in vivo and require sarcolemmal permeabilization to use in vitro (14).

The studies of PKC in preconditioning thus far have used pharmacological manipulation of PKC activity to mimic or block preconditioning and measurement of activity or translocation to infer involvement, but seldom both in a single study (13). The conclusions of these studies are difficult to reconcile because some investigators (13, 14-16) report PKC activation is both a sufficient and necessary component of the ischemic preconditioning pathway, and others (10, 11, 13) suggest there is no involvement. The reason for this dichotomy is thought to lie in the complexities and uncertainties of pharmacological manipulation of the PKC pathway (6, 12, 13). This issue is of critical importance if preconditioning is to be mimicked and exploited by specific manipulation of its signaling pathway.

Recently PKC isotypes have become available that have been rendered constitutively active by limited amino-terminal deletions within the pseudosubstrate domain (17, 18). These alterations are thought to prevent, or decrease the probability of, interactions with the catalytic site and thus reduce the potential for steric interference of substrate phosphorylation (6). Cells have been transfected with expression plasmids encoding these mutationally active PKC isotypes to specifically interrogate the PKC pathway and determine the cross-talk between this and other possible parallel signal transduction pathways (17). In addition, downstream events are preserved because there is transcriptional activation of an atrial naturetic factor promoter/reporter construct in cardiac myocytes expressing constitutively active PKC isotypes (18), an observation that suggests they, like more physiological stimuli, are capable of triggering a hypertrophic response (18). Our aim was to use these mutant constitutively active PKC isotypes to investigate ischemic preconditioning.

We have developed a system of simulated ischemia in cultured neonatal rat ventricular myocytes that shares many features of ischemic preconditioning in vivo and is similar to a previously characterized model (19). By using this system we demonstrate that -gal can be used as a marker of viability in transfected myocytes exposed to simulated ischemia. By co-transfecting expression vectors for -gal and mutant constitutively active PKC isotypes, we have examined resistance to simulated ischemia. We believe that this is the first report of the use of genetic manipulation to investigate preconditioning.

	EXPERIMENTAL PROCEDURES

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Materials-- Dulbecco's modified Eagle's medium, medium 199 (M199), bovine serum albumin, horse serum, fetal calf serum (FCS), pancreatin, and penicillin/streptomycin were from Life Technologies, Inc. (Paisley, UK). Collagenase was from Worthington. Trypan blue, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and benzamidine were from Sigma-Aldrich (Gillingham, UK). [-³²P]ATP was from Amersham Corp (Little Chalfont, UK). HEPES and the neutral phospholipid dioleoyl L--phosphatidylethanolamine (DOPE) were from Sigma (Poole, UK). Cationic amphiphile 3[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol) was synthesized as described previously (20). DE52 was from Waterman (Maidstone, UK). Monoclonal isoform-specific anti-PKC antibodies were from Affinity Research Products Ltd. (Exeter, UK). Peroxidase-conjugated rabbit anti-mouse immunoglobulins were from Dako (High Wycombe, UK).

Isolation and Culture of Rat Ventricular Cardiomyocytes-- Neonatal rat ventricular cardiomyocytes were prepared from 1- to 2-day-old Sprague-Dawley rats as described previously (21). Briefly, cells from neonatal rat ventricles were dispersed in a series of incubations at 37 °C in HEPES-buffered salt solution containing 0.6 mg/ml pancreatin and 0.5 mg/ml collagenase. The dispersed cells were preplated for at least 30 min to minimize fibroblast contamination, and the unattached cells were re-plated on 6-well gelatin-coated plates at a density of 1-1.5 million cells/well. Fibroblast contamination was less than 5%. The cardiac myocytes were cultured at 37 °C, in room air with 5% CO₂ in 4:1, Dulbecco's modified Eagle's medium:M199, supplemented with 10% horse serum, 5% FCS, and 100 units/ml penicillin/streptomycin for the first 24 h. Thereafter cells were maintained in an identical medium with a reduced serum concentration of 1% FCS. Under these conditions, in excess of 80% of cells beat spontaneously for the duration of the experiment. Experiments were performed after 1-3 days in culture.

Expression Vectors for Transfection of Neonatal Cardiomyocytes-- The high efficiency eukaryotic expression plasmid, pCAGGS, was used for all transfections (22). This plasmid contains the cytomegalovirus immediate early enhancer and chicken -actin promoter with first intron upstream of a multiple cloning site. It has been shown previously that this heterologous promoter is transcriptionally active in cardiac myocytes (23).

Transfection of Neonatal Cardiomyocytes-- Cardiocytes at 60-70% confluency were transfected by incubation with complexes of DNA with DC-Chol/DOPE cationic liposomes. These liposomes were prepared in the following way. DC-Chol (12 µmol) and a chloroform solution of DOPE (8 µmol in 0.6 ml) were combined in dichloromethane (10 ml) under nitrogen and then diluted with sterile 20 mM HEPES buffer, pH 7.8. The two-phase system under nitrogen was sonicated for 3 min at ambient temperature. Organic solvent was then removed under reduced pressure, and the resulting liposomes were sonicated for a further 3 min prior to use. The liposome-DNA complexes were prepared by mixing DNA (0.1% w/v) with liposome (0.1% w/v) in H₂O at a ratio of 1:3 (w/w). The liposome-DNA complexes were allowed to stand at room temperature for 15-30 min before use. One ml of serum-free maintenance medium with 20-40 µl of DNA/liposome mixture was added per well of a 6-well plate. Cells were then incubated at 37 °C in room air supplemented with 5% CO₂ for 1 h. Thereafter maintenance medium containing 1% FCS was gently overlaid, and the cells were returned to the incubator. Cell extracts for gene activity were assayed 48-72 h after transfection. By using pCAGGS--gal as a reporter, transfection efficiency was consistently between 5 and 12%, staining with the chromogenic substrate 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside.

Ischemia Model-- The cells were washed three times with PBS before addition of 1 ml of ischemia buffer (118 mM NaCl, 24 mM NaHCO₃, 1 mM NaH₂PO₄·H₂O, 2.5 mM CaCl₂·2H₂O, 1.2 mM MgCl₂, 0.5 mM sodium·EDTA·2H₂O, 20 mM sodium lactate, and 16 mM KCl, pH 6.2) and pre-gassed with 5% CO₂, 95% argon. On addition of ischemia buffer spontaneous contraction within the monolayer ceased. The cells were then transferred to a purpose-built ischemia chamber and incubated at 37 °C in 5% CO₂, 95% argon for up to 12 h. The O₂ content of the atmosphere inside the chamber was <1% for the duration of the experiment as measured by an on-line oxygen meter (Griffin and George, Fife, UK).

Co-culture Model-- Neonatal cardiomyocytes were prepared and cultured as described above either on standard 6-well plates or within a cell culture insert (Falcon, Oxford, UK). The inserts, designed to fit within the wells of a 6-well plate, were perforated with 1.0-µm pores allowing free communication of media between insert and well. The cells within the insert and the well were transfected and cultured apart and only combined immediately before simulated ischemia. To ensure that both cell monolayers were bathed in buffer, volumes had to be increased to 2 ml during simulated ischemia.

Evaluation of Cell Viability-- After the cells had been subjected to simulated ischemia, remaining ischemia buffer was gently aspirated and saved for LDH determination, and cells were detached by washing in PBS with trypsin. Following centrifugation a small aliquot of cells was incubated in PBS with 0.4% trypan blue for a few seconds before placing in a hemocytometer for counting under phase contrast. Dead cells, permeable to trypan blue, were counted as a percentage of the total number of cells in at least 25 grids of the hemocytometer so that at least 200 cells were counted in total. Examination of the initial ischemia buffer revealed few detached cells.

Measurement of LDH Activity-- On opening the ischemia chamber (re-oxygenation) 200-µl samples of the ischemia buffer were gently collected for the determination of LDH. The following day a spectrophotometric LDH enzyme assay was performed with a Sigma assay kit (TOX-7).

Western Blotting Analysis-- 48-72 h after transfection the cells were washed three times with PBS and harvested in 1 ml of hot electrophoresis sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, and 2% -mercaptoethanol) and then boiled for an additional 5 min. The cell extracts were then centrifuged for 5 min to remove insoluble material. The protein concentration of the diluted supernatant was determined by the Pierce method (Pierce) before adding 0.003% bromphenol blue. The samples were then loaded on a 10% polyacrylamide gel and after one-dimensional separation were transferred to nitrocellulose membranes (Hybond C, Amersham, UK). Uniform protein loading was confirmed by Coomassie staining of identically loaded gels. The blots were probed sequentially with murine monoclonal antibodies specific for PKC- or PKC- and a peroxidase-conjugated rabbit anti-mouse IgG secondary antibody prior to detection with enhanced chemiluminescence (ECL, Little Chalfont, UK).

Measurement of -Galactosidase Activity-- -Galactosidase activity was determined in the cell lysates (lysis buffer, 100 mM KH₂PO₄, 0.2% Triton X-100, 1 mM dithiothreitol, pH 7.8) using a Galacto-Light^TM assay kit (BL 330G, Insight Biotechnology Ltd, London, UK). A -gal standard was used to show that light output within the experimental range was directly proportional to -gal concentration. -Gal activity, expressed per mg of protein, was measured in cells harvested post-simulated ischemia (see evaluation of cell viability) and expressed as a percentage of activity in wells, transfected with the same expression plasmid but lacking insert (empty vector), harvested in the same manner, and subjected to simultaneous simulated ischemia.

Partial Purification of PKC-- The cells were washed with PBS and lysed in homogenization buffer (0.05 M Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM EGTA, 0.3% Triton X-100, 10 mM dithiothreitol, 10 mM benzamidine, 10 µg/ml leupeptin, 1 µg/ml pepstatin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The cells were then sonicated and centrifuged. The supernatant was collected and incubated with 0.5 ml of DEAE-cellulose (DE52) for 60 min at 4 °C with occasional gentle agitation. DE52 had been pre-equilibrated with 200 mM Tris, pH 7.5, and washed in buffer A at 4 °C (20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, and 1 mM dithiothreitol). After incubation, the lysate/DE52 slurry was loaded on a column. The column was then washed with 5 ml of buffer A and PKC eluted with 0.3 M NaCl. The first 4 ml of eluate was collected for PKC determination (24). After concentrating the fractions using Centricon^TM concentrators (Gelman Sciences, Northampton, UK), PKC activity was measured immediately or preserved in 50% glycerol and stored at 70 °C overnight for measurement the following day.

PKC Activity Assay-- PKC activity was determined using an Amersham kit (RPN-77). Measurement of enzyme activity was based on PKC-mediated transfer of the [-³²P]ATP to a synthetic peptide homologous to the PKC- pseudosubstrate domain in the presence of cofactors (i.e. calcium acetate, L--phosphatidyl-L-serine, and phorbol 12-myristate 13-acetate). The method has been optimized to exhibit maximum PKC activity.

Statistical Analysis-- All values are expressed as mean ±S.E. The cardiocytes within each well were used for three determinations, the mean of which was used as the value for that particular well. After each period of simulated ischemia, data were collected from no more than two wells for each experimental group. The "n" numbers under "Results" relate to the number of wells from which data were obtained. For each intervention mean values were pooled to allow statistical comparisons. Statistical comparisons between groups at a single time point were performed by one-way analysis of variance (ANOVA) and comparisons between the 2- and 12-h time points by two-way ANOVA. All post-hoc comparisons were by the Fischer protected least significant difference method (FPLSD). All analyses were performed using the Statview version 4.0 statistical package (Abacus Concepts Inc., Berkeley, CA). A probability value 0.05 was considered significant.

	RESULTS

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The Simulation of Ischemia-- To mimic the changes in extracellular ions occurring during ischemia in vivo, we used a bicarbonate-based buffer at pH 6.2 with 20 mM lactate, 16 mM K⁺, and no metabolic substrate (O₂ <1%), at a volume of 1 ml, just sufficient to wet the cellular monolayer. By using this technique we exposed neonatal cardiocytes to simulated ischemia of increasing duration. The rate and extent of ischemic injury were determined by an assessment of trypan blue permeability and LDH release. Fig. 1, panel A, shows that the percentage of dead cells (as assessed by trypan blue) increased in a time-dependent and reproducible manner. Similar results were obtained for LDH release (see Fig. 1, panel B).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Alterations in trypan blue permeability (A), supernatant LDH activity (B), and cellular -galactosidase activity (C) with increasing durations of simulated ischemia. Ischemia was simulated by low volumes of acidic buffer lacking substrate in an atmosphere of 95% argon, 5% CO2 at 37 °C (see "Experimental Procedures"). Panel A, percentage cell death is defined as the percentage of cells permeable to trypan blue expressed as a percentage of the total number of myocytes. Panel B, LDH activity was expressed as a percentage of activity within the supernatant after 2 h of simulated ischemia. Panel C, -gal lacking a nuclear localization sequence was introduced by liposome-mediated transfection of the expression plasmid pCAGGS--gal 2-3 days prior to simulated ischemia. After simulated ischemia -gal activity was measured using the Galactolight kit and expressed as a percentage of -gal activity at base line prior to ischemia. Each data point is the mean of values obtained from four separate experiments ± S.E. At certain time points the standard errors lie within the symbols.

Preconditioning with Simulated Ischemia and the Role of PKC-- Preconditioning with ischemia in vivo or simulated ischemia in vitro has been shown to delay necrosis/cell death in response to subsequent ischemia. We wished to investigate if similar protection occurred in our model. Cardiocytes were subjected to varying durations of simulated ischemia before returning cultures to maintenance medium in a standard incubator (room air supplemented with 5% CO₂) for 30 min. After this recovery phase cultures were exposed once again to ischemia buffer in an atmosphere of 95% argon, 5% CO₂ for periods of 6 h. These preliminary experiments demonstrated that 90 min of simulated ischemia was not a sufficiently severe stress to cause cell death but would consistently reduce injury after 6 h of simulated ischemia.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   The time course of cell death in rat neonatal cardiocytes subjected to increasing durations of simulated ischemia with and without preconditioning. Cardiocytes were preconditioned by a 90-min exposure to hypoxic ischemia buffer (see "Experimental Procedures") followed by 30 min under normal culture conditions. Percentage cell death is defined as the percentage of cells permeable to trypan blue expressed as a percentage of the total number of myocytes. Values are means ± S.E. for four separate experiments. ** p < 0.01 preconditioning versus control.

View this table: [in this window] [in a new window]   Table I The effect of preconditioning with and without PKC inhibitors on the viability of neonatal cardiocytes 6 h after simulated ischemia LDH release and -galactosidase activity in the preconditioned cells were expressed as a percentage of the value in paired (non-preconditioned) controls. Percentage cell death is defined as the percentage of cells permeable to trypan blue expressed as a percentage of the total number of myocytes. Values are mean ± S.E.

View this table: [in this window] [in a new window]   Table II The effect of PKC inhibitors on the viability of non-preconditioned neonatal cardiocytes 6 h after simulated ischemia LDH release and -galactosidase activity in the cells with inhibitors were expressed as a percentage of the value in paired (without inhibitors) controls. Percentage cell death is defined as the percentage of cells permeable to trypan blue expressed as a percentage of the total number of myocytes. Values are mean ± SE.

Expression and Activity of PKC Following Transfection-- A high efficiency eukaryotic expression plasmid was used to introduce and drive recombinant PKC expression in myocyte cultures. Transfection was achieved with cationic liposome-DNA complexes as described above. With this technique, 48-72 h post-transfection, we noticed appreciable increases in PKC immunoreactivity within protein samples derived from the whole monolayer despite transfection efficiencies of only 5-12% as assessed by staining with a chromogenic substrate (see Fig. 3). In untransfected cells PKC- (72 kDa) and PKC- (84 kDa) immunoreactivities were weak. Transfection with PKC- deletion or PKC- wild type resulted in a similar accumulation of protein (see Fig. 3).

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Immunoreactivity of PKC- (A) and PKC- (B) in samples prepared 48 h after transfection of neonatal cardiocyte cultures. Panel A, Western blot probed with anti-PKC-. Panel B, Western blot probed with anti-PKC-. In both panels 15 µg of protein prepared from neonatal cardiocytes was loaded per lane. The lanes were prepared from monolayers treated as follows. Panel A, untransfected cardiocytes, cardiocytes transfected with pCAGGS alone or encoding PKC- with a 10-amino acid deletion within the pseudosubstrate domain or wild type PKC-. Panel B, cardiocytes transfected with pCAGGS encoding PKC- with a 7-amino acid deletion, pCAGGS without an insert, untransfected cardiocytes, and a positive control consisting of 5 µg of protein from a human fibroblast cells line. Arrows correspond to the approximate molecular weights of the dominant bands derived from molecular weight markers.

The Effect of PKC Overexpression on Resistance to Simulated Ischemia-- To determine if overexpression of PKC isotypes could mediate preconditioning, we co-transfected cells with the eukaryotic expression plasmid pCAGGS encoding either wild type PKC-, PKC- deletion, PKC- deletion, or vector alone (no construct within multiple cloning site) together with pCAGGS encoding -gal (see "Experimental Procedures"). In order to ensure that neither -galactosidase expression nor the transfection procedure altered monolayer resistance to simulated ischemia, each of these components was examined in isolation (see Table IV). The rationale behind the co-transfection approach was that viability in the transfected subpopulation of cells could be measured using -gal activity as demonstrated above. This relies on the widely accepted finding that cells taking up one plasmid will also take up the others available (26). This assumption forms the basis of a variety of investigative techniques involving plasmid co-transfection (27). These experiments were performed in a blinded fashion the operator only being aware of the assignment of pCAGGS--gal and pCAGGS encoding wild type PKC-.

View this table: [in this window] [in a new window]   Table IV The effect of staurosporine on the viability after 6 h of simulated ischemia of neonatal cardiocytes transfected with constitutively active PKC- LDH release and -galactosidase activity are expressed as a percentage of activity in wells transfected with the expression vector (cAGGS) lacking insert. Percentage cell death is defined as the percentage of cells permeable to trypan blue expressed as a percentage of the total number of myocytes. Values are mean ± S.E.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   The effect of expression of recombinant PKC isotypes on cell survival after 6 h of simulated ischemia. Panel A, shows -gal activity following ischemia expressed as a percentage of activity in the wells transfected with the same vector without insert. Panel B is percent cell death calculated on the basis of the number of cells permeable to trypan blue as a percentage of the total number of cells counted. Panel C is LDH activity within the supernatant expressed as a percentage of activity in cells transfected with vector alone (pCAGGS). All plates were co-transfected with pCAGGS--gal and pCAGGS encoding the PKC isotype indicated on the abscissa or with an empty multiple cloning site (empty vector). Values are means ± S.E. All p values are for comparisons between transfections with a PKC isotype and the empty vector. **p < 0.01. All comparisons by one-way ANOVA. Post hoc comparisons were by FPLSD.

Investigation of the Bystander Effect-- To investigate further the possibility of a bystander effect, we co-cultured cardiocytes transfected with different plasmids. The cardiocyte monolayer within the wells of a standard 6-well plate were transfected with pCAGGS encoding -gal alone. The cardiocyte monolayers within an insertable well were co-transfected with pCAGGS encoding -gal and pCAGGS encoding either the PKC- deletion, PKC- wild type, or lacking any insert. These separately transfected monolayers were only combined in a single tissue culture well immediately before adding ischemia buffer and entering the ischemia chamber. After 6 h of simulated ischemia, cell viability in each monolayer was assessed separately. Fig. 5 shows viability of the monolayer transfected only with pCAGGS--gal. Despite the larger volume of ischemia buffer increasing dilution, the plasmid used to transfect the upper monolayer influenced the viability of the lower monolayer. The results of these experiments suggest that during ischemia a diffusable factor is released from cells expressing the PKC- deletion mutant that is capable of acting upon and protecting untransfected cells or cells expressing -gal alone.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Survival after 6 h of simulated ischemia of cells transfected with -gal alone but co-cultured with myocytes transfected with plasmids encoding PKC isotypes. Cellular monolayers were transfected separately and combined in a single tissue culture well immediately before simulated ischemia. Cardiocytes transfected with expression plasmid encoding -gal alone were cultured below cells transfected with the plasmids described on the abscissa. All p values are for comparisons between transfections with a vector encoding a PKC isotype and empty vector. *p  0.05; **p < 0.01. All comparisons by one-way ANOVA. Post hoc comparisons wereby FPLSD.

The Relationship between PKC Activation during Simulated Ischemia and Protection-- Protection in our model may be related to alterations in gene expression induced by PKC rather than directly to PKC pre-activation during prolonged simulated ischemia. This is because in classical preconditioning there is a short period of reperfusion between the preconditioning trigger and prolonged ischemia, while in our model there is 2 to 3 days between transfection and prolonged simulated ischemia. In an attempt to overcome these uncertainties, we examined whether protection was altered in cells transfected with constitutively active PKC- when a PKC inhibitor was present during prolonged simulated ischemia.

	DISCUSSION

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Our findings suggest that activation of PKC- consistently enhances the resistance of isolated neonatal rat cardiocytes to simulated ischemia. This finding is compatible with the observational data demonstrating PKC- translocation following brief periods of ischemia in the rat heart (16, 28, 29). However, the most surprising finding is the bystander protection conferred by the expression of constitutively active PKC-.

Classical Ischemic Preconditioning Versus Protection by Expression of Mutant PKC Isotypes-- To achieve our research objectives, we had to adopt and characterize an unorthodox model of ischemic preconditioning. Ischemic preconditioning in the strictest sense refers to brief episodes of ischemia reducing subsequent infarction in vivo (1). Although models of ischemic preconditioning in isolated adult myocytes are established and accepted, these cells are relatively resistant to transfection and are difficult to maintain in culture. In addition most models rely upon an ischemic cell pellet and hypo-osmotic shock (30), techniques best suited to freshly isolated, unplated cardiocytes. Thus far there have been two reports of ischemic preconditioning of immature cardiocytes in a similar model (19, 31). These reports, together with our observations, suggest the model shares many of the features of ischemic preconditioning. In particular it retains the ability to be preconditioned both with ischemia and a pharmacological agent. Moreover the fact that overexpression of wild type PKC- does not result in protection reinforces pharmacological studies that suggest activation/translocation of PKC must precede the prolonged period of ischemia (5).

Protection with Constitutively Active Isoforms of PKC-- Although transfection efficiency in neonatal cardiocytes was low, there was appreciable PKC accumulation on immunoblots and an increased activity of "maximally activable" phosphorylation of a PKC substrate. Transfection with wild type PKC- cDNA caused similar levels of recombinant protein expression as transfection with PKC- differed only in a limited deletion of 10 amino acids within the pseudosubstrate domain. However, protection was only seen with the deletion mutant. In addition, although there was robust accumulation of a similarly basally activated PKC-, this failed to cause consistent protection and a bystander effect was absent. The use of these structurally related proteins, differing only in a limited deletion within a single subdomain, suggests their effects on myocyte resistance to ischemia are specific and that PKC- activation causes cytoprotection in this model. Although activation of PKC- is sufficient to cause protection, it is unclear, despite the PKC inhibitor experiments we performed, whether PKC- activation is an absolute prerequisite for protection. In view of the bystander effect, to answer this question it would presumably be necessary to inhibit endogenous PKC- in greater than 95% of myocytes. Although this is possible with pharmacological inhibitors, these are nonspecific. Recently an alternative method using short peptide sequences to saturate isotype-specific receptors for activated PKCs has demonstrated the dependence of preconditioning on endogenous PKC- (19). In addition the similarities between the model used in this previous study (19) and that described here allow direct comparison of findings. Taken together they imply that PKC- is sufficient but not necessary to elicit preconditioning in isolated neonatal rat cardiocytes.

Study Limitations-- The principal limitation of the study is the surrogate nature of the model and study design. As already outlined, the purpose of the study was to address the PKC hypothesis by specific manipulation of the activity of a single PKC isotype. This was most easily achieved in cultured cardiocytes. With this in vitro system we attempted as closely as possible to mimic changes occurring during ischemia in vivo. However, the differences between true ischemia and simulated ischemia and between in vivo and in vitro end points of injury questionably reduce the relevance of our findings.

Conclusion-- In cultured rat neonatal cardiocytes it is possible to increase resistance to a prolonged period of simulated ischemia by prior exposure to a brief period of simulated ischemia. This protection can be blocked by two structurally diverse PKC inhibitors and mimicked by transfection and expression of PKC- rendered basally active by a limited deletion within the pseudosubstrate domain. The depth of protection afforded by this strategy exceeds that expected on the basis of the efficiency of transfection. This observation is supported by co-culture experiments which indicate that transfected cardiocytes are capable of protecting neighboring untransfected cells by the release of an extracellular signal during simulated ischemia.