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Am J Physiol Regul Integr Comp Physiol 280: R1399-R1406, 2001;
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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

1.   Abdel-aleem, S, El-Merzabani MM, Sayed-Ahmed M, Taylor DA, and Lowe JE. Acute and chronic effects of Adriamycin on fatty acid oxidation in isolated cardiac myocytes. J Mol Cell Cardiol 29: 789-797, 1997[Web of Science][Medline].

2.   Angel, P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, and Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49: 729-739, 1987[Web of Science][Medline].

3.   Bogoyevitch, MA, Glennon PE, Andersson MB, Clerk A, Lazou A, Marshall CJ, Parker PJ, and Sugden PH. Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. J Biol Chem 269: 1110-1119, 1994[Abstract/Free Full Text].

4.   Bogoyevitch, MA, Ketterman AJ, and Sugden PH. Cellular stresses differentially activate c-Jun N-terminal protein kinases and extracellular signal-regulated protein kinases in cultured ventricular myocytes. J Biol Chem 270: 29710-29717, 1995[Abstract/Free Full Text].

5.   Borgeson, DD, Grantham JA, Williamson EE, Luchner A, Redfield MM, Opgenorth TJ, and Burnett JC, Jr. Chronic oral endothelin type A receptor antagonism in experimental heart failure. Hypertension 31: 766-770, 1998[Abstract/Free Full Text].

6.   Cavero, PG, Miller WL, Heublein DM, Margulies KB, and Burnett JC, Jr. Endothelin in experimental congestive heart failure in the anesthetized dog. Am J Physiol Renal Fluid Electrolyte Physiol 259: F312-F317, 1990[Abstract/Free Full Text].

7.   Cheng, TH, Shih NL, Chen SY, Wang DL, and Chen JJ. Reactive oxygen species modulate endothelin-1-induced c-fos gene expression in cardiomyocytes. Cardiovasc Res 41: 654-662, 1999[Abstract/Free Full Text].

8.   Cody, RJ, Haas GJ, Binkley PF, Capers Q, and Kelley R. Plasma endothelin correlates with the extent of pulmonary hypertension in patients with chronic congestive heart failure. Circulation 85: 504-509, 1992[Abstract/Free Full Text].

9.   Doroshow, JH, and Davies KJA Redox cycling of anthracyclines by cardiac mitochondria. II. Formation of superoxide anion, hydrogen peroxide, and hydroxy radical. J Biol Chem 261: 3068-3074, 1986[Abstract/Free Full Text].

10.   Hiroe, M, Hirata Y, Fujita N, Umezawa S, Ito H, Tsujino M, Koike A, Nogami A, Takamoto T, and Marumo F. Plasma endothelin-1 levels in idiopathic dilated cardiomyopathy. Am J Cardiol 68: 1114-1115, 1991[Web of Science][Medline].

11.   Ho, Y-S, and Crapo JD. Nucleotide sequences of cDNAs coding for rat manganese-containing superoxide dismutase. Nucleic Acids Res 15: 10070, 1987[Free Full Text].

12.   Hu, RJ, Harsdorf RV, and Lang RE. Endothelin has potent inotropic effects in rat atria. Eur J Pharmacol 158: 275-278, 1988[Web of Science][Medline].

13.   Ide, T, Tsutsui H, Kinugawa S, Utsumi H, Kang D, Hattori N, Uchida K, Arimura K, Egashira K, and Takeshita A. Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing myocardium. Circ Res 85: 357-363, 1999[Abstract/Free Full Text].

14.   Ishikawa, S, Miyauchi T, Sakai S, Yamaguchi I, Sugishita Y, and Goto K. Elevated plasma endothelin-1 in young patients with pulmonary hypertension due to congenital heart diseases is decreased after successful surgical repair. J Thorac Cardiovasc Surg 110: 271-273, 1995[Free Full Text].

15.   Ishikawa, T, Yanagisawa M, Kimura S, Goto K, and Masaki T. Positive inotropic action of novel vasoconstrictor peptide endothelin on guinea pig atria. Am J Physiol Heart Circ Physiol 255: H970-H973, 1988[Abstract/Free Full Text].

16.   Iwanaga, Y, Kihara Y, Hasegawa K, Inagaki K, Yoneda T, Kaburagi S, Araki M, and Sasayama S. Cardiac endothelin-1 play a critical role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive rats. Circulation 98: 2065-2073, 1998[Abstract/Free Full Text].

17.   Julicher, RHM, van der Laase A, Sterrenberg L, Bloys van Treslong CHF, Bast A, and Noordhoek J. The involvement of an oxidative mechanism in the Adriamycin induced toxicity in neonatal rat heart cell cultures. Res Commun Chem Pathol Pharmacol 47: 35-47, 1985[Web of Science][Medline].

18.   Kramer, BK, Smith TW, and Kelly RA. Endothelin and increased contractility in adult rat ventricular myocytes: role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na+-H+ exchanger. Circ Res 68: 269-279, 1991[Abstract/Free Full Text].

19.   Kurabayashi, M, Dutta S, Jeyaseelan R, and Kedes L. Doxorubicin-induced Id2A gene transcription is targeted at an activating transcription factor/cyclic AMP response element motif through novel mechanisms involving protein kinases distinct from protein kinase C and protein kinase A. Mol Cell Biol 15: 6386-6397, 1995[Abstract].

20.   Kurabayashi, M, Jeyaseelan R, and Kedes L. Antineoplastic agent doxorubicin inhibits myogenic differentiation of C2 myoblasts. J Biol Chem 268: 5524-5529, 1993[Abstract/Free Full Text].

21.   Kurabayashi, M, Jeyaseelan R, and Kedes L. Doxorubicin represses the function of the myogenic helix-loop-helix transcription factor MyoD. J Biol Chem 269: 6031-6039, 1994[Abstract/Free Full Text].

22.   Lerman, A, Kubo SH, Tschmperlin LK, and Burnett JC, Jr. Plasma endothelin concentrations in humans with end-stage heart failure and after transplantation. J Am Coll Cardiol 20: 849-853, 1992[Abstract].

23.   Liu, LF. DNA topoisomerase poisons as antimumor drugs. Annu Rev Biochem 58: 351-375, 1989[Web of Science][Medline].

24.   Margulies, KB, Hildebrand FL, Jr, Lerman A, Perrella MA, and Burnett JC, Jr. Increased endothelin in experimental heart failure. Circulation 82: 2226-2230, 1990[Abstract/Free Full Text].

25.   Miyauchi, T, and Goto K. Heart failure and endothelin receptor antagonists. Trends Pharmacol Sci 20: 210-217, 1999[Medline].

26.   Miyauchi, T, and Masaki T. Pathophysiology of endothelin in the cardiovascular system. Annu Rev Physiol 61: 391-415, 1999[Web of Science][Medline].

27.   Miyauchi, T, Yamauchi R, Kobayashi T, Hoshino T, Sakai S, Kikkawa K, Yabana H, Murata S, Goto K, and Sugishita Y. Chronic (1 year) treatment with an endothelin receptor antagonist greatly improves survival in hamsters with heart failure due to cardiomyopathy: increased expression of endothelin-1 mRNA in the failing heart of cardiomyopathic hamsters (Abstract). Circulation 98: I-82, 1998.

28.   Miyauchi, T, Yorikane R, Sakai S, Sakurai T, Okada M, Nishikibe M, Yano M, Yamaguchi I, Sugishita Y, and Goto K. Contribution of endogenous endothelin-1 to the progression of cardiopulmonary alterations in rats with monocrotaline-induced pulmonary hypertension. Circ Res 73: 887-897, 1993[Abstract/Free Full Text].

29.   Moravec, CS, Reynolds EE, Stewart RW, and Bond M. Endothelin is a positive inotropic agent in human and rat heart in vitro. Biochem Biophys Res Commun 159: 14-18, 1989[Web of Science][Medline].

30.   Mulder, P, Richard V, Derumeaux G, Hogie M, Henry JP, Lallemand F, Compagnon P, Mace B, Comoy E, Letac B, and Thuillez C. Role of endogenous endothelin in chronic heart failure: effect of long-term treatment with an endothelin antagonist on survival, hemodynamics, and cardiac remodeling. Circulation 96: 1976-1982, 1997[Abstract/Free Full Text].

31.   Nguyen, QT, Cernacek P, Calderoni A, Stewart DJ, Picard P, Sirois P, White M, and Rouleau JL. Endothelin A receptor blockade causes adverse left ventricular remodeling but improves pulmonary artery pressure after infarction in the rat. Circulation 98: 2323-2330, 1998[Abstract/Free Full Text].

32.   Olson, RD, and Mushlin PS. Doxorubicin cardiotoxicity: analysis of prevaling hypotheses. FASEB J 47: 35-47, 1990.

33.   Osada, K, Tsunoda H, Miyauchi T, Sugishita Y, Kubo T, and Goto K. Pregnancy increases ET-1-induced contraction and changes receptor subtypes in uterine smooth muscle in humans. Am J Physiol Regulatory Integrative Comp Physiol 272: R541-R548, 1997[Abstract/Free Full Text].

34.   Packer, M, Caspi A, Charlon V, Cohen-Solal A, Kiowski W, Kostuk W, Krum H, Levine B, Massie B, McMurray J, Rizzon P, and Swedberg K. Multicenter, double blind, placebo-controlled study on long term endothelin blockade with bosentan in chronic heart failure. Results of the REACH-1 trial (Abstract). Circulation 98: I-3, 1998.

35.   Pieske, B, Beyermann B, Breu V, Loffler BM, Schlotthauer K, Maier LS, Schmidt-Schwade S, Just H, and Hasenfuss G. Functional effects of endothelin and regulation of endothelin receptors in isolated human nonfailing and failing myocardium. Circulation 99: 1802-1809, 1999[Abstract/Free Full Text].

36.   Rodeheffer, RJ, Lerman A, Heublein D, and Burnett JC, Jr. Increased plasma concentration of endothelin in congestive heart failure in humans. Mayo Clin Proc 67: 719-724, 1992[Web of Science][Medline].

37.   Rubanyi, GM, and Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 46: 325-415, 1994[Web of Science][Medline].

38.   Sakai, S, Miyauchi T, Kobayashi M, Yamaguchi I, Goto K, and Sugishita Y. Inhibition of myocardial endothelin pathway improves long-term survival in heart failure. Nature 384: 353-355, 1996[Medline].

39.   Sakai, S, Miyauchi T, Sakurai T, Kasuya Y, Ihara M, Yamaguchi I, Goto K, and Sugisgita Y. Endogenous endothelin-1 participates in the maintenance of cardiac function in rats with conjestive heart failure. Marked increase in endothelin-1 production in the failing heart. Circulation 93: 1214-1222, 1996[Abstract/Free Full Text].

40.   Shah, AJ, Lewis MJ, and Henderson AH. Inotropic effects of endothelin in ferret ventricular myocardium. Eur J Pharmacol 163: 365-267, 1989[Web of Science][Medline].

41.   Shubeita, HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, and Chien KR. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. A paracrine mechanism for myocardial cell hypertrophy. J Biol Chem 265: 20555-20562, 1990[Abstract/Free Full Text].

42.   Singal, PK, Deally CM, and Weinberg LE. Subcellular effects of adriamycin in the heart: a concise review. J Mol Cell Cardiol 19: 817-828, 1987[Web of Science][Medline].

43.   Singal, PK, and Panagia V. Direct effects of Adriamycin on the rat heart salcolemma. Res Commun Chem Pathol Pharmacol 43: 67-77, 1984[Web of Science][Medline].

44.   Solem, LE, Heller LJ, and Wallace KB. Dose-dependent increase in sensitivity to calcium-induced mitochondrial dysfunction and cardiomyocyte cell injury by doxorubicin. J Mol Cell Cardiol 28: 1023-1032, 1996[Web of Science][Medline].

45.   Souren, JEM, Van Der Mast C, and Van Wijk R. NADPH-oxidase-dependent superoxide production by myocyte-derived H9c2 cells: influence of ischemia, heat shock, cycloheximide and cytochalasin D. J Mol Cell Cardiol 29: 2803-2812, 1997[Web of Science][Medline].

46.   Suzuki, T, Hoshi H, and Mitsui Y. Endothelin stimulates hypertrophy and contractility of neonatal rat cardiac myocytes in a serum-free medium. FEBS Lett 268: 149-151, 1990[Web of Science][Medline].

47.   Suzuki, T, Kumazaki T, and Mitsui Y. Endothelin-1 is produced and secreted by neonatal rat cardiac myocytes in vitro. Biochem Biophys Res Commun 191: 823-830, 1993[Web of Science][Medline].

48.   Suzuki, T, Tsuruda A, Katoh S, Kubodera A, and Mitsui Y. Purification of endothelin from a conditioned medium of cardiac fibroblastic cells using beating rate assay of myocytes cultured in a serum-free medium. J Mol Cell Cardiol 29: 2087-2093, 1997[Web of Science][Medline].

49.   Takanashi, M, and Endoh M. Characterization of positive inotropic effect of endothelin on mammalian ventricular myocardium. Am J Physiol Heart Circ Physiol 261: H611-H619, 1991[Abstract/Free Full Text].

50.   Underwood, RD, Aarhus LL, Heublein DM, and Burnett JC, Jr. Endothelin in thoracic inferior vena caval constriction model of heart failure. Am J Physiol Heart Circ Physiol 263: H951-H955, 1992[Abstract/Free Full Text].

51.   Vigne, P, Lazdunski M, and Frelin C. The inotropic effect of endothelin-1 on rat atria involves hydrolysis of phosphatidylinositol. FEBS Lett 249: 143-146, 1989[Web of Science][Medline].

52.   Wada, A, Tsutamoto T, Fukai D, Ohnishi M, Maeda K, Hisanaga T, Maeda Y, Matsuda Y, and Kinoshita M. Comparison of the effects of selective endothelin ETA and ETB receptor antagonists in congestive heart failure. J Am Coll Cardiol 30: 1385-1392, 1997[Abstract].

53.   Wada, A, Tsutamato T, Ohnishi M, Sawaki M, Fukai D, Maeda Y, and Kinoshita MM. Effects of a specific endothelin-converting enzyme inhibitor on cardiac, renal, and neurohumoral functions in congestive heart failure: comparison of effects with those of endothelin A receptor antagonism. Circulation 99: 570-577, 1999[Abstract/Free Full Text].

54.   Yamashita, N, Hoshida S, Nishida M, Igarashi J, Taniguchi N, Tada M, Kuzuya T, and Hori M. Heat shock-induced manganese superoxide dismutase enhances the tolerance of cardiac myocytes to hypoxia-reoxygenation injury. J Mol Cell Cardiol 29: 1805-1813, 1997[Web of Science][Medline].

55.   Yamashita, N, Nishida M, Hoshida S, Igarashi J, Hori M, Kuzuya T, and Tada M. alpha 1-Adrenergic stimulation induces cardiac tolerance to hypoxia via induction and activation of Mn-SOD. Am J Physiol Heart Circ Physiol 271: H1356-H1362, 1996[Abstract/Free Full Text].

56.   Yamashita, N, Nishida M, Hoshida S, Kuzuya T, Hori M, Taniguchi N, Kamada T, and Tada M. Induction of manganese superoxide dismutase in rat cardiac myocytes increases tolerance to hypoxia 24 hours after preconditioning. J Clin Invest 94: 2193-2199, 1994.

57.   Yamashita, J, Ogawa M, and Nomura K. Plasma endothelin-1 and doxorubicin cardiotoxicity. N Engl J Med 331: 1528-1529, 1994[Free Full Text].

58.   Yamashita, J, Ogawa M, and Shirakusa T. Plasma endothelin-1 as a marker for doxorubicin cardiotoxicity. Int J Cancer 62: 542-547, 1995[Web of Science][Medline].

59.   Yamauchi-Kohno, R, Miyauchi T, Hoshino T, Kobayashi T, Aihara H, Sakai S, Yabana H, Goto K, Sugishita Y, and Murata S. Role of endothelin in deterioration of heart failure due to cardiomyopathy in hamsters: increase in endothelin-1 production in the heart and beneficial effect of endothelin A antagonist on survival and cardiac function. Circulation 99: 2171-2176, 1999[Abstract/Free Full Text].

60.   Yamazaki, T, Komuro I, Kudoh S, Zou Y, Shiojima I, Hiroi Y, Mizuno T, Maemura K, Kurihara H, Aikawa R, Takano H, and Yazaki Y. Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy. J Biol Chem 271: 3221-3228, 1996[Abstract/Free Full Text].

61.   Yanagisawa, M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411-415, 1988[Medline].

62.   Yen, H-C, Oberley TD, Vichitbandha S, Ho Y-S, and St. Clair DK. The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J Clin Invest 98: 1253-1260, 1996[Web of Science][Medline].

63.   Yonemochi, H, Yasunaga S, Teshima Y, Iwao T, Akiyoshi K, Nakagawa M, Saikawa T, and Ito M. Mechanisum of beta -adrenergic receptor upregulation induced by ACE inhibition in cultured neonatal rat cardiac myocytes. Role of bradykinin and protein kinase C. Circulation 97: 2268-2273, 1998[Abstract/Free Full Text].

64.   Young, RC, Ozols RF, and Myers CE. The anthracycline neoplastic drugs. N Engl J Med 305: 139-153, 1981[Web of Science][Medline].

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