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Am J Physiol Heart Circ Physiol 289: H477-H487, 2005; doi:10.1152/ajpheart.00879.2004
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INNOVATIVE METHODOLOGY

Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell line for apoptosis analysis

Qi Hou and Yi-Te Hsu

Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina

Submitted 25 August 2004 ; accepted in final form 11 February 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The proapoptotic protein Bax plays an important role in cardiomyocytic cell death. Ablation of this protein has been shown to diminish cardiac damage in Bax-knockout mice during ischemia-reperfusion. Presently, studies of Bax-mediated cardiac cell death examined primarily the expression levels of Bax and its prosurvival factor Bcl-2 rather than the localization of this protein, which dictates its function. Using immunofluorescence labeling, we have shown that in neonatal rat cardiomyocytes and in H9c2 cardiomyoblasts, Bax translocates from cytosol to mitochondria upon the induction of apoptosis by hypoxia-reoxygenation-serum withdrawal and by the presence of the free-radical inducer menadione. Also, we found that Bax translocation to mitochondria was associated with the exposure of an NH2-terminal epitope, and that this translocation could be partially blocked by the prosurvival factors Bcl-2 and Bcl-XL. To visualize the translocation of Bax in living cells, we have developed an H9c2 cell line that stably expresses green fluorescent protein (GFP)-tagged Bax. This cell line has GFP-Bax localized primarily in the cytosol in the absence of apoptotic inducers. Upon induction of apoptosis by a number of stimuli, including menadione, staurosporine, sodium nitroprusside, and hypoxia-reoxygenation-serum withdrawal, we could observe the translocation of Bax from cytosol to mitochondria. This translocation was not affected by retinoic acid-induced differentiation of H9c2 cells. Additionally, this translocation was associated with loss of mitochondrial membrane potential, release of cytochrome c, and fragmentation of nuclei. Finally, using a tetramethylrhodamine-based dye, we have shown that a rapid screening process based on the loss of mitochondrial membrane potential could be developed to monitor GFP-Bax translocation to mitochondria. Overall, the GFP-Bax-stable H9c2 cell line that we have developed represents a unique tool for examining Bax-mediated apoptosis, and it could be of great importance in screening therapeutic compounds that could block Bax translocation to mitochondria to attenuate apoptosis.

green fluorescent protein; cardiomyocyte; Bcl

BAX IS A PROAPOPTOTIC MEMBER of the Bcl-2 family that plays an important role in apoptosis regulation (23). Physiologically, Bax is involved in neuronal development and spermatogenesis. Animals that are deficient in Bax have an increased number of neurons, and the males are sterile (15, 24, 25). In certain cases of cancer, mutations were found in the gene that encodes this protein, presumably to enable the cancer cells to be less susceptible to cell death (29). In addition, Bax has been shown to be upregulated in heart and brain tissues subjected to ischemia-reperfusion (5, 16, 20). In Bax-knockout mice, the ablation of this protein decreased cardiac damage through the suppression of cardiomyocytic apoptosis (10).

Bax is primarily a soluble protein in healthy living cells (9, 12–14, 28). Upon apoptosis induction, Bax undergoes a conformational change that leads to the exposure of both its NH2- and COOH-terminal segments and translocates from the cytosol to the mitochondria (9, 12, 22, 28). The exposure of the NH2-terminal segment epitope can be detected by the monoclonal antibody 6A7 (14, 22), whereas the exposure of the COOH-terminal hydrophobic segment enables the insertion of Bax into mitochondrial outer membranes. Bax localization to mitochondria is associated with loss of mitochondrial membrane potential ({Delta}{Psi}m) and release of cytochrome c (6, 8, 18, 27). Cytochrome c then activates caspases through the formation of apoptosomes to induce cell death (17). Prosurvival factors Bcl-2 and Bcl-XL antagonize the proapoptotic activities of Bax by inhibiting Bax translocation from the cytosol to the mitochondria (7, 21).

Thus far, few studies have examined Bax distribution dynamics during cardiac apoptosis. For this report, we investigated Bax subcellular localization in neonatal cardiomyocytes and in an H9c2 cardiomyoblast cell line during apoptosis induction. In addition, to facilitate the examination of the roles of different apoptosis inducers in influencing Bax localization in living cells, we have developed and extensively characterized an H9c2 cell line that stably expresses green fluorescent protein (GFP)-tagged Bax. This unique cell line has enabled us to directly visualize the subcellular localization of Bax and to quantitatively assess the effects of different inducers in regulating its distribution.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. An H9c2 rat cardiomyoblast cell line was obtained from American Type Culture Collection. Fetal bovine serum (FBS) and culture supplements were purchased from Invitrogen. SuperFect and Maxi Prep Kits were from Qiagen. MitoTracker-CMXRos and tetramethylrhodamine (TMRE) were from Molecular Probes. The TMRE {Delta}{Psi}m screening kit was from Cell Technology. Rabbit anti-heat shock protein (HSP) 60 antibody was from Santa Cruz Biotechnology. Mouse anti-cytochrome c antibody was from Pharmingen. Cy3-labeled goat anti-rabbit and anti-mouse Ig, horseradish peroxidase-conjugated sheep anti-mouse Ig, and an enhanced chemiluminescence Western blot detection kit were obtained from Amersham. FITC-labeled goat-anti-mouse Ig was from Kirkegaard and Perry Laboratories, and Z-Val-Ala-DL-Asp-fluoromethylketone (z-VAD-fmk) was purchased from Axxora. All other chemicals were obtained from either Sigma or Fisher Scientific.

Preparation of neonatal rat cardiomyocytes and H9c2 cell culture. Primary cultures of neonatal rat cardiomyocytes from 1-day-old Wistar rats were prepared by using the methods of Blondel et al. (2) and Simpson and Savion (26) with minor modifications, and cells were cultured in modified Eagle's medium that contained 10% FBS, 2 mM glutamine, 1% penicillin-streptomycin, and 1% 5-bromodeoxyuridine. The H9c2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine and 10% FBS. Both cell types were maintained at 37°C in the presence of 5% CO2.

Transient transfection and development of H9c2 cell lines stably expressing GFP-Bax, Bcl-2, and Bcl-XL. For transient transfection of H9c2 cells, H9c2 cardiomyoblasts were seeded onto a six-well plate and cultured in DMEM supplemented with 10% FBS. The cells were then transfected with 2 µg/well C3-EGFP-Bax (28) with the SuperFect transfection reagent using the protocol described by the manufacturer. At 6 h posttransfection, 25 µM z-VAD-fmk was added, and at 12 h posttransfection, the cells were stained with 20 ng/ml MitoTracker-CMXRos and visualized by fluorescence microscopy.

To develop stable clones of H9c2 cells expressing GFP-Bax, human Bcl-2, or Bcl-XL, H9c2 cells were seeded onto 10-cm plates and cultured in DMEM supplemented with 10% FBS. The cells were transfected with 12 µg/plate of C3-EGFP-Bax, pcDNA3-human Bcl-2, or pcDNA3-human Bcl-XL (28) with SuperFect transfection reagent. The day after transfection, the cells were selected with 0.75 mg/ml G-418. After 1 wk of selection, the surviving cells were trypsinized, serially diluted, and plated onto 96-well plates. Fluorescence microscopy was used to screen GFP-Bax-stable clones. GFP-Bax-stable H9c2 cells were maintained in DMEM culture medium in the presence of 0.5 mg/ml G-418. To differentiate the GFP-Bax-stable H9c2 cells, these cells were grown in the presence of 1 µM all-trans-retinoic acid with reduced FBS (1%; see Ref. 3) before apoptosis treatment. For the screening of H9c2 clones that stably express human Bcl-2 and Bcl-XL, cell lysates from various clones were analyzed by Western blotting with anti-Bcl-2 and -Bcl-XL monoclonal antibodies. The resulting Bcl-2- and Bcl-XL-stable clones were amplified by using the same culture medium as described above.

To assess the efficacy of Bcl-2 and Bcl-XL in blocking Bax localization to mitochondria, H9c2 cells that stably express these proteins were plated onto six-well plates to ~70–80% confluency. The cells were then transfected with C3-EGFP-Bax (2 µg/well) by using SuperFect transfection reagent. At 6 h posttransfection, 25 µM z-VAD-fmk was added. The cells were visualized by fluorescence microscopy at 12 h posttransfection. The percentages of cells with GFP-Bax localized to mitochondria were determined from four separate visual fields.

Apoptosis induction in rat cardiomyocytes and H9c2 cardiomyoblasts. Apoptosis was induced in neonatal rat cardiomyocytes and H9c2 cells (untransfected and GFP-Bax stable) by using a variety of stimuli, including hypoxia-reoxygenation-serum withdrawal and treatment with menadione, staurosporine, and sodium nitroprusside (SNP).

Hypoxia was induced in neonatal rat cardiomyocytes and in H9c2 cells by equilibrating these cells in a 95% N2-5% CO2 environment in a hypoxic chamber (Billups-Rothenberg). In these cultures, serum was also omitted from the culture medium. After 24 h of hypoxia, cells were reoxygenated for 12 h. The treated cells were subjected to immunofluorescence labeling with anti-rat Bax-1D1 monoclonal antibody. The cells were costained with anti-HSP60 polyclonal antibody to label mitochondria.

For menadione treatment, neonatal rat cardiomyocytes and H9c2 cells were treated with 12.5 µM menadione in the presence of 25 µM z-VAD-fmk for 18 h. The treated cells were subjected to immunofluorescence labeling with either anti-Bax-1D1 or 6A7 monoclonal antibodies. The cells were costained with anti-HSP60 polyclonal antibody to label mitochondria. To compare the sensitivity of H9c2 and H9c2 GFP-Bax-stable cells to menadione, these cells were treated with 11 µM menadione in the absence of z-VAD-fmk for 17–41 h. The cells were then stained with Hoechst nuclear stain (10 µg/ml) and analyzed by fluorescence microscopy to quantitate apoptotic nuclei. To determine the effects of menadione on GFP-Bax localization, GFP-Bax-stable H9c2 cells were treated with 12.5 µM menadione in the presence of 25 µM z-VAD-fmk for 18 h. The treated cells were stained with 20 ng/ml MitoTracker for mitochondrial labeling. To determine how GFP-Bax localization to mitochondria affects cytochrome c localization, GFP-Bax-stable H9c2 cells were treated with 12.5 µM menadione in the presence of 25 µM z-VAD-fmk for 17 h. Immunofluorescence labeling with anti-cytochrome c monoclonal antibody was then carried out. To study the relationship of GFP-Bax translocation to mitochondria and {Delta}{Psi}m change, GFP-Bax-stable H9c2 cells were treated with 12 µM menadione for 17 h in the presence of 25 µM z-VAD-fmk. The treated cells were stained with TMRE (10 ng/ml; Ref. 27) and analyzed by fluorescence microscopy. To quantitatively assess the {Delta}{Psi}m loss, GFP-Bax-stable H9c2 cells were plated onto 96-well plates and treated with 17.5 or 20 µM menadione in the presence of z-VAD-fmk for 17 h. The treated cells were stained with TMRE and subjected to fluorescence quantitation.

Staurosporine was used to induce apoptosis in H9c2 and GFP-Bax-stable H9c2 cells. To study the staurosporine-induced Bax-6A7 antibody epitope exposure, H9c2 cells were treated with 62.5 nM staurosporine in the presence of 25 µM z-VAD-fmk for 18 h. The treated cells were then subjected to immunoprecipitation analysis with 6A7 antibody-bound Sepharose beads. To investigate the effects of staurosporine on GFP-Bax localization in undifferentiated and retinoic acid-differentiated GFP-Bax-stable H9c2 cells, these cells were treated with 10 nM staurosporine in the presence of 25 µM z-VAD-fmk for 18 h. The treated cells were stained with MitoTracker to visualize mitochondria. To demonstrate that GFP-Bax localization to mitochondria leads to cell death, GFP-Bax-stable H9c2 cells were treated with 10 nM staurosporine in the absence of z-VAD-fmk for 17 h. The cells were stained with Hoechst nuclear stain (10 µg/ml) and analyzed by fluorescence microscopy. To determine the effects of GFP-Bax translocation to mitochondria on cytochrome c localization, GFP-Bax-stable H9c2 cells were treated with 10 nM staurosporine in the presence of 25 µM z-VAD-fmk for 17 h. Immunofluorescence labeling with an anti-cytochrome c monoclonal antibody was then carried out. To quantitatively assess the staurosporine-induced loss of {Delta}{Psi}m, GFP-Bax-stable H9c2 cells were plated onto 96-well plates and treated with 15 and 17.5 nM staurosporine in the presence of 25 µM z-VAD-fmk for 17 h. The treated cells were stained with TMRE and subjected to fluorescence quantitation.

For SNP treatment, GFP-Bax-stable H9c2 cells were treated with 4 mM SNP in the presence of 25 µM z-VAD-fmk for 14 h. The treated cells were stained with MitoTracker and visualized by fluorescence microscopy.

Immunoprecipitation of Bax by 6A7 antibody. For immunoprecipitation of Bax by the 6A7 antibody, H9c2 cells were treated with 62.5 nM staurosporine. Both the untreated and treated cells were then solubilized in 10 mM HEPES, pH 7.4, 150 mM NaCl, and 1% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesul-fonate (CHAPS) and subjected to immunoprecipitation with 6A7 antibody-bound Sepharose beads (14). The immunoprecipitated samples were analyzed by Western blotting with anti-rat Bax-1D1 monoclonal antibody (14).

Immunofluorescence labeling and fluorescence microscopy. For rat Bax and HSP60 dual immunofluorescence labeling, neonatal rat cardiomyocytes and H9c2 cardiomyoblasts were plated onto 3.5-cm plates. The cells were subjected to either hypoxia-reoxygenation-serum withdrawal or menadione treatment. Both the treated and untreated cells were fixed in 6% paraformaldehyde for 30 min and permeabilized with 0.06% saponin in PBS for 15 min. The cells were blocked for 30 min in the blocking buffer, which contained PBS, 5% FBS, and 0.06% saponin. The cells were subsequently incubated with 2 µg/ml rabbit anti-HSP60 polyclonal antibody diluted in blocking buffer that contained 0.04% saponin for 2 h. The cells were washed and incubated with a 1:2 dilution of mouse anti-Bax-1D1 or 6A7 culture supernatant (14) in the presence of 0.04% saponin for 2 h. The cells were again washed and incubated with 6 µg/ml Cy3-labeled goat anti-rabbit Ig and 5 µg/ml FITC-labeled goat anti-mouse Ig in blocking buffer that contained 0.04% saponin for an additional 2 h. Labeled cells were then washed with PBS and treated with antifade reagent. To determine the subcellular localization of cytochrome c during apoptosis induction, untreated and menadione- or staurosporine-treated GFP-Bax-stable H9c2 cells were subjected to immunofluorescence labeling with an anti-cytochrome c antibody as previously described (11).

Cell visualization was carried out with an Olympus IX-70 fluorescence microscope by using an LCPlanFI x20 objective lens with x1.5 intermediate magnification. The images were captured with an Optronics DEI-750D digital imaging camera.

Fluorescence quantitation of {Delta}{Psi}m. To quantitate the loss of {Delta}{Psi}m, GFP-Bax-stable H9c2 cells were plated onto black 96-well plates with clear bottoms (100 µl/well at a density of 1.2 x 104 cells/ml), treated with either staurosporine or menadione, and stained using a TMRE Kit (Cell Technology) according to modified manufacturer's instructions. Before fluorescence quantitation, the culture supernatants that contained the added TMRE were removed and replaced with 100 µl of culture medium. The relative fluorescence of the samples was quantitated with a PerkinElmer Victor3 1420 multilabel counter.

SDS-polyacrylamide gel electrophoresis and Western blotting. SDS polyacrylamide gel electrophoresis (12%) and Western blotting analyses were carried out as previously described (13). For immunoblotting analysis, the blots were probed with the anti-Bax, -Bcl-2, and -Bcl-XL monoclonal antibodies including {alpha}-Bax-2C8 (epitope amino acids 43–62; 1:20 dilution in culture fluid; Ref. 14), {alpha}-rBax-1D1 (epitope amino acids 3–16; 1:10 dilution in culture fluid; Ref. 14), {alpha}-m/r-Bcl-2–10C4 and {alpha}-hBcl-2–8C8 (epitope amino acids 41–54; 1:10 dilution in culture fluid; Refs. 13, 28), and {alpha}-Bcl-XL-2H12 (epitope amino acids 3–14; 1:10 dilution in culture fluid; Ref. 13) monoclonal antibodies. Immunoblotting and enhanced chemiluminescence documentation were carried out as previously described (13, 14).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Bax translocates from cytosol to mitochondria in cardiomyocytes subjected to a variety of apoptosis-inducing agents. Bax translocation from cytosol to mitochondria culminates a key step by which it promotes cell death in a number of cellular systems. To examine how apoptosis induction affects Bax localization in cardiomyocytes, we treated both the neonatal rat cardiocytes and H9c2 cardiomyoblasts to apoptosis induction by hypoxia-reoxygenation-serum withdrawal. The treated cells were then subjected to immunofluorescence labeling with the anti-rat Bax monoclonal antibody 1D1. As shown in Fig. 1, in neonatal cardiocytes and H9c2 cells, Bax was found primarily in the cytosol as indicated by its diffuse labeling pattern. On the other hand, exposure of these cells to 24 h of hypoxia followed by 12 h of reoxygenation in the absence of serum resulted in a number of cells having a punctate Bax-labeling pattern. The punctate Bax was localized to mitochondria as determined by costaining with the mitochondrion-specific anti-HSP60 polyclonal antibody. In addition to the hypoxia-reoxygenation-serum withdrawal treatment, we found that the exposure of the neonatal rat cardiocytes and H9c2 cells to the free-radical inducer menadione (12.5 µM for 18 h) would also result in Bax localization to mitochondria. These results suggest that in cardiac cells, Bax redistribution from the cytosol to the mitochondria is an underlying event during apoptosis induction.



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  		Fig. 1. Apoptosis induction by hypoxia-reoxygenation-serum withdrawal (H/R–serum) and menadione treatment induces Bax translocation from cytosol to mitochondria in neonatal rat cardiomyocytes and H9c2 cardiomyoblasts. Immunofluorescence labeling of neonatal rat cardiocytes and H9c2 cells subjected to apoptosis induction by either hypoxia (24 h) with subsequent reoxygenation (12 h) and serum withdrawal or menadione treatment (12.5 µM for 18 h) was carried out with the anti-rat Bax-1D1 monoclonal antibody. Whereas the untreated cells displayed a cytosolic Bax labeling, many of the treated cells displayed a punctate Bax-labeling pattern. Punctate Bax colocalized with mitochondria as determined by costaining with rabbit anti-heat shock protein (HSP) 60 polyclonal antibody. Overlays of Bax and HSP60 antibody labeling are also shown. No labeling was observed in the absence of primary antibodies.

 
Bax in cardiac cells undergoes a conformational change as detected by conformation-sensitive antibody 6A7. Previously it was shown (14, 22) that in cells undergoing apoptosis, Bax exposes an NH2-terminal epitope that could be detected with the conformation-sensitive anti-Bax 6A7 antibody. To examine whether this epitope of Bax is exposed in rat cardiac cells undergoing apoptosis, we carried out an immunofluorescence labeling analysis by staining both untreated H9c2 cells and menadione-treated (12.5 µM for 18 h) H9c2 cells with the 6A7 and anti-HSP60 antibodies. As shown in Fig. 2A, although the 6A7 antibody did not label the cytosolic Bax in untreated cells, it labeled the mitochondria-bound Bax in menadione-treated cells. In addition to immunofluorescence labeling, we carried out an immunoprecipitation analysis with the 6A7 antibody. This study was performed by solubilizing both untreated and staurosporine-treated H9c2 cells in CHAPS. The resulting cell lysates were then subjected to immunoprecipitation with the 6A7 antibody, and the immunoprecipitated samples were analyzed by Western blotting with an anti-rat Bax antibody. As shown in Fig. 2B, the 6A7 antibody did not bind to the Bax protein from nonapoptotic cells. On the other hand, a significant amount of Bax from the apoptotic cells was found to bind to the 6A7 antibody. This corroborates with the immunofluorescence labeling study, which showed that upon induction of apoptosis in cardiac cells, Bax underwent a conformational change that led to the exposure of its NH2-terminal epitope.



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  		Fig. 2. Bax in H9c2 cells exposes an NH2-terminal epitope when it translocates from cytosol to mitochondria, and this translocation is partially blocked by Bcl-2 and Bcl-XL. A: H9c2 cells were treated either with (bottom) or without (top) 12.5 µM menadione for 18 h. Cells were then subjected to immunofluorescence labeling with the conformation-sensitive anti-Bax-6A7 monoclonal (left) and mitochondrion-specific HSP60 polyclonal (middle) antibodies. Overlay images of Bax and HSP60 antibody labeling are also shown (right). B: H9c2 cells were treated either with or without 62.5 nM staurosporine. Cells were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-2-hy-droxy-1-propanesulfonate (CHAPS), and the cell lysate was subjected to immunoprecipitation with anti-Bax-6A7 antibody-conjugated Sepharose beads. Immunoprecipitated samples were analyzed by Western blotting with anti-rat Bax-1D1 antibody. Lane a, CHAPS-solubilized cell lysate from untreated H9c2 cells; lane b, immunoprecipitated sample from untreated cells; lane c, CHAPS-solubilized cell lysate from staurosporine-treated H9c2 cells; and lane d, immunoprecipitated sample from staurosporine-treated cells. C: Western blotting analysis of H9c2 cells stably expressing human Bcl-2 and Bcl-XL. Cell lysates from the stable cell lines were analyzed by immunoblotting with anti-universal Bcl-XL-2H12 and anti-human Bcl-2–8C8 monoclonal antibodies. Lane a, control H9c2 cells; lane b, H9c2 Bcl-XL-stable cells; lane c, H9c2 Bcl-2-stable cells. D: efficacy of Bcl-2 and Bcl-XL in blocking Bax localization to mitochondria was assessed by transfecting green fluorescent protein (GFP)-Bax into H9c2 control and Bcl-2- and Bcl-XL-stable cells. Percentages of GFP-Bax punctate cells were determined from four separate visual fields. A minimum of 25 transfected cells was counted per visual field.

 
Bcl-2 and Bcl-XL partially block Bax localization to mitochondria. Prosurvival proteins Bcl-2 and Bcl-XL have been shown to antagonize the proapoptotic activity of Bax by blocking Bax translocation from the cytosol to the mitochondria. To determine the efficacy of Bcl-2 or Bcl-XL in inhibiting Bax in cardiac cells, we developed H9c2 cell lines that stably express these proteins. The expression levels of Bcl-2 and Bcl-XL in these stable cell lines were assessed by Western blotting with anti-Bcl-2 and and -Bcl-XL monoclonal antibodies (Fig. 2C). Control H9c2 cells and H9c2 cells that stably express Bcl-2 and Bcl-XL were then transfected with GFP-Bax. Transient transfection of GFP-Bax in mammalian cells often leads to a number of cells having Bax localized to mitochondria (11, 27). However, this mitochondrial localization of GFP-Bax could be inhibited in Cos-7 cells transiently cotransfected with Bcl-2 or Bcl-XL (11) and in HeLa cells that stably express these prosurvival proteins (Q. Hou and Y.-T. Hsu, unpublished data). We found that like other mammalian cells, transient transfection of GFP-Bax into H9c2 cells led to a number of cells having Bax localized to mitochondria (Figs. 2D and 3A). Prosurvival proteins Bcl-2 and Bcl-XL decreased the extent of Bax localization to mitochondria, as transient transfection of GFP-Bax into these stable cell lines resulted in a significant decrease in the percentages of cells having Bax localized to mitochondria (decreases from 55 to 36 and 23%, respectively; see Fig. 2D). Thus as in other cell lines, Bcl-2 and Bcl-XL can inhibit Bax localization to mitochondria in H9c2 cells.



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  		Fig. 3. Development of an H9c2 cell line stably expressing GFP-tagged Bax. A: H9c2 cells were transiently transfected with GFP-Bax and stained with mitochondrion-specific MitoTracker dye. A number of transfected cells had GFP-Bax localized to mitochondria. B: visualization of H9c2 cells stably expressing GFP-Bax by fluorescence microscopy. GFP-Bax-stable cells were stained with MitoTracker to label mitochondria. Overlay images show GFP-Bax and MitoTracker fluorescence. C: cell lysates from H9c2 cells (lane a) and H9c2 cells stably expressing GFP-Bax (lane b) were subjected to Western blotting analysis with anti-Bax-2C8, anti-Bcl-2–5B7, and anti-Bcl-XL-2H12 monoclonal antibodies for detection of endogenous rat Bax, Bcl-2, and Bcl-XL and expressed GFP-tagged human Bax. Lane RC, rat cardiomyocyte lysate. D: H9c2 cells stably expressing GFP-Bax showed an enhanced sensitivity to apoptosis induction. H9c2 cells and H9c2 cells stably expressing GFP-Bax were treated with 11 µM menadione for 17 and 41 h. At the specified time, the cells were stained with Hoechst nuclear stain, and the percentages of condensed and fragmented nuclei were quantitated from four separate visual fields. A minimum of 75 cells was counted per visual field.

 
Development and characterization of an H9c2 cardiomyoblast cell line that stably expresses GFP-Bax. By immunofluorescence labeling, we have shown that Bax translocates from the cytosol to the mitochondria in primary cardiomyocytes and in H9c2 cells during apoptosis. However, a major drawback associated with this method of assessing Bax subcellular localization is that these cells tend to round up and become loosely attached to the plate surface as they undergo apoptosis. The immunofluorescence labeling process very often removes these loosely attached cells and thus makes it difficult to quantitate cells with Bax localized to mitochondria. Previously we have shown that transient transfection of mammalian cells with GFP-tagged Bax could allow for the direct visualization of this translocation process (28). However, in mammalian cells, transient transfection of GFP-Bax often forces Bax localization to mitochondria and makes the distinction between forced and apoptosis-induced Bax redistribution to mitochondria difficult. In our study, we found that in H9c2 cells, transient transfection of GFP-Bax would result in approximately half of the transfected cells having Bax localized to mitochondria (see Figs. 2D and 3A). To overcome this problem, we attempted to develop H9c2 cardiomyoblast cell lines that stably express GFP-Bax. This was done by transfecting H9c2 cells with GFP-Bax and then selecting for stable clones that express this protein. Based on this approach, we have developed an H9c2 cardiomyoblast cell line that stably expresses GFP-Bax. These cells expressed slightly less GFP-Bax compared with transiently transfected cells when visualized by fluorescence microscopy. However, unlike transiently transfected cells, practically all of the H9c2 GFP-Bax-stable cells displayed a diffuse cytosolic GFP-Bax localization pattern, and virtually no cells had GFP-Bax localized to mitochondria (Fig. 3B). Immunoblotting with the anti-Bax antibody confirmed the expression of GFP-Bax in these H9c2 cells (Fig. 3C). These cells express equivalent levels of endogenous Bax, Bcl-2, and Bcl-XL as their H9c2 parental cells (Fig. 3C). In addition, this cell line showed an enhanced sensitivity to menadione compared with the H9c2 parental cells (Fig. 3D); this is in agreement with the previous study (23), which showed that an elevated expression of Bax promotes apoptosis.

To study the role of Bax in H9c2 cells undergoing apoptosis, we treated these GFP-Bax-stable cells with a number of well-known apoptosis inducers including menadione, hypoxia-reoxygenation-serum withdrawal, staurosporine (a protein kinase inhibitor), and SNP (a nitric oxide inducer). We found that although in the absence of apoptosis induction, GFP-Bax was primarily cytosolic, it translocated from the cytosol to the mitochondria when subjected to apoptosis induction with the above-stated inducers (Fig. 4, A and B). The mitochondrial localization of GFP-Bax in these cells during apoptosis induction was confirmed by staining with the mitochondrion-specific dye MitoTracker. Treatment of GFP-Bax-stable H9c2 cells with all-trans-retinoic acid for 1 wk in reduced serum did not change the ability of GFP-Bax to translocate to mitochondria during apoptosis induction with staurosporine (Fig. 4C). This suggests that cellular differentiation does not affect the signaling pathways that trigger Bax redistribution.



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  		Fig. 4. Apoptosis induction with menadione, staurosporine, sodium nitroprusside (SNP), or H/R/–serum withdrawal induces Bax translocation from the cytosol to the mitochondria in H9c2 cells stably expressing GFP-Bax. A: fluorescence microscopic visualization of GFP-Bax in H9c2 stable cells subjected to treatment with a number of apoptosis-inducing agents, including menadione (12.5 µM for 18 h), staurosporine (STS; 10 nM for 18 h), SNP (4 mM for 14 h), and hypoxia (24 h) with subsequent reoxygenation (12 h) and serum withdrawal. Cells were stained with MitoTracker to label mitochondria. Overlay images show GFP-Bax and MitoTracker fluorescence. B: percentages of GFP-Bax punctate cells after apoptosis induction with the above-described apoptosis inducers were quantitated from four separate visual fields. Between 30 and 80 cells were counted per visual field. C: retinoic acid differentiation of GFP-Bax-stable H9c2 cells did not affect Bax translocation to mitochondria. GFP-Bax-stable H9c2 cells grown in culture medium supplemented with all-trans-retinoic acid were subjected to apoptosis induction with 10 nM staurosporine for 18 h. Cells were then stained with MitoTracker and visualized by fluorescence microscopy. Overlay images show GFP-Bax and MitoTracker fluorescence.

 
Bax translocation to mitochondria results in release of cytochrome c (1, 18, 19) and activation of caspases that lead to cell death. Using immunofluorescence labeling with an anti-cytochrome c antibody, we found that cytochrome c was restricted primarily to mitochondria in cells that had GFP-Bax localized in the cytosol. However, in cells treated with menadione or staurosporine that had GFP-Bax localized to mitochondria, these cells displayed a cytosolic cytochrome c labeling pattern (Fig. 5A). In addition, by staining both the untreated and menadione- and staurosporine-treated cells with Hoechst nuclear stain, we found that apoptosis occurred only in the H9c2 cells that had GFP-Bax localized to the mitochondria. As shown in Fig. 5B, although cells with GFP-Bax localized in the cytosol maintained normal nuclear morphology, rounded-up cells with GFP-Bax in the mitochondria had either condensed or fragmented nuclei, which is a common feature associated with apoptosis.



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  		Fig. 5. GFP-Bax translocation to mitochondria during apoptosis is associated with the release of cytochrome c and nuclear condensation and fragmentation. A: GFP-Bax-stable H9c2 cells were subjected to treatment with menadione or staurosporine for 17 h. Untreated and treated cells were then subjected to immunofluorescence labeling with an anti-cytochrome c antibody. Labeled cells were visualized by fluorescence microscopy. B: GFP-Bax-stable H9c2 cells were subjected to treatment with 12 µM menadione or 10 nM STS for 17 h. Cells were then stained with Hoechst nuclear stain and visualized by fluorescence microscopy.

 
Bax translocation from cytosol to mitochondria is associated with the loss of {Delta}{Psi}m. This loss appears to precede the bulk of Bax translocation to mitochondria (8, 27). To examine the relationship of Bax subcellular localization vis-à-vis {Delta}{Psi}m of the GFP-Bax-stable H9c2 cells, we stained both the untreated and menadione-treated cells with the {Delta}{Psi}m-sensitive dye TMRE. As shown in Fig. 6A, untreated cells with GFP-Bax residing in the cytosol had brightly stained mitochondria, which are indicative of an intact {Delta}{Psi}m. On the other hand, for cells treated with menadione that had GFP-Bax localized to mitochondria, there was a significant loss in {Delta}{Psi}m as reflected by the weak TMRE staining. Based on these results, it appears that the translocation of Bax to mitochondria in GFP-Bax-stable H9c2 cells is associated with the loss of {Delta}{Psi}m. Using a TMRE-based {Delta}{Psi}m-screening kit, we have carried out a quantitative analysis of this cell death-associated loss of {Delta}{Psi}m. As shown in Fig. 6B, the treatment of H9c2 cells with menadione or staurosporine (which caused a large fraction of cells to have GFP-Bax localized to mitochondria) resulted in a significant loss in {Delta}{Psi}m as evidenced by a decrease in the fluorescence intensities of these samples.



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  		Fig. 6. Bax translocation to mitochondria is associated with the loss of mitochondrial membrane potential ({Delta}{Psi}m). A: GFP-Bax-stable H9c2 cells were treated with 12 µM menadione for 17 h. Both the treated and untreated cells were then stained with the {Delta}{Psi}m-sensitive dye tetramethylrhodamine (TMRE) and visualized by fluorescence microscopy. B: GFP-Bax-stable H9c2 cells were plated onto a 96-well plate. Cells were treated with the stated concentration of menadione or STS for 17 h (in triplicates). Both the treated and untreated cells were then stained with TMRE and analyzed by using a fluorescence plate reader (excitation and emission wavelengths, 530 and 590 nm, respectively).

 
Taken together, our studies indicate that the translocation of Bax from the cytosol to the mitochondria is an important step in cardiac cells undergoing apoptosis. In addition, through extensive characterization of the properties of our newly developed GFP-Bax-stable H9c2 cell line, it appears that the selection process used to develop these cells did not affect their apoptotic signaling pathways and that these cells represent an important tool for studying Bax-mediated cell death.


   DISCUSSION
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 METHODS
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Bax is a proapoptotic protein of the Bcl-2 family that plays an important role in mediating cell death under both physiological and pathological conditions. In an ischemic heart, Bax was shown to be upregulated to mediate cardiomyocytic apoptosis and cardiac damage (16, 20). Ablation of Bax, conversely, could attenuate cardiac damage through inhibition of cardiac apoptosis (10), which signifies the importance of this protein in the regulation of cardiac survival. Presently, the molecular regulation involved in Bax-mediated cardiomyocytic cell death is unclear. In a number of cellular systems, Bax translocation from the cytosol to the mitochondria culminates in a key step in Bax-mediated apoptosis. Bax translocation to mitochondria leads to the release of cytochrome c (18) and is associated with a decrease in {Delta}{Psi}m (8, 27). Thus far, only a single study has focused on Bax distribution dynamics during cardiac apoptosis; that study (4) involved the transient transfection of GFP-Bax in cardiomyocytes, and apoptosis was induced with the help of the kinase inhibitor staurosporine. In this report, we have broadened the description of Bax dynamics by showing that in primary neonatal rat cardiocytes and in H9c2 cardiomyoblast cells, translocation of endogenous Bax from the cytosol to the mitochondria is an important step in cardiac apoptosis. In addition, we have developed an H9c2 cardiomyoblast cell line that stably expresses GFP-tagged Bax and demonstrated that this cell line could be used as an important tool to study Bax redistribution in the presence of a variety of apoptotic stimuli, including a physiologically relevant model such as hypoxia-reoxygenation.

We have previously shown (14, 22) that when Bax translocates to mitochondria in murine thymocytes and in Cos-7 green monkey kidney epithelial cells, Bax undergoes a conformational change that leads to the exposure of an NH2-terminal 6A7 antibody epitope. In this study, we have shown by immunofluorescence labeling and immunoprecipitation analyses that in rat cardiac cells, Bax exposes its NH2-terminal 6A7-antibody epitope when it inserts into mitochondrial membranes. This suggests that the NH2-terminal conformational change of Bax is likely a universal event in cells undergoing apoptosis.

The primary mechanism by which the prosurvival family members such as Bcl-2 and Bcl-XL block the function of Bax appears to be the inhibition of Bax translocation from the cytosol to the mitochondria (7, 21). To determine the effectiveness of these proteins in inhibiting the proapoptotic activity of Bax in cardiac cells, we have developed H9c2 cell lines that stably express these prosurvival proteins. These cells were transiently transfected with GFP-Bax. We found that the presence of Bcl-2 and Bcl-XL significantly blocked Bax localization to mitochondria. However, the extent of Bax inhibition by Bcl-2 and Bcl-XL in H9c2 cells appeared to be lower compared with other cellular systems (Bcl-2- and Bcl-XL-stable HeLa cells and Cos-7 cells transiently transfected with Bcl-2 and Bcl-XL) in which complete inhibition of Bax localization to mitochondria was observed. This suggests that there may be a slight variation in the mechanism of Bax inhibition by Bcl-2 and Bcl-XL between H9c2 cells and other cell types.

Presently, there are three methods for determining the subcellular localization of Bax in cells. The first is by subcellular fractionation and Western blotting (12). This particular method, however, is cumbersome and requires a large quantity of cells. In addition, this method does not give a quantitative assessment of the percentage of cells within a population that have Bax localized to mitochondria. The second method is by immunofluorescence labeling with anti-Bax antibodies. This method, although it can give a clear view of Bax subcellular distribution, is not quantitative, because cells in the late stage of apoptosis (which have Bax localized to mitochondria) tend to round up and become loosely attached to the plate surface. These cells are easily removed during the washing steps of the labeling study. Finally, the third method for determining Bax localization during apoptosis is through the transient transfection of GFP-Bax in mammalian cells (28). This method also gives a very clear distinction between the cytosolic vs. mitochondrial localization of Bax in living cells. However, it has a major drawback in that transient transfection of GFP-Bax often leads to the forced localization of Bax to mitochondria in a significant number of transfected cells. This makes the assessment difficult regarding determination of whether the observed Bax localization to the mitochondria is due to the forced GFP-Bax localization to the mitochondria or the apoptosis induction.

Our development of a cardiomyoblast cell line that stably expresses GFP-Bax overcomes the above-stated drawbacks associated with detecting Bax subcellular localization. This cell line basically enables us to monitor, with great ease, Bax subcellular distribution in living cells subjected to apoptosis induction. In our study, we found that a number of well-known apoptosis stimuli, including menadione, staurosporine, hypoxia-reoxygenation-serum withdrawal, and SNP, could induce the translocation of Bax to mitochondria. In addition, we found that the differentiation of H9c2 cardiomyoblasts with retinoic acid did not affect Bax translocation to mitochondria during apoptosis induction, which suggests that the upstream signaling mechanism that triggers Bax redistribution is not regulated by cellular differentiation. Moreover, we found that Bax translocation to mitochondria in the GFP-Bax-stable H9c2 cells was associated with the release of cytochrome c, the loss of {Delta}{Psi}m, and nuclear fragmentation. Thus based on our studies, it appears that the underlying mechanisms involving Bax-mediated apoptosis are similar between the GFP-Bax-stable H9c2 cardiomyoblasts and other mammalian cells.

Overall, Bax translocation from the cytosol to the mitochondria appears to be an intrinsic event in cardiac apoptotic cell death. Our development of a GFP-Bax-stable H9c2 cell line represents a unique approach for examining the mechanism of Bax-mediated cardiac cell death. In addition, because our study and studies by others have shown that a diminishing {Delta}{Psi}m is associated with GFP-Bax localization to mitochondria, a high-throughput assay based on the {Delta}{Psi}m measurement could perhaps serve as the initial screening step to identify anti-Bax compounds. Because the loss of {Delta}{Psi}m precedes Bax translocation to mitochondria and the loss of {Delta}{Psi}m may not always be indicative of apoptosis, a subsequent step would be needed to assess the efficacy of any identified {Delta}{Psi}m-preserving compounds in blocking Bax localization to mitochondria. Taken together, this two-step approach could potentially identify therapeutic compounds that may block the signaling pathway leading to Bax translocation to mitochondria and attenuate Bax-mediated cell death.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grants COBRE P20 RR-16434 and RO1 NS-40932.


   ACKNOWLEDGMENTS
 
The authors thank Fei Qiao and Yuyu Yao for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Yi-Te Hsu, Dept. of Biochemistry and Molecular Biology, Medical Univ. of South Carolina, 173 Ashley Ave., PO Box 250509, Charleston, SC 29425 (E-mail: hsuy{at}musc.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.


   REFERENCES
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