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Am J Physiol Heart Circ Physiol 282: H1625-H1634, 2002. First published January 3, 2002; doi:10.1152/ajpheart.00783.2001
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Vol. 282, Issue 5, H1625-H1634, May 2002

Protein kinase C-delta modulates apoptosis induced by hyperglycemia in adult ventricular myocytes

Yukitaka Shizukuda1, Mary E. Reyland3, and Peter M. Buttrick1,2

1 Section of Cardiology, Department of Medicine, and 2 Department of Physiology and Biophysics, University of Illinois, Chicago, Illinois 60612; and 3 Department of Basic Science and Oral Research, University of Colorado Health Sciences Center, Denver, Colorado 80282


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We evaluated the direct effect of hyperglycemia on apoptosis of adult rat ventricular myocytes (ARVM) in vitro. Hyperglycemia (16.5 mM) for 24 h increased apoptosis by greater than threefold (48.2 ± 4.4%, by the TdT-mediated dUTP nick-end labeling method) compared with baseline (14.7 ± 2.5%). Hyperosmolarity with mannitol (11.0 mM) in the presence of 5.5 mM glucose also increased apoptosis by approximately twofold of baseline. Both glucose and mannitol treatment resulted in the membrane translocation of protein kinase C (PKC)-delta , and the activation of PKC-delta was confirmed by immune complex kinase assay. PKC-delta -specific translocation inhibitor peptide (delta V1-1) attenuated only apoptosis induced by hyperglycemia but not by mannitol. A PKC-varepsilon -specific translocation inhibitor peptide (varepsilon V1-1) affected neither type of apoptosis. Moderate overexpression of PKC-delta by adenovirus gene transfer prevented the antiapoptotic effect of delta V1-1. Furthermore, delta V1-1 attenuated the production of reactive oxygen species (ROS) by glucose. Taken together, our results indicate that increased ROS production regulated by PKC-delta is in part responsible for the induction of apoptosis by hyperglycemia and that apoptosis by hyperglycemia is mechanistically different from that by hyperosmolarity.

reactive oxygen species; glucose; mannitol; antioxidant; adenovirus


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DIABETIC PATIENTS are prone to the development of a syndrome of left ventricular (LV) dysfunction that differs from that seen in other chronic diseases (such as hypertension and chronic ischemia) in that the ventricle is not prominently hypertrophied (11, 15, 28, 40). Various factors have been proposed to contribute to this clinical situation, including microvascular abnormalities (14), increased oxidative stress (11), decreased sarcoplasmic reticular calcium uptake (35), and decreased myosin ATPase activity (31); however, molecular mechanisms that potentiate cardiac dysfunction in this context remain largely undefined.

Ventricular myocyte apoptosis has been shown to contribute to LV dysfunction in a number of disease states, including those associated with elevated catecholamines (37) and ischemia/hypoxia (4). It has been reported that hyperglycemia directly induces apoptosis of endothelial cells (2) and neural cells (1). Recently, Fiordaliso et al. (16) described increased apoptosis in the hearts of streptozotocin-induced diabetic rats, which was prevented by angiotensin II blockade in vivo. Therefore, we hypothesized that hyperglycemia might directly cause apoptosis in adult ventricular myocytes and potentially worsen cardiac function in the diabetic heart via loss of ventricular myocytes.

In the present study, we evaluated the effect of hyperglycemia and hyperosmolarity on apoptosis of cultured adult rat ventricular myocytes (ARVM). We also focused on the relationship between apoptosis and protein kinase C (PKC) because various PKC isoenzymes are known to be involved in altered regulatory functions in the hearts of diabetic animals, and PKC has been linked to apoptosis induced by other toxic stimuli such as anticancer drugs (33, 34) and myocardial ischemia/hypoxia (18).


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

Cell culture and treatments. ARVM were harvested from 7- to 9-wk-old male Wistar rats and cultured as previously described (29, 46). ARVM were cultured in ACCIT media [medium 199 with Earle's balanced salts, including 25 mM HEPES and 26 mM NaHCO3 (Sigma; St. Louis, MO) supplemented with 2 mg/ml BSA, 2 mM L-carnitine, 5 mM creatine, 5 mM taurine, 0.1 µM insulin (GIBCO-BRL; Rockville, MD), 100 IU/ml penicillin, and 100 µg/ml streptomycin (GIBCO-BRL)] for 48 h before the protocols were started. The medium was changed every 24 h before the protocols were initiated.

In experiments to investigate the effect of hyperglycemia, glucose (11.0 mM) was added to the media to bring the total glucose concentration to 16.5 mM (including the 5.5 mM glucose contained in the ACCIT culture media) for 24 h to analyze apoptosis and for 30 min to analyze the translocation of PKC. Thirty minutes of stimulation was chosen to assess the activation of PKC because early responses of PKC have been suggested by us (38, 39) and others (7, 18) to modulate subsequent cellular adaptation. Twenty-four hours were chosen to investigate the rate of apoptosis based on previous reports that assessed the cumulative impact of stress stimuli in ARVM (9, 46). In experiments to investigate the effect of hypersomolarity, mannitol (11.0 mM) was added to the media. This concentration was chosen for our experiments based on the previous investigations of cellular signaling (20, 22). In studies to investigate the role of reactive oxygen species (ROS), either ascorbic acid (100 µM) or catalase (50 U/ml) was added to cell culture. All chemical materials not specified were purchased from Sigma. In some experiments, one-half of the concentration listed above (5.5 mM) was added to the media to investigate the effect of lower concentrations of either glucose or mannitol.

PKC isoenzyme-specific translocation inhibition. PKC-varepsilon and -delta isoenzyme translocation were specifically inhibited by their specific translocation inhibitors [varepsilon V1-2 peptide (amino acid residues 14-21 of PKC-varepsilon ) (7, 18) and delta V1-1 peptide (amino acid residues 1-21 of PKC-delta ) (24), respectively; kindly provided by Dr. Daria Mochly-Rosen, Stanford University]. These peptides were cross-linked via a NH2-terminal Cys-Cys bond to the Drosophia antennapedia-derived carrier peptide to make them cell permeable (7). In some experiments, the carrier peptide alone without linking was used as a control. The peptides (750 nM final concentration) were added to media every 8 h. In our previous study (36), this concentration was shown to inhibit PKC isoenzyme-specific translocation induced by 1 nM phorbol 12-myristate 13-acetate.

Overexpression of PKC-delta by adenovirus-mediated gene transfer. After 12 h of culture, ARVM were infected by a recombinant adenovirus vector containing rat wild-type PKC-delta (6) (generous gift from Dr. Trevor J. Biden, Garvan Institute of Medical Research; Sydney, Australia) or adenovirus vector containing green fluorescent gene (GFP) as a vector control (generous gift from Dr. Mary E. Reyland, University of Colorado Health Sciences Center; Denver, CO) as previously reported (6, 47). Briefly, a half volume of full media containing adenovirus (1.5 ml for 60-mm petri dish and 1 ml for 35-mm petri dish) was added to establish viral concentration at a multiplicity of infection of 100 plaque-forming units/cell. The infected dishes were shaken every 15 min for 1 h, and an additional half volume of full media was then added. The media was replaced 12 h after infection. We observed a >90% infection rate by GFP illumination 36 h after adenovirus infection. The expression level of PKC-delta in infected ARVM was assessed 36 h after infection using immunoblotting and immune complex kinase assay as described below.

Immunoblot analysis of translocation of PKC isoenzymes. Cells were harvested with lysis buffer containing 20 mM Tris · HCl, 2 mM EGTA, 20 mM EDTA (pH 7.5), 20 µM leupeptin, 10 µM E64, 200 µM phenylmethylsulfonyl fluoride (PMSF), and 5 mM dithiothreitol and sonicated. Protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad; Hercules, CA), and 2-3 mg of the equal amount of each ventricular myocyte preparation were subject to differential centrifugation to collect the cytosolic and particulate fraction as previously described (25, 38). Immunoblots were performed using anti-PKC-alpha (Santa Cruz Biotechnology; Santa Cruz, CA), anti-PKC-beta II (Santa Cruz Biotechnology), anti-PKC-delta (Pharmingen/Signal Transduction Laboratories; San Diego, CA), or anti-PKCvarepsilon antibodies (Pharmingen/Signal Transduction Laboratories). The percentages of individual PKC isoenzymes in each fraction were calculated as previously described (25, 38).

Immunoblot analysis of whole cells. Whole cell lysates were prepared by addition of lysis buffer (PBS, 1% NP-40, 0.1% SDS, 200 µM PMSF, 2 µg/ml aprotinin, and 4.2 µM leupeptin). Immunoblots were performed using an anti-PKC-delta (Pharmingen/Signal Transduction Laboratories) and anti-actin antibody (Santa Cruz Biotechnology). The films were developed using a Supersignal chemiluminescence kit (Pierce; Rockford, IL). The photodensity of each band was quantitated using the Gel-Doc system (Bio-Rad).

Immune complex kinase assay of PKC-delta isoenzyme in particulate fractions. The particulate fraction was harvested from 3 to 4 mg of whole cell lysates as described above. The immune complex kinase assay of PKC-delta in these fractions was carried out as previously described (36, 39). Briefly, PKC-delta isoenzyme was immunoprecipitated using an anti-PKCdelta antibody (Pharmigen/Signal Transduction laboratories) from 1.5 mg protein from each particulate fraction. The reaction mixture did not contain additional calcium acetate for assay. The presence of PKC-delta isoenzyme was confirmed by immunoblotting in preliminary studies. PKC-delta isoenzyme activity in the particulate fraction was expressed as that measured relative to unstimulated ARVM.

TdT-mediated dUTP nick-end labeling assay. TdT-mediated dUTP nick-end labeling (TUNEL) assay was carried out as previously described (38). Both ARVM floating in the media and trypsinized cells were collected together and used for the assay. Approximately 5 × 104 ARVM were fixed with 0.5 ml of 4% paraformaldehyde in PBS for 10 min, centrifuged, and then resuspended with 80% ethanol for 24 h. ARVM were placed on slides and air dried overnight. TUNEL assay was performed on slides with a Trevigen TACS 2 TdT (TBL) kit (Trevigen; Gaithersburg, MD). For each slide, the number of TUNEL-positive cells was scored in 12 randomly chosen high-power fields (×400). The number of TUNEL-positive cells was normalized to the total number of cells counted.

DNA gel electrophoresis assay. Gel electrophoresis to detect the DNA ladder was carried out as previously described (38). Briefly, ARVM were washed with PBS and then digested with lysis buffer [10 mM Tris (pH 8.0), 100 mM NaCl, 25 mM EDTA, 05% SDS, and 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). Equal quantities of each sample (6-15 µg) were subjected to electrophoresis on 1.25% agarose gels containing 0.5 µg/ml ethidium bromide.

Measurements of ROS. After the 30 min of stimulation of ARVM with either glucose or mannitol, intracellular ROS levels were measured using florescence of dihydrodichlorofluorescein diacetate (DCF; Calbiochem-Novabiochem; San Diego, CA) as described previously (41, 42). Briefly, cells were loaded with 5 µg/ml DCF, and the fluorescence intensities were quantitated (41). In certain cases, cells were also treated with 50 U/ml of 2 mM N-acetyl-L-cysteine or 50 U/ml catalase for 1 h before stimulation.

Levels of lipid peroxidation in ARVM were also measured after 6 h of these stimulations using a lipid peroxidation assay kit (Calbiochem-Novabiochem) (41).

L-Lactate production. The level of lactate accumulation in cell culture media was assessed with reducing reaction of nicotinamide adenine dinucleotide by lactate as we have previously reported (5). The reaction was carried out according to the manufacturer's instructions (Sigma). The absorbance at 340 nm of each specimen was measured to calculate L-lactate concentration using complete media as the background.

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

Both hyperglycemia and hyperosmolarity induce apoptosis in ARVM. Hyperglycemia with 16.5 mM glucose for 24 h induced a significantly higher rate of apoptosis as measured with the TUNEL assay in ARVM compared with control cells (48.2 ± 4.4%, n = 6, vs. 14.7 ± 2.5%, n = 6, P < 0.05; Fig. 1A). The same level of osmolarity as hyperglycemia (produced with 11.0 mM mannitol) also increased apoptosis compared with controls but to a lesser extent (31.2 ± 3.1%, n = 6, P < 0.05 vs. controls and P < 0.05 vs. glucose-treated cells; Fig. 1A). These results were confirmed with DNA gel electrophoresis (Fig. 1B).


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  		Fig. 1.   Effects of hyperglycemia (16.5 mM) and hyperosmolarity with mannitol (11.0 mM) on apoptosis of adult rat ventricular myocytes. Cultured adult ventricular myocytes were stimulated either with 11.0 mM glucose (G) or 11.0 mM mannitol (M) in the presence of 5.5 mM glucose for 24 h. The extent of apoptosis was measured with the TdT-mediated dUTP nick-end labeling (TUNEL) assay (A) and DNA gel electrophoresis (B). Data are means ± SD. Results from 3 experiments of duplicates. C, control unstimulated cells; L, 100-bp molecular ladder. * P < 0.05 vs. control cells; § P < 0.05 vs. mannitol-treated cells.

Effects of hyperglycemia and hyperosmolarity on PKC isoenzyme translocation. The translocation of individual PKC isoenzymes by both hyperglycemia by glucose treatment and hyperosmolarity was examined to identify an isoenzyme that might play a role in apoptosis induced by these conditions. Both conditions increased PKC-delta translocation after 30 min of stimulation (glucose treatment: 49.8 ± 7.2%, n = 6, P < 0.05; mannitol treatment: 49.2 ± 7.5%, n = 7, P < 0.05; controls: 32.2 ± 7.1%, n = 6; data are the percentages of the isoenzyme in the particulate fraction; Fig. 2A). On the other hand, PKC-alpha , PKC-beta II, and PKC-varepsilon did not significantly translocate at this time point. The enzymatic activation of PKC-delta in particulate fractions by both glucose and mannitol treatment was confirmed by an immune complex kinase assay (287 ± 99%, n = 4, P < 0.05 vs. control cells; 243 ± 80%, n = 4, P < 0.05 vs. control cells, respectively; Fig. 2B).


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  		Fig. 2.   Effects of glucose (Glu) or mannitol (Man) stimulation on protein kinase C (PKC) isoenzyme translocation. A: cultured adult rat ventricular myocytes were stimulated with either glucose (11.0 mM) or mannitol (11.0 mM) in the presence of 5.5 mM glucose for 30 min. The translocation of PKC isoenyzmes was assessed by immunoblotting using isoenzyme-specific antibodies (top). Bottom, the percentage of each PKC isoenzyme in the particulate fraction. B: enzymatic activity of PKC-delta in the particulate fraction was measured with an immune complex kinase assay. The values of enzymatic activity were normalized to those of simultaneously measured unstimulated control (Cont) cells. C, cytosolic fraction; P, particulate fraction. Data are means ± SD. Results are from 4 to 7 separate experiments in A and 4 separate experiments in B. * P < 0.05 vs. control cells.

Time course of PKC-delta translocation by hyperglycemia or hyperosmolarity in ARVM. PKC-delta translocation induced by either glucose or mannitol returned to the baseline level at 1 h after these stimulations (Fig. 3). No significant translocation of PKC-delta was seen at 12 h after stimulation by either glucose or mannitol. Because apoptosis induced by glucose was significantly increased after 12 h of stimulation in ARVM (24.4 ± 3.6%, n = 4 at 12 h, P < 0.05 vs. control; 37.6 ± 2.4%, n = 4 at 16 h, P < 0.05 vs. control; apoptosis was assessed by the TUNEL method) and mannitol also significantly increased apoptosis after 16 h of stimulation (26.2 ± 3.9%, n = 4 at 16 h, P < 0.05 vs. control), we did not perform translocation analyses later than these time points to avoid contamination with large populations of nonviable cells in this assay.


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  		Fig. 3.   PKC-delta translocation was evaluated at 1 and 12 h after stimulation by either 11.0 mM glucose or 11.0 mM mannitol in the presence of 5.5 mM glucose as described in MATERIALS AND METHODS. Data are means ± SD. Results are from 4 to 5 separate experiments.

Effects of low-dose glucose or mannitol stimulation on PKC-delta translocation and apoptosis in ARVM. To investigate whether PKC-delta activation is associated with induction of apoptosis in a different concentration of glucose or mannitol stimulation, ARVM were stimulated with either 5.5 mM glucose or 5.5 mM mannitol in the presence of 5.5 mM glucose in the complete media. With this low concentration, only glucose (45.5 ± 7.4%, n = 4, P < 0.05 vs. control) but not mannitol [34.4 ± 7.9%, n = 5, P = not significant (NS) vs. control] significantly translocated PKC-delta after 30 min of stimulation (Fig. 4A). Interestingly, apoptosis was only significantly increased by glucose treatment (22.6 ± 2.8%, n = 6, P < 0.05 vs. control; apoptosis was measured by TUNEL method; Fig. 4B) but not by mannitol treatment (14.4 ± 2.2%, n = 6) after 24 h of stimulation at this concentration.


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  		Fig. 4.   Effects of low concentration of either glucose or mannitol on PKC-delta translocation and apoptosis. Culture adult rat ventricular myocytes were stimulated either 5.5 mM glucose or 5.5 mM mannitol in the presence of 5.5 mM glucose. A: translocation of PKC-delta , which was measured at 30 min of stimulation. B: apoptosis measured with the TUNEL method after 24 h of stimulation. Data are means ± SD. Results are from 4 to 6 separate experiments in A and from 3 duplicate experiments in B. * P < 0.05 vs. controls.

Effects of brief stimulation of hyperglycemia or hyperosmolarity on apoptosis in ARVM. To establish whether brief stimulation with either glucose or mannitol is sufficient to induce apoptosis in ARVM, cells were stimulated for 30 min by either 11.0 mM glucose or 11.0 mM mannitol subsequent to which the cell culture media were replaced with fresh complete media and cells were cultured for another 23 h and 30 min. This brief stimulation by either glucose or mannitol failed to increase apoptosis, as measured with the TUNEL method (16.0 ± 1.8%, n = 6, P = NS vs. control; 13.9 ± 1.5%, n = 6, P = NS vs. control, respectively). Therefore, sustained stimulation from either glucose or mannitol is essential to increase apoptosis in our cell culture model. This finding is consistent with the time course of observation described above in which apoptosis gradually accumulated, especially after 12 h of stimulation, in the presence of these stimuli.

Effects of specific translocation inhibition of PKC-delta and -varepsilon on apoptosis induced by hyperglycemia or hyperosmolarity in ARVM. The PKC-delta -specific peptide translocation inhibitor (delta V1-1) was used to investigate the role of PKC-delta in apoptosis induced by both hyperglycemia and hypersomolarity. To confirm the specific effect of delta V1-1 on PKC-delta translocation, ARVM were stimulated with either glucose or mannitol in the presence of peptide inhibitors. delta V1-1 inhibited the translocation of PKC-delta by both glucose (glucose alone: 49.8 ± 7.2%, n = 6; glucose with delta V1-1: 28.7 ± 5.5%, n = 4, P < 0.05; data are the percentages of isoenzymes in particulate fraction) and mannitol (mannitol alone: 49.2 ± 7.5%, n = 7; mannitol with delta V1-1: 30.9 ± 8.8%, n = 3, P < 0.05; Fig. 5). In contrast, the PKC-varepsilon -specific translocation inhibitor (varepsilon V1-2) had no effect on PKC-delta translocation (Fig. 5).


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  		Fig. 5.   Effects of PKC-delta - and -varepsilon -specific translocation inhibitor peptides on 11.0 mM glucose- or 11.0 mM mannitol-induced PKC-delta translocation in the presence of 5.5 mM glucose. The percentage of each PKC isoenzyme in the particulate fraction is shown in bottom. delta V1-1, PKC-delta -specific peptide translocation inhibitor; varepsilon V1-2, PKC-varepsilon -specific peptide translocation inhibitor. Data are means ± SD. Results are representative of 3-7 separate experiments. * P < 0.05 vs. controls.

The effects of PKC-delta - and PKC-varepsilon -specific translocation inhibition on apoptosis induced by glucose and mannitol were investigated. PKC-delta -specific translocation inhibition by delta V1-1 prevented apoptosis by hyperglycemia compared with that in cells treated with carrier control peptides (25.8 ± 3.7%, n = 5, vs. 50.4 ± 1.2%, n = 6, P < 0.05; data represent the percentage of apoptotic cells measured with the TUNEL method; Fig. 6A), whereas the PKC-varepsilon -specific translocation inhibitor had no effect (43.5 ± 7.1%, n = 4). In contrast, neither PKC-delta -specific translocation inhibition nor PKC-varepsilon -specific translocation inhibition suppressed apoptosis induced by mannitol compared with cells treated with carrier control peptides (delta V1-1 treatment: 31.5 ± 3.4%, n = 4; varepsilon V1-1 treatment: 32.7 ± 5.8%, n = 4; control peptide treatment: 28.4 ± 2.7%, n = 4; Fig. 7A). These results were confirmed with DNA gel electrophoresis (Figs. 6B and 7B).


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  		Fig. 6.   Effects of PKC-delta translocation inhibition on glucose-induced apoptosis of cultured adult rat ventricular myocytes. Cultured adult rat ventricular myocytes were stimulated with 11.0 mM glucose with 5.5 mM glucose contained in media for 24 h in the presence of control carrier peptides (CP), PKC-delta -specific translocation inhibitor (delta V1-1), or PKC-varepsilon -specific translocation inhibitor (varepsilon V1-2). The extent of apoptosis was measured with both the TUNEL assay (A) and DNA gel electrophoresis (B). Data are means ± SD. Results are from 2 to 3 duplicate experiments. * P < 0.05 vs. control cells.



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  		Fig. 7.   Effects of PKC-delta translocation inhibition on mannitol-induced apoptosis of cultured adult rat ventricular myocytes. Cultured adult rat ventricular myocytes were stimulated with mannitol (11.0 mM) with 5.5 mM glucose contained in media for 24 h in the presence of control carrier peptides, PKC-delta -specific translocation inhibitor (delta V1-1), or PKC-varepsilon -specific translocation inhibitor (varepsilon V1-2). The extent of apoptosis was measured with both the TUNEL assay (A) and DNA gel electrophoresis (B). Data are means ± SD. Results are from 2 to 3 duplicate experiments. * P < 0.05 vs. control cells.

These findings suggest that the membranous translocation of PKC-delta is essential for the apoptosis signal induced by hyperglycemia, but it is not required for apoptosis induced by hyperosmolarity alone.

Effects of PKC-delta overexpression on apoptosis induced by hyperglycemia and hyperosmolarity in ARVM. To investigate the role of PKC-delta on apoptosis induced by glucose and mannitol, adenovirus-mediated PKC-delta overexpression was used. An adenovirus vector encoding GFP alone was used as a control. Infection of adenovirus vector encoding wild-type PKC-delta resulted in a moderate level of PKC-delta overexpression compared with vector-infected controls (384 ± 116%, n = 3, P < 0.05; Fig. 8A). This level of overexpression increased PKC-delta kinase activity in the particulate fraction by ninefold (901 ± 307%, n = 3, in PKC-delta overexpression vs. 102 ± 17%, n = 3, in vector control infection, P < 0.05; values are normalized to unstimulated control cells). This moderate level of PKC-delta overexpression did not influence the rate of apoptosis induced by either glucose or mannitol (Fig. 8B). However, strikingly, the overexpression of PKC-delta was sufficient to override the antiapoptotic effect of delta V1-1 in the presence of hyperglycemia (46.9 ± 2.6%, n = 4, vs. 24.6 ± 3.1%, n = 4, P < 0.05; Fig. 8B). These findings further indicate that PKC-delta per se, not a nonspecific effect of delta V1-1 peptide, is involved in the apoptosis signal induced by hyperglycemia.


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  		Fig. 8.   Effects of adenovirus vector-mediated PKC-delta overexpression on apoptosis induced by either glucose or mannitol. Cultured adult rat ventricular myocytes were infected with either control vector adenovirus (vector) or adenovirus vector encoding wild-type rat PKC-delta for 36 h before cells were stimulated with either glucose (11.0 mM) or mannitol (11.0 mM) in the presence of 5.5 mM glucose for 24 h. A: expression level of PKC-delta by immunoblotting (top) and PKC-delta kinase activity in the particulate fraction (values are normalized to unstimulated control cells). Open bars, control vector-infected cells; solid bars, PKC-delta wild-type-infected cells. * P < 0.05 vs. control. B: rate of apoptosis measured with the TUNEL method of cultured adult rat ventricular myocytes treated with either 11.0 mM glucose or 11.0 mM mannitol with 5.5 mM glucose in the presence or absence of PKC-delta -specific translocation inhibitor (delta V1-1). Data are means ± SD. * P < 0.05 vs. control vector-infected cells.

Involvement of ROS in apoptosis induced by hyperglycemia. Treatment of ARVM with the antioxidant ascorbic acid and catalase significantly attenuated the apoptosis induced by hyperglycemia (36.3 ± 1.6%, n = 4, P < 0.06; 37.6 ± 1.8%, n = 4, P < 0.05, respectively; data represent the percentage of apoptotic cells measured with the TUNEL method; Fig. 9A). However, both treatments failed to reduce apoptosis induced by mannitol (Fig. 9A). Thus increased ROS is in part responsible for increased apoptosis by hyperglycemia but not mannitol.


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  		Fig. 9.   Involvement of reactive oxygen species (ROS) in apoptosis induced by hyperglycemia. Cultured adult rat ventricular myocytes were stimulated with either glucose (11.0 mM) or mannitol (11.0 mM) in the presence or absence of antioxidant drugs. A: level of apoptosis assessed with the TUNEL method. * P < 0.05 vs. cells treated with glucose alone. B: level of ROS assessed with fluorescence of dihydrodichlorofluorescein diacetate (DCF). * P < 0.05 vs. unstimulated control cells; § P < 0.05 vs. cells treated with glucose. Asc, ascorbic acid (100 µM) treatment; Cat, catalase (50 U/ml) treatment. The media used contained 5.5 mM glucose. Results are from 2 to 3 duplicate experiments.

Effects of specific translocation inhibition of PKC-delta and -varepsilon on production of ROS. The level of ROS was significantly elevated in ARVM treated with glucose (Fig. 9B). This finding was also supported by increased lipid peroxidation in ARVM treated with glucose (Fig. 10). In contrast, mannitol treatment did not increase the level of ROS. Inhibition of PKC-delta translocation, but not PKC-varepsilon translocation, in the presence of hyperglycemia resulted in significant attenuation of the ROS levels measured with DCF (Fig. 9B) and lipid peroxidation (Fig. 10). In contrast, neither delta V1-1 nor varepsilon V1-2 affected the production of ROS in the presence of mannitol. In addition, both pretreatment with 2 mM N-acetyl-L-cysteine and 50 U/ml catalase of ARVM suppressed glucose-induced ROS production (data not shown). Therefore, translocation of PKC-delta is required for increased ROS production in the presence of hyperglycemia.


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  		Fig. 10.   Lipid peroxidation of cultured adult rat ventricular myocytes. The extent of lipid peroxidation was measured with optical absorbance at 586 nm and normalized to the amount of protein used for each assay. The media used contained 5.5 mM glucose. Results are from 4 to 5 separate experiments. Data are means ± SD. * P < 0.05 vs. unstimulated control cells; § P < 0.05 vs. cells treated with glucose.

Production of L-lactate in the presence of either hyperglycemia or hyperosmolarity in ARVM. The production of L-lactate, the byproduct of glucose metabolism, was assessed in cell culture media in the presence of either glucose or mannitol. After 12 h of stimulation, L-lactate was significantly elevated in the glucose treatment group (0.52 ± 0.16 mM, n = 6) compared with unstimulated cells (0.21 ± 0.12 mM, n = 6; Fig. 11) but not in the mannitol group (0.35 ± 0.12 mM, n = 6). Our finding suggests that intermediate metabolites from glucose utilization are significantly elevated in the glucose-treated group. Despite the increase in lactate accumulation, pH of the media was similar among these groups (at 1 h: 7.33 ± 0.015 in controls, n = 3; 7.32 ± 0.010 with glucose treatment, n = 3; 7.33 ± 0.015 with mannitol treatment, n = 3; at 12 h: 7.31 ± 0.006 in controls, n = 3; 7.30 ± 0.010 with glucose treatment, n = 3; 7.30 ± 0.012 with mannitol treatment, n = 3).


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  		Fig. 11.   The lactate concentration of media was analyzed as described in MATERIALS AND METHODS at 1 and 12 h after either 11.0 mM glucose or 11.0 mM mannitol stimulation in the presence of 5.5 mM glucose. Data are means ± SD. Results are from 2 triplicate experiments. * P < 0.05 vs. control cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that both hyperglycemia and hyperosmolarity by mannitol induces apoptosis in cultured ARVM. The extent of apoptosis is much greater in cells subjected to hyperglycemia than to hyperosmolarity. Both stimuli translocate PKC-delta to the membrane fractions, and their activation of PKC-delta was confirmed by an immune complex kinase assay of the particulate fractions; however, PKC-delta translocation is required for apoptosis induced by glucose but not by mannitol. In addition, PKC-delta translocation is related to the increased production of ROS, and ROS are in part responsible for the apoptosis induced by hyperglycemia. Taken together, these findings indicate that the increased ROS production related to PKC-delta translocation is in part responsible for the increased apoptosis in the presence of hyperglycemia and that hyperosomolarity alone is not sufficient to increase ROS. Therefore, the mechanisms mediating apoptosis induced by hyperglycemia significantly differ from those induced by hyperosmolarity alone.

Two different and complementary approaches (PKC isoenzyme-specific peptide translocation inhibition and adenovirus-mediated overexpression) were used to elucidate the requirement of PKC-delta for apoptosis in the presence of hyperglycemia. In addition, PKC-varepsilon seems not to be involved in this type of apoptosis. While our study is the first to link PKC-delta and hyperglycemia-associated apoptotic cell death of ventricular myocytes, PKC-delta has been reported to participate in both apoptosis (13, 44) and nonapoptotic cell death (38) in other cell models. PKC-delta overexpression significantly enhances apoptosis induced by stress stimuli such as ultraviolet light and anticancer drugs in a variety of cell culture models (13, 34). In addition, we have previously reported that PKC-delta overexpression in endothelial cells significantly increases nonapoptotic cell damage induced by hypoxia (38). The lack of a PKC-varepsilon translocation inhibition effect is an important negative result because the activation of this isoenzyme has been shown to both induce and protect cells from apoptotic cell damage (8, 19, 27, 32) depending on the nature of initial stimuli and the cell types involved.

In our study, moderate overexpression of PKC-delta did not further increase apoptosis beyond that seen with either glucose or mannitol. This further supports the finding that mannitol-induced apoptosis is not PKC-delta dependent. However, PKC-delta overexpression could conceivably have enhanced the apoptosis induced by glucose, but did not. This may indicate either that the apoptosis induced by hyperglycemia and PKC-delta was already maximized or that other biochemical sequelae of hyperglycemia are rate-limiting steps so that apoptosis cannot be enhanced further.

We have shown that the translocation of PKC-delta resulted in increased ROS production in the presence of glucose and that apoptosis induced by hyperglycemia is in part related to elevated ROS. The association of the specific PKC isoenzyme PKC-delta as a modulator of ROS production in ARVM is a novel observation made possible by the use of highly specific translocation inhibitors with PKC inhibitors. This approach using PKC isoenzyme-specific translocation inhibitors is now well established and provides a major advantage over the use of nonspecific pharmacological blockers or the as-yet theoretical approach of overexpression of dominant negative constructs in our cell culture system. Hyperglycemia has been reported to increase ROS production in other cell types (10, 12). PKC has been reported to participate in the generation of ROS through NAD(P)H activation in cultured smooth muscle cells (23). Importantly, ROS are also known to induce apoptosis (45). In addition, PKC could further enhance the toxicity of ROS because PKC activation is reported to reduce the activity of nuclear factor-kappa B, which is a survival signal against apoptosis (3, 21). Therefore, our findings further support these previous results obtained from experiments using nonventricular myocytes.

While our data clearly describe a role for ROS production in hyperglycemia-induced apoptosis, this does not totally explain the effect of PKC-delta . We can speculate as to possible additional explanations. It has been reported in primary rat aortic vascular smooth muscle cells that 16.5 mM glucose activates p38 mitogen-activated protein kinase (MAPK) in a PKC-delta -dependent manner (22), whereas mannitol (11.0 mM) does not (22). p38 MAPK has been reported to promote apoptosis of cardiac myocytes induced by ischemia-reperfusion (30) and doxorubicin (26), and similar proapoptotic effects by p38 MAPK activation have been reported in various cell types including neurons (17) and adipocytes (43). If the differential activation of p38 MAPK between glucose and mannitol treatment is seen in our model (and if this activation is PKC-delta dependent), this could explain why PKC-delta is linked to apoptosis induced by glucose but not by mannitol. We did try to investigate this hypothesis; however, the level of p38 MAPK in our cell system was extremely low (data not shown) compared with other MAPK subfamily members such as extracellular signal-regulated kinase or c-Jun NH2-terminal kinase, which makes further investigation difficult. It is also possible that other molecular signals specifically induced by hyperglycemia that are different from p38 MAPK might be required for enhanced apoptosis by hyperglycemia because PKC-delta is also activated by mannitol without further enhancing apoptosis with this stimulus. The evidence that a brief period of exposure to hyperglycemia (30 min) is not sufficient to induce significant apoptosis further suggests that persistent glucose-mediated cellular consequences such as stress-activated intracellular kinases or related molecules are required in addition to PKC-delta activation to enhance apoptosis in our model. Such factors may also be driven by metabolites such as elevated L-lactate seen in our model.

An important caveat is that we used a primary cell culture model to investigate the effect of hyperglycemia and hyperosmolarity on apoptosis of ventricular myocytes. This does not reflect the complex nature of an in vivo organ system; however, it allows investigation of the cell-specific effects of hyperglycemia on apoptosis and intracellular signaling independent of cell-to-cell interactions and circulating neurohumoral factors. However, it is clear that our in vitro observation needs to be scrutinized in an in vivo animal model in which hyperglycemia and PKC-delta activation can be independently manipulated to evaluate the clinical importance of our findings.

In summary, we demonstrated that both hyperglycemia and hypersomolarity induce apoptosis in ARVM, although hyperglycemia enhances apoptosis to a far greater extent. Both hyperglycemia and hyperosmolarity translocate and activate PKC-delta ; however, only apoptosis induced by glucose is PKC-delta dependent, whereas that induced by hyperosomolarity is not. PKC-delta regulates the production of ROS in the presence of hyperglycemia, and this effect is in part responsible for the increased apoptosis by hyperglycemia. These findings raise the possibility that isoenzyme-specific translocation inhibition of PKC-delta and/or antioxidant therapies may have a salutary effect to attenuate diabetes-induced cardiac dysfunction by preventing apoptosis in clinical settings.


    ACKNOWLEDGEMENTS

We sincerely appreciate the technical support of Dr. Tuan A. Pham (Section of Cardiology, University of Illinois at Chicago). We thank Dr. Trevor J. Biden (Garvan Institute of Medical Research, Sydney, Australia) for the generous gift of adenovirus vector containing rat wild-type PKC-delta , Dr. Daria Mochly-Rosen (Stanford University) for the generous gift of cell-permeable delta V1-1 and varepsilon V1-2 peptides and helpful comments, and Dr. Lawrence A. Frohman (Department of Medicine, University of Illinois at Chicago) for valuable comments. We sincerely appreciate the technical advice of Angela Matassa (Department of Basic Science and Oral Research, University of Colorado).


    FOOTNOTES

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

Address for reprint requests and other correspondence: Y. Shizukuda, Sect. 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.

First published January 3, 2002;10.1152/ajpheart.00783.2001

Received 30 August 2001; accepted in final form 24 December 2001.


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Am J Physiol Heart Circ Physiol 282(5):H1625-H1634
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