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Am J Physiol Heart Circ Physiol 291: H854-H860, 2006. First published February 24, 2006; doi:10.1152/ajpheart.01205.2005
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Inhibition of phospholipase C-{gamma}1 augments the decrease in cardiomyocyte viability by H2O2

Rabban Mangat,1 Tushi Singal,2 Naranjan S. Dhalla,2 and Paramjit S. Tappia1,3

1Department of Human Nutritional Sciences, Faculty of Human Ecology, 2Department of Physiology, Faculty of Medicine, and 3Department of Human Anatomy and Cell Science, University of Manitoba, and Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Winnipeg, Canada

Submitted 14 November 2005 ; accepted in final form 23 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was conducted to examine the role of a major cardiac phospholipase C (PLC) isozyme, PLC-{gamma}1, in cardiomyocytes during oxidative stress. Left ventricular cardiomyocytes were isolated by collagenase digestion from adult male Sprague-Dawley rats (250–300 g) and treated with 20, 50, and 100 µM H2O2 for 15 min. A concentration-dependent (up to 50 µM) increase in the mRNA level and membrane protein content of PLC-{gamma}1 was observed with H2O2 treatment. Furthermore, PLC-{gamma}1 was activated in response to H2O2, as revealed by an increase in the phosphorylation of its tyrosine residues. There was a marked increase in the phosphorylation of the antiapoptotic protein Bcl-2 by H2O2; this change was attenuated by a PLC inhibitor, U-73122. Although both protein kinase C (PKC)-{delta} and -{epsilon} protein contents were increased in the cardiomyocyte membrane fraction in response to H2O2, PKC-{epsilon} activation, unlike PKC-{delta}, was attenuated by U-73122 (2 µM). Inhibition of PKC-{epsilon} with inhibitory peptide (0.1 µM) prevented Bcl-2 phosphorylation. Moreover, different concentrations (0.05, 0.1, and 0.2 µM) of this peptide augmented the decrease in cardiomyocyte viability in response to H2O2. In addition, a decrease in cardiomyocyte viability, as assessed by trypan blue exclusion, due to H2O2 was also seen when cells were pretreated with U-73122 and was as a result of increased apoptosis. It is therefore suggested that PLC-{gamma}1 may play a role in cardiomyocyte survival during oxidative stress via PKC-{epsilon} and phosphorylation of Bcl-2.

cardiomyocyte viability; phospholipase C; signal transduction

THE PHOSPHOINOSITIDE-SPECIFIC phospholipase C (PLC) isozymes associated with the cardiac sarcolemma (SL) membrane play an important role in activating intracellular signal transduction pathways for the regulation of various cell functions (12, 48, 50). PLC-{gamma}1, a predominant PLC isozyme expressed in the heart (59), is known to be activated by tyrosine phosphorylation (62) and to convert its substrate, phosphatidylinositol 4,5-bisphosphate, into two messenger molecules, inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). While IP3 may serve to enhance the sarcoplasmic reticulum Ca2+ release (19, 26), DAG functions as a potent activator of protein kinase C (PKC) isozymes, which in turn phosphorylate several cardiac proteins (31) that influence cardiomyocyte function (31, 46).

Ischemia-reperfusion (I/R) injury is known to occur during clinical procedures such as coronary bypass surgery, angioplasty, thrombolytic therapy, and cardiac transplantation (9, 16), resulting in myocardial abnormalities (7, 24, 28, 52, 57, 66). We have previously shown that I/R of the isolated rat heart is associated with changes in PLC isozymes (4). Specifically, PLC-{gamma}1 was activated in the first minute of reperfusion of heart subjected to a 30-min period of global ischemia. Some studies have shown that PLC-{gamma}1 undergoes phosphorylation in response to H2O2 in lymphocytes (8), platelets (47), and mouse embryonic fibroblasts (5, 65). Recent evidence has suggested an antiapoptotic role of PLC-{gamma}1 activation in oxidative stress in mouse embryonic fibroblasts (5, 65). Furthermore, PKC has been implicated in the PLC-{gamma}1-mediated survival signaling in these cells (65). However, the functional significance of PLC-{gamma}1 activation in the cardiomyocyte during oxidative stress is not known.

The present study was therefore undertaken to determine the role of PLC-{gamma}1-mediated signal transduction processes in isolated adult rat left ventricular (LV) cardiomyocytes exposed to different concentrations of H2O2, a major oxidant molecule known to be generated during cardiac I/R and considered to contribute significantly to the cellular injury seen during reperfusion (37, 40, 43, 63). The results of this study show for the first time that H2O2 induces activation of PLC-{gamma}1 that mediates the phosphorylation of Bcl-2 via PKC-{epsilon} in adult rat cardiomyocytes. Furthermore, inhibition of PLC-{gamma}1 augments the decrease in cardiomyocyte viability by H2O2, suggesting that PLC-{gamma}1 may play a role in cardiomyocyte survival during oxidative stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cardiomyocyte isolation and stimulation. All experimental protocols for animal studies were approved by the Animal Care Committee of the University of Manitoba, following the guidelines established by the Canadian Council on Animal Care. The LV cardiomyocytes were isolated from male Sprague-Dawley rats (250–300 g) as described previously (54, 60, 61). The final pellet was resuspended in medium-199 (M-199) containing 10% fetal calf serum and plated out onto laminin-coated 100-mm petri dishes at 1 x 106 cells/plate. After a period of 3 h, cells were washed with M-199 and then incubated overnight with 0.5% serum-containing M-199 in a 5% CO2 humidified incubator at 37°C. After 24 h incubation, cells were exposed to H2O2 (20, 50, and 100 µM) for 15 min. At the end of incubation, the medium was removed by aspiration, and the dishes were placed on ice. Cardiomyocytes were scraped off in 2 ml of PBS and collected by centrifugation at 27 g for 1 min. A total membrane fraction was then isolated by homogenizing cardiomyocytes followed by centrifugation at 100,000 g. The final membrane pellet was resuspended and homogenized in 500 µl of buffer containing 250 mM sucrose and 10 mM histidine, pH 7.4, frozen in liquid N2, and stored at –80°C until use. Protein concentrations were determined by the Lowry method as described elsewhere (59).

RNA isolation and semiquantitative PCR. Total RNA was isolated from LV cardiomyocytes by using RNA isolation kit (Life Technologies, ON, Canada) according to the manufacturer's procedures. Reverse transcription (RT) was conducted for 45 min at 48°C by using the Superscript Preamplification System for first-strand cDNA synthesis (Life Technologies) as previously described (3, 4, 15). Primers used for amplification were synthesized as follows: PLC-{gamma}1: 5'- CCTCTATGGAATGGAATTCCG-3' (forward) and 5'- CTAGGGAGGACTCGCTGGAGAACT-3' (reverse). Temperatures used for PCR were as follows: denaturation at 94°C for 30 s, annealing at 62°C for 60 s, and extension at 68°C for 120 s, with a final extension for 7 min; 25 amplification cycles for each individual primer set were carried out. For the purpose of normalization of the data, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers, 5'-TGA AGG TCG GTG TCA ACG GAT TTG-3' (forward) and 5'-GCA TGT CAG ATC CAC AAC GGA TAC-3' (reverse), were used to amplify GAPDH gene as a multiplex with the target genes. The PCR products were analyzed by electrophoresis in 2% agarose gels. The intensity of each band was photographed and quantified by using a Molecular Dynamics STORM scanning system (Amersham Biosciences) as a ratio of a target gene over GAPDH (3, 4, 15).

Western blot. High-molecular-weight markers (Bio-Rad, Hercules, CA) and 20 µg total membrane proteins were separated on SDS-PAGE gels as previously described (4, 15). Separated proteins were transferred onto 0.45-µm polyvinylidene difluoride (PVDF) membrane. PVDF membrane was blocked overnight at 4°C in Tris-buffered saline (TBS) containing 5% skim milk and probed with either mouse monoclonal PLC-{gamma}1 antibody (Upstate Biotechnology, NY) diluted in TBS-T (1:2,000) or with rabbit polyclonal phospho-Bcl-2 antibody (Cell Signaling Technology) diluted in TBS-T (1:1,000) containing 2% skim milk or with mouse monoclonal PKC-{epsilon} antibody (Santa Cruz Biotechnology) diluted in TBS-T (1:1,000) for 1 h at room temperature. Appropriate secondary horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG (Bio-Rad) was diluted 1:3,000 in TBS-T and used as secondary antibody. Protein bands were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Boehringer Mannheim, Laval, Canada). Band intensities of the Western blot were quantified by using a CCD camera imaging densitometer, Bio-Rad GS 800 (Bio-Rad), and corrected for background. In some experiments, Western blotting with PLC-{gamma}1 was performed with immunoprecipitated membrane phosphotyrosyl proteins as previously described (15). Immunoprecipitation was performed with anti-phosphotyrosyl monoclonal antibodies, PY99 (Santa Cruz Biotechnology), 5 µg of antibody to 855 µg membrane extract. In other experiments, Western blotting for phospho-Bcl-2 antibody was conducted after cardiomyocyte pretreatment for 30 min before the addition of H2O2 (50 µM) for 15 min, with 0.1 µM of a PKC-{epsilon} inhibitor peptide [Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr] (29) (Santa Cruz Biotechnology), which was conjugated to Tat carrier peptide [Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg] via a cysteine S-S bond, for efficient transfer of PKC-{epsilon} inhibitor peptide into cells (29, 55).

Assessment of cardiomyocyte viability by trypan exclusion. For cell survival assays, cardiomyocytes were pretreated with the PLC inhibitor U-73122 (0.5, 1, and 2 µM) or with different concentrations (0.05, 0.1, and 0.2 µM) of the PKC-{epsilon} inhibitor peptide-Tat carrier peptide for 30 min and then exposed to H2O2 (50 µM) for 15 min. After treatment, cardiomyocytes were harvested and stained with 0.25% trypan blue for 2 min, and live cells were counted by using a hemocytometer as described elsewhere (65). The percentage of viable cardiomyocytes in the treated cells was determined from cell counts in treated cardiomyocytes divided by the number of cardiomyocyte counts in untreated cells. The reduction in the number of viable cardiomyocytes reflected cell death (65).

Quantification of cardiomyocyte apoptosis and necrosis. Cardiomyocyte apoptosis and necrosis were quantitatively determined by using the Cell Death Detection ELISA kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer's instructions. Briefly, 1 ml of cardiomyocyte suspension (1 x 104 cells) was centrifuged at 300 g for 5 min. The resultant supernatant containing necrotic cardiomyocytes was used for the assessment of necrosis. The resultant cell pellet was incubated in 200 µl lysis buffer for 30 min at room temperature, and the supernatant containing cytoplasmic histone-associated DNA fragments was used for measuring cardiomyocyte apoptosis. Twenty microliters each of these supernatant fractions were added to a streptavidin-coated microtiter plate, followed by 80 µl of the immunoreagent containing anti-histone biotin against histone and anti DNA-peroxidase against DNA, to each well. The plate was gently shaken (300 rpm) for 2 h at 15–25°C, and the unbound antibodies were removed by washing the plate 3x with 250 µl of incubation buffer. One hundred microliters of 2,2'-azino-di(3-ethylbenzthiazolin-sulfonate) reagent were added as a substrate, and the fluorescence was measured, by using a microtiter plate reader, at emission wavelength 450 nm with 490 nm as reference wavelength. Apoptosis was taken as the difference of absorbance (A405nm – A490nm) in the lysed cell supernatant, whereas necrosis was taken as the difference of absorbance (A405nm – A490nm) in the cell supernatant obtained from the first step.

Statistical analysis. All values are expressed as means ± SE. The differences between two groups were evaluated by Student's t-test. The data from more than two groups were evaluated by one-way ANOVA followed by Duncan's multiple comparison test. A probability of 95% or more (P < 0.05) was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of PLC attenuates cardiomyocyte viability in the presence of H2O2. Although treatment of isolated LV cardiomyocytes with different concentrations (20, 50, and 100 µM) of H2O2 resulted in a concentration-dependent decrease (67, 53, and 18%) in the number of viable cardiomyocytes remaining after H2O2 treatment, as assessed by the trypan blue exclusion method (Fig. 1A), pretreatment of cardiomyocytes with different concentrations (0.5, 1, and 2 µM) of the PLC inhibitor U-73122 for 30 min before the addition of H2O2 (50 µM) resulted in a progressive concentration-dependent augmentation of the decrease (55, 33, and 19%) in cardiomyocyte viability due to H2O2 (Fig. 1B). It should be noted that U-73122 (2 µM) in the absence of H2O2 had no effect on the cell viability (Fig. 1B). Furthermore, use of a cell death ELISA kit that distinguishes between cellular apoptosis and necrosis revealed that while H2O2 caused a loss in cardiomyocyte viability due to apoptosis and necrosis, further loss in cardiomyocyte viability due to H2O2 in U-73122-pretreated cells was due only to increased apoptosis (Fig. 1C). These data suggested that PLC activity may have a role in cardiomyocyte viability during oxidative stress. Thus, because PLC-{gamma}1 has been reported to be phosphorylated in response to H2O2 in some cell types (5, 8, 50, 65), it was decided to examine the status of PLC-{gamma}1, by measuring its mRNA level and protein content as well as its phosphorylation in cardiomyocytes in response to H2O2.


Figure 1
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  		Fig. 1. Cardiomyocyte viability after exposure to H2O2 in the absence and presence of U-73122. Values are means ± SE of 5 experiments. Cardiomyocyte viability was determined by trypan blue exclusion after treatment with different concentrations (20, 50, and 100 µM) of H2O2 for 15 min (A) and in cells pretreated for 30 min with different concentrations (0.5, 1.0, and 2.0 µM) of U-73122 before exposure to 50 µM H2O2 for 15 min (B). Cardiomyocyte apoptosis and necrosis were determined after treatment of cardiomyocytes with 50 µM H2O2 for 15 min in cells pretreated for 30 min with and without 2.0 µM U-73122, by a cell death ELISA kit (C) as described in MATERIALS AND METHODS. *P < 0.05 vs. control. #P < 0.05 vs. H2O2 value in the absence of U-73122.

 
Effect of H2O2 on PLC-{gamma}1 in cardiomyocytes. While a concentration-dependent (up to 50 µM) increase in the cardiomyocyte membrane PLC-{gamma}1 protein content was observed (Fig. 2A), a H2O2 concentration-dependent (up to 50 µM) increase in the activation of PLC-{gamma}1, as revealed by an increase in phosphorylation of PLC-{gamma}1 tyrosine residues, was also detected in the cardiomyocyte membrane fraction, with a peak effect occurring at 50 µM H2O2 (Fig. 2B). To understand if the change in the PLC-{gamma}1 protein content could be due to a change in the cardiomyocyte PLC-{gamma}1 mRNA level, RT-PCR was conducted. It can be seen in Fig. 3 that H2O2 treatment of cardiomyocytes resulted in an increase in the PLC-{gamma}1 mRNA level in a concentration-dependent (up to 50 µM) manner.


Figure 2
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  		Fig. 2. Phospholipase C (PLC)-{gamma}1 protein content and phosphorylation in cardiomyocytes treated with and without H2O2. Representative Western blot and quantified data of the membrane PLC-{gamma}1 protein content (A) and the phosphorylated form of PLC-{gamma}1 (B) in cardiomyocytes treated with different concentrations (20, 50, and 100 µM) of H2O2 for 15 min as described in MATERIALS AND METHODS. Values are means ± SE of 5 experiments. *P < 0.05 vs. control (CON).

 

Figure 3
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  		Fig. 3. PLC-{gamma}1 mRNA levels in cardiomyocytes treated with and without H2O2. Representative blot showing the PLC-{gamma}1 mRNA level (% of control) vs. GAPDH in cardiomyocytes treated with different concentrations (20, 50, and 100 µM) of H2O2 for 15 min as described in MATERIALS AND METHODS. Values are means ± SE of 5 experiments. *P < 0.05 vs. control.

 
H2O2 induced phosphorylation of Bcl-2 mediated by PLC-{gamma}1 via PKC-{epsilon}. To examine the signal transduction processes associated with PLC-{gamma}1 activation as well as to investigate if PLC-{gamma}1 has a protective role during cardiomyocyte oxidative stress, the relationship between PLC-{gamma}1 activation and the antiapoptotic protein Bcl-2 in cardiomyocytes treated with different concentrations of H2O2 was studied. Figure 4A shows that H2O2 induced a concentration-dependent (up to 50 µM) increase in the phosphorylation of Bcl-2, with a peak effect occurring at 50 µM H2O2. The phosphorylation of Bcl-2 induced by this concentration of H2O2 was markedly attenuated by the PLC inhibitor U-73122 (2 µM) (Fig. 4B). To identify the mediator of the PLC-{gamma}1 signal to phosphorylation of Bcl-2, the activation of PKC isozymes ({delta} and {epsilon}) was investigated in cardiomyocytes treated with H2O2 (50 µM) in the presence and absence of U-73122 (2 µM). While PKC-{delta} protein content was increased in the cardiomyocytes membrane fraction, after H2O2 treatment, this activation of PKC-{delta} was not prevented by U-73122 (Fig. 5A). However, in contrast, while the PKC-{epsilon} protein content was increased in the membrane fraction, in response to H2O2, its activation was markedly attenuated by U-73122 (Fig. 5B). Furthermore, Fig. 5C shows that the phosphorylation of Bcl-2 induced by H2O2 was prevented by pretreatment of cardiomyocytes with 0.1 µM of a selective PKC-{epsilon} inhibitor peptide. Furthermore, it was also demonstrated that pretreatment of cardiomyocytes with different concentrations of the PKC-{epsilon} inhibitor peptide (0.05, 0.1, and 0.2 µM) for 30 min before the addition of H2O2 (50 µM) resulted in a progressive concentration-dependent augmentation of the decrease in cardiomyocyte viability due to H2O2, as revealed by trypan blue exclusion (Fig. 5D).


Figure 4
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  		Fig. 4. Bcl-2 phosphorylation in cardiomyocytes treated with and without H2O2 and in the absence and presence of U-73122. Representative blot and quantified data show Bcl-2 protein phosphorylation in cardiomyocytes treated with H2O2 (20, 50, and 100 µM) for 15 min (A) and in cardiomyocytes pretreated for 30 min with U-73122 (2.0 µM) before exposure to 50 µM H2O2 for 15 min (B). Values are means ± SE of 5 experiments. *P < 0.05 vs. control. #P < 0.05 vs. H2O2 value in the absence of U-73122.

 

Figure 5
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  		Fig. 5. Protein kinase C (PKC) isozyme and phosphorylated Bcl-2 protein contents in cardiomyocytes treated with and without H2O2 in the presence and absence of U-73122 and PKC-{epsilon} inhibitor peptide. Protein contents of PKC-{delta} (A), PKC-{epsilon} (B), and phosphorylated Bcl-2 (C) were measured in isolated cardiomyocytes pretreated with U-73122 (2 µM) for 30 min or PKC-{epsilon} inhibitor peptide (0.1 µM) for 5 min before the addition of H2O2 (50 µM) to the cardiomyocytes culture medium and further incubated for 15 min as described in MATERIALS AND METHODS. Cardiomyocyte viability in response to 50 µM H2O2 for 15 min (D) was determined by trypan blue exclusion in cells pretreated for 30 min with different concentrations of PKC-{epsilon} inhibitor peptide (IP; 0.05, 0.1, and 0.2 µM). Values are means ± SE of 5 experiments. *P < 0.05 vs. control. #P < 0.05 vs. H2O2 value in the absence of PKC-{epsilon} inhibitor peptide.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously reported that cardiac I/R induces differential changes in PLC isozyme activities, SL protein contents, and mRNA expression levels. Specifically, PLC-{gamma}1 was activated in the first minute of reperfusion of hearts subjected to a 30-min period of global ischemia (4). However, the distinct function of each PLC isozyme in the adult cardiomyocyte has yet to be completely understood. The present study was therefore undertaken to determine some of the mechanisms responsible for the activation of PLC-{gamma}1, as well as to investigate the functional significance of this change under conditions of oxidative stress. While the upregulation of PKC-{epsilon} has been shown to transduce a powerful cell survival signal involving Bcl-2 in response to apoptotic stimuli in a number of different cell types (10, 22, 33, 41, 56), our findings are the first to report that stimulation of PLC-{gamma}1 is required for cardiomyocyte survival during oxidative stress response and involves PKC-{epsilon}-mediated phosphorylation of Bcl-2. The pharmacological blockade of PLC-{gamma}1 with U-73122 resulted in a further loss of cardiomyocytes due to apoptosis. In addition, our findings indicate that PKC-{epsilon} contributes to cell survival during oxidative stress and may be a downstream target of PLC-{gamma}1. Also, our study has shown that myocardial PLC-{gamma}1 is activated in response to H2O2 treatment in a tyrosine kinase-dependent manner. Although cardiomyocyte survival signals downstream of PLC-{gamma}1 are already known (13, 14), this study provides new information on cardiomyocyte survival response and demonstrates that activation of the antiapoptotic pathway is initiated by PLC-{gamma}1 and occurs rapidly in response to H2O2.

A variety of reactive oxygen species (ROS) and oxidant molecules are generated during reperfusion of the ischemic heart, including H2O2 (16, 17). In a clinical setting in which the heart is exposed to transient ischemia followed by coronary reflow, infiltrating polymorphonuclear leukocytes (neutrophils and eosinophils) can also produce H2O2 (18, 20). Some studies have shown mitochondria-dependent production of H2O2 during ischemia (53) and reperfusion (62). The concentrations of H2O2 employed in our study are compatible with those detected in vivo during myocardial I/R (32). It is therefore likely that our present in vitro observations in response to H2O2 may occur in cardiomyocytes during I/R. It should be noted that the degree of activation of PLC-{gamma}1 was less with 100 µM H2O2 compared with lower H2O2 concentrations. It is possible that this concentration is harmful to cardiomyocytes. In fact, low concentrations of ROS and oxidants or exposure for a transient period may stimulate the signal transduction mechanisms for cardiomyocyte survival, whereas high concentrations of ROS and oxidants or exposure for a prolonged period seem to result in cardiomyocyte death (17).

The present study has also shown that PLC-{gamma}1 mRNA level during oxidative stress was increased. Although it is known that genes may be sensitive to regulatory elements or upregulated by transcription factors, which are activated during the ischemic phase, some transcription factors are expressed acutely and chronically in response to hypoxia and I/R (11) and have numerous targets, including, possibly, PLC-{gamma}1. Because our data represent the steady-state level of the PLC-{gamma}1 gene, the enhanced expression could also be due to either increased rate of transcription and/or increased mRNA stability. On the other hand, the H2O2-mediated increase in PLC-{gamma}1 mRNA level was significantly attenuated with high concentration of H2O2 that could represent an increased mRNA degradation and subsequent decreased amounts of PLC-{gamma}1 nonphosphorylated and phosphorylated protein. While such issues could be resolved by using PLC-{gamma}1 small interfering RNA or PLC-{gamma}1 knockouts, these technologies were not used in the present study, and therefore caution should be exercised in the interpretation of the mRNA data. Also it is pointed out that the increase in the membrane contents of PLC-{gamma}1 and PKC-{delta} and -{epsilon} isozymes in response to low concentration of H2O2 most likely represents translocation from the cytosol to membrane compartments (4, 45).

The role of PKC in cardiac I/R is well documented (1, 23, 27, 45, 46, 51, 58). PKC-{epsilon} activation is considered to be crucial to cardioprotection during I/R because isozyme-specific inhibitory peptides are able to abolish protection in response to ischemic preconditioning (27). Furthermore, the targeted disruption of PKC-{epsilon} gene abolishes the infarct size reduction that follows ischemic preconditioning (51). In addition, inhibition of PKC-{delta} during reperfusion provides protection from I/R injury (23, 27). While a similar activation of PLC-{gamma}1 and Bcl-2 phosphorylation has been reported in mouse embryonic fibroblasts in response to H2O2 (5, 65), it has also been demonstrated that the mediator of the signal from PLC-{gamma}1 to Bcl-2 phosphorylation is PKC (5), but the identity of the PKC isoform involved was not determined. However, our results show that PKC-{epsilon} and PKC-{delta} are activated during cardiomyocyte oxidative stress, although it appears that PKC-{epsilon} may be activated by PLC-{gamma}1; inhibition of PLC-{gamma}1 with U-73122 almost completely prevented the activation of PKC-{epsilon}, whereas PKC-{delta} activation was not prevented by U-73122. Furthermore, pretreatment of cardiomyocytes with a PKC-{epsilon} inhibitor peptide prevented Bcl-2 phosphorylation and augmented the loss in cardiomyocyte viability in response to H2O2. Although U-73122 is a nonselective inhibitor of PLC, these data provide evidence that a functional link between PLC-{gamma}1 and PKC-{epsilon} and a protective role during cardiac oxidative stress may exist; however, to further validate this relationship it would be interesting to overexpress both PLC-{gamma}1 and PKC-{epsilon} in cardiomyocytes and examine the effect on viability. The question, however, arises as to why the activation of PKC-{delta} is not prevented by U-73122. It is possible that PKC-{delta} activation is independent of PLC-{gamma}1 under our experimental conditions. We have recently reported that the activities of the major cardiac sarcolemmal phospholipase D (PLD) isozyme, PLD2, and phosphatidate phosphohydrolase (PAP) are increased during I/R (2), suggesting that DAG derived from the PLD-PAP pathway may be involved in activating PKC-{delta}. This possibility warrants further investigation as PKC isozymes have been suggested to be activated specifically by PLC-derived DAG (25, 44), and the in vivo significance of PLD-PAP derived DAG remains to be determined (64). Another possibility is that PKC-{delta} activation in cardiomyocytes may occur through a tyrosine-dependent phosphorylation, a DAG-independent mechanism similar to that reported in other cell types in response to H2O2 (21, 34).

It should be mentioned that in view of our earlier observation that inhibition of PLC improves postischemic recovery of the heart (3), it could be difficult to reconcile this with the findings of the present study and the suggestion that PLC-{gamma}1 may be protective of cardiomyocyte viability during oxidative stress. However, this can be explained on the basis that in hearts subjected to I/R, there is a specific increase in PLC-beta1 activity during the ischemic phase (3, 4), which may be more deleterious for postischemic recovery. Because hearts were pretreated with U-73122 and it was present in the reperfusate, PLC isozyme changes were attenuated in both the ischemic as well as reperfusion phases. Although inhibition of PLC-beta1 improved the recovery of the heart, it is reasonable to assume, given the findings of the present study, that a better recovery would have been observed if PLC-{gamma}1 activity was not inhibited, which, as already indicated, is activated in the first minute of reperfusion (3, 4). In this regard, the adverse effects of PLC-beta1 are indicated because the fibrosis, which occurs in I/R (42), may be mediated by PLC-beta1 (30). Also, prazosin, an {alpha}1-adrenoceptor blocker, has been reported to attenuate myocardial injury in I/R (39) and may be linked to attenuation of PLC-beta1. It is pointed out that PLC-{delta}1, the major cardiac PLC isozyme, is also activated during reperfusion; however, we have recently suggested that this may be due to Ca2+ overload (3).

Although the rapid cell death analyzed by trypan blue exclusion is considered to be mainly due to necrosis, as apoptosis happens much slower, our data have revealed that short-term exposure of cardiomyocytes to H2O2 results in cardiomyocyte death due to necrosis as well as apoptosis. However, pretreatment of cardiomyocytes with U-73122 resulted in a further loss of cardiomyocyte viability due to apoptosis, whereas necrosis was unaffected, implying that PLC may not be involved in cardiomyocyte necrosis in response to H2O2. Therefore, we consider that activation of the antiapoptotic pathway involving Bcl-2 is initiated by PLC-{gamma}1 and occurs rapidly in cardiomyocytes treated with H2O2. In fact, the antiapoptotic role of PLC-{gamma}1 activation, which starts 2 min after H2O2 treatment of mouse embryonic fibroblasts, has been reported (5, 65). It would, however, be interesting to measure changes in early indicators of apoptosis, such as mitochondrial membrane depolarization, under the conditions employed in our studies to fully comprehend the significance of Bcl-2 phosphorylation.

A few other studies have addressed the protective role of PLC-{gamma}1 under conditions of oxidative stress in other cell types. Overexpression of PLC-{gamma}1 in PC12 cells has resulted in suppression of UV-induced apoptosis (6). On the other hand, no protective effect of PLC-{gamma}1 overexpression has been reported in NIH-3T3 cells subjected to H2O2 treatment (35). In addition, epidermal growth factor has been reported to provide protection against oxidants in human epithelial (Caco-2) cells via a PLC-{gamma}1-dependent signaling pathway (36). In conclusion, our findings suggest that exposure of cardiomyocytes to low concentrations of H2O2 for short periods of time initiates a cell survival event that may be mediated by a PLC-{gamma}1-dependent signaling pathway and therefore indicate that PLC-{gamma}1 could emerge as a novel target for cardioprotection during conditions of oxidative stress.


   ACKNOWLEDGMENTS
 
This study was supported by a grant from the Canadian Institutes of Health Research in partnership with the St. Boniface Hospital Research Foundation. T. Singal is a recipient of a University of Manitoba Graduate Fellowship.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. S. Tappia, Laboratory of Cardiac Membrane Biology, Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre (R3020), 351 Tache Ave., Winnipeg, Manitoba, Canada R2H 2A6 (e-mail: ptappia{at}sbrc.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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