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Cardiovascular Research 2001 50(1):65-74; doi:10.1016/S0008-6363(00)00322-9
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through {epsilon} protein kinase C

Katrina Mackay and Daria Mochly-Rosen*

Department of Molecular Pharmacology, Stanford University School of Medicine, 269 Campus Drive, CCSR 3145, Stanford, CA 94305-5332, USA

* Corresponding author. Tel.: +1-650-725-7720; fax: +1-650-725-2952 mochly{at}stanford.edu

Received 29 June 2000; accepted 12 December 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Arachidonic acid is a second messenger which activates protein kinase C (PKC) and is released from the heart during ischaemic preconditioning. The purpose of this study was to examine the effect of arachidonic acid on activation of PKC in cardiac myocytes and the cellular consequences. Methods: Neonatal rat cardiac myocytes were isolated and maintained in culture. Arachidonic acid-induced activation of PKC was examined by cell fractionation and western blot analysis. Contraction frequency was measured by visual inspection under a microscope. Ischaemia was simulated by subjecting cells to an atmosphere of lower than 0.5% oxygen in the absence of glucose and cell damage determined by release of cytosolic lactate dehydrogenase or direct cell viability assay. Results: Arachidonic acid resulted in translocation of {delta} and {epsilon}PKC but not {alpha}, βII, {eta} or {zeta}PKC isozymes, indicating activation of only {delta} and {epsilon}PKC. Arachidonic acid induced a dose-dependent decrease in spontaneous contraction rate of cardiac myocytes which was blocked by a selective peptide translocation inhibitor of {epsilon}PKC. Pretreatment with arachidonic acid partially protected cardiac myocytes against ischaemia. Down-regulation of PKC with 24 h 4β-phorbol,12-myristate,13-acetate treatment, inhibition of PKC by chelerythrine and selective inhibition of {epsilon}PKC translocation all decreased the protective effect of arachidonic acid. Pretreatment with eicosapentaenoic acid or oleic acid also protected cardiac myocytes against ischaemia. Conclusions: These results demonstrate that arachidonic acid selectively activates {delta} and {epsilon}PKC in neonatal rat cardiac myocytes, leading to protection from ischaemia. We suggest this is a potential mechanism of PKC activation during PC. In addition, our results suggest that different classes of free fatty acid directly exert cardioprotection from ischaemic injury in cardiac myocytes.

KEYWORDS AA, arachidonic acid (20:4n-6); EPA, eicosapentaenoic acid (20:5n-3); FFA, free fatty acid; LDH, lactate dehydrogenase; OA, oleic acid (18:1n-9); PC, preconditioning; PKC, protein kinase C; PMA, phorbol,12-myristate,13-acetate


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Subjecting the heart to brief periods of sub-lethal ischaemia, termed ischaemic preconditioning (PC), is an effective method of protecting the heart against subsequent ischaemia-induced cell injury and death. The protective ability of PC was first demonstrated by decreasing infarct size in dogs subjected to regional cardiac ischaemia [1], and has subsequently been shown in pigs, rats, rabbits and humans [2–5]. In addition, PC leads to improved functional recovery of the heart after reperfusion [4]. PC also decreases ischaemia-induced cell death in freshly isolated adult rabbit cardiac myocytes [6] and cultured neonatal rat cardiac myocytes [7,8], useful models to study cellular events in ischaemia and PC.

The protein kinase C (PKC) family of serine/threonine kinases has long been implicated in mediating the protective effect of PC — protection afforded by PC is abolished or partly prevented by inhibitors of PKC (including staurosporine, polymyxin B and chelerythrine), and protection is induced by activators of PKC e.g. 4β-phorbol,12-myristate,13-acetate (4β-PMA) or 1,2-dioctanoyl-sn-glycerol [6,9]. In addition, stimulation with several endogenous agonists coupled to PKC provides protection mimicking PC (reviewed in Ref. [10]).

More recently, it has been shown that PKC isozymes are differentially activated by PC, although the isozymes activated vary in different models. For example, Ping et al. [11] show that {epsilon} and {eta}PKC, but no other isozymes, are activated in rabbit heart whereas Gray et al. [8] find {delta} and {epsilon}PKC, but not {alpha} or βPKC, to be activated by PC in cultured neonatal rat cardiac myocytes. {epsilon}PKC has specifically been implicated by the finding that a selective peptide inhibitor of {epsilon}PKC translocation abolishes PC protection [8] and that {epsilon}PKC selective agonist provides protection from ischaemia in isolated myocytes, as well as in vivo, in transgenic mice [12]. However, activation of {delta}PKC may also provide protection, since overexpression of constitutively active {delta}PKC decreases cell death in neonatal rat cardiac myocytes [13].

Our study examines the mechanism of PKC activation during PC. As mentioned, activation of several PKC-coupled receptors is cardioprotective. These include receptors for adrenergic agonists, bradykinin, endothelin-1, vasodilator prostaglandins and adenosine. However, use of inhibitors of these receptors do not support any one agonist as being responsible [10]. Alternatively, second messenger pathway involves arachidonic acid (AA) and may provide protection. AA activates PKC in vitro [14–17] and selectively activates PKC isozymes [18,19] or PKC-dependent signaling pathways [20,21] in different cells. During ischaemia, phospholipase A2 is activated, resulting in release of AA and its metabolites from phospholipids [22,23]. These products protect against cell death and improve post-ischaemic cardiac function in a model of global ischaemia in perfused rat heart [24,25].

Here, we have demonstrated that AA selectively activates {delta}PKC and {epsilon}PKC in cultured neonatal rat cardiac myocytes. AA decreased the spontaneous contraction rate of neonatal cardiac myocytes through activation of {epsilon}PKC. In addition, we demonstrated that AA protects against cell death due to simulated ischaemia and have shown that this effect is at least partly through activation of {epsilon}PKC. Therefore, AA, or its metabolites, released during PC is a potential mechanism for PKC activation, resulting in protection from ischaemia, and pretreatment with fatty acids may have direct beneficial effects on ischaemic myocardium.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture
Care of rats in this investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Primary cultures of ventricular cardiac myocytes from 1 day old Sprague–Dawley rats were performed by gentle, serial trypsinization, as described previously [26] with modifications [27]. A preplating step was included to reduce the number of contaminating non-myocytes. Myocytes represented 90–95% of total adhering cells. Cells were maintained at 37°C in a 1% CO2 incubator in M-199 medium (Gibco-BRL) containing 10% fetal bovine serum (Hyclone), 50 U/ml penicillin, 80 µM vitamin B12 and 0.1 mM bromodeoxy uridine for the first 4 days. Vitamin C (80 µM) was present from day 2. On day 4, myocytes were placed in defined (10 µg/ml insulin, 10 µg/ml transferrin, 80 µM vitamin C, 50 U/ml penicillin and 80 µM vitamin B12) M-199. Myocytes exhibited a spontaneous contraction rate of 250–300 beats/min and cultures with a slower, or irregular, contraction rate were not used. Experiments were performed on days 5 and 6 of culture.

2.2 Cell fractionation and Western blot analysis
This method is adapted from one described previously [28]. Cardiac myocytes cultured in one 100-mm dish per treatment were treated as appropriate. Cells were washed twice with cold PBS, then scraped into 1 ml PBS. Cells were pelleted at 1000xg and PBS removed. The pellet was resuspended in 300 µl cell lysis buffer (4 mM ATP, 100 mM KCl, 10 mM imidazole, 2 mM EGTA, 1 mM MgCl2, 20% glycerol, 0.05% Triton X-100, 17 µg/ml PMSF, 20 µg/ml soybean trypsin inhibitor, 25 µg/ml leupeptin, 25 µg/ml aprotinin) and then centrifuged at 4°C at 1000xg for 10 min. The supernatant (the soluble fraction) was carefully taken off and recentrifuged at 16 000xg for 15 min to get rid of any contaminating pellet material. The initial pellet was resuspended in 200 µl cell lysis buffer containing 1% Triton X-100 and was extracted on ice for 60 min. Samples were centrifuged at 16 000xg for 15 min. The supernatant is the membrane fraction. The pellet was again resuspended in 200 µl cell lysis buffer containing 1% Triton X-100 and designated the insoluble fraction. Each fraction was analyzed for protein content by Bradford assay and 30 µg protein from each fraction separated by 12% SDS–PAGE. Protein was transferred to nitrocellulose and incubated with the following antibodies, according to the manufacturer's instructions: anti-{alpha}PKC (Santa Cruz Biotech), βIIPKC (Santa Cruz Biotech), {delta}PKC (Gibco BRL), {epsilon}PKC (Gibco BRL), {eta}PKC (Santa Cruz Biotech), {zeta}PKC (Gibco BRL). Immunoreactivity was detected using enhanced chemiluminescence. Autoradiographs were scanned using an ArcusII flatbed scanner (AGFA) with FotoLookPS 2.07.2 and band density analyzed by NIH ImageTM.

2.3 Monitoring of cardiac myocyte contraction rate
Cardiac myocytes cultured in 35-mm dishes (Corning) were placed in a temperature-regulation apparatus at 37°C (Medical Systems Corp.) on the stage of an inverted microscope (Carl Zeiss Inc.). Cells were brought to 37°C and equilibrated for 15 min before monitoring. In each experiment, four cells were monitored in one microscopy field. Counting of the number of contractions was staggered for each cell such that each cell was monitored for 15 s in a 2-min period. Basal rate of contraction was monitored for 8 min before commencing treatment. Contraction rate was expressed as percent of basal.

2.4 Induction of simulated ischaemia
Ischaemia was induced in a humidified 37°C incubator within an air-tight glove box (Anaerobic Systems) maintained with 0.2–0.5% O2, 1% CO2 and the balance N2. Medium (defined MEM Hank's balanced salt solution without glucose) was equilibrated to low O2 within the glove box for at least 90 min before commencing experiments. Inside the glove box, cells were washed twice with warm, pre-equilibrated medium before addition of incubation medium (1.5 or 10 ml per 35- or 100-mm dish, respectively).

2.5 Measurement of lactate dehydrogenase release
After ischaemic or normoxic treatments, incubation medium was stored at 4°C and the same volume of cold buffer (10 mM Tris–HCl, pH 7.4, 1 mM EDTA) added to cells. Cells were scraped and lysed by trituration. Lysates were centrifuged at 4°C at 16 000xg for 15 min and supernatant stored at 4°C. Lactate dehydrogenase (LDH) activity was measured from both medium (released LDH) and cell lysate (retained LDH) using a spectrophotometric assay (Sigma).

2.6 Measurement of creatine kinase release
Where indicated, supernatants and cell lysates obtained as for the LDH assay were used to measure creatine kinase release, by Sigma CK10 assay kit, as a measure for cell damage. Previous studies show that both assays give similar results [8,12].

2.7 Cell viability assay
Cell death was assessed using the LIVE/DEAD® Viability/Cytotoxicity kit (Molecular Probes). Calcein acetoxymethyl ester (calcein AM) and ethidium homodimer were added to incubation medium at final concentrations of 2 and 1 µM, respectively, and dishes incubated at 37°C for 15 min (ischaemic samples were maintained under ischaemic conditions during this incubation). Cells were viewed using a Zeiss microscope and a 40x objective and were scored as live (green cytosolic fluorescence) or dead (red nuclear fluorescence).

2.8 Chronic treatment with PMA
After the medium was changed to defined medium on day 4, myocytes were allowed to recover to normal contraction rate, then 100 nM 4β-PMA (to down-regulate PKC) or 4{alpha}-PMA (inactive phorbol ester control) was added. Final DMSO vehicle concentration was 0.05%). Experiments were initiated 24 h after PMA treatment started.

2.9 Peptide and fatty acid treatment
Peptides, {epsilon}V1-2 [(C)EAVSLKPT, residues 7–14 of {epsilon}PKC] or βC2-4 [(C)SLNPEWNET, residues 218–226 of βPKC], were synthesized, cross-linked via N-terminal cysteine disulfide bond to the Drosophila Antennapedia homeodomain-derived carrier peptide [(C)RQIKIWFQNRRMKWKK] [29,30] and purified (<95%) at the Stanford Protein and Nucleic Acid Facility. Control peptide was a dimer conjugate of the carrier peptide. Peptides were added to final concentration of 150 nM to the medium 20 min prior to AA treatment to allow delivery into myocytes.

Fatty acids (Sigma) were dissolved in 95% ethanol to a concentration of 50 mM, dispensed into aliquots and stored at –20°C. Aliquots were used within 24 h of being prepared. Medium containing AA was replaced by conditioned medium prior to subjecting cells to ischaemia.

2.10 Statistics
Experiments were performed in duplicate unless otherwise stated. Results were expressed as mean±S.E.M. Significance was considered at P<0.05, using Student's t-test.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Selective translocation of {delta}PKC and {epsilon}PKC induced by AA
PKC, specifically {epsilon}PKC, has been implicated as mediating PC [8,11,12]. Activation of PKC is associated with translocation from the soluble to particulate fraction of cells [12,31]. Therefore, to test if AA activates PKC in neonatal rat ventricular cardiac myocytes, the effect of AA on subcellular localization of PKC isozymes was determined by cell fractionation and Western blot analysis. In these cells, the isozymes {alpha}, βII, {delta}, {epsilon}, {eta} and {zeta}, but not {gamma}PKC, are found [32–34]. The subcellular fractionation method used separates proteins into a soluble fraction, a fraction to remove contaminants in the soluble fraction, particulate material (nucleus, filaments and detergent extractable membrane) and insoluble material. The contaminating particulate and insoluble fractions contained less than 5% of total PKC for each isozyme and the amounts did not vary significantly in response to different treatments (data not shown). Therefore, to determine sub-cellular localization of PKC isozymes, only the soluble and particulate fractions were quantitated. Of the isozymes present, all but the atypical, 4β-PMA-insensitive isozyme, {zeta}PKC translocated in response to 100 nM 4β-PMA, as expected (Fig. 1A,B). However, in response to 5-min treatment with 25 µM AA, only {delta} and {epsilon}PKC translocated from the soluble to the particulate fraction (Fig. 1A,B). Relative to vehicle-treated cells, {delta} and {epsilon}PKC decreased in the soluble fraction by 32±13 and 28±7%, respectively, with a corresponding increase in the particulate fraction (n = 3, Fig. 1B). These results indicate that AA activates only two PKC isozymes, {delta} and {epsilon}, in neonatal rat cardiac myocytes.


Figure 1
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  		Fig. 1 Immunoblot of PKC isozymes in control, 4β-PMA- and AA-treated cardiac myocytes. (A) Neonatal rat cardiac myocytes were treated with 13.3 mM (0.05%) ethanol vehicle (C), 100 nM 4β-PMA (P) or 25 µM AA (A) for 5 min, then underwent sub-cellular fractionation and Western blot analysis, probed with antibodies detecting the PKC isozymes indicated. The soluble and particulate fractions are shown. One experiment representative of three is shown. (B) Immunoblots as represented in (A) were subjected to densitometric analysis and percent translocation from soluble to particulate fraction determined. Results are expressed as percent of total PKC in soluble fraction for each sample, which was then expressed as percent of control, vehicle-treated sample. For a decrease in soluble fraction there is a corresponding increase in particulate (not shown). Results are expressed as mean±S.E.M. of three experiments. *P<0.05 vs. control.

 
3.2 AA causes a dose-dependent decrease in spontaneous contraction rate of cardiac myocytes, which is dependent on {epsilon}PKC
Cultured neonatal rat cardiac myocytes contract spontaneously at a rate of approximately 250–300 beats/min. AA decreases contraction rate, also termed negative chronotropy, in neonatal rat cardiac myocytes [35]. To confirm this in our cells, we determined the effect of exogenous AA on spontaneous contraction rate. AA rapidly decreased contraction rate in a dose-dependent manner, whereas vehicle treatment had no effect (Fig. 2A).


Figure 2
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  		Fig. 2 AA is a negative chronotrophic factor, dependent on {epsilon}PKC. (A) The spontaneous contraction rate of neonatal cardiac myocytes was measured. The arrow indicates time of addition of AA, the concentration of which is indicated on the right, or 0.05% ethanol vehicle (vehicle). Results are expressed as percent of basal contraction rate, as mean±S.E.M. of four independent experiments. (B) Spontaneous contraction rate was again measured and 150 nM peptide was added at the time indicated by the first arrow. {epsilon}V1-2-Cr, {epsilon}PKC selective inhibitor peptide linked to carrier peptide; βC2-4-Cr, βPKC selective inhibitor linked to carrier peptide; Cr-Cr, carrier dimer. Then, 16 µM AA was added at the time indicated by the second arrow. Results are expressed as average (±S.E.M.) of four cells from one experiment representative of two experiments. In a further three experiments, {epsilon}V1-2-Cr prevented AA-induced negative chronotropy when compared to no peptide control. Peptides were added in a blinded fashion to prevent observers' bias.

 
Previous results from this laboratory demonstrated that 4β-PMA-induced negative chronotropy in cardiac myocytes is mediated by {epsilon}PKC [36]. Therefore, we determined if AA-induced negative chronotropy is also mediated through {epsilon}PKC. A peptide inhibitor of {epsilon}PKC translocation, and therefore activity, was utilized [36]. Peptide was delivered to cells using a cell permeable carrier peptide, derived from the Drosophila Antennapedia homeodomain, which delivers biologically active peptides into cells [29,30]. We previously found that the intracellular concentration of the peptides is approximately 10% of that applied [37]. {epsilon}V1-2-carrier (150 nM) delayed but did not prevent negative chronotropy induced by 25 µM AA (results not shown). However, {epsilon}V1-2-carrier (150 nM) prevented the decrease in contraction rate induced by 16 µM AA whereas carrier dimer, or βC2-4-carrier, an inhibitor of βPKC translocation [38], had no effect (Fig. 2B). None of these peptides had any effect on the basal contraction rate. Thus, AA reduces spontaneous contraction rate through translocation and activation of {epsilon}PKC.

3.3 AA decreases injury of cardiac myocytes by simulated ischaemia
To test the hypothesis that AA-induced PKC activation mediates protection from ischaemia, we determined the effect of AA on cellular damage due to simulated ischaemia (0.2–0.5% oxygen in the absence of glucose). Release of the cytosolic enzyme LDH is a measure of membrane leakiness, which increases both with necrosis and apoptosis in cultured cells, although at a later stage of apoptotic cell death. We have observed previously that subjecting neonatal rat cardiac myocytes to simulated ischaemia results in significant damage to myocytes, with release of approximately 60% of cellular LDH in 7–9-h incubation [27]. Because activation of PKC with 25 µM AA occurred within 5 min (Fig. 1), cells underwent a pretreatment of 10 min with 25 µM AA. This treatment significantly decreased ischaemia-induced LDH release by 28±9% in comparison to control, vehicle-treated cells (Fig. 3A).


Figure 3
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  		Fig. 3 AA decreases injury of cardiac myocytes from simulated ischaemia. Cardiac myocytes were pretreated with 25 µM AA or 0.05% ethanol vehicle for 10 min, then incubated under simulated ischaemic or normoxic conditions for 8–9 h. (A) Cell damage was assayed by measuring release of cytosolic LDH into the medium and LDH retained within cells. Results are expressed as released LDH as percent of total LDH activity, then were normalized to 100% for vehicle-treated ischaemic samples. *P<0.00002. Results are mean±S.E.M. of five experiments performed in duplicate. In these experiments, LDH release in vehicle controls ranged from 39.0 to 64.1% total cellular LDH. (B) Cell death was assayed using a cell viability assay, LIVE/DEAD®. Viable cells (stained green by calcein AM) and non-viable cells (nuclei stained red by ethidium homodimer) were counted and non-viable cells expressed as percent of total cells. Data were then normalized to 100% for vehicle-treated ischaemic samples. Results are mean±S.E.M. of three experiments performed in duplicate, with more than 600 cells counted in each experiment. *P<0.002 vs. vehicle-treated ischaemic samples. In vehicle controls, non-viable cells as % total ranged from 34.0 to 67.8.

 
In addition, a direct assay of cell viability was used. In this assay, intracellular esterases of live cells convert cell-permeable calcein AM to fluorescent calcein, which is viewed as green cytosolic fluorescence, while the intact cell membrane excludes ethidium homodimer. In contrast, dead cells do not retain the esterase and therefore do not show green fluorescence but the damaged membrane allows uptake of ethidium homodimer which causes nuclei to produce red fluorescence. Pretreatment with AA significantly increased cell viability after prolonged ischaemia (Fig. 3B). Therefore, using two different assays for cell damage and death, a short 10-min pretreatment with 25 µM AA significantly protects neonatal rat cardiac myocytes from ischaemia-induced damage.

3.4 Inhibition of PKC decreases AA-induced protection of cardiac myocytes from simulated ischaemia
We determined the contribution of PKC to the AA-induced protection initially by down-regulation of PKC with 24-h pretreatment of cells with 100 nM 4β-PMA. This treatment decreases the cellular amount of 4β-PMA-sensitive PKC isozymes to between 12±2 and 32±5% of basal (n = 5, not shown).

PKC down-regulation did not affect viability under normoxic conditions (Fig. 4A), but it increased susceptibility of the cells to ischaemic damage by 36±8% and reduced protection induced by AA. When expressed as percent of AA-induced protection in control cells, down-regulation of PKC significantly inhibited protection to 56±9% (Fig. 4B).


Figure 4
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  		Fig. 4 Inhibition of PKC decreases AA-induced protection. (A) Cardiac myocytes were treated with 100 nM 4{alpha}-PMA or 4β-PMA for 24 h. Cells were then pretreated with 25 µM AA or 0.05% ethanol vehicle for 10 min. Cells were incubated under normoxic or simulated ischaemic conditions for 8–9 h and LDH release measured. Results are mean±S.E.M. of three experiments performed in duplicate. *P<0.0002, **P<0.002. (B) From data represented in panel A, the percent protection induced by AA in 4{alpha}-PMA- or 4β-PMA-treated cells was determined then normalized to 100% for 4{alpha}-PMA-treated samples. *P<0.005. (C) Cardiac myocytes were treated with 2 µM chelerythrine (CHE) for 20 min prior to treatment with 25 µM AA or 0.05% ethanol vehicle for 10 min. Cells were incubated in simulated ischaemia, in the presence of 2 µM chelerythrine for 8–9 h and LDH release measured. Results are mean±S.E.M. of three experiments performed in duplicate. *P<0.05 vs. all other treatments. The effects of other treatments did not significantly differ from each other.

 
To confirm that inhibition of protection was not due to the higher basal damage from ischaemia in cells subjected to PKC down-regulation, similar experiments were performed except that myocytes with down-regulated PKC were exposed to ischaemia for 1 h less than control cells. Thus, PKC down-regulated and control cells showed the same amount of total injury. Down-regulation still resulted in significant inhibition of AA-induced protection to 55±16% of control (n = 3, not shown). The incomplete inhibition may be due to the amount of PKC remaining after down-regulation, which is 14±6 and 30±4% of basal for {delta} and {epsilon}PKC, respectively (n = 5, not shown). Alternatively, it may be due to a PKC-independent mechanism.

To further determine the role of PKC, chelerythrine, an inhibitor of PKC catalytic activity, was used [39]. Chelerythrine (2 µM) was added 20 min prior to AA treatment and, because of the medium change required to simulate ischaemia, a further 2 µM chelerythrine was added at the start of the ischaemic incubation. Chelerythrine significantly blocked AA-induced protection by 66±9% (Fig. 4C). Together these results demonstrate that PKC is required for AA-induced protection of neonatal rat cardiac myocytes from simulated ischaemia. Because AA resulted in translocation of only {delta} and {epsilon}PKC, and {epsilon}PKC has been implicated in PC [8], we examined if selective inhibition of {epsilon}PKC blocked AA-induced protection. Although 150 nM {epsilon}V1-2-carrier peptide was an amount sufficient to inhibit 25 mM AA-induced contraction in myocytes (Fig. 2), this amount added as a pre-treatment and/or as co-treatment to AA did not result in a significant inhibition of 25 mM AA-induced protection (not shown). However, pre-incubation for 20 min with 1 µM {epsilon}V1-2-carrier and a 10-min co-incubation with 25 µM AA followed by a change to inhibitor-free media demonstrated that AA-induced protection is likely mediated by {epsilon}PKC (Fig. 5) (Note that no more than 10% of the applied peptides enter the cells using this method [40]). Cell damage was determined by the release of creatine kinase (CK). We have previously shown that similar data are obtained using this assay as compared with the LDH release assay. We also showed that cell viability was unaffected by 1 µM {epsilon}V1-2-carrier pre-treatment if cells were kept under normoxic conditions for the duration of the experiment (not shown). Therefore, AA-induced protection from ischaemic injury is likely mediated by a strong and/or long-term activation of {epsilon}PKC.


Figure 5
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  		Fig. 5 Inhibition of {epsilon}PKC decreases AA-induced protection. Cardiac myocytes were treated with 1 µM {epsilon}V1-2-carrier ({epsilon}V1-2) or with carrier alone (control) for 20 min prior to treatment with 25 AA or 0.05% ethanol vehicle for 10 min. Cells were incubated in simulated ischaemia as above in the absence of peptides for 12 h, and creatine kinase release was determined. *P<0.05 vs. all other treatments.

 
3.5 Effect of other fatty acids
The effect of other unsaturated fatty acids, eicosapentaenoic acid (EPA) and oleic acid (OA), was investigated. Again, EPA and OA were added as a 10-min pretreatment to ischaemia and were not present during the ischaemic period. Significant protection of cardiac myocytes from ischaemia was observed with 40 but not 25 µM OA and with both 25 and 40 µM EPA (Fig. 6).


Figure 6
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  		Fig. 6 OA and EPA decrease injury of cardiac myocytes from simulated ischaemia. Cardiac myocytes were pretreated with 25 or 40 µM OA, EPA or 0.05% ethanol vehicle for 10 min, then incubated under simulated ischaemic conditions for 8–9 h and LDH release measured. Results represent released LDH as percent of total LDH activity, then were normalized to 100% for vehicle-treated ischaemic samples. Results are mean±S.E.M. of three experiments performed in duplicate. *P<0.05 vs. vehicle-treated sample.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Ischaemia causes degradation of membrane phospholipids, probably via the action of phospholipase A2, resulting in accumulation of unesterified fatty acids including arachidonic acid [22]. This accumulation correlates with cellular injury [23,41]. Recent studies, however, have demonstrated that AA is also released early in ischaemia and by PC treatment [24,25]. AA acts as an intracellular second messenger (reviewed in Ref. [17]) and can activate PKC both in vitro [14–16] and in many cellular systems [18–21]. PKC has been demonstrated to be a major signal transduction pathway leading to cardioprotection by PC. Our study addressed the possibility that AA contributes to the activation of PKC in neonatal rat cardiac myocytes, thereby mediating cardioprotection. We demonstrated that AA results in the selective activation of {delta}PKC and {epsilon}PKC, the two PKC isozymes activated by PC in these cells [8]. In addition, our results showed that AA is cardioprotective to neonatal rat cardiac myocytes and that this effect is mediated at least partly through {epsilon}PKC activation. An important implication of this study is that fatty acids delivered to the ischaemic or preischaemic heart in vivo may provide protection from cell death.

Chronic treatment of myocytes with 4β-PMA to down-regulate PKC was used as a method for PKC inhibition and we found that this increased basal susceptibility to ischaemia-induced damage. Chelerythrine did not increase susceptibility to ischaemia, therefore it is unlikely that inhibition of PKC per se causes the additional damage. The increase in damage that follows PMA down-regulation may be due to the fact that this method of inhibiting PKC initially goes through a potent activation of PKC isozymes by the high levels of PMA. This may result in expression of a gene or regulation of a protein that causes damage under the additional stress of ischaemia. These results suggest that strong activation of some 4β-PMA-sensitive PKC isozymes may be deleterious during ischaemia. Therefore, the selective activation of isozymes seen during PC or AA treatment appears to be important to the cardioprotective mechanism, suggesting that substrates specific to the isozymes activated mediate protection. Our study and others [8,11,12] suggest that {epsilon}PKC mediates protection and so substrates of {epsilon}PKC will be important.

There is growing evidence that the ATP-sensitive potassium channel (KATP), specifically the mitochondrial and not the sarcolemmal channel, is the end effector of preconditioning protection [42,43]. PKC can lead to activation of mitochondrial KATP channels [44], although the link may be indirect, involving a tyrosine kinase or p38 mitogen-activated protein kinase [45].

It has been suggested that sub-threshold amounts of several PKC activators can act together to result in PC protection [10]. In vivo, therefore, AA may act during PC in combination with other PKC activators. Alternatively, AA may be produced by PKC activators, e.g. angiotensin, endothelin or bradykinin [46–48], and act in synergy with diacylglycerol to result in activation of selective PKC isozymes.

Cultured neonatal myocytes can respond differently from adult myocytes. For example, neonatal cells are inherently less susceptible to ischaemia [49]. In infarction models in the intact heart, the ischaemic period is typically 20–40 min and in freshly isolated adult myocytes, ischaemia of 90 min gives significant injury. In the neonatal rat myocyte model used in this study and others [7,8], times of at least 6 h are required to show significant damage. In addition to technical reasons due to differences in experimental models, metabolic differences could alter sensitivity to ischaemia and responses of cells to injury. Also, levels of PKC and ion channel expression vary between neonatal and adult myocytes. In this study, we demonstrated the presence of {alpha}, βII, {delta}, {epsilon}, {eta} and {zeta}PKC in neonatal rat cardiac myocytes. The isozymes {alpha}, {delta} and {epsilon}PKC are consistently detected in neonatal and adult ventricular myocytes [32,50,51] and {eta}PKC has been detected in adult rat heart extract [52] and in adult rabbit heart [11]. The presence of β and {zeta}PKC is more controversial [32,53–55]. The expression of PKC isozymes on a per cell basis increases in the adult [53], which may alter response to ischaemia or PKC activators. However, it has been demonstrated that neonatal myocytes are similar to adult cells in that they can be preconditioned and show PMA-induced protection against ischaemia [7,8] and that KATP channel opening increases tolerance to ischaemia protection [49]. Our study also show that {epsilon}PKC activation is required and sufficient to mediate this protection in both neonatal and adult myocytes [8,12]. In addition, it has been shown that AA causes {epsilon}PKC translocation in adult rat myocytes [19]. Therefore, we believe the results demonstrated here with neonatal myocytes will be applicable to adult myocytes and the whole heart.

Previously, it has been shown that polyunsaturated fatty acids protect against fatal arrhythmias induced by ischaemia in animal models [56,57]. In addition, in cultured cells, arachidonic acid and other polyunsaturated fatty acids, but not monounsaturated or saturated fatty acids, have anti-arrhythmic activity [58]. The anti-arrhythmic effect of at least one fatty acid, EPA, appears to be by directly interacting with, and inhibiting, both voltage-sensitive Na2+ channels and voltage-gated L-type Ca2+ channels through partitioning into the hydrophobic environment of the membrane [59,60]. AA is complex in its effects on rhythmicity, however, because as a free fatty acid it has anti-arrhythmic effects whereas certain oxidized metabolites appear to cause arrhythmia [35]. Indeed, release of AA late in ischaemia is thought to contribute to deleterious arrhythmia [41]. Because of these complex effects of AA, we also tested the effects of other fatty acids, EPA and OA. We found that both EPA and OA protected cardiac myocytes from prolonged ischaemia. Together these data indicate that the cardioprotective effect of fatty acids is likely to be separate from the anti-arrhythmic effect shown previously because of different concentrations required and because OA does not show anti-arrhythmic properties [35,58], but is protective against cell damage. The effect of fatty acids on contraction may be different in neonatal as opposed to adult myocytes because the effect is likely to be through PKC-mediated phosphorylation of ion channels, some of which are down-regulated in primary cultures of neonatal myocytes. This may result in a weaker response in neonatal cells if the channel responsible is down-regulated or in a stronger response if an opposing channel is down-regulated.

This study did not address whether the protective effect is directly through AA or oxidized metabolites produced by, e.g. lipoxygenases, which can act when oxygen is present during reperfusion [24]. In an isolated perfused rat heart model, inhibition of lipoxygenase blocked the protective effect of preconditioning on post-ischaemic functional recovery and infarct size [24,25]. Because in this study AA is added as a pretreatment to ischaemia, oxygen will be present and therefore oxygenated metabolites may be formed. However, the protective effect of OA and EPA suggests that fatty acids themselves or a combination of fatty acid and AA metabolites may protect.

In conclusion, AA treatment causes a PKC-mediated negative chronotropy in cultured cardiac myocytes which are beating rhythmically and spontaneously. AA also has a direct cardioprotective effect against simulated ischaemia and may be one mediator of PKC activation during PC, either directly as free fatty acid or as metabolites.

Time for primary review 19 days.


   Acknowledgements
 
This work was supported by the National Institutes of Health grant AA11147.


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

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