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Endocrinology Vol. 143, No. 9 3515-3521
Copyright © 2002 by The Endocrine Society

ARTICLE

Adrenomedullin Inhibits Doxorubicin-Induced Cultured Rat Cardiac Myocyte Apoptosis via a cAMP-Dependent Mechanism

Takeshi Tokudome, Takeshi Horio, Fumiki Yoshihara, Shin-ichi Suga, Yuhei Kawano, Masakazu Kohno and Kenji Kangawa

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that adrenomedullin produced by cardiac myocytes acts as a local modulator in some cardiac disorders. However, the role of adrenomedullin (AM) in cardiomyocyte apoptosis remains to be clarified. The present study investigated the effect of AM on doxorubicin-induced cardiac myocyte apoptosis. Doxorubicin increased the number of cells with pyknotic nuclei and lactate dehydrogenase release, and AM dose-dependently (10-10–10-86 M) inhibited these increases produced by doxorubicin. Treatment with AM also suppressed doxorubicin-induced DNA fragmentation and caspase-3 activation. 8-Bromo-cAMP, a cAMP analog, mimicked these antiapoptotic effects of AM. An AM/calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8–37) and a protein kinase A inhibitor H89 attenuated the antiapoptotic effect of AM. CGRP-(8–37) and H89 had no apoptotic effect alone, but accelerated doxorubicin-induced apoptosis. Under serum-free conditions, AM secretion into the culture medium and expression of AM mRNA were significantly increased after treatment with doxorubicin. Hydrogen peroxide scavenger catalase and antioxidant N-acetyl-L-cysteine inhibited the doxorubicin-mediated increase in AM secretion and its gene expression. These results indicate that AM inhibits doxorubicin-induced cardiac myocyte apoptosis through a cAMP-dependent mechanism and suggest that augmented production of AM by doxorubicin has an endogenous antiapoptotic effect. AM, as an autocrine factor, may play a protective role against cardiomyocyte injury by doxorubicin.


   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).

Adrenomedullin (AM) is a 52-amino acid vasoactive peptide that was originally isolated from human pheochromocytoma (8). AM shares 24% amino acid homology with calcitonin gene-related peptide (CGRP) and also has a biological activity profile similar to that of CGRP (9). In some systems, including cardiac myocytes, AM and CGRP share a common receptor and signaling mechanism (10, 11, 12). Accumulating evidence has revealed that AM has more biological effects than initially expected as a vasodilating reagent (13), and that AM is synthesized in various organs, including the heart (14, 15). A previous study showed that AM immunoreactivity was markedly increased in failing human ventricles (16), and we also demonstrated augmented levels of the AM gene and peptide in the hearts of rats with heart failure (17, 18). In addition, our in vitro study demonstrated that AM was produced and secreted in cultured rat cardiac myocytes and nonmyocytes stimulated by IL-1ß and TNF{alpha} (19). These observations suggested that AM was produced from normal hearts, and that its production was accelerated in cardiac disorders such as heart failure. However, it remains to be elucidated whether AM produced from the failing heart has a protective effect on the heart itself. In particular, there has been no study that examined the effect of AM on cardiomyocyte injury and apoptosis.

Therefore, we conducted the present study to investigate the effect of AM on DOX-induced myocyte apoptosis and the effect of DOX on peptide release and gene expression of AM in cultured ventricular myocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
DMEM, fetal calf serum, and TRIzol LS reagent were purchased from Life Technologies, Inc. (Grand Island, NY). Recombinant human AM and DOX were provided by Shionogi & Co. Ltd. (Osaka, Japan) and Kyowa Hakko Kogyo Co. Ltd. (Tokyo, Japan), respectively. Human CGRP-(8–37) was purchased from Peptide Institute (Osaka, Japan). N-Acetyl-L-cysteine (NAC), 8-bromo-cAMP (Br-cAMP), and Hoechst 33342 were purchased from Sigma (St. Louis, MO). Catalase, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide), and N-nitro-L-arginine methyl ester (L-NAME) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Cell culture
Primary cultures of neonatal rat cardiac myocytes were prepared as previously described (19). Briefly, apical halves of cardiac ventricles from 1- to 2-d-old Wistar rats were separated, minced, and dispersed with 0.1% collagenase type II. To segregate myocytes from nonmyocytes, a discontinuous gradient of Percoll was prepared. After centrifugation, the upper layer consisted of a mixed population of nonmyocyte cell types, and the lower layer consisted almost exclusively of cardiac myocytes. After the myocytes were incubated twice on uncoated 10-cm culture dishes for 30 min to remove any remaining nonmyocytes, the nonattached viable cells were plated on gelatin-coated culture plates or culture dishes and then cultured in DMEM supplemented with 10% FCS. After 72 h of incubation in DMEM with 10% FCS, cardiac myocytes were serum-starved for 12 h before the experiments.

Morphological examination
Cardiac myocytes cultured in 12-well plates were treated with DOX, AM, and/or Br-cAMP for 24 h. After incubation, cells were stained with Hoechst 33342 and observed under a microscope equipped with phase contrast and epifluorescence optics according to the method previously reported (20). Pyknotic nuclei under Hoechst 33342 staining were counted and expressed as the percentage of total nuclei. At least 500 nuclei were counted from randomly selected fields in each experiment.

Cytotoxicity assay
Cardiac myocytes cultured in 24-well plates were treated with DOX, AM, Br-cAMP, CGRP-(8–37), H89, and/or L-NAME for 24 h. The cytotoxicity of cardiac myocytes was evaluated by lactate dehydrogenase (LDH) release from injured cells. For this assay the culture medium was collected, and the amount of LDH in each medium was measured using colorimetric assay kits (LDH-Cytotoxic Test, Wako Pure Chemical Industries) (21).

DNA fragmentation
Cardiac myocytes cultured in 60-mm dishes were treated with DOX, AM, and/or Br-cAMP for 24 h. To examine the DNA laddering formation, we used the apoptosis ladder detection kit (Wako Pure Chemical Industries) as described previously (22). Additionally, histone-associated DNA fragments were quantified by ELISA (Roche Molecular Biochemicals, Mannheim, Germany) as described previously (23, 24).

Measurement of caspase-3 activity
Cardiac myocytes cultured in 60-mm dishes were treated with DOX, AM, and/or Br-cAMP for 24 h. The activities of caspase-3 were determined with CPP32/caspase-3 fluorometric protease assay kit (MBL, Nagoya, Japan) by the detection of 7-amino-4-trifluoromethyl coumarin (AFC) after cleavage from the labeled substrate Asp-Glu-Val-Asp-AFC as previously described (25). In brief, 1 x 106 cells were solubilized, and equal amounts of lysates were reacted with 50 µM Asp-Glu-Val-Asp-AFC substrate at 37 C for 1 h. The activity was read in a spectrophotometer at 505 nm.

Measurement of immunoreactive (ir-) AM and ir-atrial natriuretic peptide (ANP)
The culture medium (1.5 ml) was acidified with acetic acid, boiled for 5 min to inactivate intrinsic proteases, and lyophilized. RIAs for rat AM and ANP were performed as previously reported (19, 26). These assays were performed in duplicate.

Northern blot analysis
Total RNA was extracted from cultured cells with TRIzol LS reagent as previously reported (26). Total RNA (10 µg/lane) was electrophoresed on a 1% agarose gel and then transferred to a nylon membrane. Hybridization and washing of the membrane were carried out with cDNA probes for rat AM and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes as described previously (19). Band intensity was estimated using a radioimage analyzer (BAS-5000, Fuji Photo Film Co., Ltd., Tokyo, Japan).

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
 
Effect of AM on DOX-induced cardiac myocyte apoptosis
DOX (10-6 M) treatment displayed an apoptotic morphology characterized by cell shrinkage (Fig. 1AGo) and nuclear pyknosis (Fig. 1BGo). A quantification study revealed that 27.0 ± 1.3% of the nuclei were pyknotic after DOX treatment vs. 4.9 ± 0.6% in the controls (Fig. 1CGo; P < 0.0001). When cultured in the presence of AM (10-8 M), the number of cells that showed the apoptotic phenotype decreased significantly (16.9 ± 1.4%; P < 0.0001 vs. DOX alone), and the inhibition by AM of cardiac myocyte apoptosis was dose dependent (Fig. 1CGo). To elucidate whether this antiapoptotic effect of AM is causally linked to the increase in cellular cAMP induced by AM, we examined the effect of Br-cAMP, a membrane-permeable analog of cAMP. Treatment with Br-cAMP (10-3 M) significantly reduced the number of apoptotic cells (16.5 ± 2.0%; P < 0.0001 vs. DOX alone), and the inhibition by Br-cAMP of cardiac myocyte apoptosis was also dose dependent (Fig. 1DGo).



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  		Figure 1. Effect of AM or Br-cAMP on DOX-induced cultured cardiac myocyte apoptosis. Myocytes were incubated for 24 h with or without DOX (10-6 M), AM (10-8–10-10 M), and/or Br-cAMP (10-3–10-5 M) and observed under a phase contrast microscope (A). Cells were stained with Hoechst 33342 and observed under a fluorescent microscope (B). Pyknotic nuclei under Hoechst 33342 staining were counted and expressed as the percentage of total nuclei (C and D). At least 500 nuclei were counted from randomly selected fields in each experiment. Values are given as the mean ± SD of five measurements. *, P < 0.0001 (vs. control). #, P < 0.001; {dagger}, P < 0.0001 (vs. DOX alone).

 
Cytotoxicity was also assessed by LDH release from injured cells. As shown in Fig. 2AGo, DOX (10-6 M) increased LDH release from cardiac myocytes. When cultured in the presence of AM (10-10–10-8 M), this increase was significantly attenuated. As shown in Fig. 2BGo, treatment with Br-cAMP (10-5–10-3 M) also decreased DOX-induced LDH release. AM-suppressed LDH release was restored by the AM/CGRP receptor blockade with CGRP type 1 receptor antagonist CGRP-(8–37) and the inhibition of protein kinase A (PKA) with H89, but not by the inhibition of nitric oxide with L-NAME (Fig. 2CGo). CGRP-(8–37) and H89 had no cytotoxic effect alone, but enhanced LDH release induced by DOX.



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  		Figure 2. Effect of AM (A) or Br-cAMP (B) on the DOX-induced cytotoxicity evaluated with LDH release into the medium from cultured cardiac myocytes. Myocytes were incubated for 24 h with or without DOX (10-6 M), AM (10-8–10-10 M), and/or Br-cAMP (10-3–10-5 M). C, Effect of CGRP-(8–37), H89, or L-NAME on AM-suppressed LDH release into the medium from cultured cardiac myocytes. Myocytes were incubated for 24 h with or without DOX (10-6 M), AM (10-8 M), CGRP-(8–37) (10-6 M), H89 (10-6 M), and/or L-NAME (10-3 M). Values are given as the mean ± SD of six measurements. *, P < 0.0001 (vs. control). , P < 0.05; #, P < 0.01; §, P < 0.001; {dagger}, P < 0.0001 (vs. DOX alone). {ddagger}, P < 0.0001 (vs. DOX plus AM).

 
DNA fragmentation was assessed by agarose gel electrophoresis (Fig. 3AGo) and quantified by histone-associated DNA fragment-specific ELISA (Fig. 3BGo). Treatment of DOX increased histone-associated DNA fragments with exhibition of the ladder pattern of apoptosis, and both AM (10-8 M) and Br-cAMP (10-3 M) significantly suppressed DOX-induced DNA fragmentation.



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  		Figure 3. Effect of AM or Br-cAMP on DOX-induced cultured cardiac myocyte DNA fragmentation. Myocytes were incubated for 24 h with or without DOX (10-6 M), AM (10-8 M), and/or Br-cAMP (10-3 M). A, Each 1-µg DNA sample was separated by electrophoresis on 1.5% agarose gel. B, Quantitative analysis of DNA fragmentation by histone-associated DNA fragment-specific ELISA. Values are given as the mean ± SD of six measurements. *, P < 0.0001 (vs. control). #, P < 0.001 (vs. DOX alone).

 
As caspase-3 is an important component of the final pathway leading to the occurrence of cell death, the effects of DOX and AM on its activity were also evaluated. DOX (10-6 M) treatment induced a 2.5-fold increase in caspase-3 activity compared with that in the control cells (Fig. 4Go). Treatment with AM (10-8 M) and Br-cAMP (10-3 M) significantly reduced this augmented enzyme activity.



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  		Figure 4. Effect of AM or Br-cAMP on DOX-induced caspase-3 activation in cultured cardiac myocytes. Myocytes were incubated for 24 h with or without DOX (10-6 M), AM (10-8 M), and/or Br-cAMP (10-3 M). Values are given as the mean ± SD of five measurements. *, P < 0.0001 (vs. control). #, P < 0.0001 (vs. DOX alone).

 
Effect of DOX on the secretion and gene expression of AM in cardiac myocytes
In cultured cardiac myocytes, the secretion level of ir-AM was significantly increased after 6- to 24-h incubation with 10-6 M DOX (Table 1Go). In contrast, the secretion of ir-ANP was markedly decreased by DOX (Fig. 5AGo). As DOX is known to produce reactive oxygen intermediates such as hydroxyl radicals, superoxide radicals, and hydrogen peroxide in cardiac myocytes (4, 5, 6), we examined whether suppression of oxidative stress attenuates DOX-induced secretion of AM. A hydrogen peroxide scavenger, catalase (10 U/ml), and an antioxidant, NAC (10-5 M), significantly blunted the increase in ir-AM secretion mediated by DOX (Fig. 5BGo).


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  		Table 1. Time course of the effect of DOX on the secretion of ir-AM in cultured cardiac myocytes

 


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  		Figure 5. A, Effect of DOX on the secretion of ir-AM or ir-ANP after 24-h incubation in cultured cardiac myocytes. B, Effects of antioxidants on DOX-mediated up-regulation of AM secretion in cultured cardiac myocytes. Myocytes were incubated for 24 h with or without DOX (10-6 M), catalase (10 U/ml), and/or NAC (10-5 M). Values are given as the mean ± SD of six measurements. *, P < 0.0001; #, P < 0.001.

 
The expression of rat AM mRNA in cultured rat cardiac myocytes was examined by Northern blot analysis. As shown in Fig. 6AGo, a single band hybridizing to the rat cDNA probe was found in myocytes. Figure 6BGo shows a quantitative analysis of these blots corrected for the levels of GAPDH mRNA as an internal standard. The expression of AM mRNA in myocytes was increased after 24-h incubation with 10-6 M DOX. Catalase (10 U/ml) and NAC (10-5 M) significantly attenuated the increase in AM mRNA expression mediated by DOX.



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  		Figure 6. Effect of DOX on the expression of rat AM mRNA and effects of antioxidants on DOX-mediated up-regulation of AM transcripts in cultured cardiac myocytes. Myocytes were incubated for 24 h with or without DOX (10-6 M), catalase (10 U/ml), and/or NAC (10-5 M). Representative experiments are presented in A. Values shown were corrected using the density of the corresponding GAPDH mRNA in B. Values are given as the mean ± SD of four measurements. *, P < 0.0001; #, P < 0.001; {dagger}, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has demonstrated for the first time that AM prevents DOX-induced cultured cardiac myocyte apoptosis evaluated by the increase in the number of pyknotic nuclei and LDH release from the cells. AM also prevented DOX-induced DNA strand breaks. This study demonstrated that a cAMP analog, Br-cAMP, mimicked the antiapoptotic effect of AM on cardiac myocytes. Furthermore, the cytoprotective effect of AM was attenuated by CGRP-(8–37), a CGRP type 1 receptor antagonist, and by a PKA inhibitor, H89. As our previous studies showed that AM elevated intracellular cAMP levels in cultured cardiac myocytes, and cAMP accumulation was inhibited by CGRP-(8–37) (11, 27), the present findings suggest that AM elicits the antiapoptotic effect via the cAMP-dependent mechanism likely to involve a specific AM receptor with which CGRP interacts. A previous study showed that AM had an antiapoptotic effect via nitric oxide-dependent and cAMP-independent pathway in cultured vascular endothelial cells (20). In the present study, however, the antiapoptotic effect of AM was not attenuated by the inhibition of nitric oxide with L-NAME. The discrepant findings regarding the involvement of nitric oxide between these studies may be partly due to a cell-specific difference. Therefore, the antiapoptotic effect of AM on endothelial cells might be mediated by nitric oxide production from those cells stimulated by AM (20). On the other hand, a recent study by Tomoda et al. (28) showed that in cultured cardiac myocytes AM did not stimulate NOx synthesis at concentrations of 10-9–10-8 M. Our observations were consistent with the findings of their study.

As for the effect of cAMP on cardiac myocytes, contrasting findings have been reported. Iwai-Kanai et al. (29) demonstrated that an extremely high dose (3 x 10-2 M) of Br-cAMP induced apoptosis in cardiac myocytes via the cAMP-PKA pathway. In contrast to their observations, Henaff et al. (30) demonstrated that a low concentration of catecholamine (10-7 M epinephrine) promoted myocyte survival via an effect mediated through the cAMP pathway and extracellular signal-regulated kinase activation. Although the exact reason for these discrepant findings remains unexplained, cAMP may have complex effects on cardiac myocyte growth and survival; that is, cAMP may stimulate a pathway involving extracellular signal-regulated kinase activation, while it may be a possible trigger of apoptosis at high concentrations, suggesting that a delicate balance in the levels of cAMP determines pro- or antiapoptotic pathways in cardiac myocytes.

It should be noted that the doses of Br-cAMP (10-5–10-3 M) used in the present study were much higher than the doses of AM (10-10–10-8 M) required to protect myocytes from DOX-induced apoptosis. However, several previous studies also showed that the same high concentrations of Br-cAMP (10-5–10-3 M) reproduced the effects of AM (12, 27, 31, 32). Although the exact reason why high doses of Br-cAMP were needed to mimic the effects of AM is unknown, accumulating intracellular cAMP levels mediated by this cAMP analog might be considerably lower than those by AM.

How AM regulates apoptotic signaling is not well understood, but the present study suggested that the inhibitory action of AM on caspase-3 activation may represent a critical step through which AM modulates apoptotic signaling in cardiac myocytes. Chae et al. (33) also reported that dibutyryl cAMP, a cAMP analog, blocked S-nitroso-N-acetylpenicillamine-induced caspase-3 activation, and H89 reversed the cAMP-induced regulation of caspase-3. Recently, using AM knockout mice, Shimosawa et al. (34) reported that endogenous AM possesses a protective action against cardiovascular damage, possibly through the inhibition of oxidative stress production. However, further investigations are needed to clarify the precise mechanism of the antiapoptotic effect of AM in cardiac myocytes.

The present study has demonstrated that DOX increases the secretion of AM peptide and the expression of AM mRNA in cultured cardiac myocytes. DOX has been reported to produce reactive oxygen intermediates, including hydroxyl radicals, superoxide radicals, and hydrogen peroxide (4, 5, 6). Therefore, an increase in oxidative stress is suggested to be linked to the molecular mechanism by which DOX induces the increase in AM secretion and AM gene transcription. In fact, we and other investigators showed that hydrogen peroxide stimulated AM production in cultured cardiac myocytes, vascular endothelial cells, and smooth muscle cells (21, 35, 36, 37). In addition, hydrogen peroxide scavenger catalase and antioxidant NAC significantly blocked DOX-induced AM production and secretion in the present study. These findings raise the possibility that reactive oxygen intermediates participate in the stimulatory action of DOX on AM secretion and AM mRNA expression. However, as catalase and NAC were partially effective in counteracting the DOX-mediated increase in the secretion and gene expression of AM, oxidative stress-independent mechanisms might also be involved in the induction of AM production by DOX. Furthermore, we cannot rule out the possibility that the action of antioxidants may simply reflect neutralization of DOX toxicity and thereby less induction of AM.

In contrast to the stimulatory effect of DOX on AM production, DOX decreased ANP peptide release from cardiac myocytes. Recently, Chen et al. (38) reported that DOX suppressed ANP secretion and mRNA levels, related to its prooxidant properties. Our observation was consistent with that of their study.

In the present study a significant antiapoptotic effect of AM was observed at doses of 10-10–10-8 M. Although baseline AM levels in the culture medium of cardiac myocytes were relatively low, the release levels of AM after DOX treatment in the medium (50 fmol/105 cells) contained approximately 10-10 M. Moreover, the local concentration of endogenous AM that can serve as an autocrine/paracrine factor appears to be higher than the amount of AM released into the medium. Therefore, endogenous AM augmented by DOX may act locally as an antiapoptotic factor in cardiac myocytes. This possibility was supported by the present findings that CGRP-(8–37) and H89 accelerated DOX-induced myocyte apoptosis even in the absence of exogenous AM.

In summary, the present study demonstrated that AM protected cultured ventricular myocyte cells from DOX-induced apoptosis by inhibiting caspase-3 activation via a cAMP-dependent pathway. As DOX stimulated the production and secretion of AM in cardiac myocytes, AM may work as a potent survival factor that attenuates DOX-induced myocardial damage, acting in an autocrine manner.


   Acknowledgments
 
We thank Yoko Saito for her technical assistance.


   Footnotes
 
This work was supported by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research of Japan.

Abbreviations: AFC, 7-Amino-4-trifluoromethyl coumarin; AM, adrenomedullin; ANP, atrial natriuretic peptide; Br-cAMP, 8-bromo-cAMP; CGRP, calcitonin gene-related peptide; DOX, doxorubicin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ir-, immunoreactive; LDH, lactate dehydrogenase; L-NAME, N-nitro-L-arginine methyl ester; NAC, N-acetyl-L-cysteine; PKA, protein kinase A.

Received February 27, 2002.

Accepted for publication May 29, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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