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Endocrinology, doi:10.1210/en.2003-1322
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Endocrinology Vol. 145, No. 5 2458-2466
Copyright © 2004 by The Endocrine Society

Ventricular Nonmyocytes Inhibit Doxorubicin-Induced Myocyte Apoptosis: Involvement of Endogenous Endothelin-1 as a Paracrine Factor

Takeshi Tokudome, Takeshi Horio, Megumu Fukunaga, Hiroyuki Okumura, Jun Hino, Kenji Mori, Fumiki Yoshihara, Shin-Ichi Suga, Yuhei Kawano, Masakazu Kohno and Kenji Kangawa

Research Institute (T.T., H.O., J.H., K.M., S.S., K.K.) and Department of Medicine (T.H., F.Y., Y.K.), National Cardiovascular Center, Osaka 565-8565, Japan; and Department of Integrated Medicine (M.F.) and Second Department of Internal Medicine (M.K.), Kagawa University Faculty of Medicine, Kagawa 761-0793, Japan

Address all correspondence and requests for reprints to: Takeshi Horio, M.D., Division of Hypertension and Nephrology, Department of Medicine, National Cardiovascular Center, 5-7-1, Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: thorio{at}ri.ncvc.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A cross-talk between cardiac myocytes and nonmyocytes via humoral factors plays an important role in the development of cardiac growth. However, it remains to be elucidated whether humoral factors produced from nonmyocytes have a protective effect on acute myocardial injury. The present in vitro study investigated the antiapoptotic effect of nonmyocytes on doxorubicin (DOX)-induced myocyte apoptosis and its molecular mechanism. Myocyte-nonmyocyte coculture and treatment with nonmyocyte-conditioned media significantly attenuated DOX-induced myocyte apoptosis. Treatment with nonmyocyte-conditioned media stimulated the phosphorylation of ERK, Akt, and cAMP response element-binding protein (CREB) in myocytes. Nonmyocyte-conditioned media also increased protein levels of Bcl-2 but not Bcl-xL and decreased caspase-3 activation induced by DOX. MAPK kinase-specific inhibitor PD98059, phosphatidylinositol-3 kinase-Akt inhibitor LY294002, and CREB antisense oligonucleotide significantly blocked the antiapoptotic effect of nonmyocyte-conditioned media. A considerable amount of endothelin (ET)-1 production was detected in nonmyocytes but not in myocytes. Exogenous ET-1 mimicked nonmyocyte-conditioned media-mediated ERK and CREB phosphorylation and Bcl-2 protein increase but not Akt phosphorylation. In addition, ET-A receptor antagonists BQ123 and BQ485 partially blocked nonmyocyte-conditioned media-mediated antiapoptotic effect, ERK and CREB phosphorylation, and Bcl-2 protein increase. Nonmyocyte-conditioned media and exogenous ET-1 unchanged protein levels of manganese superoxide dismutase and oxidative stress-related product levels augmented by DOX. The present findings demonstrate that cardiac nonmyocytes inhibit DOX-induced myocyte apoptosis, at least in part, via ET-1 secretion-mediated CREB activation independent of the decrease in oxidative stress.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH DOXORUBICIN (DOX), a quinone-containing anthracycline antineoplastic agent, is used in treating a wide spectrum of human neoplasms; the development of severe cardiac toxicity in humans compromises its clinical effectiveness (1) Recently DOX was found to induce apoptosis in isolated ventricular myocytes (2, 3). It has been demonstrated that DOX is metabolically activated to a free radical state and interacts with molecular oxygen to generate superoxide radicals (4, 5, 6). These highly toxic reactive oxygen species react with cellular molecules including nucleic acids, protein, and lipids, thereby causing cell damage (7).

Cardiac myocytes make up most of the adult myocardial mass, but they make up only 30% of the total cell number present in the heart; the rest is composed of nonmyocytes (8, 9). Nonmyocytes are reported to consist primarily of fibroblasts and small amounts of other cell types, including vascular endothelial cells, smooth muscle cells, and macrophages (8, 9). Recently it was reported that many growth factors and cytokines, acting as autocrine/paracrine factors, are involved in cardiac remodeling, and the cross-talk between myocytes and nonmyocytes via such humoral factors appears to play an important role in the pathophysiology of cardiac disorders (10, 11). However, it remains to be elucidated whether humoral factors produced from nonmyocytes have a protective effect against myocardial injury. Endothelin (ET)-1 is a 21-amino acid peptide secreted by many types of cells including cardiac normocytes (12, 13). Some studies reported on the antiapoptotic effect of exogenous ET-1 on cardiac myocyte apoptosis (14, 15, 16, 17). However, the precise molecular mechanism of endogenous ET-1-mediated antiapoptotic effect on cardiac myocyte apoptosis remains unclear.

Therefore, we conducted the present study to investigate the paracrine effect of humoral factors secreted from cardiac nonmyocytes including ET-1 on DOX-induced myocyte apoptosis. We also examined which intracellular mechanism is involved in the antiapoptotic effect.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
DMEM and fetal calf serum (FCS) were purchased from Invitrogen (Grand Island, NY). Recombinant human ET-1 was purchased from Peptide Institute (Osaka, Japan). DOX was provided by Kyowa Hakko Kogyo (Tokyo, Japan). BQ123 and BQ485 were purchased from Sigma (St. Louis, MO). BQ788 was purchased from Calbiochem (San Diego, CA). PD98059, wortmannin, and LY294002 were purchased from Wako Pure Chemical Industries (Osaka, Japan). Anti-{alpha}-sarcomeric actin antibody and tetramethylrhodamine isothiocyanate-conjugated second antibody for immunocytochemistry was purchased from Sigma and Dako (Glostrup, Denmark), respectively. The following antibodies were used in the present Western immunoblot analyses: anti-phospho ERK, anti-ERK, anti-phospho Akt, anti-Akt, anti-phospho cAMP response element-binding protein (CREB), anti-CREB, anti-phospho Bad, anti-Bad, anti-Bcl-xL, horseradish peroxidase-conjugated antirabbit IgG (Cell Signaling Technology, Beverly, MA), anti-manganese superoxide dismutase (Mn-SOD, Upstate Biotechnology, Lake Placid, NY), anti-Bcl-2, and anti-{alpha}-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA). In antisense experiments, CREB antisense (5'-GCTCCAGAGTCCATGGTCAT-3') and CREB sense (5'-ATGACCATGGACTCTGGAGC-3') oligonucleotides (18) were purchased from Sigma. Transfection reagent Lipofectamine Plus was purchased from Invitrogen.

Cell culture
All animal experimental procedures were carried out in accordance with the institutional and national ethical guidelines for animal experimentation. Primary cultures of neonatal rat cardiac myocytes and nonmyocytes were prepared as described previously (19). The separation of myocytes from nonmyocytes was performed using the discontinuous Percoll gradient method (19). After 36 h of incubation in DMEM with 10% FCS, cardiac myocytes were serum starved for 24 h before the experiments.

Cocultures of myocytes and nonmyocytes were prepared as previously reported (13). Cultured cardiac nonmyocytes were removed by tripsinization and added to the myocyte culture prepared as described above, resulting in an equal number of myocytes in the myocyte-nonmyocyte coculture and the myocyte culture. After 36 h of incubation in DMEM with 10% FCS, cells were serum starved for 24 h before the experiments.

For preparation of nonmyocyte-conditioned medium, cardiac nonmyocytes were cultured on 10-cm dishes. After incubation in DMEM with FCS, the medium was changed to fresh serum-free DMEM, and cells were incubated for 24 h. The medium was then collected as nonmyocyte-conditioned medium.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining
In situ labeling of fragmented DNA was performed as previously described using the Apop tag apoptosis detection kit (Intergen, Purchase, NY) (20). Cardiac myocytes and/or nonmyocytes cultured on collagen-coated chamber slides were treated with DOX and/or ET-1 for 24 h. After cells were fixed with 4% paraformaldehyde in PBS, terminal deoxynucleotidyl transferase enzyme reaction was performed for 1 h at 37 C. Anti-DIG conjugate was added to the slide and incubated for 30 min at 37 C, and then cells were stained with propidium iodide for counterstaining. Thereafter, for detection of myocyte cytoplasm, cells were incubated with monoclonal anti-{alpha}-sarcomeric actin and tetramethylrhodamine isothiocyanate-conjugated second antibody. The cells were analyzed using a confocal laser microscopy.

Cytotoxicity assay
The cytotoxicity of cardiac myocytes was evaluated by lactate dehydrogenase (LDH) release from injured cells as described previously (21).

DNA fragmentation
To examine the DNA laddering formation, we used the apoptosis ladder detection kit (Wako) as described previously (22). In addition, histone-associated DNA fragments were quantified by ELISA (Roche Molecular Biochemicals, Mannheim, Germany) as described previously (23).

Caspase-3 activity
The activities of caspase-3 were determined with CPP32/caspase-3 fluorometric protease assay kit (MBL, Nagoya, Japan) as described previously (24).

Immunoreactive ET-1 and atrial natriuretic peptide (ANP) release
RIA for rat ET-1 was performed with endothelin-1, 2 [125I] high sensitivity RIA system (Amersham Bioscience, Tokyo, Japan). RIA for rat ANP was performed as described previously (19, 25).

Western immunoblot analysis
After stimulation for selected periods, cells were rapidly rinsed with ice-cold PBS and harvested in a sample buffer [62.5 mmol/liter Tris-HCl, 2% sodium dodecyl sulfate, 10% glycerol, 0.01% bromophenol blue, protease inhibitor cocktail (pH 6.8)]. The lysates were centrifuged, and the supernatants were then analyzed by Western blotting as described previously (26).

8-iso-Prostaglandin F2{alpha} (PGF2{alpha}) determination
Determination of 8-iso-PGF2{alpha} was performed as previously described (27) with partial modification. After stimulation for selected periods, ethanol was added to the media to stop reaction and cells were harvested. After centrifugation, supernatants were hydrolyzed with 15% KOH. Its pH was adjusted to 3.0 with hydrochloric acid. Acidified samples were loaded into the Sep-Pak C18 columns (Waters Associates, Milford, MA) followed by consecutive washing with hydrochloric acid and heptanes. The samples were eluted onto 1 g of sodium sulfate with a mixture of heptane and ethyl acetate (1:1). The samples were then loaded into Sep-Pak Silica column (Waters Associates). The eluted samples were dried under nitrogen gas and reconstituted with assay buffer supplied in an 8-isoprostane enzyme immunoassay (Cayman Chemical Co., Ann Arbor, MI), in which the concentration of total 8-iso-PGF2{alpha} was evaluated.

Statistical analysis
All values are shown as the mean ± SD. Statistical significance between the two groups was determined using unpaired t test. For multiple comparisons, data were subjected to one-way ANOVA followed by Fisher’s multiple comparison test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibitory effects of cardiac nonmyocytes and ET-1 on DOX-induced cardiac myocyte apoptosis
As shown in Fig. 1Go, a significant increase in apoptotic cells was identified after 24-h incubation with DOX. In myocyte-nonmyocyte coculture, the number of apoptotic cells was significantly decreased. When cultured in the presence of nonmyocyte-conditioned media or ET-1, the number of apoptotic cells was also markedly decreased. Although apoptotic cells were slightly observed in untreated myocytes (control cells), myocyte-nonmyocyte coculture, nonmyocyte-conditioned media, or ET-1 had no significant effect on this proportion.



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  		FIG. 1. Effects of myocyte (MC)-nonmyocyte (NMC) coculture, nonmyocyte-conditioned media (CM), and ET-1 on DOX-induced cardiac myocyte apoptosis. Cardiac myocytes were plated onto chamber slides and incubated for 24 h with or without DOX (10–6 mol/liter), nonmyocyte-conditioned media (90% replacement), and/or ET-1 (10–7 mol/liter). In myocyte-nonmyocyte coculture, 10% number of nonmyocytes was added to the myocyte culture. Cardiac myocytes were stained with the anti-{alpha}-sarcomeric actin antibody as a marker for cardiac myocytes. Nuclei of the cells were stained with the TUNEL and propidium iodide. The apoptotic nuclei were stained in yellow and nonapoptotic nuclei were stained in red.

 
We previously reported that the decrease in ANP secretion from myocytes is a sensitive marker of DOX-induced cardiomyocyte toxicity (28). As shown in Fig. 2Go, A and B, the secretion of ir-ANP was significantly decreased after 24-h incubation with DOX. Myocyte-nonmyocyte coculture (5–10% number of nonmyocytes) and nonmyocyte-conditioned media (30–90% replacement) attenuated the decrease in ANP release. DNA fragmentation was assessed by agarose gel electrophoresis (Fig. 2CGo) and quantified by histone-associated DNA fragment-specific ELISA (Fig. 2DGo). Treatment of DOX increased histone-associated DNA fragments with exhibition of the ladder pattern of apoptosis in myocytes, and both nonmyocyte-conditioned media and ET-1 significantly suppressed the DOX-induced DNA fragmentation.



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  		FIG. 2. A and B, Effects of myocyte-nonmyocyte coculture and nonmyocyte-conditioned media on DOX-induced cytotoxicity evaluated with the decrease in ANP secretion from cultured cardiac myocytes. Cardiac myocytes were incubated for 24 h with or without DOX (10–6 mol/liter) and/or nonmyocyte-conditioned media (3–90%). In myocyte-nonmyocyte coculture, 1–10% of nonmyocytes was added to the myocyte culture. ir, Immunoreactive. C and D, Effects of nonmyocyte-conditioned media and ET-1 on DOX-induced DNA fragmentation in cultured cardiac myocytes. Cardiac myocytes were incubated for 24 h with or without DOX (10–6 mol/liter), nonmyocyte-conditioned media (90%), and/or ET-1 (10–7 mol/liter). Each 1 µg of DNA sample was separated by electrophoresis on 1.5% agarose gel (C). DNA fragmentation was quantified with histone-associated DNA fragment-specific ELISA (D). Values are given as the mean ± SD of six measurements. *, P < 0.0001 vs. control; #, P < 0.0001 vs. DOX alone.

 
Cytotoxicity was assessed by LDH release from injured cells. As shown in Fig. 3AGo, DOX markedly increased LDH release from cardiac myocytes. When cultured in the presence of nonmyocyte-conditioned media or ET-1, this increase was significantly attenuated in a dose-dependent manner. Nonmyocyte-conditioned media or ET-1 per se had no significant effect on the control level of LDH release.



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  		FIG. 3. Effects of nonmyocyte-conditioned media (CM) and ET-1 on DOX-induced cytotoxicity evaluated with LDH release into the medium from cultured cardiac myocytes. A, Cardiac myocytes were incubated for 24 h with or without DOX (10–6 mol/liter), nonmyocyte-conditioned media (3–90%), and/or ET-1 (10–10 to 10–7 mol/liter). B, Cardiac myocytes were pretreated for 1 h in the absence or presence of BQ123 (10–6 mol/liter), BQ485 (10–6 mol/liter), or BQ788 (10–6 mol/liter). Then cells were incubated for 24 h with or without DOX (10–6 mol/liter), nonmyocyte-conditioned media (90%), and/or ET-1 (10–7 mol/liter). Values are given as the mean ± SD of six measurements. *, P < 0.0001 vs. control; #, P < 0.0001 vs. DOX alone; {dagger}, P < 0.0001 vs. DOX plus CM; §, P < 0.0001 vs. DOX plus ET-1.

 
A significant amount of ET-1 was detected in nonmyocyte-conditioned media (7.6 x 10–10 mol/liter) but not in myocyte-conditioned media (<6.3 x 10–13 mol/liter). Thus, the involvement of endogenous ET-1 in the antiapoptotic effect of nonmyocyte-conditioned media was examined using ET-A receptor antagonists BQ123 and BQ485 and an ET-B receptor antagonist BQ788. Both BQ123 and BQ485 partially or completely blocked the suppression of LDH release by nonmyocyte-conditioned media or ET-1, respectively, although BQ788 had no significant effect (Fig. 3BGo).

Intracellular mechanisms of the antiapoptotic effect mediated by nonmyocyte-conditioned media
To determine the pathway through which nonmyocyte-conditioned media transduce signals in cardiac myocytes, we examined the phosphorylation of ERK, Akt, CREB, and Bad by Western blot analysis using phospho-specific antibodies. ERK was maximally phosphorylated within 5 min of exposure to nonmyocyte-conditioned media and ET-1 (Fig. 4Go, A and B). Akt was phosphorylated within 5 min of exposure to nonmyocyte-conditioned media but not to ET-1. Nonmyocyte-conditioned media-mediated phosphorylation of Akt was sustained over 60 min. The phosphorylation of CREB was activated within 5 min of exposure to nonmyocyte-conditioned media and ET-1, and the activation sustained over 60 min. In contrast, neither nonmyocyte-conditioned media nor ET-1 phosphorylated Bad serine112 and serine136.



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  		FIG. 4. Effects of nonmyocyte-conditioned media (CM, A) and ET-1 (B) on ERK, Akt, CREB, and Bad phosphorylation in cultured cardiac myocytes. Cardiac myocytes were incubated with nonmyocyte-conditioned media (90%) or ET-1 (10–7 mol/liter) for 15–60 min. C, Effects of several inhibitors on nonmyocyte-conditioned media-mediated ERK, Akt, and CREB phosphorylation. Cardiac myocytes were pretreated for 1 h in the absence or presence of BQ123 (10–6 mol/liter), BQ485 (10–6 mol/liter), BQ788 (10–6 mol/liter), PD98059 (2 x 10–5 mol/liter), wortmannin (2 x 10–6 mol/liter), or LY294002 (2 x 10–6 mol/liter). Then cells were incubated for 24 h with or without DOX (10–6 mol/liter) and/or nonmyocyte-conditioned media (90%). Western immunoblot analyses were performed using antibodies against phospho-ERK (P-ERK), ERK, phospho-Akt (P-Akt), Akt, phospho-CREB (P-CREB), CREB, phospho-Bad (P-Bad) S112 and S136, and Bad. Results are representative of two independent experiments.

 
Nonmyocyte-conditioned media-mediated ERK phosphorylation was blocked partially by ET-A receptor antagonists but not by ET-B receptor antagonist (Fig. 4CGo). ET-A and ET-B receptor antagonists did not affect Akt phosphorylation. Phosphatidylinositol-3 kinase (PI3K) inhibitors wortmannin and LY294002 had no effect on ERK phosphorylation, and MAPK kinase (MEK-1)-specific inhibitor PD98059 did not affect Akt phosphorylation, suggesting that there was no cross-talk between ERK and Akt phosphorylation mediated by nonmyocyte-conditioned media. However, CREB phosphorylation was inhibited by ET-A receptor antagonists, MEK inhibitor, and PI3K inhibitors.

Next, we examined the involvement of these pathways in the nonmyocyte-conditioned media-mediated antiapoptotic effect. As shown in Fig. 5Go, inhibition of MEK-ERK pathway by PD98059 or inhibition of PI3K-Akt pathway by LY294002 partially blocked the nonmyocyte-conditioned media-treated decrease in LDH release, and both inhibitions of MEK-ERK and PI3K-Akt pathways completely blocked the effect of nonmyocyte-conditioned media. The protective effect of exogenous ET-1 was completely inhibited by PD98059, but LY294002 showed no effect. To determine whether CREB activation is involved in the nonmyocyte-conditioned media-mediated antiapoptotic effect, the effect of CREB antisense was tested. The antisense, but not sense oligonucleotide, significantly blocked the nonmyocyte-conditioned media- and ET-1-mediated decrease in LDH release.



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  		FIG. 5. Effects of MEK inhibitor, Akt inhibitor, and CREB antisense on nonmyocyte-conditioned media (CM)- or ET-1-mediated attenuation of DOX-induced cytotoxicity in cultured cardiac myocytes. Cardiac myocytes were pretreated for 1 h in the absence or presence of PD98059 (2 x 10–5 mol/liter) and/or LY294002 (2 x 10–6 mol/liter) or for 24 h in the absence or presence of CREB antisense (AS, 2 x 10–6 mol/liter) or CREB sense (S, 2 x 10–6 mol/liter). Then cells were incubated for 24 h with or without DOX (10–6 mol/liter), nonmyocyte-conditioned media (90%), and/or ET-1 (10–7 mol/liter). Values are given as the mean ± SD of eight measurements. *, P < 0.0001 vs. control; #, P < 0.0001 vs. DOX alone; {dagger}, P < 0.05 vs. DOX plus CM; §, P < 0.05 vs. DOX plus ET-1.

 
We further examined the effect of nonmyocyte-conditioned media on Bcl-2 and Bcl-xL protein expression as antiapoptotic members of Bcl-2 families (29). DOX decreased both Bcl-2 and Bcl-xL protein levels, and nonmyocyte-conditioned media and ET-1 significantly attenuated DOX-induced Bcl-2 protein decrease but not Bcl-xL protein decrease (Fig. 6AGo). ET-A antagonists and CREB antisense blocked nonmyocyte-conditioned media-mediated Bcl-2 protein increase (Fig. 6BGo).



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  		FIG. 6. A, Effects of nonmyocyte-conditioned media (CM) and ET-1 on DOX-induced decrease in Bcl-2 and Bcl-xL protein levels in cultured cardiac myocytes. Cardiac myocytes were incubated for 24 h with or without DOX (10–6 mol/liter), nonmyocyte-conditioned media (90%), and/or ET-1 (10–7 mol/liter). Values shown were corrected using the density of {alpha}-tubulin. Values are given as the mean ± SD of three experiments. *, P < 0.001 vs. control; #, P < 0.01 vs. DOX alone. B, Effects of ET-1 receptor antagonists and CREB antisense on nonmyocyte-conditioned media-mediated increase in Bcl-2 protein. C, Effects of ET-1 receptor antagonists and CREB antisense on nonmyocyte-conditioned media- or ET-1-mediated attenuation of DOX-induced caspase-3 activation. Cardiac myocytes were pretreated for 1 h in the absence or presence of BQ123 (10–6 mol/liter), BQ485 (10–6 mol/liter), or BQ788 (10–6 mol/liter) or for 24 h in the absence or presence of CREB antisense (AS, 2 x 10–6 mol/liter) or CREB sense (S, 2 x 10–6 mol/liter). Then cells were incubated for 24 h with or without DOX (10–6 mol/liter), nonmyocyte-conditioned media (90%), and/or ET-1 (10–7 mol/liter). Values are given as the mean ± SD of six measurements. *, P < 0.0001 vs. control; #, P < 0.0001 vs. DOX alone; {dagger}, P < 0.001 vs. DOX plus CM; §, P < 0.01 vs. DOX plus ET-1.

 
Because caspase-3 is an important component of the final pathway leading to the occurrence of cell death, we determined whether endogenous ET-1 and CREB activation contribute to nonmyocyte-conditioned media-mediated attenuation of DOX-induced caspase-3 activation. As shown in Fig. 6CGo, DOX-induced increase in caspase-3 activity in cardiac myocytes was significantly reduced by treatment with nonmyocyte-conditioned media and ET-1. The inhibitory effect of nonmyocyte-conditioned media was blocked with BQ123 and CREB antisense.

Finally, to clarify whether antiapoptotic effect of nonmyocyte-conditioned media is mediated by the decrease in DOX-induced oxidative stress, we investigated whether nonmyocyte-conditioned media increase the protein level of Mn-SOD, which is a well-recognized antiapoptotic enzyme, and decrease the production of 8-iso-PGF2{alpha}, one of oxidative stress-related products. The Mn-SOD protein level was not significantly changed 4–24 h after incubation with nonmyocyte-conditioned media or ET-1 (Fig. 7AGo). The 8-iso-PGF2{alpha} production was significantly increased after 24-h incubation with DOX (Fig. 7BGo). Treatment with nonmyocyte-conditioned media and ET-1 tended to increase PGF2{alpha} production, but not significantly. Pretreatment with BQ123 tended to decrease PGF2{alpha} production, but this was also not significant.



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  		FIG. 7. A, Effects of nonmyocyte-conditioned media (CM) and ET-1 on the expression of Mn-SOD protein. Cardiac myocytes were incubated for 4–24 h with nonmyocyte-conditioned media (90%) or ET-1 (10–7 mol/liter). B, Effects of DOX, nonmyocyte-conditioned media and ET-1 on the production of 8-iso-PGF2{alpha}. Cardiac myocytes were pretreated for 1 h in the absence or presence of BQ123 (10–6 mol/liter) or BQ788 (10–6 mol/liter). Then cells were incubated for 24 h with or without DOX (10–6 mol/liter), nonmyocyte-conditioned media (90%), and/or ET-1 (10–7 mol/liter). Values are given as the mean ± SD of six measurements. *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated a significant decrease in TUNEL-positive cardiac myocytes and attenuation of DOX-induced decrease in ANP release in the myocyte-nonmyocyte coculture. These results clearly indicate that nonmyocytes contribute to the inhibition of DOX-induced myocyte apoptosis. Nonmyocyte-conditioned media also decreased TUNEL-positive cells and attenuated DOX-induced decrease in ANP release. Furthermore, nonmyocyte-conditioned media prevented DOX-induced DNA strand breaks and blunted LDH release from myocytes. These findings suggest that nonmyocytes secrete some humoral factors regulating myocyte apoptosis. We also demonstrated that the ET-A receptor antagonists significantly blocked the antiapoptotic effect of nonmyocyte-conditioned media. The ET-1 level detected in nonmyocyte-conditioned media was comparable with the concentration of exogenous ET-1 that prevents cardiac myocyte apoptosis. These results suggest that ET-1 might be involved in the antiapoptotic effect of nonmyocyte-conditioned media on myocytes and that this effect of ET-1 is probably mediated by the ET-A receptor. The partial blockade of the nonmyocyte-conditioned media-treated antiapoptotic effect by ET-A receptor antagonists suggests the possibility that other humoral factor(s) are secreted form nonmyocytes and also regulate myocyte survival. One of the candidates is cardiotrophin-1. Secretion of cardiotrophin-1 from nonmyocytes and phosphorylation of Akt by cardiotrophin-1 in cardiac myocytes were previously reported (30). Further investigations are needed to clarify the involvement of factor(s) other than ET-1 in the interaction between myocytes and nonmyocytes.

We analyzed the intracellular mechanism of nonmyocyte-conditioned media-mediated cell survival. MEK-ERK pathway and PI3K-Akt have been shown to be involved in the antiapoptotic effect of many growth factors and cytokines (31, 32, 33, 34, 35). In this study, nonmyocyte-conditioned media phosphorylated ERK and Akt. Inhibition of MEK-ERK and PI3K-Akt pathways blocked the antiapoptotic effect of nonmyocyte-conditioned media. Therefore, our findings show the crucial participation of the ERK and Akt pathways in nonmyocyte-conditioned media-induced survival signaling in cardiac myocytes. Exogenous ET-1 also phosphorylated ERK but not Akt. ET-A receptor antagonists partially inhibited nonmyocyte-conditioned media-mediated ERK phosphorylation but not Akt. These results suggest that nonmyocyte-conditioned media-mediated ERK phosphorylation is dependent, at least in part, on endogenous ET-1, but Akt phosphorylation is independent of the action of ET-1. Other humoral factor(s) secreted from nonmyocytes may survive cardiac myocytes via Akt-dependent pathway. Nonmyocyte-conditioned media also activated the transcription factor CREB via an ERK- and Akt-dependent mechanism. CREB has been shown as a target of several signaling molecules, including MAPK and Akt (32, 36). In the present study, we demonstrated that CREB antisense blocked nonmyocyte-conditioned media- and ET-1-mediated decreases in caspase-3 activity and LDH release. These results suggest that CREB is an important transducer of nonmyocyte-conditioned media- and ET-1-mediated antiapoptotic effect on cardiac myocytes.

In the present study, we demonstrated that nonmyocyte-conditioned media and exogenous ET-1 augmented Bcl-2 protein expression but not Bcl-xL. The Bcl-2 gene product is a 25-kDa membrane protein that functions to prevent apoptosis by various stimuli (37). Prevention of apoptosis by increased Bcl-2 expression has been shown in adult cardiac myocytes (38). A recent study (16) showed that stimulation of cardiac myocytes with ET-1 increased the expression of Bcl-2 protein. Our observations were consistent with the findings of that study. Moreover, existence of a cAMP response element site in the Bcl-2 promoter and its important role in Bcl-2 expression have been reported (39, 40). In our study, ET-A receptor antagonists and CREB antisense attenuated nonmyocyte-conditioned media-mediated Bcl-2 protein increase. These results suggest that humoral factors including ET-1 secreted from nonmyocytes up-regulate Bcl-2 expression through CREB activation. However, a recent study by Ogata et al. (17) reported opposite findings about ET-1-mediated antiapoptotic protein expression and subcellular signaling pathway. First, they showed that exogenous ET-1 up-regulated Bcl-xL but not Bcl-2 using H9c2 cardiac myoblast cells (embryonic rat heart-derived cell line). Second, MEK-specific inhibitor PD98059 failed to block ET-1-mediated antiapoptotic effect in their study. The exact reason for these discrepant findings is not clear, but it may be partly due to the difference of cultured cells used in respective experiments. Therefore, in contrast to the experiments by Ogata et al., our observations and the findings by Kakita et al. (16), which also showed the Bcl-2 protein induction mediated by ET-1, were obtained from primary cultures of neonatal cardiac myocytes. As for the effect of PD98059, the concentration of the agent used in the study by Ogata et al. (10–5 mol/liter) was different from that in our study. In our preliminary examination, 10–5 mol/liter PD98059 was not sufficient to block 10–7 mol/liter ET-1-induced ERK phosphorylation.

In the present study, attenuation by CREB antisense of nonmyocyte-conditioned media- and ET-1-mediated LDH decrease and inhibition of caspase-3 activation were not complete. A previous study demonstrated that the calcineurin pathway is required for ET-1-mediated protection against oxidative stress-induced apoptosis in cardiac myocytes (16). In addition, nuclear factor of activated T cells transcription factors were shown to be survival factors in cardiac myocytes (41). It has been recently reported that c-Src and Pyk2 are also involved in the ET-1-mediated antiapoptotic effect (17). According to these findings, the antiapoptotic effect of endogenous ET-1 in cardiac myocytes may be elicited through the pathways of calcineurin-nuclear factor of activated T cells and tyrosine kinase in addition to the ERK-CREB pathway.

Bad is known as a proapoptotic factor recently found to be phosphorylated by Akt. Several studies showed PI3K/Akt/Bad pathway might be one of the mechanism by which growth factors and cytokines promote cell survival (31, 32, 33). In the present study, nonmyocyte-conditioned media phosphorylated Akt but not Bad. Although the reason that nonmyocyte-conditioned media did not phosphorylate Bad is unclear, one possibility is that the response of Bad to the Akt phosphorylation may be different among several ligands that phosphorylate Akt.

DOX-induced cardiac myocyte apoptosis is mediated in part by oxidative stress. Although exogenous ET-1 clearly inhibited DOX-induced apoptosis, augmentation of oxidative stress by ET-1 in vascular cells was reported (42, 43). For investigation of the discrepant phenomenon, we examined whether the antiapoptotic effect of endogenous ET-1 is dependent on the decrease in DOX-induced oxidative stress. In the present study, nonmyocyte-conditioned media and ET-1 did not increase the protein expression of Mn-SOD and did not decrease the production of 8-iso-PGF2{alpha} induced by DOX. Therefore, it is probable that endogenous ET-1 protects cardiac myocytes against DOX via increase in cytoprotective proteins such as Bcl-2 and inhibition of caspase-3 activation but not via decrease in oxidative stress.

Although it was previously reported that exogenous ET-1 prevents cardiac myocyte apoptosis (15, 16, 17), the role of endogenous ET-1 on myocardial disorder has been unknown. Moreover, several studies reported the effectiveness of the blockade of ET-1 receptors on chronic heart failure in experimental animals and patients (44, 45), suggesting the harmful role of endogenous ET-1 in such cardiac disorders. However, our in vitro study demonstrated that ET-1 secreted from nonmyocyte prevented DOX-induced cardiac myocyte apoptosis. Therefore, the present findings suggest that endogenous ET-1, as a paracrine factor, may have a protective effect on injured cardiac myocytes in some cardiac disorders, especially in the acute myocardial injury.

Figure 8Go shows a scheme representing the role of cardiac nonmyocytes during the process of inhibition of myocyte apoptosis and its intracellular mechanisms suggested by the present study. ET-1 secreted from nonmyocytes phosphorylates ERK via ET-A receptor activation. Additional unknown factor(s) phosphorylate ERK and Akt. Transcription factor CREB is phosphorylated by ERK- and Akt-dependent pathways. The activated CREB increases Bcl-2 protein expression and inhibits caspase-3 activation, and finally myocyte apoptosis is attenuated.



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  		FIG. 8. Inhibitory effect of cardiac nonmyocytes on myocyte apoptosis through humoral factors including ET-1 and its intracellular mechanisms.

 


   Footnotes
 
This work was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan, and Grants-in-Aid for Scientific Research (14571044) from the Japan Society for the Promotion of the Science.

Abbreviations: ANP, Atrial natriuretic peptide; CREB, cAMP response element-binding protein; DOX, doxorubicin; ET, endothelin; FCS, fetal calf serum; LDH, lactate dehydrogenase; MEK, MAPK kinase; Mn-SOD, manganese superoxide dismutase; PGF2{alpha}, prostaglandin F2{alpha}; PI3K, phosphatidylinositol-3 kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling.

Received October 2, 2003.

Accepted for publication January 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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