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Am J Physiol Heart Circ Physiol 282: H320-H327, 2002;
0363-6135/02 $5.00
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Vol. 282, Issue 1, H320-H327, January 2002

Protein kinase C-zeta modulates thromboxane A2-mediated apoptosis in adult ventricular myocytes via Akt

Yukitaka Shizukuda1 and Peter M. Buttrick1,2

1 Program in Cardiovascular Sciences, Section of Cardiology, Department of Medicine, and 2 Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that thromboxane A2 (TxA2) receptor stimulation directly induces apoptosis in adult cardiac myocytes. To investigate this, we exposed cultured adult rat ventricular myocytes (ARVM) to a TxA2 mimetic [1S-[1alpha ,2alpha (Z),3beta (1E,3S*),4alpha ]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) for 24 h. Stimulation with I-BOP induced apoptosis in a dose-dependent manner and was completely prevented by a TxA2 receptor antagonist, SQ-29548. We further investigated the role of protein kinase C (PKC) in this process. TxA2 stimulation resulted in membrane translocation of PKC-zeta but not PKC-alpha , -beta II, -delta , and -epsilon at 3 min and 1 h. The activation of PKC-zeta by I-BOP was confirmed using an immune complex kinase assay. Treatment of ARVM with a cell-permeable PKC-zeta pseudosubstrate peptide (zeta -PS) significantly attenuated apoptosis by I-BOP. In addition, I-BOP treatment decreased baseline Akt activity and its decrease was reversed by treatment with zeta -PS. The inhibition of phosphatidylinositol 3-kinase upstream of Akt by wortmannin or LY-294002 abolished the antiapoptotic effect of zeta -PS. Therefore, our results suggest that the activation of PKC-zeta modulates TxA2 receptor-mediated apoptosis at least, in part, through Akt activity in adult cardiac myocytes.

atypical protein kinase C; eicosanoids; Akt/protein kinase B; mitogen activated protein kinases


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THROMBOXANE A2 (TxA2), a vasoactive eicosanoid released by activated platelets (13), directs a spectrum of physiological functions, including platelet aggregation, vasoconstriction, proliferation of smooth muscle cells, and inhibition of endothelial cell migration (26). These are largely mediated via receptor-coupled activation of Gq/G11, with downstream engagement of other second messengers, including protein kinase C (PKC) (16, 27, 37). Several reports in diverse cell types have linked TxA2 activation to apoptosis, or programmed cell death (15, 20, 39). The mechanism by which this occurs has not been well established, although one group has described TxA2-dependent Akt inhibition as a potential cause of apoptosis in endothelial cells (15). Like many other cells, cardiac myocytes are also known to have TxA2 receptors (11, 38), but the functional role of the receptor in the cardiac myocyte context is unknown.

Apoptosis of cardiac myocytes is induced by acute ischemic insults (3, 18, 29). This phenomenon occurs in regions of hypoxia (5) where the local expression of a number of cytokines and cytotoxic substances has been reported, including TxA2, which is released locally both from damaged vessels and from activated platelets (26). Because TxA2 has been reported to trigger apoptosis in a number of cell types (15, 20, 39), establishing a role for TxA2 in cardiac myocyte apoptosis would have both biologic and therapeutic importance. The possible pathophysiological role of TxA2 in myocardial ischemia is supported by reports indicating improvement in postischemic cardiac function (28, 40) and reduction of myocardial infarct size (40) with the use of TxA2 antagonists in in vivo animal models. Accordingly, the focus of this investigation was to determine whether TxA2 receptor stimulation is associated with cardiac myocyte apoptosis and, if so, to identify the relevant second messenger pathways.

Our data in isolated adult cardiac myocytes demonstrate that TxA2 receptor engagement does directly induce cardiac myocyte apoptosis and that this is at least, in part, a result of reduction of Akt activity by PKC-zeta .


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and treatments. Adult rat ventricular myocytes (ARVM) were harvested from 7- to 9-wk-old male Wistar rats and cultured as previously described (24). ARVM were cultured in ACCIT media of medium 199 with Earle's balanced salts, including 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 26 mM NaHCO3 (Sigma; St. Louis, MO), supplemented with 2 mg/ml bovine serum albumin (BSA), 2 mM L-carnitine, 5 mM creatinine, 5 mM taurine, 0.1 µM insulin (GIBCO BRL; Grand Island, NY), 100 IU/ml penicillin, and 100 µg/ml streptomycin (GIBCO BRL) for 48 h before the protocols were started. The medium was changed 2 h after initial plating to remove unattached cells as well as every 24 h, thereafter.

A TxA2 mimetic, [1S-[1alpha ,2alpha (Z),3beta (1E,3S*),4alpha ]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP, 10-100 nM), and/or a highly specific TxA2 receptor inhibitor, SQ-29548 (10 µM), was added to the media for 24 h depending on the experiments. Both chemicals were purchased from Cayman Chemical; Ann Arbor, MI. In experiments to investigate the role of Akt, phosphatidylinositol 3-kinase (PI3-K) was blocked by specific inhibitors, i.e., wortmannin (100 nM) and LY-294002 (1 µM). These drug concentrations were chosen based on previous studies that investigated TxA2 receptor-mediated signaling transduction (15, 41) and Akt signaling transduction (7). All chemical materials not specified were purchased from Sigma; St. Louis, MO.

PKC-zeta isoenzyme-specific inhibition. PKC-zeta isoenzyme was inhibited by a specific pseudosubstrate peptide (4, 23) (zeta -PS, SIYRRGARRWRKLYRAN) that corresponds to amino acid residues 113-129 of human PKC-zeta (kindly provided by Dr. Daria Mochly-Rosen, Stanford University). These peptides were modified by cross-linking NH2-terminal Cys-Cys bond to the Drosophila antennapedia-derived carrier peptide to make them cell-permeable. The peptides (1 µM, final concentration) were added to culture media every 8 h. This approach to isoenzyme-specific inhibition of PKC-zeta has been detailed previously (4, 12, 23) in a variety of cell types. To inhibit PKC-epsilon translocation, 750 nM epsilon V1-2 peptide (a generous gift from Dr. Mochly-Rosen) was used at a dose known to inhibit phorbol 12-myristate 13-acetate-induced translocation (data not shown) as well as to prevent apoptosis induced by beta -adrenergic stimulation (33).

Immunoblot analysis. Whole cell lysates were prepared by the addition of the lysis buffer [PBS, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 200 µM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml aprotinin, and 4.2 µM leupeptin]. Immunoblots were performed using an antiphosphorylated Akt, anti-Akt, antiphosphorylated extracellular regulated kinase (ERK) (all New England Bio Labs; Beverly, MA), or anti-ERK (Upstate Biotechnology; Lake Placid, NY) antibody. The films were developed using a Supersignal chemiluminescence kit (Pierce; Rockford, IL), and photodensity of each band was quantitated using the Gel-Doc system (Bio-Rad, Hercules, CA).

Immunoblot analysis and translocation of PKC isoenzymes. Cells were harvested in the presence of lysis buffer containing 20 mM Tris · HCl (pH 7.5), 2 mM EGTA, 20 mM EDTA, 20 µM leupeptin, 10 µM E64, 200 µM PMSF, and 5 mM dithiothreitol and sonicated. Protein concentration was determined with Bio-Rad protein assay kit (Bio-Rad), and 3 mg of each ventricular myocyte preparation was subject to differential centrifugation to collect the cytosolic and particulate fraction as described (21, 32, 34). The percentages of individual PKC isoenzymes in each fraction were calculated as previously reported (21, 32). Briefly, both cytosolic and particulate fractions were precipitated by sodium trichloroacetate and resuspended with the sample buffer containing 63 mM Tris · HCl (pH 6.8), 2% SDS, 10% glycerol, 1% 2-mercaptoethanol, and 5 µg/ml bromophenol blue (200 µl for cytosolic fraction and 100 µl for particulate fraction). Equal amounts of protein from resuspended fractions (20-30 µl) were subjected to immunoblotting using PKC isoenzyme-specific antibodies. The relative distribution of each PKC isoenzyme in different fractions was calculated from the photodensity, and the sample buffer volume was used for resuspension of each fraction as previously described (21).

Immune complex kinase assay of individual PKC isoenzyme activity. Immune complex kinase assay of PKC-alpha , -beta II, -delta , and -epsilon was performed as previously described (32, 34). Briefly, ARVM were lysed in ice-cold lysis buffer for 10 min and were homogenized by repeated aspiration through a 21-gauge needle. The individual PKC isoenzyme was immunoprecipitated using an anti-PKC-alpha or -beta II or -epsilon specific polyclonal antibody (Santa Cruz Biotechnology; Santa Cruz, CA) or an anti-PKC-delta antibody (Pharmigen/Transduction Laboratories, San Diego, CA) from 2 mg whole cell lysate. The reaction mixture did not contain additional calcium acetate for assay of PKC-delta , -epsilon , or -zeta kinase activity. The presence of each PKC isoenzyme was confirmed by immunoblotting. Individual PKC isoenzyme activity was expressed as the PKC isoenzyme activity measured relative to unstimulated ARVM.

Immune complex kinase assay of Akt. The kinase activity of Akt1/protein kinase Balpha was measured as previously described using a kit from Upstate Biotechnology (Lake Placid, NY) (2). Briefly, Akt1 was immunoprecipitated from 1 mg whole cell lysates. The kinase reaction was carried out according to the manufacturer's instructions. The activity of 32P bound to p81 cellulose paper was measured with a scintillation counter (model LS6500, Beckman). The values were normalized to those of simultaneously measured unstimulated cells.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was carried out as previously described (32). Both ARVM floating in the media and trypsinized were collected together and used for the assay. ARVM were placed on slides and air-dried overnight. TUNEL assay was performed on slides with a TACS 2 terminal deoxynucleotidyl transferase (TBL) kit (Trevigen, Gaithersburg, MD). For each slide, the number of TUNEL-positive cells was scored in 12 randomly chosen high-power fields (×400) and was normalized to the total number of cells counted as previously described (31, 32).

DNA gel electrophoresis assay. Gel electrophoresis to detect the DNA ladder was carried out as previously described (32). Briefly, ARVM were washed with PBS and then digested with DNA extraction cell lysis buffer [10 mM Tris · HCl (pH 8.0), 100 mM NaCl, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml protease K (GIBCO BRL)] overnight at 37°C. Genomic DNA was precipitated with isopropanol after extraction with phenol:chloroform:isoamyl alcohol (25:24:1, vol/vol/vol). Equal quantities of each sample (6 to 15 µg) were subjected to electrophoresis on 1.25% agarose gels containing 0.5 µg/ml ethidium bromide (GIBCO BRL).

Statistical analysis. All data are presented as means ± SD. The effects of different treatment groups were compared by ANOVA. Multigroup comparison was carried out with Bonferroni-modified t-tests. When the comparison was made between only two treatment groups, unpaired Student's t-tests were used. Probability values <0.05 were accepted as statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

I-BOP induces TxA2 receptor-mediated apoptosis in ARVM. I-BOP induced apoptosis in a dose-dependent fashion as measured with TUNEL assay compared with control cells (Fig. 1). The proapoptotic effect of I-BOP was abolished by concomitant treatment with TxA2 receptor antagonist treatment by SQ-29548 (10 µM) at each concentration (Fig. 1). SQ-29548 (10 µM) treatment alone did not affect the rate of apoptosis in the absence of I-BOP (13.9 ± 1.3%, n = 4). Therefore, the proapoptotic effect of I-BOP is TxA2 receptor mediated.


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  		Fig. 1.   Dose-response relations of thromboxane A2-induced apoptosis in cultured adult rat ventricular myocytes. Cultured adult rat ventricular myocytes were stimulated with a thromboxane A2 mimetic, I-BOP, for 24 h in the presence (open circles) or absence (closed circles) of a thromboxane A2 receptor-specific inhibitor, 10 µM SQ-29548 (SQ). Apoptosis was assessed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Data are means ± SD. Results are from 2 to 6 duplicate experiments. *P < 0.05 vs. untreated control cells. §P < 0.05 vs. I-BOP treatment with SQ-29548.

Effects of TxA2 stimulation on PKC isoenzyme translocation. To identify the specific PKC isoenzyme that might play a role in TxA2-induced apoptosis, translocation of individual PKC isoenzymes by TxA2 stimulation was examined. I-BOP translocated PKC-zeta significantly as measured by immunoblotting (11.6 ± 7.4%, unstimulated, n = 4 vs. 24.4 ± 6.1% with I-BOP, n = 4, P < 0.05, data expressed as percentages of PKC isoenzyme located in the particulate fraction, Fig. 2) but not PKC-alpha , -beta II, -delta , and -epsilon . To examine the effect of I-BOP on possible reversible rapid and transient translocation of PKC isoenzymes, the translocation of individual PKC isoenzymes was examined 3 min after stimulation. Translocation of PKC-zeta was even more prominent (41.6 ± 7.5%, n = 3) at this time point; however, alpha -, beta 2-, delta -, and epsilon -isoenzymes of PKC were not translocated at this time point (Fig. 3A). In addition, the total amount of these PKC isoenzymes (in whole cell lysates) was not altered after up to 1 h of stimulation with I-BOP (Fig. 3B).


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  		Fig. 2.   Effects of thromboxane A2 receptor stimulation on the membrane translocation of protein kinase C (PKC) isoenzymes. Translocation was assessed with immunoblotting at 1 h after 100 nM I-BOP stimulation. The percentage of each PKC isoenzyme in the particulate fraction is shown. Open bars, control cells; filled bars, cells treated with 100 nM I-BOP for 1 h; cont, control cells; C, cytosolic fraction; P, particulate fraction. Data are means ± SD. Results are from 3 to 5 separate experiments. *P < 0.05 vs. unstimulated control cells.



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  		Fig. 3.   Effects of thromboxane A2 receptor stimulation on the membrane translocation of PKC isoenzymes. The translocation was assessed with immunoblotting at 3 min after 100 nM I-BOP stimulation. A: the percentage of each PKC isoenzyme in the particulate fraction is shown. Open bars, unstimulated control cells (the same control groups represented in Fig. 2); filled bars, cells treated with 100 nM I-BOP for 3 min. B: time course of protein expression level of individual PKC isoenzymes in which whole cell lysates over 60 min were demonstrated. Equal amounts of protein (20-30 µg) were subject to immunoblotting. Data are means ± SD. Results are from 3 to 5 separate experiments in A and 3 separate experiments in B. *P < 0.05 vs. unstimulated control cells.

Effects of TxA2 on immune complex kinase activity of PKC isoenzymes. In addition to translocation of PKC-zeta isoenzyme, I-BOP (100 nM) also increased PKC-zeta enzyme activity (261.6 ± 92.8% of control activity, n = 4, Fig. 4A) at 1 h of stimulation. This activation of PKC-zeta was completely prevented by 10 µM SQ-29548 (95.8 ± 22.6%, n = 6), suggesting that it is TxA2 receptor dependent. zeta -PS, a cell-permeable pseudosubstrate peptide for PKC-zeta (1 µM) also attenuated I-BOP-induced PKC-zeta activation (131.2 ± 42.5%, n = 7).


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  		Fig. 4.   Effects of thromboxane A2 receptor stimulation on individual PKC isoenzyme activity. Individual PKC isoenzyme activity was measured with immune complex kinase assay as described in MATERIALS AND METHODS. Enzyme activity was measured after 1 h I-BOP (100 nM) stimulation. PKC-zeta activity in the presence (+) or absence (-) of PKC-zeta -specific pseudosubstrate (zeta -PS) is shown in A. PKC-alpha , -beta II, -delta , and -epsilon enzyme activity in the presence (+) or absence (-) of zeta -PS is shown in B. Values are normalized to the activity of unstimulated control cells in A and B. ND, PKC-beta II isoenzyme-specific activity was not detected by treatment. Data are means ± SD. Results are from 4 to 7 separate experiments in A and 3-4 separate experiments in B. *P < 0.05 vs. controls. §P < 0.05 vs. I-BOP treated cells.

I-BOP did not affect PKC-alpha , -delta , or -epsilon specific enzyme activity in both the absence and presence of zeta -PS (Fig. 4B). Our method was not sufficiently sensitive to detect PKC-beta II-specific isoenzyme activity although translocation studies indicated the presence of this isoenzyme in ARVM.

Effects of PKC-zeta -specific inhibition by a PKC-zeta pseudosubstrate peptide on apoptosis induced by I-BOP in ARVM. A cell-permeable pseudosubstrate-blocking peptide for PKC-zeta , zeta -PS, significantly attenuated I-BOP (100 nM) induced apoptosis (23.3 ± 3.8%, n = 6, P < 0.05 vs. I-BOP treated cells, Fig. 5A). As expected, 10 µM SQ-29548 also blocked apoptosis by I-BOP. These effects were confirmed by DNA gel electrophoresis assay (Fig. 5B). The PKC-epsilon translocation inhibitor peptide, epsilon V1-2 did not affect the rate of apoptosis induced by I-BOP (37.7 ± 1.2%, n = 4). In addition, a carrier peptide of zeta -PS alone did not affect baseline rate of apoptosis (13.9 ± 0.9%, n = 4) or that induced by 100 nM I-BOP (38.9 ± 1.8%, n = 4).


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  		Fig. 5.   The effect of PKC-zeta -specific inhibition by PKC-zeta pseudosubstrate peptide (zeta -PS) on apoptosis induced by thromboxane A2 stimulation by I-BOP (100 nM). The extent of apoptosis was measured by TUNEL assay (A) and DNA gel electrophoresis assay (B). +, Treatment; -, no treatment. M, 100 bp DNA ladder marker. Data are means ± SD. Results are from 2 to 6 duplicate experiments in A and 3 separate experiments in B. *P < 0.05 vs. control cells. §P < 0.05 vs. I-BOP treatment alone.

Is ERK or Akt activity modulated by PKC-zeta in the presence of TxA2 receptor stimulation in ARVM? Two well-established signals (Akt and ERK) that can prevent apoptosis in various cell types (10, 19, 30) were investigated to see whether they are modulated by PKC-zeta in the presence of TxA2 receptor stimulation. I-BOP stimulation (100 nM) gradually suppressed Akt phosphorylation over a 1-h period (Fig. 6). On the other hand, ERK phosphorylation was not affected by this stimulation (Fig. 6). The effect of I-BOP on Akt phosphorylation was statistically significant at 1 h (61.2 ± 19.7%, n = 6, P < 0.05, the value was normalized to the intensity of unstimulated cells, Fig. 7A), but the effect on ERK was not (93.0 ± 10.2%, n = 4). Strikingly, the suppression of Akt phosphorylation was prevented by zeta -PS treatment at the 1-h point (108.4 ± 18.4%, n = 6, P < 0.05 vs. I-BOP treatment, Fig. 7A). As expected, SQ-29548 also prevented the reduction of Akt phosphorylation by I-BOP (106.4 ± 14.7%, n = 4, P < 0.05 vs. I-BOP treatment, Fig. 7A). These findings were confirmed by Akt immune complex kinase assay (Fig. 7C). The activity of Akt was suppressed to 45.6 ± 6.4% (n = 4, P < 0.05 vs. control cells) by I-BOP stimulation for 1 h using this assay, suggesting that the immune complex kinase assay is more sensitive than immunoblotting to assess Akt activity. To further investigate the role of Akt, we employed two pharmacological inhibitors of PI3-K, wortmannin (100 nM) and LY-294002 (1 µM). Both agents significantly blocked the effect of zeta -PS on Akt phosphorylation (51.1 ± 20.3%, n = 5; 43.3 ± 8.7%, n = 4, respectively, Fig. 7B). Both wortmannin and LY-294002 at this dose decreased Akt kinase activity slightly (73.4 ± 11.8%, n = 3, P < 0.05 vs. control activity; 78.0 ± 5.7%, n = 3, P < 0.05 vs. control activity, respectively; data are normalized to baseline Akt activity) without a significant change in Akt phosphorylation measured by immunoblotting (data not shown).


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  		Fig. 6.   Effects of thromboxane A2 stimulation on phosphorylation of Akt/protein kinase B (Akt) and extracellular regulated kinase (ERK). The extent of phosphorylated form of Akt (p-Akt) and phosphorylated ERK (p-ERK) was assessed with immunoblotting over 60 min. Results are representative of 2 separate experiments.



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  		Fig. 7.   Effects of zeta -PS on Akt phosphorylation in the presence of I-BOP (100 nM). WR, wortmannin (100 nM); LY, LY-294002 (1 µM); phospho-Akt, phosphorylated Akt. The intensity of p-Akt at 1 h of I-BOP stimulation (normalized to control band intensities) in the presence (+) or absence (-) of various pharmacological interventions is shown (A and B). Akt enzymatic activity was measured with immune complex kinase assay with various pharmacological interventions (C). The enzymatic activity was normalized to that of simultaneously measured unstimulated cells. Data are means ± SD. Results are from 3 to 6 separate experiments in A and B and results from 4 separate experiments in C. *P < 0.05 vs. control cells in A and C and vs. I-BOP with zeta -PS treatment in B. §P < 0.05 vs. I-BOP treatment alone in A and C.

Akt is involved in the antiapoptotic effect of PKC-zeta inhibition. Treatment of cells with PI3-K inhibitors abolished the antiapoptotic effect by zeta -PS (39.3 ± 1.5%, n = 4, 39.8 ± 2.8%, n = 4, respectively, values are the percentage of apoptotic cells by TUNEL assay, Fig. 8A). These effects were also confirmed by DNA gel electrophoresis (Fig. 8B). These inhibitors at the concentration chosen for these experiments did not affect baseline apoptosis in the absence of I-BOP (15.3 ± 1.1%, n = 4 for wortmannin, 14.3 ± 1.4%, n = 4 for LY-294002).


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  		Fig. 8.   Effects of PI 3-K inhibitors on thromboxane A2 receptor-induced apoptosis. The extent of apoptosis was measured with TUNEL assay (A) and DNA gel electrophoresis (B). +, Treatment; -, no treatment; I-BOP, 100 nM I-BOP. Data are means ± SD. Results are from 2 to 3 duplicate experiments in A and 4 separate experiments in B. *P < 0.05 vs. I-BOP and zeta -PS treatment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that apoptosis induced by TxA2 receptor stimulation is modulated by PKC-zeta in cultured AVRM. PKC-zeta is specifically translocated by TxA2 receptor stimulation, and PKC-zeta isoenzyme-specific inhibition prevents apoptosis induced by TxA2 stimulation. In addition, the antiapoptotic effect of PKC-zeta suppression appears to be largely mediated by preserved Akt activity. Thus our results indicate that the activation of PKC-zeta by TxA2 receptor stimulation likely promotes apoptosis by suppressing the activation of a critical antiapoptotic factor, Akt. These data imply that isoenzyme-specific suppression of PKC-zeta and/or activation of Akt might be an effective tool to prevent apoptosis of cardiac myocytes in conditions associated with TxA2 stimulation.

Activation of PKC as a result of TxA2 receptor stimulation is not surprising; indeed, PKC has been implicated in various TxA2-linked cellular functions, including increased motility of rabbit tracheal epithelial cells, induction of hypertrophy, endothelin-1 production of rat smooth muscle cells (8, 9), alteration of neuron transmission of rat hippocampus (16), and induction of adhesion molecules in human endothelial cells (17). Interestingly, PKC also mediates agonist-induced TxA2 receptor downregulation in mesangial cells (36). Although extensive work has been done in noncardiac myocytes, little is known about the role of PKC in TxA2 receptor-mediated functions in cardiac myocytes. One report indicates TxA2 mediates bradykinin-induced inositol 1,4,5-trisphosphate production, which results in increased calcium mobilization and contractility, and PKC is involved in this process (25). Our data suggest that PKC is also involved in apoptotic signaling induced by TxA2 stimulation.

The PKC family consisted of 12 different isoenzymes, many of which have common activation domains but observe very different cellular functions. Most studies investigating the role of PKC in TxA2-mediated functions mentioned above have used nonisoenzyme-specific PKC agonists or antagonists, and, therefore, almost no information is available linking TxA2 stimulation to specific PKC isoenzymes. One report using CHO cells suggested that TxA2 receptor desensitization is linked to calcium-sensitive PKC isoenzymes, but this study did not employ PKC isoenzyme-specific agents (41). We have found that PKC-zeta is activated by TxA2 receptor stimulation and this isoenzyme is involved in the process mediating apoptosis. We employed an isoenzyme-specific strategy using cell-permeable PKC-zeta pseudosubstrate, reported to be effective and specific for PKC-zeta inhibition (4, 23). The participation of PKC-zeta in apoptosis has been reported (6) in neuroblastoma cells, and PKC-zeta protein expression is reported (22) to increase in parallel with apoptosis in the same cell type. Recently, digestion and activation of PKC-zeta has been reported (35) as a result of caspase processing, which is postulated to be a mechanism undergoing apoptosis in Hela cells.

We have found that Akt, a universal cell survival factor, is regulated by PKC-zeta . This process is influenced by activation of PI3-K, because inhibition of PI3-K activity prevented the PKC-zeta effect on Akt activity. Akt has been reported to mediate the antiapoptotic effect of calcineurin in adult cardiac myocytes (10) and also that related to beta 2-adrenergic receptor stimulation in neonatal cardiac myocytes (7). The fact that a similar mechanism is described in our study further strengthens the conclusion that Akt serves a potent antiapoptotic function in cardiac myocytes. Although Akt is one of the major signals regulated by PI3-K, our results do not preclude the possibility that the other downstream signals regulated by PI3-K that are different from Akt might have additional survival benefits for apoptosis induced by TxA2 receptor stimulation.

Recently, some reports indicate that TxA2 receptor stimulation modulates ERK activity (14, 28). ERK also is reported to be a potent antiapoptotic factor in cardiac myoyctes (1, 19, 30). However, our experiments did not demonstrate increased ERK activity assessed by phosphorylated forms by immunoblotting in the presence of TxA2 stimulation in our cell culture system. This finding certainly does not exclude the possibility that the subtle ERK activity changes by TxA2 receptor stimulation might be detected by more sensitive ERK activity measurements such as immune complex kinase assay. It is also noteworthy that the reversal of Akt activity suppression by zeta -PS only explains ~60% of suppression of apoptosis induced by TxA2 receptor. This suggests that other antiapoptotic factors and/or other PKC isoenzymes might be involved, albeit to a lesser degree in this process. It is also possible that activation of other PKC isoenzymes not detected at the time points that we used for our study might influence this apoptosic process.

An important caveat is that we used cultured AVRM to investigate mechanisms involved in apoptosis induced by TxA2 receptor stimulation. The exact level (or range) of TxA2 receptor stimulation seen in vitro in cardiac tissue is unknown, because the biological half-life of TxA2 is extremely short (<30 s). Although the culture system is useful to dissect pathways involved as a result of TxA2 receptor activation, the true physiological relevance of our findings needs to be established in intact animal models. In addition, such an investigation has scientific merit, because TxA2 has been reported to modulate ischemia/perfusion induced cardiac dysfunction (28, 40) and myocardial infarct size (40) in intact animal models.

In conclusion, we demonstrate that TxA2 receptor stimulation induces cardiac myocyte apoptosis via activation of PKC-zeta and inhibition of Akt and that the inhibition of PKC-zeta prevents apoptosis via restoration of Akt activity. This observation suggests specific inhibition of PKC-zeta and/or activation of Akt may be potent tools to prevent apoptosis in patients with acute coronary syndromes who demonstrate elevated local TxA2 release.


    ACKNOWLEDGEMENTS

We thank Dr. Jayant Bagai of the Section of Cardiology, University of Illinois at Chicago for technical assistance. We also thank Dr. Daria Mochly-Rosen, Stanford University, for the generous gift of cell-permeable PKC-zeta pseudosubstrate peptide and cell-permeable epsilon V1-2 peptide as well as for helpful comments.


    FOOTNOTES

This study was supported by a Grant-in-Aid from the American Heart Association, Midwest Affiliate (to Y. Shizukuda), National Heart, Lung, and Blood Institute Grant HL-62230 (to P. M. Buttrick), and funds dedicated to the program in cardiovascular sciences at the University of Illinois at Chicago, Chicago, IL.

Address for reprint requests and other correspondence: Y. Shizukuda, Section of Cardiology, Univ. of Illinois at Chicago, M/C 787, 840 S. Wood St., Chicago, IL 60612 (E-mail: shizukud{at}uic.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 June 2001; accepted in final form 14 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 282(1):H320-H327
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