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Am J Physiol Regul Integr Comp Physiol 280: R1399-R1406, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 5, R1399-R1406, May 2001

A novel pharmacological action of ET-1 to prevent the cytotoxicity of doxorubicin in cardiomyocytes

Takahiko Suzuki and Takashi Miyauchi

Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305 - 8575, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that cardiomyocytes produce endothelin (ET)-1 and that the tissue level of ET-1 markedly increased in failing hearts in rats with chronic heart failure. Because the level of plasma ET-1 also increased progressively in patients with breast cancer who received doxorubicin (Dox; Adriamycin), which possesses cardiotoxicity, we hypothesized that ET-1 plays a role in the pathophysiology of cardiomyocytes injured by Dox. In this study, we investigated the effect of ET-1 on the cytotoxicity of Dox in primary cultured neonatal rat cardiomyocytes. The results showed that ET-1 effectively attenuated Dox-induced acute cardiomyocyte cytotoxicity (24-h incubation with Dox) evaluated by in vitro cell toxicity assay {3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase release}. The cytoprotective effect of ET-1 was mediated via ETA receptors, because pretreatment with the ETA-receptor antagonist BQ123 completely suppressed the cytoprotective effect of ET-1, whereas the ETB-receptor antagonist BQ788 did not. The cytoprotective effect of ET-1 was abolished by pretreatment with cycloheximide or staurosporine. These results suggest that a protein molecule(s), which is synthesized de novo by the stimulation of protein kinase pathway, is involved in the cytoprotective effect of ET-1. ET-1 increased the expression of an endogenous antioxidant, manganese superoxide dismutase (Mn-SOD), in the cardiomyocytes, as demonstrated by a Western blotting analysis. Pretreatment with an antisense oligodeoxyribonucleotide of Mn-SOD markedly attenuated the cytoprotective effect of ET-1 on the Dox-induced cytotoxicity. However, under conditions of prolonged incubation with Dox (48 h), ET-1 did not affect Dox-induced cardiomyocyte cytotoxicity in culture. These results suggest that ET-1 prevents the early phase of Dox-induced cytotoxicity via the upregulation of the antioxidant Mn-SOD through ETA receptors in cultured cardiomyocytes.

cultured cardiomyocytes; antioxidant; endothelin type A-receptor antagonist; protein kinase C; antisense oligodeoxyribonucleotide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIN (ET)-1 is a potent vasoconstrictor peptide first identified from the conditioned medium of vascular endothelial cells (61). We previously demonstrated in primary cultures that ET-1 is synthesized and secreted by cardiac myocytes (47). ET-1 acts not only on vascular smooth muscles, but also on myocardium. ET-1 has been shown to have a positive inotropic effect on the myocardium (12, 15, 29, 40, 49) through activation of phospholipase C and hydrolysis of phosphatidylinositol (18, 51). Furthermore, we and other groups have reported that ET-1 induces hypertrophy of cardiomyocytes (28, 41, 46). Receptors for ET peptides have been subclassed as ETA and ETB receptors (26, 37), and the above cardiac effects of ET-1 are mediated primarily by ETA receptors (25, 26).

We previously reported that tissue levels of both ET-1 peptide and mRNA markedly increased in failing hearts in rats with chronic heart failure (CHF) (38, 39) and in hamsters with cardiomyopathy (59). Moreover, we and other groups have reported that plasma ET-1 levels are increased in patients with CHF (8, 10, 14, 22, 36). These findings suggest a possibility that myocardial ET-1 is involved in the pathophysiology of heart diseases. Because ET-1 increases cardiac muscle contractility (12, 15, 29, 40, 49), an increase in ET-1 expression in the failing heart may possess some adaptive aspect of supporting contractility in the failing heart (39). However, persistent and progressive increase in cardiac ET-1 expression in the failing heart (27) possesses a maladaptative aspect. Indeed, it has been reported that cardiac ET-1 is involved in the progression of heart failure (25, 26, 38). There are several reports that show the effectiveness of the blockade of ET receptors in improving survival (16, 30, 38, 59) and hemodynamic features (5, 30, 38, 52, 53, 59) in heart failure models. We also reported that repeated treatment with an ETA-receptor antagonist improved the survival and hemodynamics of rats with CHF when started 10 days after the onset of myocardial infarction (38). On the other hand, Nguyen et al. (31) reported that the effect of an ETA-receptor antagonist initiated immediately after the onset of myocardial infarction was detrimental in cardiac dysfunction in rats. Thus the increase in myocardial ET-1 in heart diseases seems to have an adaptive (beneficial) role and a maladaptive (harmful) role in various stages of heart diseases.

Doxorubicin (Dox; Adriamycin), an anthracycline anticancer drug, is widely used for the treatment of various human malignancies, including several leukemias, lymphomas, and solid tumors (64). However, the clinical use of Dox is limited because of its serious cumulative dose-dependent cardiotoxicity, which leads to irreversible degenerative cardiomyopathy (42). There are at least two major processes causing cardiotoxicity of Dox. One is free-radical formation in cardiomyocytes (1, 17, 32), and the other is direct effect on the DNA and other cellular components in cardiomyocytes (9, 19-21, 23, 43, 44). It has been reported that the level of plasma ET-1 increased progressively in patients with breast cancer who had received Dox (57, 58). However, the pathophysiological effect of increasing the plasma level of ET-1 in patients who receive Dox treatment has not been elucidated.

Because it has been reported that plasma ET-1 becomes a marker for Dox-induced cardiotoxicity in patients with breast cancer in whom congestive heart failure developed (58), we hypothesized that ET-1 plays a role in the pathophysiology of cardiomyocytes injured by Dox. To test this hypothesis, in the present study, we investigated the effect of ET-1 on the cytotoxicity of Dox in primary cultured cardiomyocytes. We herein report a new pharmacological action of ET-1 on cardiomyocytes: ET-1 rescues the cultured cardiomyocytes from the Dox-induced early phase of cardiotoxicity via upregulation of manganese superoxide dismutase (Mn-SOD), an endogenous antioxidative molecule, through ETA receptors, suggesting that myocardial ET-1 has an adaptive (beneficial) aspect in injured cardiomyocytes in the early phase of Dox-induced cytotoxicity in culture.


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

Isolation and primary culture of cardiac myocytes. Ventricular cardiomyocytes were isolated from 2- to 3-day-old Sprague-Dawley rats as described previously (48). For the purification of myocytes, the isolated heart cells were suspended in a culture medium [DMEM-Ham's F-12 medium (1:1) supplemented with 5 µg/ml of insulin and transferrin] containing 10% fetal bovine serum (FBS) and preincubated twice in tissue culture flasks (75 cm2) for 20 min to separate nonadhering myocytes from adhering nonmyocytes. The purified myocytes (>95%) were suspended in the fresh culture medium supplemented with 5% FBS and seeded into fibronectin-coated culture plates and were incubated in a humidified 5% CO2-air incubator. The medium was changed on the first and third days and was changed to serum-free medium on the fifth day. Dox was added to the cells on the seventh day.

Cytotoxicity assays. Cytotoxicity was assessed using a colorimetric assay system (Boehringer Manheim, Manheim, Germany). Briefly, cardiac myoctes were cultured in 48-well culture plates at a density of 105 cells/well. Twenty-four hours after the addition of Dox to the myocytes, yellow labeling reagent, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) solution, was added to each well and incubated for 4 h. After that, the culture medium was removed and the cells were solubilized with solubilizing solution (10% SDS in 0.01 M HCl). The purple formazan crystals were formed from yellow MTT by succinate dehydrogenase in viable cells, and optical density of the purple formazan solution at 584 nm was measured with a spectrophotometer. Cytotoxicity was also assessed by lactate dehydrogenase (LDH) release from injured cells. For this assay, culture medium was collected, and the amount of LDH in each medium was measured using colorimetric assay kits (MTX "LDH," Kyokuto Pharmaceutical Industrial, Tokyo, Japan).

Immunoblot analyses. Preparation of cell lysate and immunoblotting was performed as follows. The cells were washed twice with ice-cold PBS and dissolved with SDS buffer [125 mM Tris, 50% (wt/vol) glycerol, 10% (wt/vol) SDS, pH 6.8], then heated for 5 min at 98°C on a block heating bath. Ten micrograms of protein of the cell lysate per lane was loaded on a 12.5% polyacrylamide gel and electrophoresed at 15 mA for 1.5 h. For the Western blotting, proteins were transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA) at 2 mA/cm2 for 1 h. After that, the membranes were stained with 0.5% ponseau S solution to confirm the uniformity of electroblotting efficiency of the proteins. The blots were then incubated in PBS containing 5% skim milk to block nonspecific binding sites on the membranes. Blots were first immunoreacted with a 1:10,000 dilution of purified polyclonal antibody against rat Mn-SOD produced in rabbits (a generous gift from Dr. Kumagai, University of Tsukuba). Second, blots were incubated in a 1:2,500 dilution of horseradish peroxidase conjugated donkey anti-rabbit immunoglobulins (Amersham Pharmacia Biotech, Little Chalfont, UK). Third, the blots were treated using an enhanced chemiluminescence detection kit (ECL detection system, Amersham Pharmacia Biotech), and chemiluminescence from the reaction products was detected with X-ray film (Hyperfilm-ECL, Amersham Pharmacia Biotech). The developed films were subjected to a densitometer for quantification.

Pharmacological treatment of cardiomyocytes. To supress the ET receptors, we used slective ET-receptor antagonists, BQ123 for type A receptors and BQ788 for type B receptors (33). BQ123 (0.1-10 µM) and BQ788 (1-10 µM) were added to the cardiomyocytes 30 min before addition of ET-1 (10 nM). Cycloheximide was used to inhibit de novo syntheses of proteins (45) in the cardiomyocytes. Doses ranging fom 0.1 to 50 µM were evaluated, the cell toxicity of cycloheximide itself by MTT assay, and we used 0.1 to 1.0 µM cycloheximide in this study. Cycloheximide was added 6 h before ET-1. For the inhibition of protein kinase C of cardiomyocytes, we used staurosporine (0.1% dimethyl sulfoxide) (63). Doses ranging from 0.1 to 100 nM of staurosporine were also evaluated, the cell toxicity by MTT assay, and 0.1-10 nM of staurosporine were used in this study. Staurosporine was added to the cardiomyocytes 1 h before ET-1.

Suppression of Mn-SOD induction by antisense oligodeoxyribonucleotide. Antisense and sense 22-mer phosphothioate oligodeoxyribonucleotides (AS-ODN and S-ODN, respectively) directed to the translation initiation site of rat Mn-SOD transcripts were designed and synthesized (11). (AS-ODN: 5'-CACGCCGCCCGACACAACATTG-3' and S-ODN: CAATGTTGTGTCGGG-CGGCGTG). Each ODN (1.15 µmol/l) was added as a complex with lipofection reagent (Lipofectin, GIBCO BRL Life Technologies, Gaithersburg, MD) 9 h before the addition of ET-1.

Protein assay. Protein was determined using a BCA protein assay system (Pierce, Rockford, IL) with bovine serum albumin as a standard.

Materials. ET-1 was purchased from Peptide Institute (Osaka, Japan). Dox was purchased from Kyowa Hakko Kogyo (Tokyo, Japan). BQ123 and BQ788 were a generous gift from Banyu Pharmaceutical (Tokyo, Japan). Cycloheximide and staurosporine were obtained from Sigma Chemical (St. Louis, Mo).

Statistical analysis. All data except those of Western blotting are expressed as means ± SD. Statistical analysis was carried out by ANOVA followed by Scheffé's test for multiple comparisons with a commercially available statistical package (StatView, version 5.0; Abacus Concepts, Berkley, CA). The results were considered statistically significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cardiomyocyte cell toxicity of Dox and the effects of ET-1 on Dox-induced cardiotoxicity. Dox showed cell toxicity in primary cultured cardiomyocytes. As shown in Fig. 1A, 24-h incubation with Dox ranging from 10 to 20 µM inhibited MTT activity in a dose-dependent manner. Twenty-four-hour treatment before Dox with ET-1 ranging from 0.1 to 10 nM reversed the MTT activity inhibited by 20 µM of Dox dose dependently (Fig. 1A). Furtherrmore, we also observed that the duration of survival term of the cardiomyocytes was greatly improved by the ET-1 treatment under the 24-h Dox-treated cultures (data not shown). However, no dose of ET-1 inhibited the decrease of MTT activity induced by a further 24 h incubation with Dox (total, 48 h; Fig. 1B). Conversely, LDH released from the myocytes increased after treatment with Dox. LDH release from the cardiomyocytes markedly increased after 12 h of incubation with 20 µM Dox. At the same time point, pretreatment with ET-1 inhibited LDH release from the cells by Dox. After 24 h of incubation with Dox, the LDH release increased about sixfold compared with the control and increased about threefold even with existing ET-1 (Table 1). Figure 2 shows morphological changes in cardiomyocytes induced by 20 µM of Dox and the effect of 10 nM of ET-1 on Dox-induced cell toxicity. Pretreatment with ET-1 attenuated the cytotoxic effect of Dox when the cells were incubated with Dox for 24 h. However, many vacuoles were formed in the myocytes by Dox treatment despite the existing ET-1 (Fig. 2, bottom left). A further 24 h of incubation (total, 48 h) of the cells with Dox eventually caused cell death, even with existing ET-1 (Fig. 2, bottom right).


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  		Fig. 1.   Effect of endothelin (ET)-1 on the doxorubicin (Dox)-induced cytotoxicity evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay (see MATERIALS AND METHODS). ET-1 was added 24 h before Dox, and MTT assay was performed 24 (A) and 48 h (B) after the addition of Dox. The average of optical densities at 584 nm in control wells is expressed as 100%. Each column and bar represents the mean ± SD of relative activity in 6 samples.


                              
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  		Table 1.   Relative concentrations of LDH in the culture medium of cardiomyocytes



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  		Fig. 2.   Photographs show the morphological cytotoxic effect of Dox on primary cultured cardiomyocytes and the effect of pretreatment with ET-1 on Dox-induced cell toxicity. Cells were photographed 24 (left) or 48 h (right) after the addition of Dox or vehicle. Top: control cells. Middle: cytotoxic effect of 20 µM of Dox on the cardiomyocytes. Twenty-four hours of incubation of cardiomyocytes with Dox induced an increase in round dead cells, and a further 24 h incubation caused complete cell death. Bottom: effect of 24 h of pretreatment of 10 nM ET-1 on Dox-induced cytotoxicity. ET-1 inhibited cytotoxicity induced by 24 h of incubation with Dox. However, a further 24 h (total, 48 h) incubation with Dox caused complete cell death, even with existing ET-1 in the medium.

Determination of the ET-receptor subtype involved in the cytoprotective effect of ET-1. To determine which ET-receptor subtype is involved in the cytoprotective effect of ET-1, we examined the effects of ET-receptor antagonists on the cytoprotection of ET-1 against Dox-induced cell toxicity. An ETA-receptor antagonist, BQ123, attenuated the cytoprotective effect of ET-1, and 10 µM of BQ123 completely suppressed the effect of ET-1. On the other hand, an ET type B (ETB)-receptor antagonist, BQ788, did not show any significant effect on the cytoprotective effect of ET-1 on the Dox-induced cell toxicity estimated by MTT assay (Fig. 3).


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  		Fig. 3.   Determination of the ET-receptor subtype involved in the cytoprotective effect of ET-1 on the Dox-induced cardiomyocyte cell toxicity evaluated by MTT assay. Each receptor antagonist was added to the cardiomyocytes 30 min before the ET-1. ET-1 was added 24 h before Dox, and MTT assay was performed 24 h after the addition of Dox. The average of optical densities at 584 nm in control wells is expressed as 100%. Each column and bar represents the mean ± SD of relative activity in 6 samples. P < 0.05 and P < 0.01, statistical significance. NS, no significance.

Protein synthesis and protein kinase inhibition. Cytoprotection of ET-1 was effective when the ET-1 was added 24 h before the Dox treatment of the cardiac myocytes in the former studies. Pretreatment with 1 µM cycloheximide, a protein synthesis inhibitor, markedly suppressed the cytoprotective effect of ET-1 on Dox-induced cytotoxicity estimated by MTT assay (Fig. 4). On the other hand, a protein kinase inhibitor, staurosporine, also inhibited the cytoprotective effect of ET-1 on Dox-induced cardiotoxicity estimated by MTT assay (Fig. 4).


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  		Fig. 4.   Inhibitory effects of cycloheximide (CHX; A) and staurosporine (Stauro; B) on cytoprotection of ET-1 against Dox-induced cytotoxicity in cardiomyocytes evaluated by MTT assay. CHX or Stauro was added 6 h or 1 h before the addition of ET-1, respectively. ET-1 was added 24 h before the addition of Dox, and MTT assay was performed 24 h after the addition of Dox. The average of optical densities at 584 nm in control wells is expressed as 100%. Each column and bar represents the mean ± SD of relative activity in 6 samples. P < 0.01, statistical significance.

Induction of Mn-SOD by ET-1. To clarify the mechanism of the cytoprotective effect of ET-1 on Dox-induced cardiomyocyte cell death, we investigated whether upregulation of Mn-SOD, a mitochondrial antioxidative molecule, occurred after treatment with ET-1. As shown in Fig. 5A, protein levels of Mn-SOD increased with incubation time by 10 nM of ET-1 estimated by Western blotting analysis. Similar results were obtained in three other independent experiments.


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  		Fig. 5.   A: Western blotting analysis of manganese superoxide dismutase (Mn-SOD) induction induced by ET-1. Each column represents the relative density of each blot indicated in the inset. Blotting analysis was performed 4 times, and similar results were obtained from all the experiments. B: effect of ET-receptor antagonists on the induction of Mn-SOD by ET-1. Each receptor antagonist was added to the cardiomyocytes 30 min before the ET-1, and the cell lysate for the Western blotting analysis was prepared 12 h after ET-1. Lanes 1 and 2: control; lanes 3 and 4: 10 nM ET-1; lanes 5 and 6: 10 µM BQ123 and 10 nM ET-1; lanes 7 and 8: 10 µM BQ788 and 10 nM ET-1. Duplicate lanes were prepared from independent samples. The blotting analysis was performed 2 times, and similar results were obtained from the experiments.

The induction of Mn-SOD by ET-1 was attenuated by the pretreatment with the ETA-receptor antagonist BQ123 but not by the ETB-receptor antagonist BQ788 (Fig. 5B).

Suppression of Mn-SOD by antisense ODN. To suppress the induction of Mn-SOD by ET-1 selectively, we used an AS-ODN, which corresponds to the initiation site of Mn-SOD translation. Pretreatment with the AS-ODN (1.15 µM) attenuated ~80% of the cytoprotective effect of ET-1. However, complete inhibition was not obtained even when the dose of AS-ODN was increased. In contrast, S-ODN showed no effect on the cytoprotective effect of ET-1 (Fig. 6).


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  		Fig. 6.   Inhibition of the cytoprotective effect of ET-1 on Dox-induced cytotoxicity by pretreatment with the antisense oligodeoxyribonucleotide (AS-ODN) of Mn-SOD evaluated by MTT assay. The average of optical densities at 584 nm in control wells is expressed as 100%. Each column and bar represents the mean ± SD of relative activity in 6 samples. P < 0.05 and P < 0.01, statistical significance. S-ODN, sense oligodeoxyribonucleotide.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrated that ET-1 prevented Dox-induced acute cytotoxicity (after 24 h of incubation with Dox) in a dose-dependent manner, as evaluated by MTT assay and LDH release from the cells in primary cultured rat cardiomyocytes. The cytoprotective effect of ET-1 was completely suppressed by pretreatment with the ETA-receptor antagonist BQ123 but not with the ETB-receptor antagonist BQ788, indicating that the ETA-receptor system is involved in the cytoprotective effect of ET-1 on the cytotoxicity of Dox. The cytoprotective effect of ET-1 was abolished by pretreatment with cycloheximide and staurosporine. These results suggest that some protein molecule(s), which is synthesized de novo by the stimulation of the protein kinase pathway, is involved in the cytoprotective effect of ET-1.

It has been reported that the formation of free radicals is an important process that contributes to cardiotoxic effects of Dox (1, 17, 32). Moreover, Dox-induced acute cardiotoxicity is attenuated in Mn-SOD transgenic mice (62). Therefore, we hypothesized that ET-1 affects the expression of an endogenous antioxidant molecule(s) in cardiomyocytes. Then, we investigated whether ET-1 regulates the expression of Mn-SOD in the cultured cardiomyocytes. The results showed that ET-1 increased the expression of Mn-SOD in the cardiomyocytes, as demonstrated by Western blotting analysis. Moreover, the increased expression of Mn-SOD by ET-1 was suppressed by BQ123, an ETA-receptor antagonist. Then, to investigate whether this upregulation of Mn-SOD is actually involved in the cytoprotective effect of ET-1, we investigated whether the transfection of the AS-ODN, designed to work against rat Mn-SOD mRNA to cultured cardiomyocytes, affects the cytoprotective effect of ET-1 on Dox-induced cardiotoxicity. Pretreatment with the AS-ODN markedly attenuated the cytoprotective effect of ET-1, whereas the S-ODN did not affect this effect of ET-1. These results suggest that ET-1 has a cytoprotective effect mainly via the upregulation of Mn-SOD levels in the cardiomyocytes. However, because complete inhibition was not obtained by the pretreatment with AS-ODN, these results also imply that the cardioprotective effect of ET-1 is mediated through some other mechanism(s) in addition to that mediated by Mn-SOD.

Recently, it has been reported that ET-1 stimulates c-fos gene expression via the Ras pathway (7) and also activates the phosphorylation cascade of Raf-1 and the extracellular signal-regulated kinase of mitogen-activated protein kinase (MAPK) (60) in neonatal rat cardiomyocytes. Moreover, ET-1 also activates phosphorylation of MAPK/Jun amino-terminal kinase in cardiomyocytes (3, 4). It is known that c-fos and c-jun make the heterodimer complex activating protein-1 (AP-1), which preferentially binds to many genes that have a 12-O-tetradecanoyl-phorbol-13-acetate-responsive element in their promoter region (2). Rat Mn-SOD gene contains an AP-1 binding site in its regulatory sequence (11). Therefore, ET-1 could upregulate the transcription of Mn-SOD via the stimulation of the AP-1 site in the gene. Yamashita et al. (54-56) demonstrated in their series of reports that Mn-SOD was induced by treatment with ischemic preconditioning, norepinephrine, and heat shock and these upregulations of Mn-SOD contributed to tolerance to hypoxia or hypoxia-reperfusion injury in cardiomyocytes. Furthermore, they also suggested that the Mn-SOD induction observed in their system was due to C-kinase activation and subsequent phosphorylation of AP-1. Our present findings that some protein molecule(s), which is synthesized de novo by the stimulation of the protein kinase pathway, is involved in the cytoprotective effect of ET-1 on injured cardiomyocytes by Dox and that Mn-SOD is the most important candidate for this molecule are consistent with the above reports.

However, although ET-1 prevented Dox-induced acute cytotoxicity, ET-1 did not affect the cytotoxicity under conditions of prolonged incubation with Dox over 48 h. These results suggest that ET-1 blocked the early phase of cytotoxicity induced by Dox in culture. Cardiotoxicity induced by Dox has multiple steps, including free-radical formation (1, 17, 32), and complicates direct effects on DNA and other cellular components (9, 19-21, 23, 43, 44). Therefore, ET-1 may have suppressed the free radicals produced by Dox through the upregulation of Mn-SOD and reduced the cytotoxicity of Dox. However, ET-1 could not antagonize the other effects of Dox on the cellular components that gradually progress in the cells.

In summary, the present study revealed a new pharmacological action on cardiomyocytes: ET-1 rescued cultured cardiomyocytes from the early phase of Dox-induced cardiotoxicity and, furthermore, revealed the molecular mechanism that this cytoprotective action was mainly attributable to upregulation of the antioxidant Mn-SOD through ETA receptors. Therefore, it is possible that this action of ET-1 on cardiomyocytes would affect the pathophysiological condition of injured hearts in Dox-treated patients with cancer.

Perspectives

We and other groups have reported an increase in plasma levels of ET-1 in patients (8, 10, 14, 22, 36) and experimental animals (6, 24, 38, 39, 50) with CHF and also reported a marked increase in tissue levels of ET-1 in failing hearts in patients (35) and in experimental animals (38, 39, 59) with CHF, suggesting that the heart is one of the major origins of the increase in plasma ET-1 in CHF. It has also been reported that plasma ET-1 was progressively increased in Dox-treated patients with breast cancer in whom congestive heart failure developed (58). Therefore, it can be speculated that myocardial ET-1 plays a role in failing hearts in these patients. As described in the introduction, the increase in myocardial ET-1 in heart diseases seems to have an adaptive (beneficial) role and a maladaptive (harmful) role in various stages of heart diseases. The present study suggests that ET-1 has a beneficial aspect in injured cardiomyocytes in the early phase of Dox-induced cytotoxicity in culture.

Although there are several reports showing the effectiveness of the blockade of ET receptors on CHF in experimental animals (5, 16, 30, 38, 52, 53, 59) and in patients (34), there is no report on how the blockade of ET receptors affects (deteriorates or ameliorates) pathophysiological conditions in vivo of experimental animals or patients with CHF caused by Dox treatment. In other words, there is a possibility that the ET-1 in cardiomyocytes induced by Dox attenuates the acute cardiotoxicity of Dox, and the blockade of ET receptors at early stages of the Dox treatment suppresses the antioxidative effect of ET-1 and accelerates cardiotoxicity. In this regard, an in vivo study that investigates the effects of an ET-receptor antagonist initiated at different stages of heart disease on hemodynamics and survival of CHF animals caused by Dox treatment is considered to be very important. Furthermore, because oxidative stress in the myocardium has been reported to play an important role in the pathogenesis of CHF by various etiologies (13), it is interesting and important to determine whether upregulation of myocardial Mn-SOD by ET-1, as shown in this study, occurs in the diseased heart at some stage of CHF by various etiologies (ischemic heart diseases, valvular heart diseases, cardiomyopathy, etc.) in animals and patients.


    ACKNOWLEDGEMENTS

This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (10670629, 11357019) and by a grant from the Miyauchi project of Tsukuba Advanced Research Alliance at the Univ. of Tsukuba.


    FOOTNOTES

Present address for T. Suzuki: Radiation Research Institute, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Address for reprint requests and other correspondence: T. Suzuki, Radiation Research Institute, Graduate School of Medicine, Univ. of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail tsuzuki{at}m.u-tokyo.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.

Received 30 June 2000; accepted in final form 9 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 280(5):R1399-R1406
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