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Am J Physiol Heart Circ Physiol 293: H3111-H3121, 2007. First published September 14, 2007; doi:10.1152/ajpheart.00328.2007
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Doxorubicin-induced cardiotoxicity: direct correlation of cardiac fibroblast and H9c2 cell survival and aconitase activity with heat shock protein 27

Samir Turakhia,1 C. D. Venkatakrishnan,1 Kathy Dunsmore,2 Hector Wong,2 Periannan Kuppusamy,1 Jay L. Zweier,1 and Govindasamy Ilangovan1

1Division of Cardiovascular Medicine, Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio; 2Division of Critical Care Medicine, Cincinnati Children's Hospital and the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio

Submitted 15 March 2007 ; accepted in final form 14 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The use of doxorubicin (Dox) and its derivatives as chemotherapeutic drugs to treat patients with cancer causes dilated cardiomyopathy and congestive heart failure due to Dox-induced cardiotoxicity. In this work, using heat shock factor-1 wild-type (HSF-1+/+) and HSF-1 knockout (HSF-1–/–) mouse fibroblasts and embryonic rat heart-derived cardiac H9c2 cells, we show that the magnitude of protection from Dox-induced toxicity directly correlates with the level of the heat shock protein 27 (HSP27). Western blot analysis of normal and heat-shocked cells showed the maximum expression of HSP27 in heat-shocked cardiac H9c2 cells and no HSP27 in HSF-1–/– cells (normal or heat-shocked). Correspondingly, the cell viability, measured [with (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] after treatment with various concentrations of Dox, was the highest in heat-shocked H9c2 cells and the lowest in HSF-1–/– cells. Depleting HSP27 in cardiac H9c2 cells by small interfering (si)RNA also reduced the viability against Dox, confirming that HSP27 does protect cardiac cells against the Dox-induced toxicity. The cells that have lower HSP27 levels such as HSF-1–/–, were found to be more susceptible for aconitase inactivation. Based on these results we propose a novel mechanism that HSP27 plays an important role in protecting aconitase from Dox-generated O2bullet–, by increasing SOD activity. Such a protection of aconitase by HSP27 eliminates the catalytic recycling of aconitase released Fe(II) and its deleterious effects in cardiac cells.

superoxide dismutase

TREATMENT OF PATIENTS WITH CANCER with higher doses of the chemotherapeutic drug doxorubicin (Dox) and its derivatives causes dilated cardiomyopathy (DCM) and congestive heart failure (CHF) (36) as serious side effects and thus the dose is limited to less than required for optimum killing of cancer cells. Fortunately, the mechanisms of killing cancer cells (intended effect) and causing DCM and CHF (unwanted effect) by Dox and its analogs are found to be different, indicating that the mode of action of these drugs depends on the nature of tissues. Dox-based chemodrugs kill cancer cells primarily by DNA intercalation and damage and inhibition of topoisomerase II (36). On the other hand, cardiotoxicity is caused by mitochondrial apoptosis, which is triggered and mediated by reactive oxygen species (ROS), generated in the redox reactions of Dox (10, 18). This difference in action gives hope that by selectively targeting some of the pathways through new approaches, the inevitable occurrence of DCM and CHF among the Dox-treated patients with cancer, could be avoided. Thus there is renewed interest to identify and selectively target these pathways.

ROS, generated by Dox redox reactions, are aggravated by many factors in cardiac cells. For example, free redox cycling iron species have been found to play important roles in the development of Dox-induced cardiomyopathy (1, 4, 26, 36). Important evidence of iron participation was obtained from the addition of membrane-permeable iron chelators and their ability to attenuate the Dox-induced apoptosis (25). Similar to the suppression of Dox-activated NF-{kappa}B in the presence of GSH-peroxidase-1 (GSHPx-1), the addition of these iron chelators reduced the NF-{kappa}B levels (54), suggesting that proapoptotic effects of ROS are mediated by the iron. Although there is not much free iron available in cardiomyocytes, redox reactions of Dox have been proposed to induce sequestration of iron from intracellular sources such as ferritin and cytoplasmic aconitase. Especially, O2bullet– formed in the Dox redox reaction and the metabolites, notably the Dox-ol, have been found to target aconitase and release Fe(II) from its [4Fe-4S] clusters (36). Thus retaining integrity of both cytoplasmic and mitochondrial aconitases in the presence of Dox can potentially reduce Dox-induced cardiotoxicity.

Apart from the Fe(II) release by the Dox-generated O2bullet–, many studies in the literature have shown that free radical species such as O2bullet– and .OH, generated during redox cycling of Dox, can directly cause apoptosis and cell death by favoring cytochrome c release (36). It has also been reported that Dox induces apoptosis by activating p38 mitogen-activated protein kinases (p38 MAPK-{alpha} and -beta). Inhibition of p38 MAPK by selective cardiac-specific overexpression of cysteine-rich metallothioneins reduced the apoptosis (23). Interestingly, heat shock proteins (HSPs), which are induced as a stress response, have been found to be phosphorylated by MAPK activated protein-2 (MAPKAP-2) (downstream of p38 MAPK), and phosphorylated HSPs prevent apoptosis (2, 3, 5, 6). Thus expression of p38 MAPK-phosphorylatable HSPs, such as HSP27, should regulate the Dox-activated p38 MAPK and may inhibit the Dox-induced apoptosis and cell death, although this hypothesis still remains untested.

In the present work, we hypothesize that HSP27 may reduce Dox-induced cardiac cell death by reducing the O2bullet–-induced inactivation of aconitase and iron redox cycling and by increasing SOD activity; that is, HSP27 may effectively act as an endogenous antioxidant against Dox. To evaluate this hypothesis, we use three different cardiac cell lines, namely, cardiac H9c2 cells, heat shock factor-1 wild-type (HSF-1+/+), and HSF-1 knockout (HSF-1–/–) fibroblasts, because it is extremely difficult to modulate the HSP27 level in isolated adult cardiomyocytes. Moreover, two other major concerns preclude the use of adult cardiomyocytes in the present study. Heat-shock treatment is used as a nonspecific mode of HSP27 expression and its phosphorylation. Adult cardiomyocytes in culture may not be viable after heat shock. Moreover, the total time required to complete a typical heat-shock treatment and Dox treatment is more than 72 h. Adult cardiomyocytes may not be viable for more than 3 days. Cardiac H9c2 cells are from a clonal muscle cell line derived from embryonic rat hearts. Although these cells display certain features of skeletal muscle (20, 24), they retain many features of cardiac muscle such as expression of a cardiac isoform of creatine phosphokinase, L-type calcium channels, and the tissue-specific splicing protein smN (16, 21). Previous studies reported in the literature (11, 28) have used this cell line as a model system to evaluate various characteristics of cardiomyocytes, including cardiotoxicity caused by Dox. Also, previous studies on this cell line revealed that various HSPs can be induced by heat shock (38, 39), pharmacological induction (42, 49), or viral vector transfection (9). On the other hand, both HSF-1+/+ and HSF-1–/– embryonic fibroblasts have been demonstrated as good models to study HSP effects on various cell signaling (32, 41, 55).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and viability measurements. HSF-1+/+ and HSF-1–/– mouse embryonic fibroblasts (a gift from Dr. Ivor Benjamin, University of Texas Southwestern Medical Center, Dallas, TX) were maintained in DMEM containing 10% FBS, 55 µM beta-mercaptoethanol, 0.1 mM MEM nonessential amino acid solution, 2 mM L-glutamine, and 10 ml/l of antibiotic/antimyotic solution containing 10,000 U/ml penicillin G, 10,000 µg/ml streptomycin sulfate, and 25 µg/ml amphotericin B (55). Cardiac H9c2 cells [CRL 1446; American Type Culture Collection (ATCC), Rockville, MD] were cultured in DMEM (ATCC) supplemented with 10% fetal bovine serum (ATCC) and 1% antibiotic/antimycotic solution (100x, Sigma).

Toxicity measurements (MTT Assay). Cell viability against Dox-induced toxicity was determined using (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in 96-well plates. Cells were treated with a supplemental medium containing the desired concentration of Dox (in the range 0–10 µM) for 6 h and then switched to regular medium. After 24 h, MTT assay was carried out as described previously (4). The Dox concentration range was chosen based on the fact that the present model is an acute model (36).

Synthesis and transfection of HSF-1 siRNA. HSF-1 small interfering (si)RNA was synthesized by Ambion (Austin, TX) for the targeted sequence (5'-AAGAGAAAGATCCCTCTGATG-3'). The targeted sequence resides within the open reading frame of the rat HSF-1 gene (accession, XM_343270) 613 nucleotides downstream of the start codon. With the use of a Cenix Bioscience-designed algorithm, a 21-mer sequence (sense strand: 5'CCCUGCAGGUUGUUCAUAAtt: antisense strand 5'UUAUGAACAACCUGCAGGGtc), which closely matched the target sequences, was synthesized. The supplied siRNA was guaranteed to be >80% pure by analytical HPLC.

The 21-mer siRNA for HSF-1 was transfected in H9c2 using the siPORT NeoFX transfecting agent (Ambion). Two aliquots of OptiMEM Medium (Invitrogen), 1.5 ml each, were added to 20 µl of SiPORT NeoFX and HSF-1 siRNA (final concentration, 2 nM), respectively, and incubated for 10 min at room temperature. The two were mixed (final volume, 3.0 ml), incubated for a further 10 min at room temperature for the formation of transfection complex, and dispensed in a 10-cm petri dish. H9c2 (1 x 105 cells/ml) cells were added immediately and gently mixed, and the plates were then incubated at 37°C for 24 h for the effective transfection. After 24 h, cells were used either for a toxicity assay or Western blot analysis.

HSP27 expression in HSF-1–/– cells. HSF-1–/– cells were transfected as follows. The cultures with 50% confluency were washed and treated with 2 µg of either empty vector or HSP27 cDNA (Invitrogen)-cloned vector pcDNA-HSP27. The cells were added with G418 (500 µg/ml) since the vector had the geneticin resistance gene. The G418-resistant colonies were singly cloned and propagated for 2 wk in G418-containing medium. In control experiments, the empty vector was transfected. These cells were used for either Western blot analysis or Dox-induced toxicity assays.

Western blot analysis. Cells, after appropriate treatment, were lysed in radioimmunoprecipitation assay (RIPA) buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µl of 1x protease inhibitor cocktail, 1 mM of PMSF, and 1 mM of NaVO3). Normalized lysates were used for SDS gel electrophoresis. Twenty to thirty micrograms of total proteins (30 µl) were resolved on a precasted 4–12% Bis-Tris PAGE gel (Invitrogen) and transferred onto a polyvinylidene difluoride (PVDF) membrane. After being blocked [5% fat-free milk in Tris-buffered saline with Tween (TBST)], the membranes were treated overnight at 4°C with respective primary antibody at appropriate dilution, followed by the addition of HRP-linked secondary antibody (1:3,000 dilution). GAPDH was used as the loading control in all Western blot analysis because the level of actin (normally used as loading control) was found to be affected by the HSP27 level. Densometric analyses of Western blot analyses were carried out using PDQuest software (Bio-Rad).

Aconitase assay. The aconitase activity assay in the whole cell lysate was carried out using an established procedure (26). Briefly, the cells were treated with a desired Dox concentration for 6 h and then switched to regular medium. After 24 h, the cells were lysed using a RIPA buffer containing 0.2% Triton, 100 µM diethylenetriamine pentaacetic acid (DTPA), and 5 mM citrate in PBS. Aconitase activity was measured for 100 µg of protein in 1 ml of 100 mM Tris·HCl (pH 8.0), containing 20 mM of DL-trisodium isocitrate. The rate of change of absorbance was followed for 5 min at 240 nm in a UV spectrometer.

SOD activity. SOD activity in the whole cell lysate was measured using a spectrophotometric ferricytochrome c assay (35). The normal and heat-shocked H9c2, HSF-1+/+, and HSF-1–/– cells (2 x 106 cells/ml) were treated with the desired concentration of Dox for 6 h. After 24 h, the cells were lysed using a RIPA buffer and the total protein concentration was determined. The activity of SOD was measured in terms of change in absorption at 240 nm due to the reduction of cytochrome c by the superoxide generated by the xanthine/xanthine oxidase (X/XO) system.

Flow cytometry. The ROS generation in cardiac H9c2, HSF-1+/+, and HSF-1–/– cells was measured by chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) staining. For DCFDA staining, cells treated with Dox at appropriate conditions were washed with ice-cold PBS and treated with 10 µM CM-H2DCFDA (Molecular Probes, Invitrogen) in serum-free medium for 45 min. Cells were then trypsinized and suspended in PBS, and the fluorescence was studied at 538 nm.

Data analysis. Data are presented as means ± SE. Statistical analysis was performed using Student's t-test and analysis of variance (one-way ANOVA). The general acceptance level of significance was P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HSP27 expression in cardiac H9c2, HSF-1+/+, and HSF-1–/– cells. Figure 1 demonstrates the Western blot analysis and quantitative data on HSP27 expression in these cells. As shown in Fig. 1A, HSP27 expression was completely suppressed in HSF-1 knockout cells (HSF-1–/–). Among the three cell lines studied, the cardiac H9c2 cells showed a higher expression of HSP27 than other cells at normal conditions (Fig. 1A). The quantitative plots (Fig. 1B) show that the HSP27 expression was about two times higher in H9c2 cells compared with the HSF-1+/+ cells. However, there was no significant change in the expression of other HSPs such as HSP70 and HSP90 (data not shown). For example, although there was no change in HSP70 for HSF-1–/–after heat shock, the HSF-1+/+ cells showed only a 10% increase compared with normal cells.


Figure 1
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  		Fig. 1. Analyses of heat shock protein 27 (HSP27) in cardiac H9c2, heat shock factor-1 knockout (HSF-1–/–) and HSF-1 wild-type (HSF-1+/+) cells. A: Western blot analysis of HSP27, phospho s-15 HSP27, phospho s-82 HSP27, and GAPDH (loading control) for these cells both in normal and heat-shocked conditions. In heat-shocked cases, the cells were heat shocked for 4 h at 42°C, 24 h prior to analysis. B: quantitative plots of total HSP27 expressed in these cells. White bars represent cells at normal conditions and black bars heat-shocked cells. C: quantitative plots for phospho s-15 HSP27. D: quantitative plots for phospho s-82 HSP27. Error bars in quantitative plots are the SEs from 3 independent (n = 3) measurements. P values were obtained by comparison between normal and heat-shocked H9c2 cells. au, Arbitrary units.

 
When subjected to heat shock at 42°C, H9c2 cells overexpressed HSP27 compared with the level at normal conditions (Fig. 1A). The quantitative plots (Fig. 1B) showed that HSP27 is expressed about two times higher compared with normal (non-heat-shocked cells). Similarly, HSF-1+/+ showed an increase (about 2 times) in HSP27 but was still lower than the cardiac H9c2 cells. On the other hand, there was no trace of HSP27 in HSF-1–/– cells even after heat shock. A similar trend was observed in HSP70 and HSP90 after heat shock.

Phosphorylated HSP27 was analyzed by Western blot analysis using specific antibodies of phosphorylated serine residues in all three cell lines, both in normal and heat-shocked conditions. Phosphorylation of s-15 and s-82 have been found to functionalize HSP27 (27, 43, 51). The Western blot analysis obtained with respective antibodies for phospho s-15 and s-82 are also shown in Fig. 1A. Neither HSF-1–/– nor HSF-1+/+ cells showed any significant amount of phospho s-15, whereas the cardiac H9c2 cells showed only a trace at normal condition. However, heat shock increased the amounts of phospho s-15 and phospho s-82 HSP27. We recently reported that heat shock increased the phosphorylation of these residues in HSP27 in cardiac H9c2 cells, by activating MAPKAP-2, which is downstream of p38 MAPK (51).

Cell viability versus HSP27 level in cardiac cells. Dox-induced toxicity in all three cardiac cell lines was assessed using an MTT assay. In normal conditions, cell viability was higher in cardiac H9c2 cells compared with other cells (Fig. 2). In the concentration range 0–10 µM, the cell viability decreased to 70% in H9c2 cells. However, in both HSF-1+/+ and HSF-1–/– cells, the viability decreased to <40% and 20%, respectively.


Figure 2
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  		Fig. 2. Cell viability determination [using (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] of doxorubicin (Dox)-induced toxicity in cardiac H9c2, HSF-1–/–, and HSF-1+/+ cells. The cells (in 96-well plates) were treated with various concentrations of Dox for 6 h, and the medium was then changed to normal medium. The cell viability was assessed by MTT after 24 h. In the case of heat-shocked cells, the cells were given heat shock 24 h before Dox treatment. Each data point presented was an average of measurements from 10 wells. This is a representative set of 3 independent measurements.

 
Upon heat-shock treatment, the H9c2 cells showed increased viability compared with normal (Fig. 2) H9c2 cells. However, there was no such increase observed in the case of either the HSF-1+/+ or the HSF-1–/– cells. The trend, observed in the viability, was parallel to the HSP27 expression measured by Western blot analysis (shown in Fig. 1), showing that the cell viability against the Dox directly correlates with the level of HSP27 in these cells.

For further confirmation of the protective effect of HSP27 against the Dox-induced toxicity, the HSF-1–/– cells can be overexpressed with HSP27, and one can then check whether the wild-type phenotype is rescued. Alternatively, in the HSF-1 positive cells (H9c2 or HSF-1+/+) HSP27 can be selectively silenced using appropriate siRNA (8), and one can then check whether such a selective silencing sensitizes the toxicity. In the present work we used both approaches. Based on a basic local alignment search tool (BLAST) search, three different siRNA were designed to specifically target the HSP27 genes. Among these siRNA, the one with the specific sequence described in MATERIALS AND METHODS gave significant reduction in HSP27, without altering the contents of HSP70 and HSP90. Variations in both siRNA concentration (2–20 nM) and transfection time (12–76 h) were carried out (data not shown). These experiments showed that 2 nM of the siRNA for a 24-h treatment period was optimum to achieve the maximum suppression of HSP27 in cardiac H9c2 cells, as shown in Fig. 3A. However, the other HSPs such as HSP90 and HSP70 remained the same (Fig. 3A). The quantitative plots (Fig. 3B) showed that the HSP27 is attenuated to one-third of the unsilenced H9c2 cells. Dox-induced toxicity assays on these HSP27-silenced cells were carried out in the concentration range 0–10 µM, and the results are shown in Fig. 3C, along with the results obtained with siRNA-untreated cells. The viability for siRNA-treated cells was observed to be consistently lower compared with that of the untreated cells. Additional confirmation on the protective effects was obtained by overexpressing HSP27 in HSF-1–/– cells. Human HSP27 cDNA was cloned in the pcDNA3.1D vector and stably transfected in HSF-1–/– cells. Western blot analysis showed a high level of HSP27 expression in transfected cells (Fig. 4, A and B). The Dox-induced toxicity, carried out using the MTT assay, showed higher survival (measured after 24 h of postdrug treatment) of HSP27 overexpressed HSF-1–/– cells. These results once again confirm that HSP27 is playing a protective role against the Dox-induced toxicity in cardiac cells.


Figure 3
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  		Fig. 3. Viability measurements in HSP27-silenced cardiac H9c2 cells, using small interfering (si)RNA. A: Western blot analysis of HSP27, HSP70, and HSP90 in normal and siRNA-treated H9c2 cells. Cells were plated in 75-cm2 plates with 2 nM of siRNA (details in MATERIALS AND METHODS). After the cells were maintained with siRNA for 24 h, the cells were either used for Western blot analysis or a toxicity assay. B: quantitative plots of HSPs, obtained from densometric analyses of blots. Error bars are SE from 3 independent measurements. C: cell viability (MTT assay) for normal (open symbols) and siRNA-treated cells (closed symbols), measured at 24 h postdrug treatment.

 

Figure 4
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  		Fig. 4. Dox-induced toxicity in HSP27-overexpressed HSF-1–/– cells. A: Western blot analysis showing overexpression of HSP27 in control (empty vector transfected) and pcDNA-HSP27-transfected HSF-1–/– cells. B: quantitative analysis of Western blot analysis shown in A. C: viability measurements (MTT) in control and HSP27-overexpressing HSF-1–/– cells, treated with various concentrations of Dox. MTT assay was carried out 24 h after Dox treatment.

 
The effect of desferrioxamine, an Fe(III) chelator, on the Dox-induced toxicity in cardiac H9c2, HSF-1+/+, and HSF-1–/– cells was studied. This chelator has been previously demonstrated to reduce Dox-induced cardiotoxicity in in vivo models (52). A dose-dependent study was carried out to evaluate the toxicity in all three cell lines. Up to 1 mM, there was no significant toxicity observed (data not shown). Based on these results and previously used doses in vivo (52), 200 µM concentration was chosen in the present study. The addition of 200 µM desferrioxamine showed a significantly increased viability in all Dox concentrations for all three cell lines (Fig. 5, A–C).


Figure 5
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  		Fig. 5. Effect of desferrioxamine (DFO), an Fe(III) chelator, on the Dox-induced toxicity in H9c2 (A), HSF-1+/+ (B), and HSF-1–/– (C) cells. Cells, in 96-well plates, were treated with DFO (200 µM) for 30 min before the addition of Dox at different concentrations and maintained in the medium for the next 24 h until the cells were analyzed for cell viability by MTT assay.

 
The above results (Figs. 1–4) establish two facts: the observed Dox-induced toxicity is mediated by iron in all three cell lines studied, and this iron-mediated toxicity could be suppressed by the increased expression of HSP27. Although the fact that chelation of iron by addition of desferrioxamine has been previously shown to be effective in minimizing the Dox-induced toxicity (17, 57), it is important to infer from our studies that the magnitude of reduction in toxicity depends on the HSP27 levels. Further experiments were designed to elucidate the mechanism of such attenuation by HSP27 overexpression as described below in Aconitase activity: negative correlation with the levels of HSP27.

Aconitase activity: negative correlation with the levels of HSP27. Aconitase, an enzyme that converts citrate to isocitrate in the Krebs cycle, has been recently shown to become a target of Dox-generated O2bullet– (36). It was found that [4Fe-4S] of aconitase is inactivated by O2bullet– when treated with Dox (26, 37). In the present work we carried out experiments to determine whether there is any correlation between the HSP27 level and aconitase activity in these cells, that is, whether excess HSP27 can reduce aconitase inactivation. In the first set of experiments, aconitase activity measurements were carried out in both normal and heat-shocked cells (no Dox treatment). Under normal conditions, with no Dox treatment, H9c2 cells showed the lowest activity among the three cell lines (Fig. 6A). Heat shock was found to slightly reduce the activity. A quantitative plot of aconitase activity versus the overall content of HSP27 showed an inverse correlation (Fig. 6B), indicating that HSP27 induction preserves the aconitase activity.


Figure 6
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  		Fig. 6. Aconitase measurements in whole cell lysates of H9c2, HSF-1+/+, and HSF-1–/– cells. A: both normal (Nor) and heat-shocked (HS) cells were lysed, and the whole cell lysates were used for aconitase activity measurements in terms of citrate to isocitrate conversion. The assay medium contained 100 µg of protein concentration, and absorbance was measured at 550 nm at a time interval of 10 s. The slope of absorbance change with respect to time was converted into activity. B: quantitative correlation of aconitase activity, measured in the whole cell lysate, to HSP27 level in H9c2, HSF-1+/+, and HSF-1–/– cells. C: effect of Dox treatment (at concentrations 0, 0.25, 5.0, and 10 µM) on the aconitase activity in these cells (normalized with respect to untreated with Dox in each cell line).

 
Aconitase activity measurements were also carried out in all three cell lines, which were treated with various concentration of Dox, both at normal and heat-shocked conditions. When treated with Dox, the three cell lines showed different magnitudes of aconitase susceptibility. Although the aconitase activity was low in H9c2 cells (Fig. 6A), after treatment with Dox, the activity was retained in close to 80–90% of untreated cells (Fig. 6C). In the case of heat-shocked H9c2 cells, there was no inactivation by Dox (Fig. 6C). On the other hand, however, the HSF-1–/– cells, which had the highest aconitase activity in the absence of Dox (Fig. 6A), showed the highest inactivation by Dox, i.e., 70% (Fig. 6C). The HSF-1+/+ cells also displayed a reduction of up to 60% after treatment with Dox. The activity loss after Dox treatment was also studied using these three cell lines after subjecting them to heat shock (Fig. 6). Unlike the normal cells, the heat-shocked H9c2 cells did not show any reduction in aconitase activity after Dox treatment. On the other hand, large decreases were observed in both the heat-shocked HSF-1–/– and HSF-1+/+ cells, similar to the behavior observed with these cell lines under normal conditions (Fig. 6C). Altogether, these results show that heat shock or HSP27 overexpression protects the cells by preserving the aconitase activity, presumably blocking Fe(II) release, which is known to aggravate the toxicity.

SOD activity: a positive correlation to the level of HSP27. Since the Dox-generated O2bullet– is responsible for the aconitase inactivation in Dox-treated cells, we carried out further experiments to determine SOD activity and correlate it to the HSP27. Total SOD activity (both Cu-Zn and MnSOD) was measured in whole cell lysates of these cells treated with various concentrations of Dox. The SOD activity measured both at normal and heat-shocked conditions in all three cell lines is summarized in Fig. 7. Overall, the trend observed in these data shows the reverse of what was observed with aconitase activity, shown in Fig. 6; that is, the SOD activity was found to directly correlate with the HSP27 levels (Fig. 7A). The SOD activity was observed to be higher in H9c2 cells compared with HSF-1–/– cells. The HSF-1+/+ cells did not show any significant change. When the cells were subjected to heat shock, the H9c2 cells showed an increase in the SOD activity (Fig. 7), confirming our previous report that heat shock increases the SOD activity (51). However, there was no significant change noticed in the other HSF-1+/+ and HSF-1–/– cells. Once again, the trend observed here is parallel to the HSP27 expression in these cells.


Figure 7
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  		Fig. 7. SOD activity in whole cell lysates of H9c2, HSF-1+/+, and HSF-1–/– cells. A: both normal and heat-shocked cells were lysed, and the whole cell lysates were used to measure SOD activity, as described in MATERIALS AND METHODS. The absorbance change with respect to time was measured, and the activity was defined as the rate of 0.0125 absorbance unit/min as 1 unit of activity. B: quantitative plot of correlation between SOD activity and HSP27 levels in these cells. C: effect of Dox treatment (at concentrations 0, 0.25, 5.0, and 10 µM) on the SOD activity in these cells (normalized with respect to untreated with Dox in each cell line).

 
Treatment with Dox was also observed to show an opposing trend in SOD activity (Fig. 7C) compared with the aconitase activity (Fig. 6C). Unlike the aconitase where a decrease in activity was found after treatment with Dox, the SOD activity increased in all three cell lines upon treatment with Dox (Fig. 7C). However, the magnitude of increase differed, depending on the cell line. Treatment of H9c2 cells with Dox increased the SOD activity roughly about 25% compared with untreated cells. On the other hand, a huge increase in activity was observed in HSF-1–/– cells (about 2.5 times that of the no-drug treatment level). The HSF-1+/+ cells also showed a significant increase in SOD activity.

Flow cytometry: HSP27 overexpression reduced ROS generation. Since the SOD activity was found to be different in the three cell lines both at control and Dox-treated conditions, further studies were carried out on these cells using flow cytometry [fluorescence-activated cell sorting (FACS)], to assess the magnitude of ROS (including nonradical species such as H2O2) generation. Both normal and heat-shocked cells, which were treated with Dox, were stained with DCFDA, fixed, and then analyzed using FACS. DCFDA has been reported to show high sensitivity to H2O2 (product of SOD reaction with O2bullet–). The histogram of cell distribution is illustrated in Fig. 8, A and B. For normal untreated cells, the lowest fluorescence intensity (left-most histogram) was observed for HSF-1–/–, and the highest (right-most histogram) was observed for H9c2 cells. This is consistent with SOD activity measured in these cells at normal conditions (Fig. 7), that is, in HSF-1–/– cells less SOD activity and correspondingly less dichlorofluorescein (DCF) fluorescence. On the other hand, in normal H9c2 cells, higher SOD activity was observed, and correspondingly, higher DCF fluorescence (a rightward shift of the histogram in Fig. 8A). In heat-shocked cells, the histogram shifts more toward the right side (higher fluorescence) for H9c2 cells compared with the other cell lines (Fig. 8B). This trend is also in parallel with SOD activity as well as with HSP27 expression in these cells. Figure 8C shows the percentages of cells in the mean range, defined as M1 (nonfluorescent cell population) in Fig. 7A. Overall, there is an inverse correlation of nonfluorescent cell population with HSP27 levels in these cells, indicating that the magnitude of ROS generation is directly proportional to the HSP27 levels shown in Fig. 1.


Figure 8
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  		Fig. 8. Dichlorofluorescein diacetate (DCFDA) staining and fluorescence-activated cell sorting (FACS) analyses of H9c2, HSF-1+/+, and HSF-1–/– cells. Both control and heat-shocked cells, grown in 75-cm2 plates, were stained with DCFDA at 10 µM, fixed, and subjected to FACS analyses. A: histograms for cells at normal conditions. B: histograms for heat-shocked cells. Comparison of normal and heat-shocked cells shows that in the case of H9c2 cells, the histogram shifts more toward higher fluorescence (right) than the HSF-1+/+ and HSF–/– cells. C: quantitative plot of cell population in the limit M1 (as marked in section A) for both normal and heat-shocked cells with the level of HSP27.

 
Figure 9 shows the flow cytometry histograms for Dox-treated cells [both normal (left) and heat shocked (right)]. These histograms are actually corrected for respective histograms obtained for untreated cells (the histograms were subtracted from respective untreated cells, shown in Fig. 8). As illustrated in Fig. 9, on treatment with Dox, the histogram shifts to the right side in all the concentrations of Dox for all three cell lines, which is consistent with previous results (51). It is interesting to note that the histogram shifts more in the cases of HSF-1+/+ and HSF-1–/– cell lines (Fig. 9, A–F) both in normal and heat-shocked conditions compared with H9c2 cells. This once again confirms our hypothesis that the difference in the HSP27 level is responsible for the different levels of ROS generated after Dox treatment.


Figure 9
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  		Fig. 9. Effect of Dox treatment on ROS generation in H9c2, HSF-1+/+, and HSF-1–/– cells. Both normal (A–C) and heat-shocked cells (D–F), grown in 75-cm2 plates, were treated with Dox (0, 0.25, 5, 10 µM concentrations as indicated) for 6 h and then switched to normal medium. After 24 h, the cells were stained with DCFDA at 10 µM, fixed, and subjected to FACS analyses. The presented histograms were subtracted from respective histograms obtained from untreated cells (presented in Fig. 8, A and B).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of the present work demonstrate that HSP27 could reduce the Dox-induced toxicity in cardiac cells by enhancing the SOD activity and thus protecting the integrity of aconitases from superoxide-induced inactivation. Cardiotoxicity, caused by Dox and its derivatives, is a serious limitation for use of these drugs to treat patients with cancer. Both in vitro and in vivo studies (14, 29, 31, 46, 50) have shown that Dox causes apoptotic and necrotic cardiac cell deaths, depending on the concentration of Dox being used. Recent studies on the mechanism(s) of cardiac cell death have confirmed that the ROS generation by Dox and its exacerbation by various intracellular sources such as iron lead to mitochondrial apoptosis as a major pathway of cell death (4). One of the reasons why cardiomyocytes are more susceptible to ROS-induced apoptosis is that cardiomyocytes exhibit low levels of catalase (which detoxifies the H2O2) and readily undergo inactivation of selenium-dependent GSHPx1 (13, 47). Previous studies have established that this apoptotic pathway could be suppressed either by external supplementation of antioxidants or by induction of endogenous antioxidants such as cardiac-specific overexpression of cysteine-rich metallothioneins (53). Thus various methods have been devised to selectively target the undesired ROS-mediated cardiac cell deaths, and hence the deleterious side effect could be avoided (36). Overall, we have shown in the present work that HSP27 could act beneficially in avoiding Dox-induced cardiotoxicity.

Our results in this work, with HSF-1–/–and HSF-1+/+ along with cardiac H9c2 cells, show that there is a direct correlation between the amount of HSP27 expressed and the cell survival against Dox-induced toxicity (Figs. 1–3). In our earlier study, we showed that heat-shock treatment could protect cardiac H9c2 cells from Dox-induced toxicity and proved that phosphorylation of HSP27 by heat shock-activated MAPK AP-2 was playing a critical role in such protection (51). Other laboratories have also reported that small heat-shock proteins such as HSP10/60 could protect cardiac cells from Dox-induced toxicity (45). Although previous studies including our recent study (51) have found the antiapoptotic effect of small HSPs such as HSP27, the actual mechanism of the protective effect of HSP27 against Dox-induced cardiac cell death remains unsolved. The cell viability, observed in the present work, directly correlates with HSP27 (Figs. 1–3) in these cells (H9c2, HSF-1–/–, and HSF-1+/+). Especially selective silencing of HSP27 in H9c2 cells showed an increase in viability (Fig. 3), proving that HSP27 does protect the cells from Dox-induced toxicity. This observation ruled out the argument that the difference in the cell viability against Dox among the three cells (Fig. 2) could be due to difference in the nature of cells. Also similar correlation of cell viability is found with both s-15 and s-82 phosphorylated HSP27 (Figs. 1 and 2). In our previous work, we showed that phosphorylation of s-15 and s-82 was essential to protect from Dox-induced toxicity (51). Thus the increased viability in cells with higher HSP27 is likely due to high levels of phosphorylated HSP27. Based on the results of the present work, we propose a mechanism where increased expression and phosphorylation of HSP27 could increase the SOD activity to quench the Dox-derived O2bullet– (Fig. 10). Such an increased superoxide dismutation prevents the O2bullet–-induced inactivation of aconitase. Thus the Fe(II) release from aconitase and its subsequent aggravation of ROS can be avoided to reduce the Dox-induced toxicity, as illustrated in Fig. 10.


Figure 10
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  		Fig. 10. Schematic illustration of the mechanism of HSP27-offered protection of cardiac cells against the Dox-induced toxicity (1). Dox generates O2bullet– in a redox reaction with NADPH. The generated O2bullet– can potentially lead to necrosis (1) or apoptosis (2) pathways as depicted, depending on the quantity of O2bullet– being generated. In cardiac cells, the aconitase becomes the target for the O2bullet–, which releases an Fe(II) from the [4Fe-4S] complex. The released iron redox cycles the NADP+, generating more superoxide (4). If HSP27 is present, it increases the SOD activity, which will stop the aconitase disintegration (4) because the O2bullet– is dismutated at a faster rate, so that cells can survive. However, the accumulated H2O2 can inactivate the aconitase without the disintegration pathway (3). RSH, thiols in general.

 
The first part of the work (with no addition of Dox) addresses how different levels of HSP27 correlate to three different oxidant-related parameters, namely the magnitude of ROS generation (known to be the cause for cardiac cell death), the aconitase enzyme activity (known for the release of free Fe and its aggravation of the ROS), and the SOD activity (which can quench the cardiotoxic O2bullet–). Our results have shown that the magnitude of HSP27 in these cells corresponds to these three parameters either directly or inversely (Figs. 6–8). The SOD activity was found to be in the order of H9c2 > HSF-1+/+ > HSF-1–/–, the same order as the magnitude of HSP27 measured in these cells, revealing a direct correlation (Fig. 8B). In our previous study we found that heat shock can increase the MnSOD activity in H9c2 cells (51). Using in vivo models, Li and Singal (30) reported that a decline in SOD and GSHPx activities is an early event after Dox treatment. Whereas the SOD activity was found to recover after 24 h, to normal or even higher than the control, the GSHPx activity continued to decline even after 24 h. Although we did not evaluate the GSHPx activity in the present work, the SOD results are consistent with those results in which, after 24 h of Dox treatment, the SOD activity was higher (Fig. 7C). Thus the present finding not only confirms our previous results and others but also expands to other cell lines to establish that SOD activity depends on the HSP27 content in cardiac cells.

On the other hand, the aconitase activity was found to be in the order HSF-1–/– > HSF-1+/+ > H9c2, opposite of the SOD activity with respect to the HSP27 level. As far as we know, there is no such comparison of any HSP to aconitase activity that has been reported previously, except a few indirect evidences. Nanda et al. (40) reported that HSPs can assist other proteins to bind the transportation of m-aconitase to mitochondria. Heme oxygenase, one of the low molecular weight HSPs, has been found to modulate the aconitase activity in lens epithelial cells (44). We reported previously that heat-shock treatment decreased .OH radical generation by preventing the iron release from aconitase (22). Since hyperthermia is expected to induce HSP27 along with other HSPs, we proposed that HSP27 played a significant role. The present results, with the use of HSP27-suppressed cells such as HSF-1–/–, confirm that HSP27 plays an important role in reducing the aconitase inactivation.

It is important to note that the H9c2 cells, which showed higher SOD activity, showed lower aconitase activity. This finding is against the current understanding that aconitase inactivation is considered as a quantitative index of the steady-state levels of O2bullet– in a cell (15, 19). Accordingly, one would expect more aconitase activity in H9c2 due to increased dismutation of O2bullet– by SOD. One possible explanation is that the cardiac H9c2, HSF+/+, and HSF–/– cells had different levels of aconitase. The other possibility is that the dismutation product H2O2 can also inactivate the aconitase. Indeed, as discussed later, this is found to be the case.

Many studies in the literature have found that generation of ROS resulted in the induction of HSP27 (48). Similarly, overexpression of HSP27 has been known to reduce ROS (56). The ROS measured with flow cytometry by staining with DCF (which primarily indicates the H2O2) also showed a trend similar to the trend observed with aconitase activity. At basal condition, lower ROS were observed in HSF-1–/– cells and higher in H9c2 cells. This establishes the fact that the aconitase enzyme is inactivated in the presence of H2O2. Recently, it was found that the H2O2 can inactivate the aconitase without release of Fe(II) (7). In this condition, aconitase is inactivated, but there is no redox cycling leading to aggravation of oxidants (Fig. 10). The results of the present work show that overexpression of HSP27 might protect the integrity of aconitase.

The second part of the work addresses how the effects of Dox on the aconitase activity, SOD activity, and ROS generation can be modulated by HSP27. The [4Fe-4S] cluster in aconitase has been found to be susceptible to O2bullet– attack, knocking out one of the Fe(II) resulting in a [3Fe-4S]+ species and a free Fe(II). This aconitase-released Fe is active for redox conversion (36). On the other hand, the Dox metabolites knock all four Fe(II) from the aconitase, turning it into the iron-regulating protein (IRP)-1. When activated, IRPs enhance transferrin receptor mRNA stability, thereby facilitating iron uptake over sequestration. The direct correlation of protection of the aconitase activity (Fig. 6C) to the amount of HSP27 (Fig. 1A) shows that HSP27 protects the aconitase activity from the Dox-derived O2bullet–. It is interesting to note that although the untreated HSF-1–/– fibroblasts had higher aconitase activity, these cells were found to be more susceptible to Dox-induced O2bullet– (Fig. 6, A and C), unlike the H9c2 cells, which had lower aconitase activity but were not inactivated by Dox treatment. This can be explained as follows. The mitochondrial aconitase is known to be inactivated primarily by O2bullet–. On the other hand, the H2O2 is also found to inactivate the aconitase. However, the magnitude of such an inactivation by H2O2 is very low compared with O2bullet–. Recently, it was found that the H2O2-mediated aconitase inactivation could be increased by the presence of Fe(II). For such an Fe(II)-catalyzed H2O2-induced inactivation of aconitase, the rate was comparable to the O2bullet–-mediated inactivation (7). Based on this fact, we propose that H2O2 formed by higher SOD activity (also confirmed by DCF staining) in HSF-1–/– and HSF-1+/+ cells may be responsible for higher inactivation of aconitase compared with the H9c2 cells.

The question of how the aconitase is protected in the presence of HSP27 is addressed in terms of SOD activity. The trend of inactivation of aconitase (Fig. 6C) corroborates the reverse trend observed with SOD activity and the total ROS generated in Dox-treated cells (Fig. 7C). Unlike the aconitase activity, the SOD showed almost three times higher activity in HSF-1–/– cells, whereas a moderate increase in activity was observed in H9c2 cells. This result can be interpreted as follows. When higher O2bullet– is generated in the HSF-1–/– cells due to the increased release of Fe(II) by higher inactivation of aconitase, the SOD activity is increased. Indeed in HSF-1–/– cells, higher ROS generation (possibly O2bullet–-dismutated species such as H2O2) was found especially at higher concentrations of Dox, as illustrated in Fig. 9 (5 and 10 µM). Overall, HSP27 regulates the aconitase activity and protects from the O2bullet– attack.

Conclusions. The present study has revealed that there is a direct correlation between HSP27 levels and the amount of protection from Dox-induced toxicity in cardiac cells. Thus cardiac-specific overexpression of HSP27 is expected to reduce Dox-induced DCM and CHF. Such a resistance to Dox- induced toxicity is obtained in the form of the increased protection of aconitase enzyme, which has been found to release Fe(II) and aggravate the ROS-induced toxicity in Dox-treated cells. It was also observed that the SOD activity is higher in the cells that have HSP27, giving a notion that HSP27 may also regulate SOD activity in these cells.

A limitation of the present work, however, is that both the DCM and CHF due to Dox are mainly because of loss of cardiomyocytes in the heart (36). In the present work we have used cultured cardiac cells and fibroblasts as models to study the Dox-induced toxicity. Thus the extent to which the conclusions of the present work may be applicable to native cardiomyocytes in vivo heart will require further study. Perhaps, the objective of this study is to evaluate whether modulation of HSP27 can alter the cardiac cell survival. The direct correlation of HSP27 levels with cell survival, found in the present work, would be expected to be applicable to cardiomyocytes since cardiomyocytes have been shown to express HSP27 and leading cellular protection from oxidative stresses, such as ischemia-reperfusion (12, 33, 34). This study did not distinguish between mitochondrial and cytoplasmic aconitase and SOD, though the magnitude of inactivation is largely different in these cases.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We acknowledge the financial support from National Institutes of Health Grants R21-EB-004658 and R01-HL-078796-02 (to G. Ilangovan) and American Heart Association Grant BGIA 0365203B (to G. Ilangovan).


   ACKNOWLEDGMENTS
 
We thank Nancy Trigg for a critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Ilangovan, Rm. 116A, TMRF, The Ohio State Univ., 420 W. 12th Ave., Columbus, OH 43210 (e-mail: Govindasamy.Ilangovan{at}osumc.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.


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293/5/H3111    most recent
00328.2007v1
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