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Am J Physiol Cell Physiol 290: C208-C221, 2006. First published August 31, 2005; doi:10.1152/ajpcell.00075.2005
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Role of calreticulin in the sensitivity of myocardiac H9c2 cells to oxidative stress caused by hydrogen peroxide

Yoshito Ihara,1,2 Yoshishige Urata,1 Shinji Goto,1 and Takahito Kondo1

1Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki; and 2Core Research for Evolutional Science and Technology, Japan Science & Technology Agency, Kawaguchi, Japan

Submitted 22 February 2005 ; accepted in final form 29 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Calreticulin (CRT), a Ca2+-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac apoptosis in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In the present study, the effect of overexpression of CRT on susceptibility to apoptosis under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. Under oxidative stress due to H2O2, the CRT-overexpressing cells were highly susceptible to apoptosis compared with controls. In the overexpressing cells, the levels of cytoplasmic free Ca2+ ([Ca2+]i) were significantly increased by H2O2, whereas in controls, only a slight increase was observed. The H2O2-induced apoptosis was enhanced by the increase in [Ca2+]i caused by thapsigargin in control cells but was suppressed by BAPTA-AM, a cell-permeable Ca2+ chelator in the CRT-overexpressing cells, indicating the importance of the level of [Ca2+]i in the sensitivity to H2O2-induced apoptosis. Suppression of CRT by the introduction of the antisense cDNA of CRT enhanced cytoprotection against oxidative stress compared with controls. Furthermore, we found that the levels of activity of calpain and caspase-12 were elevated through the regulation of [Ca2+]i in the CRT-overexpressing cells treated with H2O2 compared with controls. Thus we conclude that the level of CRT regulates the sensitivity to apoptosis under oxidative stress due to H2O2 through a change in Ca2+ homeostasis and the regulation of the Ca2+-calpain-caspase-12 pathway in myocardiac cells.

apoptosis; calcium; endoplasmic reticulum

CALRETICULIN (CRT) is a Ca2+-binding molecular chaperone expressed in the endoplasmic reticulum (ER) of a wide variety of eukaryotic cells (35). CRT is involved in many biological processes, including the regulation of Ca2+ homeostasis and intracellular signaling, glycoprotein folding, cell adhesion, gene expression, and nuclear transport (17, 23, 35).

CRT is well expressed in the embryonic rat heart, but its expression is suppressed after birth (21). It has been shown that CRT is essential for cardiac development in mice (33, 45). CRT-deficient embryonic cells showed an impaired nuclear import of nuclear factor of activated T-cell type 3 (NF-AT3), a transcription factor, indicating that CRT functions in cardiac development as a component of the Ca2+/calcineurin/NF-AT/GATA-4 transcription pathway (33). On the other hand, CRT-transgenic mice experience complete heart block and sudden death (42). The CRT-dependent cardiac block involves an impairment of both the L-type Ca2+ channel and gap junction connexins 40 and 43 (Cx40 and Cx43, respectively). Also observed was a decrease in phosphorylated Cx43 in the CRT-transgenic heart, suggesting that the functions of protein kinases are altered via the regulation of Ca2+ homeostasis. CRT is also overexpressed in rat cardiomyocytes under pressure overload cardiac hypertrophy, implying some dysfunction of cardiomyocytes related to the overexpression (51). Furthermore, in cultured myocardiac H9c2 cells, overexpression of CRT after gene transfection promoted apoptosis during cardiac differentiation (24). In that study, the expression of protein phosphatase 2A (PP2A), a Ser/Thr protein phosphatase, was involved in altering the regulation of Akt signaling in H9c2 cells overexpressing CRT via an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i). Recently, we also reported that Akt signaling is important for cytoprotection against oxidative stress (39) and that a long-term change in [Ca2+]i regulates PP2Ac-{alpha} gene transcription via the cAMP response element, resulting in a change in the activation status of Akt and leading to altered susceptibility to apoptosis (56). These studies suggest that CRT plays a vital role in myocardiac development and function, although the mechanism of this phenomenon has not been clarified fully.

An increasing body of evidence suggests that apoptosis plays an important role in cardiac development and disease (9, 12). Apoptosis occurs during reperfusion after ischemia in a variety of organs, including the heart (4). Oxidative stress with reactive oxygen species generated during ischemia-reperfusion injury is implied in the mechanism of cardiac damage (4). However, the biological significance of CRT expression levels in cardiomyocytes under oxidative stress has not been revealed to date.

In the present study, we have investigated the biological role of CRT using rat myocardiac H9c2 cells transfected with the CRT gene. We show that the level of CRT alters the sensitivity to apoptosis under oxidative stress with H2O2 through a change in Ca2+ homeostasis and Ca2+-dependent signaling of the calpain-caspase-12 pathway in myocardiac cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Antibodies against CRT, calnexin (CNX), binding protein (BiP; glucose-regulating protein 78, Grp78), and ER-specific protein 57 (ERp57)/Grp58 were purchased from Stressgen (Victoria, BC, Canada). Antibodies against GAPDH, caspase-12, and caspase-3 were obtained from Chemicon International (Temecula, CA), MoBiTec (Gottingen, Germany), and Cell Signaling Technology (Beverly, MA), respectively. Peroxidase-conjugated secondary antibodies against IgG of rabbit and mouse were purchased from Dako (Glostrup, Denmark). The other reagents used in the study were all of high grade and were obtained from Sigma or Wako Pure Chemicals (Osaka, Japan).

Cell lines and culture. H9c2 cells, a clonal cell line derived from embryonic rat heart, were obtained from the American Type Culture Collection (no. CRL-1446). H9c2 cells that had been transfected with the expression vector for mouse CRT cDNA were described previously (24). Two cell lines (CRT-S2 and CRT-S8) expressing high levels of CRT protein were used in the study. A 0.6-kb restriction fragment with EcoRI containing the translation initiation site was cut from the vector pcDNA3.1/mCRT (24) and inserted in the reverse orientation into pcDNA3.1 (Invitrogen) to obtain antisense CRT (20). The antisense cDNA expression vector was also transfected into H9c2 cells to establish a cell line (CRT-AS) in which the expression of CRT was suppressed (20). The established cell lines were used between passages 12 and 18. Cells were cultured in DMEM supplemented with 10% FCS in a humidified atmosphere of 95% air-5% CO2 at 37°C. To induce oxidative stress, cells were cultured with media containing different concentrations of H2O2.

Immunoblot analysis. Cultured cells were harvested and lysed in lysis buffer A (20 mM Tris·HCl, pH 7.2, 130 mM NaCl, and 1% Nonidet P-40) including protease inhibitors (in µM: 20 4-amidinophenylmethanesulfonyl fluoride, 50 pepstatin, and 50 leupeptin). Protein samples were electrophoresed on 10% SDS-polyacrylamide gels under reducing conditions and then transferred onto a nitrocellulose membrane as described previously (19). The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS; in mM: 10 Tris·HCl, pH 7.5, and 150 NaCl) and then incubated at 4°C overnight with primary antibody in TBS containing 0.05% Tween 20. The blots were coupled with the peroxidase-conjugated secondary antibodies, washed, and then developed using the ECL detection kit (Amersham Biosciences) according to the manufacturer's instructions. The intensity of protein bands was quantified densitometrically, and the value was estimated relative to that for GAPDH.

Fluorescence microscopy. Cells (50,000/ml) were grown on Lab-Tek chamber slides (Nunc) for 24 h. They were fixed with 4% paraformaldehyde in PBS (pH 7.2) and permeabilized for 10 min with PBS containing 1% Triton X-100. The cells were then blocked with 1% BSA in PBS, incubated with the antibody for 1 h, and washed with PBS containing 1% BSA. The immunoreactive primary antibodies were visualized using FITC-conjugated anti-rabbit immunoglobulins (Cappel). After being washed, the stained cells were mounted in VectaShield medium. A Zeiss Axioskop2 (Zeiss, Jena, Germany) with illumination for epifluorescence was used for fluorescence microscopic analysis.

Cell viability assay. The viability of cultured cells was evaluated by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (38). Cells (5,000–10,000) were placed into 100 µl of medium per well in 96-well plates and cultured overnight. After cells were treated with H2O2, 10 µl of 0.5% MTT solution was added and the cells were incubated for 4 h. The reaction was stopped by adding 100 µl of lysis buffer B (20% SDS and 50% N,N-dimethyl formamide, pH 4.7), and then cell viability was evaluated by measuring the absorbance at 570 nm using a microplate reader.

Lactate dehydrogenase release assay. After H2O2 treatment, the incubation medium was collected and centrifuged at 10,000 g for 20 min, and the supernatant was stored at 4°C for the lactate dehydrogenase (LDH) activity assay. In untreated cells, the medium was removed and the same volume of lysis buffer A was added to the cells. The cells were lysed by trituration and centrifuged as described above, and then the supernatant was used to assay the LDH activity of all cells. LDH activity was measured spectrophotometrically using an LDH assay kit (MTX "LDH"; Kyokuto Pharmaceutical, Tokyo, Japan) according to the manufacturer's instructions.

Apoptosis assay. Apoptosis was detected by performing flow cytometry using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) method (11) with the ApopTag Plus fluorescein in situ apoptosis detection kit (Chemicon International) as described previously (56). Morphological changes of nuclei in apoptotic cells were also characterized using fluorescence microscopy. Cells (50,000/ml) were grown on Lab-Tek chamber slides for 24 h. After undergoing H2O2 treatment, cells were fixed with 4% paraformaldehyde in PBS. The cells were stained with 2 µg/ml Hoechst 33342 (Molecular Probes, Eugene, OR) in PBS for 5 min so that we could visualize the nuclei. After being rinsed with PBS, the slides were examined using fluorescence microscopy as described above.

Measurement of cytoplasmic free Ca2+. [Ca2+]i was measured using a dual-excitation wavelength spectrofluorophotometer (RF-5500; Shimadzu, Kyoto, Japan) with fura-2 essentially as described previously (6, 36), but with a slight modification. Briefly, cultured cells on glass coverslips were loaded with 5 µM fura-2 AM (Dojindo, Kumamoto, Japan) for 20 min in Earle's balanced salt solution (EBSS; in mM: 26 NaHCO3, pH 7.4, 1 NaH2PO4, 5.4 KCl, 116 NaCl, 5.5 glucose, and 2 CaCl2) in the presence of 0.01% Pluronic acid F-127. After being washed four times with EBSS, the coverglass was positioned at a 45° angle to both excitation and emission light paths in a quartz cuvette containing 3.5 ml of fresh EBSS. Fura-2 fluorescence was determined at 37°C using the spectrofluorophotometer operating at an emission wavelength of 505 nm and excitation wavelengths of 340 and 380 nm. The maximal signal (Rmax) was obtained by adding ionomycin at a 4 µM final concentration. Subsequently, the minimal signal (Rmin) was obtained by adding EGTA at a 10 mM final concentration, followed by Tris-free base to a 30 mM final concentration, to increase the pH to 8.3. R is the ratio (F1/F2) of the fluorescence of excitation at 340 nm and emission at 505 nm (F1) to that of excitation at 380 nm and emission at 505 nm (F2). The actual [Ca2+] was calculated as Kd x (R – Rmin)/(Rmax – R) x Sf2/Sb2, with Kd = 224 nM (16). The Sf2-to-Sb2 ratio is the ratio of fura-2 fluorescence at 380 nm in Ca2+-free medium to that in Ca2+-replete medium.

Assays for uptake and release of Ca2+ in the cell. The uptake of Ca2+ was measured radiometrically using the Millipore filtration technique as described previously (49), with a slight modification. The cells were cultured with the medium containing H2O2 for the periods indicated and then washed with EBSS and cultured for 10 min in EBSS containing 45Ca2+ (5 µCi/ml). Cells were detached from the culture wells using trypsinization buffer (0.25% trypsin and 0.02% EDTA in EBSS), and the cell suspension was filtered through a 0.45-µm nitrocellulose filter (Bio-Rad Laboratories, Hercules, CA) under vacuum conditions. The filters were rinsed twice with 0.5 ml of washing buffer (in mM: 10 HEPES, pH 7.4, 150 KCl, 2 EGTA, and 2.5 MgCl2). 45Ca2+ uptake was calculated by measuring radioactivity and standardized according to protein concentrations. For the 45Ca2+ release assay, cells were cultured for 48 h with the medium containing 45Ca2+ (1 µCi/ml). After being washed with EBSS, the cells were incubated with EBSS containing H2O2. Aliquots were collected at the time points indicated and centrifuged. The radioactivity was measured in the supernatant as the amount of Ca2+ released from the cell.

Enzyme assay. Caspase-12 activity was measured with Ala-Thr-Ala-Asp-7-amino-4-trifluoromethylcoumarin (AFC) as a substrate by using a caspase-12 fluorometric assay kit (BioVision, Mountain View, CA) according to the manufacturer's protocol. The assay is based on the detection of cleavage of the substrate, and the activity was quantified using a spectrofluorophotometer to measure fluorescence (excitation, 400 nm; emission, 505 nm) derived from free AFC. Calpain activity was measured using the calpain substrate succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-Leu-Leu-Val-Tyr-AMC) as described by Glading et al. (13), with a slight modification. Cultured cells were treated with or without H2O2 and/or thapsigargin and then harvested and washed with EBSS. Cells were resuspended in EBSS at 2.5 x 105 cells/ml and kept on ice for up to 1 h. In each sample, 250 µl were added to a 2.5-ml quartz cuvette with stirring and allowed to warm to 37°C in the spectrofluorophotometer. At time –1 min, ionomycin in DMSO was added to a final 2.5 µM concentration. DMSO alone was added as a control. At time 0, Suc-Leu-Leu-Val-Tyr-AMC was added to a final 50 µM concentration, and fluorescence (excitation, 360 nm; emission, 460 nm) was measured immediately for 3 min.

Statistical analysis. Statistical analysis was performed as previously recommended (6a) using Student’s t-test or ANOVA (StatView software). Significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Overexpression of CRT enhances cytotoxic sensitivity of H9c2 cells to oxidative stress caused by H2O2. Rat myocardiac H9c2 cells were transfected with the expression vector for mouse CRT cDNA to obtain cell lines overexpressing CRT (24). Figure 1A shows that the expression of CRT increased in the overexpressed cells to ~2.7-fold the level in the parental and mock-transfected (control) H9c2 cells. The transfection had no apparent effect on the expression of other ER proteins such as CNX, BiP, and ERp57. The intracellular localization of CRT was examined using indirect immunofluorescence. As shown in Fig. 1B, immunoreactive signals showed a perinuclear reticular pattern in all cases, including the control and CRT-overexpressing cells, although the signal intensity was increased in the transfectants compared with the control cells. To investigate the effect of overexpression of CRT on the cytotoxic sensitivity of H9c2 cells to oxidative stress, control and CRT-overexpressing cells were exposed to different concentrations of H2O2 for 1 h and then cell viability was examined by performing an MTT assay as described in MATERIALS AND METHODS. One hour of exposure to H2O2 caused cell damage in a dose-dependent manner, and the cytotoxic effect was enhanced more in the gene-transfected cells (i.e., CRT-S2 and CRT-S8) than in the controls (i.e., parental and control vector-transfected cells) (Fig. 2A, left). As shown in Fig. 2A, right, in CRT-overexpressing cells, the cell viability was markedly reduced after 1-h exposure to 50 µM H2O2, although less reduction was observed in control cells. However, in both control and gene-transfected cells, viability was similarly suppressed after 4 h of exposure to 50 µM H2O2. Next, control and CRT-overexpressing cells were exposed to different concentrations of H2O2 for 2 h, and then the cytotoxic effect of H2O2 was examined by assaying LDH release as described in MATERIALS AND METHODS. Two hours of exposure to H2O2 caused cell damage in a dose-dependent manner, and the cytotoxic effect was enhanced more in the gene-transfected cells than in the controls (Fig. 2B, left). In Fig. 2B, right, the cells were treated with 50 µM H2O2 for the periods indicated, and the LDH released into the medium was quantified and expressed as a ratio to the total intracellular LDH content. The LDH release was enhanced more in the gene-transfected cells than in the controls during the treatment. Figure 2C shows that morphological change was observed using phase-contrast microscopy in control and CRT-overexpressing cells treated with 50 µM H2O2 for 2 h. In gene-transfected cells treated with H2O2, the cell shape was apparently round and had shrunk along with some bleblike structure, although no change was observed in control cells treated with H2O2. Collectively, these results indicate that overexpression of CRT enhances the cytotoxic sensitivity to oxidative stress caused by H2O2 in myocardiac H9c2 cells.



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  		Fig. 1. A: expression levels for calreticulin (CRT), calnexin (CNX), binding protein (BiP; glucose-regulating protein 78, Grp78), ERp57/Grp58, and GAPDH were estimated in control (parental and vector) and CRT gene-transfected H9c2 (CRT-S2 and CRT-S8) cells using immunoblot analysis with specific antibodies (Ab) as described in MATERIALS AND METHODS. Data represent 3 independent experiments. B: intracellular localization of CRT and CNX was evaluated in control and CRT gene-transfected H9c2 cells using indirect immunofluorescence (IF) microscopy with specific antibodies. Background signals were obtained in cells stained with normal rabbit IgG. Data represent 3 independent experiments. Bar, 10 µm.

 


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  		Fig. 2. Overexpression of CRT promotes cell damage in H9c2 cells under oxidative stress due to H2O2. A: control (H9c2 and H9c2-Vector) and CRT gene-transfected (H9c2-CRT-S2 and H9c2-CRT-S8) cells were exposed to different concentrations of H2O2 for 1 h, and then cell viability was examined by performing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described in MATERIALS AND METHODS (left). Cells were exposed to 50 µM H2O2 for the periods indicated, and then cell viability was examined by performing an MTT assay (right). Each value represents the mean ± SD of 4–6 independent experiments. Statistical analysis was performed using a factorial ANOVA test. *P < 0.05, #P < 0.05 vs. value at same H2O2 concentration (left) or time point (right) for H9c2 cells treated with H2O2. B: control and CRT gene-transfected cells were exposed to different concentrations of H2O2 for 2 h, and then cell damage was examined by performing a lactate dehydrogenase (LDH) release assay as described in MATERIALS AND METHODS (left). Cells were treated with 50 µM H2O2 for the periods indicated, and the LDH released in the medium was quantified as described in MATERIALS AND METHODS and expressed as a percentage of total intracellular LDH content (right). Each value represents the mean ± SD of 4–6 independent experiments. *P < 0.05, #P < 0.05 vs. value at same H2O2 concentration (left) or time point (right) as H9c2 cells treated with H2O2. C: control (H9c2-Vector), CRT gene-transfected (H9c2-CRT-S8) cells were exposed to 50 µM H2O2 for 2 h, and then cell morphology was examined using phase-contrast microscopy. Bar, 10 µm.

 
Overexpression of CRT enhances apoptosis of H9c2 cells under oxidative stress due to H2O2. To examine whether apoptosis contributed to the cell damage observed in the transfectants under oxidative stress, a TUNEL assay was performed using cells treated with H2O2. In the study by Turner et al. (52), maximal fragmentation of DNA was observed in H9c2 cells treated with 250 µM H2O2 for 4 h. We compared the extent of DNA strand breaks between control and gene-transfected cells treated with 50 µM H2O2 for 1 and 4 h. The TUNEL assay (Fig. 3A) showed that an increase in fluorescence intensity derived from DNA strand breaks in the transfectants but not in the control cells after H2O2 treatment. After the nucleus was stained with Hoechst 33342 (Fig. 3B), chromatin condensation and nuclear fragmentation were observed in the gene-transfected cells treated with H2O2, but not in the control cells treated with H2O2. Altogether, these results indicate that overexpression of CRT significantly enhances apoptosis in H9c2 cells under oxidative stress caused by H2O2.



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  		Fig. 3. Overexpression of CRT promotes apoptosis in H9c2 cells under oxidative stress due to H2O2. A: terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay was performed for control (top) and gene-transfected (bottom) H9c2 cells under oxidative stress caused by H2O2. DNA strand breaks were detected using the TUNEL method as described in MATERIALS AND METHODS. Cells were treated with 50 µM H2O2 for the periods indicated. Data represent 3 independent experiments. B: morphological changes to nuclei were characterized in Hoechst-stained cells using a fluorescence microscope. Control and gene-transfected cells (50,000/ml) were grown on Lab-Tek chamber slides for 24 h and then treated with 50 µM H2O2 for 2 h. After being fixated with 4% paraformaldehyde in PBS, cells were stained with Hoechst 33342 and then visualized using fluorescence microscopy as described in MATERIALS AND METHODS. Bar, 10 µm.

 
Overexpression of CRT increases [Ca2+]i in H9c2 cells under oxidative stress due to H2O2. To investigate whether the intracellular Ca2+ homeostasis was affected in the cells under oxidative stress, we measured [Ca2+]i after H2O2 treatment (50 or 75 µM). To observe the effect of extracellular Ca2+ on [Ca2+]i, we treated the cells with H2O2 in the presence of 2 mM Ca2+ (+Ca2+) or 10 mM EGTA (+EGTA). The measurement was based on the fluorescence intensity of cells loaded with fura-2 AM as described in MATERIALS AND METHODS. Figure 4 shows that in the presence of extracellular Ca2+ (+Ca2+), control cells demonstrated no change in [Ca2+]i level during treatment with 50 µM H2O2, but CRT-overexpressing cells increased to 180 nM after 35 min of treatment. Moreover, with 75 µM H2O2 treatment, the difference in the [Ca2+]i increase was greater. On the other hand, in the absence of extracellular Ca2+ (+EGTA), the H2O2-induced [Ca2+]i increase was not observed even in the CRT-overexpressing cells, suggesting that the increase was dependent on the influx of Ca2+ from the extracellular space. Altogether, the results indicate that [Ca2+]i was apparently increased by oxidative stress due to H2O2 in CRT-overexpressing cells compared with controls, suggesting some modification of cellular Ca2+ homeostasis occurred because of CRT overexpression.



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  		Fig. 4. Cytoplasmic free Ca2+ concentration ([Ca2+]i) increases in CRT-overexpressing H9c2 cells under oxidative stress due to H2O2. After being loaded with 5 µM fura-2 AM, control and gene-transfected cells cultured on glass coverslips were treated with H2O2 (50 or 75 µM) for the periods indicated. [Ca2+]i was quantified by measuring fura-2 fluorescence as described in MATERIALS AND METHODS. To observe the effect of extracellular Ca2+ on [Ca2+]i, we treated the cells with H2O2 in the presence of 2 mM Ca2+ (+Ca2+) or 10 mM EGTA (+EGTA). Each value represents the mean ± SD of 4 independent experiments. Statistical analysis was performed using a factorial ANOVA test. *P < 0.05 vs. value at same time point for control cells (Ca2+) treated with 50 µM H2O2. #P < 0.05 vs. value at same time point for control cells (Ca2+) treated with 75 µM H2O2.

 
Alteration of Ca2+ flux in CRT-overexpressing H9c2 cells under oxidative stress. To investigate the effect of overexpression of CRT on Ca2+ influx in cells under oxidative stress, we examined the 45Ca2+ uptake in control and CRT gene-transfected cells during treatment with 75 µM H2O2 as described in MATERIALS AND METHODS. As shown in Fig. 5A, in both control and gene-transfected cells, the rate of 45Ca2+ uptake was suppressed within the first 30 min and then increased during oxidative stress. The uptake rate was higher in the control than in the gene-transfected cells after 40 min. Next, to investigate the effect of overexpression of CRT on Ca2+ efflux in the cell under oxidative stress, we examined the 45Ca2+ release in control and gene-transfected cells during treatment with 75 µM H2O2 as described in MATERIALS AND METHODS. After being labeled with 45Ca2+, the cells were treated with H2O2 and the amount of 45Ca2+ released was measured as described in MATERIALS AND METHODS. As shown in Fig. 5B, although the amount of 45Ca2+ released did not differ between untreated control and gene-transfected cells, the release was increased in gene-transfected cells compared with controls after 30-min treatment with H2O2. Collectively, in CRT-overexpressing cells, Ca2+ influx seemed to be suppressed and the efflux was increased in the cells under oxidative stress caused by H2O2 compared with control cells treated with H2O2. Although these results were not consistent with the finding that [Ca2+]i was highly elevated in gene-transfected cells treated with H2O2 (Fig. 4), they also suggested that an alteration of responses in the intracellular Ca2+ stores might lead to increased [Ca2+]i in CRT-overexpressing cells.



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  		Fig. 5. A: uptake of Ca2+ in CRT-overexpressing H9c2 cells under oxidative stress due to H2O2. The cells were cultured with the medium containing 75 µM H2O2 for the indicated periods, washed with Earle's balanced salt solution (EBSS), and then cultured for 10 min in EBSS containing 45Ca2+ (5 µCi/ml) as described in MATERIALS AND METHODS. After being washed with EBSS, the cells were harvested and 45Ca2+ uptake was measured as described in MATERIALS AND METHODS. Each value represents mean ± SD of 3 independent experiments. Statistical analysis was performed using a factorial ANOVA test. *P < 0.05 compared with value at same time point for control cells treated with 75 µM H2O2. B: release of Ca2+ from CRT-overexpressing H9c2 cells under oxidative stress due to H2O2. Cells were cultured for 48 h with 45Ca2+ as described in MATERIALS AND METHODS. After being washed with EBSS, the cells were incubated with EBSS containing 75 µM H2O2. Aliquots were collected at the time points indicated and then centrifuged. Radioactivity was measured in the supernatant as the amount of Ca2+ released from the cell. Each value represents mean ± SD of counts per minute (cpm) recovered in the supernatant normalized to the protein in the total cell pellets. *P < 0.05 vs. same time point for CRT-S8 cells treated without H2O2. C: effect of Ca2+ modulators on [Ca2+]i in CRT-overexpressing H9c2 cells under oxidative stress with H2O2. After being loaded with 5 µM fura-2 AM, control and gene-transfected cells cultured on glass coverslips were pretreated with various modulators containing Ni2+ (5 mM), FCCP (1 µM), ryanodine (100 µM), thapsigargin (5 µM), and xestospongin C (1 µM) and then were treated with H2O2 (75 µM) for 30 min. [Ca2+]i was quantified by measuring fura-2 fluorescence as described in MATERIALS AND METHODS. Each value represents mean ± SD of 3 independent experiments. *P < 0.05 vs. corresponding control cells treated without H2O2. #P < 0.05 vs. untreated control cells (None). **P < 0.05 vs. corresponding CRT-S8 cells treated without H2O2. ##P < 0.05 vs. untreated CRT-S8 cells (None).

 
Effect of Ca2+ modulators on [Ca2+]i in CRT gene-transfected H9c2 cells under oxidative stress due to H2O2. To investigate whether intracellular Ca2+ pools contribute to the altered Ca2+ homeostasis, we examined the effect of Ca2+ modulators on [Ca2+]i in cells treated with H2O2 (Fig. 5C). Ni2+ (5 mM) was used to block Ca2+ influx from the extracellular space via Ca2+ channels and Na+/K+ exchangers in the plasma membrane (26, 27). FCCP (1 µM) is a mitochondrial uncoupler that collapses the mitochondrial membrane potential that drives Ca2+ uptake into mitochondria (5). A high concentration of ryanodine (100 µM) was used as an antagonist for the ryanodine receptor (55). Thapsigargin (5 µM) was used to inhibit the function of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) to block the uptake of Ca2+ into the ER/sarcoplasmic reticulum (SR) (50). Xestospongin C (1 µM) is a cell-permeable inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor (10). The cells were treated with each Ca2+ modulator for 10 min after being loaded with fura-2 AM and then were incubated with 75 µM H2O2 for 30 min, and [Ca2+]i was measured as described in MATERIALS AND METHODS. The uptake of Ca2+ into mitochondria and the ER was disrupted by treatment with FCCP and thapsigargin, respectively. In control cells, FCCP did not enhance the H2O2-induced increase of [Ca2+]i. In CRT-overexpressing cells, [Ca2+]i was similarly increased by H2O2 both cases, with and without FCCP. These results indicate no influence of FCCP on the H2O2-induced change of [Ca2+]i in control and CRT-overexpressing cells. This finding also suggests that the enhancement of H2O2-induced increase of [Ca2+]i observed in CRT-overexpressing cells may not be explained solely by dysfunction of mitochondrial Ca2+ uptake. In contrast, the H2O2-induced increase in [Ca2+]i was apparently enhanced in control cells treated with thapsigargin. This indicates that the thapsigargin-sensitive pool (i.e., ER) is involved in the enhancement of H2O2-induced increase of [Ca2+]i and also suggests that dysfunction of SERCA may have a promoting effect on the H2O2-induced increase of [Ca2+]i. The ryanodine and IP3 receptors may be involved in the increase in [Ca2+]i in response to H2O2. However, high concentrations of ryanodine and xestospongin C did not suppress the increase in [Ca2+]i in CRT-overexpressing cells treated with H2O2, suggesting that the ryanodine and IP3 receptors were not necessarily the main sources of the H2O2-induced increase of [Ca2+]i in CRT-overexpressing cells. Furthermore, it was noteworthy that the H2O2-induced increase of [Ca2+]i was clearly suppressed by Ni2+ in gene-transfected cells. This finding is also consistent with the result that the increase was inhibited in gene-transfected cells in the absence of extracellular Ca2+ (Fig. 4). These findings indicate that the influx of Ca2+ from the extracellular space is important for the H2O2-induced increase of [Ca2+]i in CRT-overexpressing cells, despite the fact that the rate of influx was not increased in the gene-transfected cells treated with H2O2 (Fig. 5A). Altogether, these results indicate that the ER-stored Ca2+ pool plays an important role in the enhancement of the H2O2-induced increase of [Ca2+]i in CRT-overexpressing cells, although Ca2+ influx from the extracellular space was also an important contributor to the increase.

[Ca2+]i level is implicated in the susceptibility of H9c2 cells to apoptosis under oxidative stress due to H2O2. To determine whether the increase in [Ca2+]i is part of the causative mechanism of apoptosis in the gene-transfected cells, H2O2-dependent cytotoxicity was examined in CRT-overexpressing cells in the presence or absence of BAPTA-AM, a cell-permeable Ca2+ chelator. As shown in Fig. 6A, top, cell viability was assessed by performing an MTT assay in gene-transfected cells exposed to H2O2 treatment (50 µM for 2 h) in the absence or presence of BAPTA-AM (10 µM). The viability was suppressed by H2O2 to 28.0 ± 3.4% of that in untreated cells in the absence of BAPTA-AM but returned to 50.5 ± 6.5% in its presence. As shown in Fig. 6A, bottom, cell damage was also assessed by performing a LDH release assay in gene-transfected cells exposed to H2O2 treatment (50 µM for 2 h) in the absence or presence of BAPTA-AM. The release was increased by H2O2 to 22.0 ± 5.4% that of untreated cells in the absence of BAPTA-AM but was remitted to 4.7 ± 2.5% in its presence. These results suggest that BAPTA-AM mitigates cell damage due to H2O2 by suppressing the increase of [Ca2+]i. The effect of BAPTA-AM on apoptosis was also examined using the TUNEL method in the gene-transfected cells after H2O2 treatment (Fig. 6B). In the absence of BAPTA-AM, TUNEL-positive cells increased after treatment with 50 µM H2O2 for 2 h but were not detected in the presence of BAPTA-AM even 2 h after treatment with H2O2. Conversely, the effect of an [Ca2+]i increase on H2O2-induced apoptosis was examined in control H9c2 cells in the absence or presence of thapsigargin (5 µM), an inhibitor for SERCA (50). As shown in Fig. 6C, top, cell viability was suppressed by H2O2 (50 µM for 2 h) to 60.0 ± 4.4% of that in untreated cells in the absence of thapsigargin but was suppressed to 17.1 ± 4.6% in its presence. In Fig. 6C, bottom, cell damage was assessed by performing a LDH release assay in control cells exposed to H2O2 treatment (50 µM for 2 h) in the absence or presence of thapsigargin. The release was slightly increased by H2O2 to 7.5 ± 2.2% that of untreated cells in the absence of thapsigargin but was enhanced to 32.7 ± 2.5% in its presence. Furthermore, the effect of thapsigargin on apoptosis was examined using the TUNEL method in control cells with H2O2 treatment (Fig. 6D). In the presence of thapsigargin, TUNEL-positive cells increased in number after exposure to 50 µM H2O2 for 2 h, whereas in its absence, no increase was observed. These results suggest that thapsigargin enhances apoptosis caused by H2O2 treatment by increasing the [Ca2+]i level. This finding was also consistent with our recently reported results (56). Altogether, these results indicate that the increase in [Ca2+]i plays a causative role in the apoptosis of CRT-overexpressing cells under oxidative stress caused by H2O2.



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  		Fig. 6. Effect of Ca2+ modulators on apoptosis due to oxidative stress caused by H2O2. A: CRT-overexpressing (CRT-S8) cells were cultured in the presence or absence of 10 µM BAPTA-AM during the H2O2 treatment. The cells were exposed to 50 µM H2O2 for 2 h and then cell viability was examined by performing an MTT assay as described in MATERIALS AND METHODS. Each value represents the mean ± SD of 4 independent experiments. Statistical analysis was performed using a paired Student's t-test. *P < 0.05, **P < 0.01 vs. CRT-S8 cells treated with H2O2 without BAPTA-AM. B: TUNEL assay for CRT-overexpressing cells treated with or without BAPTA-AM and/or H2O2. Cells were cultured in the presence or absence of 10 µM BAPTA-AM during the H2O2 treatment. Thin line, no H2O2 for 2 h; thick line, 50 µM H2O2 for 2 h. DNA strand breaks were detected using the TUNEL method as described in MATERIALS AND METHODS. Data represent 3 independent experiments. C: control cells were cultured in the presence or absence of 5 µM thapsigargin during the H2O2 treatment. Cells were exposed to 50 µM H2O2 for 2 h, and then cell viability was examined by performing an MTT assay as described in MATERIALS AND METHODS. Each value represents the mean ± SD of 4 independent experiments. #P < 0.01 vs. control cells treated with H2O2 without thapsigargin. D: TUNEL assay for control cells treated with or without thapsigargin and/or H2O2. Cells were cultured in the presence or absence of 5 µM thapsigargin during H2O2 treatment. Thin line, no H2O2 for 2 h; thick line, 50 µM H2O2 for 2 h. DNA double-stranded breaks were detected using the TUNEL method as described in MATERIALS AND METHODS. Data represent 4 independent experiments.

 
Suppression of CRT expression by transfection with antisense CRT gene enhances cytoprotection of H9c2 cells against oxidative stress due to H2O2. To confirm whether the expression level of CRT influences susceptibility to oxidant-induced apoptosis in H9c2 cells, we transfected the cells with the antisense CRT gene expression vector and a cell line (CRT-AS) in which CRT expression was suppressed to a level lower than that in controls as described in MATERIALS AND METHODS (20). The expression level of CRT was compared with that in control and CRT-overexpressing cells by immunoblot analysis, and the results showed that the expression level was decreased to ~30% of the control level (Fig. 7A). Figure 7B shows that control, CRT-overexpressing, and CRT-underexpressing cells were exposed to different concentrations of H2O2 for 1 h, and then cell viability was examined by performing an MTT assay as described in MATERIALS AND METHODS. The results showed that cell viability was well maintained in CRT-underexpressing cells treated with H2O2 compared with the decreased viability in control and CRT-overexpressing cells. As shown in Fig. 7C, control, CRT-overexpressing, and CRT-underexpressing cells were exposed to 50 µM H2O2 for 4 h, and then the cytotoxic effect of H2O2 was also examined by assaying LDH release as described in MATERIALS AND METHODS. The results showed that the LDH release was suppressed more in CRT-underexpressing cells than in controls during the treatment, although the release was apparently increased in CRT-overexpressing cells. Altogether, the results show that suppression of CRT apparently enhanced cytoprotection against oxidative stress compared with controls, although overexpression of CRT increased the susceptibility to H2O2-induced cytotoxicity. This observation indicates that the expression level of CRT is a key factor in determining the susceptibility to H2O2-induced apoptosis in H9c2 cells. As shown in Fig. 7D, [Ca2+]i was measured in control, CRT-overexpressing, and CRT-underexpressing cells after H2O2 treatment (75 µM) for 30 min. After the treatment with H2O2, the [Ca2+]i increase was apparently suppressed in CRT-underexpressing cells compared with the increase in control and CRT-overexpressing cells. Collectively, the results indicate that the H2O2-induced [Ca2+]i increase is influenced by the expression level of CRT in the cell.



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  		Fig. 7. Suppression of CRT expression showing cytoprotective effects in H9c2 cells under oxidative stress. To observe the effect of the suppressed expression of CRT on cytotoxicity under conditions of oxidative stress, we introduced the antisense gene for CRT into H9c2 cells to obtain cells underexpressing CRT as described in MATERIALS AND METHODS. A: expression levels of CRT, CNX, BiP, and GAPDH were examined in control, CRT-overexpressing (CRT-S8), and CRT-underexpressing (CRT-AS) cells using immunoblot analysis with specific antibodies. Data represent 4 independent experiments. B: control, CRT-overexpressing, and CRT-underexpressing cells were exposed to different concentrations of H2O2 for 1 h, and then cell viability was examined by performing an MTT assay as described in MATERIALS AND METHODS. Each value represents mean ± SD of 4 independent experiments. Statistical analysis was performed using a factorial ANOVA test. *P < 0.05 vs. same concentration of H2O2 for control cells treated with H2O2. C: control, CRT-overexpressing, and CRT-underexpressing cells were exposed to 50 µM H2O2 for 4 h, and then cell damage was examined by performing an LDH release assay as described in MATERIALS AND METHODS. Each value represents mean ± SD of 4 independent experiments. *P < 0.01 vs. control cells treated with H2O2. D: after being loaded with 5 µM fura-2 AM, control, CRT-overexpressing, and CRT-underexpressing cells cultured on glass coverslips were treated with 75 µM H2O2 for 30 min. [Ca2+]i was quantified by measuring fura-2 fluorescence as described in MATERIALS AND METHODS. #P < 0.01, ##P < 0.05 vs. control cells treated with H2O2.

 
Overexpression of CRT enhances processing and activation of caspase-12 through activation of calpain in H9c2 cells under oxidative stress due to H2O2. To further investigate the mechanism of the CRT-dependent enhancement of apoptosis through the alteration of Ca2+ homeostasis, we focused on caspase-12, which is activated by calpain, a Ca2+-dependent cysteine protease, in ER stress-induced apoptosis (40). As shown in Fig. 8A, the expression levels of caspase-12 and caspase-3 and ER stress-related chaperones were examined using immunoblot analysis in control and CRT-overexpressing cells treated with 75 µM H2O2. In control cells, a pro-caspase form of caspase-12 (55 kDa) showed no change after 1 h of treatment with H2O2 but was diminished after 2 h of treatment. The level of 35-kDa proteolytic fragment of caspase-12 gradually increased during the 2-h treatment. In CRT-overexpressing cells, the level of the 55-kDa pro-caspase form was low and that of the 35-kDa fragment was rather high even under nonstressed conditions. During treatment with H2O2, the 55-kDa band gradually decreased in intensity. On the other hand, the strength of the 35-kDa band was slightly increased after 1 h of treatment with H2O2 but was diminished after 2 h of treatment, suggesting enhanced proteolytic processing of the fragment in CRT-overexpressing cells treated with H2O2. The level of the 40-kDa band did not show significant change in control and CRT-overexpressing cells during treatment. These results indicate that proteolytic processing or degradation of caspase-12 was more accelerated in CRT-overexpressing cells than in control cells after treatment with H2O2. H2O2-induced processing also seemed to be enhanced by thapsigargin in both control and CRT-overexpressing cells. In the case of caspase-3, although the proteolytic cleavage was observed in control and CRT-overexpressing cells under stress due to H2O2, proteolysis was relatively accelerated in CRT-overexpressing cells compared with controls. These results indicate that proteolytic processing or degradation of caspase-12 and caspase-3 is apparently accelerated in CRT-overexpressing cells under stress caused by H2O2. In contrast, no significant change in the expression levels of ER chaperones such as CNX, BiP, and CRT seemed to be induced by H2O2. Next, to investigate the relationship between proteolytic processing and the activity of caspase-12, we examined the enzyme activity of caspase-12 in cells after 1 h with or without 75 µM H2O2 and/or 5 µM thapsigargin (Fig. 8B). In CRT-overexpressing cells, activity was increased solely by H2O2, compared with no significant increase in activity in the control cells. Although activity was increased by thapsigargin in both control and CRT-overexpressing cells, it was elevated more in CRT-overexpressing cells during the combined treatment with H2O2. As shown in Fig. 8C, the activity of calpain was also examined in the cells after 1 h with or without 75 µM H2O2 and/or 5 µM thapsigargin. The activity of calpain was significantly increased by H2O2 in CRT-overexpressing cells compared with the small increase observed in control cells. In control cells, treatment with thapsigargin synergistically enhanced the effect of H2O2, although the activity was slightly increased solely by thapsigargin. In CRT-overexpressing cells, although calpain activity was markedly increased by thapsigargin, a synergistic effect was not observed. To determine whether the activity of calpain contributes to the processing of caspase-12, we examined the effect of N-acetyl-leucyl-leucyl-norleucinal (ALLN), a calpain inhibitor (57), or BAPTA-AM on processing in CRT-overexpressing cells during treatment with H2O2 (Fig. 8D). CRT-overexpressing cells were treated for 1 h with or without 75 µM H2O2 in the presence or absence of 50 µM ALLN or 10 µM BAPTA-AM, and then the expression levels of caspase-12 and caspase-3 and ER chaperones were examined using immunoblot analysis. The H2O2-induced processing of caspase-12 was apparently suppressed in the presence of ALLN or BAPTA-AM, resulting in a slight decrease in the level of the 35-kDa fragment and an increase in that of the 55-kDa pro-caspase form. Proteolytic processing of caspase-3 was also suppressed by ALLN or BAPTA-AM in gene-transfected cells treated with H2O2. Altogether, these results suggest that the Ca2+-calpain pathway is involved in the activation of caspase-12 in CRT-overexpressing cells under oxidative stress caused by H2O2.



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  		Fig. 8. The calpain-caspase-12 pathway is activated in CRT-overexpressing H9c2 cells under oxidative stress. A: control and CRT-overexpressing (CRT-S8) cells were exposed to 75 µM H2O2 for 1 and 2 h or to 75 µM H2O2 and 5 µM thapsigargin for 1 h, and then the expression levels of caspase-12 and caspase-3, CNX, BiP, and CRT were examined using cell lysates by performing immunoblot analysis with specific antibodies. Data represent 3 independent experiments. B: cells were exposed to 75 µM H2O2 and/or 5 µM thapsigargin for 1 h. Activity of caspase-12 was assayed with cell lysates using the substrate Ala-Thr-Ala-Asp-7-amino-4-trifluoromethylcoumarin (AFC) as described in MATERIALS AND METHODS. Each value represents the mean ± SD of 3 independent experiments. Statistical analysis was performed using a factorial ANOVA test. *P < 0.05 vs. untreated control cells. **P < 0.05 vs. control cells treated with H2O2. #P < 0.05 vs. untreated CRT-S8 cells. NS, not significant vs. untreated control cells. C: cells were exposed to 75 µM H2O2 and/or 5 µM thapsigargin for 1 h. Activity of calpain was assayed using the calpain substrate succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-Leu-Leu-Val-Tyr-AMC) as described in MATERIALS AND METHODS. Each value represents mean ± SD of 3 independent experiments. *P < 0.05 vs. untreated control cells. **P < 0.05 vs. control cells treated with H2O2. #P < 0.05 vs. untreated CRT-S8 cells. D: CRT-overexpressing (CRT-S8) cells were treated for 1 h with or without 75 µM H2O2 in the presence or absence of 50 µM N-acetyl-leucyl-leucyl-norleucinal (ALLN) or 10 µM BAPTA-AM, and then the expression levels of caspase-12, caspase-3, CNX, BiP, and CRT were examined using cell lysates by performing immunoblot analysis with specific antibodies. Data represent 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we used myocardiac H9c2 cells that overexpressed CRT to investigate the effect of overexpression on H2O2-induced apoptosis in cardiac myocytes. When exposed to H2O2, the CRT-overexpressing cells showed increases in LDH release and DNA strand breaks, indicating that these cells were highly susceptible to apoptosis compared with control cells. Nakamura et al. (41) reported that overexpression of CRT resulted in increased sensitivity of HeLa cells to both thapsigargin- and staurosporine-induced apoptosis. These authors suggested that overexpression of CRT affected communication between the ER and the mitochondria to increase the sensitivity to apoptosis via the altered Ca2+ homeostasis, and their hypothesis has been supported by the study of Arnaudeau et al. (1). Pinton et al. (43) reported that the releasable [Ca2+] in the ER is important for ceramide-induced apoptosis and also showed that overexpression of CRT enhanced the ceramide-induced apoptosis in HeLa cells. Recently, it was also reported that CRT controls the susceptibility to apoptosis by regulating p53 functions (34). Furthermore, a necrosis-promoting effect of CRT has been reported to occur in Caenorhabditis elegans (53). In contrast, overexpression of CRT provided resistance to oxidant-induced cell death in renal epithelial LLC-PK1 cells treated with iodoacetamide (28), tert-butylhydroperoxide (29), or H2O2 (18). The function of CRT in the regulation of apoptosis may differ in specific cell types and is still controversial, so further investigation is required.

Ca2+ is one of the most versatile biological factors and regulates a variety of cellular events, such as cell development, cell proliferation, and cell death (3). The elevation of [Ca2+]i has been thought to be an important signal in the mechanism of apoptosis (31). In the present study, we found that the level of [Ca2+]i increased significantly in CRT-overexpressing cells treated with H2O2, although only a slight increase was observed in controls (Fig. 4). As shown in Fig. 6, H2O2-induced apoptosis was suppressed by the Ca2+ chelator BAPTA-AM in CRT gene-transfected cells but was promoted by thapsigargin in control cells, indicating that the increase in [Ca2+]i was an important trigger for apoptosis in H9c2 cells under stress due to H2O2.

In previous reports, overexpression of CRT led to an increase in the intracellular store of Ca2+ (2, 8, 32, 54). CRT also appears to modulate store-operated Ca2+ influx (1, 2, 8, 32, 46, 54) and to alter Ca2+ transport by SERCA2b (22). In the case of H9c2 cells overexpressing CRT, the total cellular Ca2+ content examined on the basis of measuring equilibrium 45Ca2+ uptake was ~160% of that in control cells and was found mainly within the thapsigargin-sensitive store. However, [Ca2+]i levels in the resting state were not significantly different between control and CRT-overexpressing cells. On the other hand, store-operated Ca2+ influx was examined spectrofluorometrically using cells labeled with fura-2 AM and was suppressed in the CRT-overexpressing cells compared with controls (data not shown). These results indicate that overexpression of CRT influences intracellular Ca2+ homeostasis, and the effect in resting H9c2 cells seems to be similar to that in other cell types described in the literature. Recently, Scorrano et al. (48) reported that Ca2+ reserved in the ER was an important gateway for apoptosis via the influence on mitochondrial Ca2+ homeostasis. Overexpression of CRT also influences the mitochondrial Ca2+ homeostasis (1). Altogether, the enhanced susceptibility to H2O2-induced apoptosis in CRT-overexpressing H9c2 cells may also be due to the modulation of mitochondrial Ca2+ homeostasis by the altered responses in the ER overexpressing CRT.

Under oxidative stress, reactive oxygen and nitrogen can disrupt normal physiological pathways and cause cell death via altered Ca2+ homeostasis (7, 30). The [Ca2+]i level is regulated by Ca2+ transport into and out of the ER or SR, in which Ca2+ can be stored, as well as by Ca2+ transport through the plasma membrane between the cytoplasm and the extracellular space (3). It was reported that oxidative stress causes a [Ca2+]i increase in a variety of cell types (7). The initial increase in [Ca2+]i results in part from a rapid release of ER Ca2+ through IP3 receptors after receptor-mediated activation of PLC, and the subsequent generation of IP3 and the sustained component results from the influx of extracellular Ca2+ (54). It is also known that the uptake of Ca2+ from the cytoplasm to the ER/SR by SERCA can be inhibited by O2– and H2O2 in smooth muscle cells (14, 15). It seems that SERCA can be inhibited both by oxidation of its sulfhydryl residues and by a direct attack of oxidants on the ATP-binding site (7).

In the present study, the influx and efflux of 45Ca2+ were examined in control and CRT-overexpressing cells with or without H2O2 treatment (Fig. 5, A and B). The results showed that Ca2+ influx was suppressed and that efflux was enhanced in the gene-transfected cells during H2O2 treatment, suggesting that the H2O2-induced increase in [Ca2+]i might not be caused simply by the change of Ca2+ flux between the cytoplasm and extracellular space, but rather by the alteration in intracellular Ca2+ pools such as those in the ER and the mitochondria. To investigate the involvement of intracellular Ca2+ pools in the H2O2-induced increase in [Ca2+]i, we examined the effect of Ca2+ modulators on [Ca2+]i in cells treated with H2O2 (Fig. 5C). The inhibition of mitochondrial function by FCCP did not enhance the H2O2-induced [Ca2+]i increase in control cells, indicating that suppressed mitochondrial function was not a main cause of the enhancement of [Ca2+]i observed in CRT-overexpressing cells. On the other hand, thapsigargin, an inhibitor for SERCA, apparently enhanced the H2O2-induced [Ca2+]i increase in control cells, strongly suggesting that the Ca2+ store in the ER might be a cause of the [Ca2+]i elevation observed in CRT-overexpressing cells. This observation also suggested that dysfunction of SERCA2a has a promoting effect on the H2O2-induced [Ca2+]i increase in H9c2 cells. Although ER residents such as the ryanodine and IP3 receptors may possibly be involved in raising [Ca2+]i levels in response to H2O2, the inhibition of both did not suppress the H2O2-induced [Ca2+]i increase in CRT-overexpressing cells. These results suggest that the H2O2-induced [Ca2+]i increase may be due to a dysfunction of SERCA2a and not to the release of Ca2+ from the ER through the ryanodine or IP3 receptors. However, it is noteworthy that the H2O2-induced increase of [Ca2+]i was clearly suppressed by Ni2+, an inhibitor of Ca2+ influx in gene-transfected cells. Collectively, these results indicate that the ER-stored Ca2+ pool plays an important role in the enhancement of the H2O2-induced [Ca2+]i increase in CRT-overexpressing cells, although Ca2+ influx from the extracellular space was also an important contributor to the increase.

In a recently published report (15), we focused on the function of SERCA2a in CRT-overexpressing H9c2 cells under oxidative stress caused by H2O2 because SERCA is an ER/SR resident protein that is highly susceptible to peroxide stress. In that study, we found that in vitro activities of SERCA2a and 45Ca2+ uptake into the ER were both suppressed by H2O2 in CRT-overexpressing H9c2 cells compared with controls (20). This finding indicates that the inactivation of SERCA2a was accelerated by the overexpression of CRT in the microsomes treated with H2O2. We also found that CRT transiently interacted with SERCA2a during H2O2-induced oxidative stress and that H2O2-induced degradation of SERCA2a was apparently enhanced in gene-transfected cells compared with controls. On the other hand, interaction between CRT and the IP3 or ryanodine receptor was not detected in the cells under the same conditions (data not shown), suggesting that other Ca2+-regulating proteins in the ER had little physical interaction with CRT under oxidative stress. These findings suggest that the increase in [Ca2+]i may be due partly to the loss of Ca2+-pumping activity of SERCA2a in the ER of CRT-overexpressing cells under oxidative stress.

Sustained elevation of the CRT level in the ER may be a consequence of ER stress. ER stress, also known as the unfolded protein response, is a physiological cellular response against accumulated misfolded proteins in the ER (25). However, prolonged ER stress is known to lead to apoptosis and to be linked to the pathogenesis of several disorders, including genetic diseases (e.g., type 1 diabetes mellitus), neurodegenerative diseases (e.g., Alzheimer disease, Parkinson disease), and metabolic diseases (e.g., hyperhomocysteinemia) (25). Caspase-12, which is associated with the ER, is specifically involved as a cell death effector via ER stress (40, 44). ER stress-induced activation of caspase-12 occurred through proteolytic processing by calpain via [Ca2+]i elevation in the stressed cell (40). In the present study, we have shown that caspase-12 was highly activated in the CRT-overexpressing cells under oxidative stress through the activation of the Ca2+-calpain pathway (Fig. 8). The results strongly suggest that a Ca2+-calpain-caspase-12 pathway is involved in the mechanism of accelerated susceptibility to H2O2-induced apoptosis in CRT-overexpressing cells. Although Morishima et al. (37) did not clarify fully the precise activation mechanism for the caspase-12-related pathway, they reported that ER stress could trigger a specific cascade involving caspase-12, caspase-9, and caspase-3 in a cytochrome c-independent manner. This finding may be consistent with our findings that the H2O2-induced processing of both caspase-12 and caspase-3 was accelerated in CRT-overexpressing cells under stress and was suppressed in the presence of a calpain inhibitor, ALLN. Furthermore, the processing of caspase-12 and caspase-3 was suppressed by a Ca2+ chelator, BAPTA-AM, in the H2O2-treated, CRT-overexpressing cells, suggesting an activated linkage of the Ca2+-calpain-caspase-12 signaling cascade in the apoptotic process of CRT-overexpressing cells under oxidative stress.

We also have shown that overexpression of CRT promotes apoptosis during cardiac differentiation in H9c2 cells (24). In that study, we showed that Akt signaling was suppressed in H9c2 cells overexpressing CRT via [Ca2+]i increase. In addition, we recently reported (56) that cAMP response element-dependent transcriptional upregulation of the PP2Ac-{alpha} gene is involved in the inactivation of Akt, leading to the enhancement of oxidant-induced apoptosis in H9c2 cells under conditions in which [Ca2+]i elevation is prolonged. With regard to the differentiation of cardiomyocytes, the importance of the intracellular generation of reactive oxygen species is implicated (47). In this respect, the altered Ca2+ homeostasis leading to accelerated apoptosis in CRT-overexpressing cells during differentiation may be related to a similar mechanism in cells to which reactive oxygen species are exposed. Moreover, in addition to the mechanism related to Akt signaling, the results of the present study also suggest that the Ca2+-calpain-caspase-12 pathway is part of another mechanism of the differentiation-induced apoptosis of CRT-overexpressing H9c2 cells (24).

In conclusion, the results of the present study indicate that the level of CRT regulates susceptibility to oxidative stress through a change in Ca2+ homeostasis and a Ca2+-dependent calpain-caspase-12 pathway in myocardiac H9c2 cells, suggesting a pathophysiological significance of CRT in myocardiac disorders under conditions of oxidative stress.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by Grants-in-Aid for the 21st Century Centers of Excellence program from the Ministry of Education, Science, Sports, Culture, and Technology of Japan, and by grants from the Ministry of Health, Labor, and Welfare, Japan.


   ACKNOWLEDGMENTS
 
We are grateful to Midori Ikezaki and Akiko Emura for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Ihara, Dept. of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki Univ. Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan (e-mail: y-ihara{at}net.nagasaki-u.ac.jp)

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
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 DISCUSSION
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