Effects of angiotensin II receptor blocker (candesartan) in daunorubicin-induced cardiomyopathic rats

International Journal of Cardiology 110 (2006) 378 – 385 www.elsevier.com/locate/ijcard

Effects of angiotensin II receptor blocker (candesartan) in daunorubicin-induced cardiomyopathic rats Mayako Soga a,1, Fadia A. Kamal a,1, Kenichi Watanabe a,*, Meilei Ma a, Suresh Palaniyandi a, Paras Prakash a, Punniyakoti Veeraveedu a, Sayaka Mito a, Megumi Kunisaki a, Hitoshi Tachikawa b, Makoto Kodama b, Yoshifusa Aizawa b a b

Department of Clinical Pharmacology, Niigata University of Pharmacy and Applied Life Sciences, Niigata City, Japan First Department of Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Japan Received 6 June 2005; received in revised form 23 August 2005; accepted 29 August 2005 Available online 1 December 2005

Abstract Background: Daunorubicin is an anthracycline anti-tumor agent; anthracycline chemotherapy in cancer can cause severe cardiomyopathy leading to a frequently fatal congestive heart failure; the first-line treatment is diuretics and digoxin. Recently, angiotensin-converting enzyme inhibitors have been shown to be effective in the treatment of such toxicity. The purpose of this study was to investigate the effects of angiotensin II type-1 receptor antagonist (candesartan) in a rat model of daunorubicin-induced cardiomyopathy. Methods: Rats were treated with a cumulative dose of 9 mg/kg body weight daunorubicin (i.v.). 28 days later, after the development of cardiomyopathy, animals were randomly assigned to candesartan-treated (5 mg/kg/day, p.o.) or vehicle-treated groups; age-matched normal rats were used as the control group. Candesartan treatment was continued for 28 days. Hemodynamic and echocardiographic parameters were measured, cardiac protein and mRNA were analyzed, and histopathological analyses of myocardial fibrosis, cell size and apoptosis were conducted. Results: Following cardiomyopathy, left ventricular end diastolic pressure and left ventricular systolic dimension were significantly elevated; while % fractional shortening and Doppler E/A ratio were significantly reduced. Cardiomyopathic hearts showed significant increases in % fibrosis, % apoptosis, and myocyte diameter/body weight ratio; candesartan treatment reversed these changes. Fas-L protein overexpression in myopathic hearts was significantly suppressed by treatment with candesartan. Moreover, SERCA2 mRNA and protein expression were both down-regulated in myopathic hearts and restored to normal by candesartan treatment, significantly. Conclusions: Our findings suggest that candesartan treatment significantly improved the left ventricular function and reversed the myocardial pathological changes investigated in this model of daunorubicin-induced cardiomyopathy; suggesting its potentials in limiting daunorubicin cardiotoxicity. D 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Anthracycline; Cardiomyopathy; Angiotensin receptor blocker; Apoptosis; Fas-L; SERCA2

1. Introduction The anthracycline daunorubicin (DNR) is one of the major anti-tumor agents widely used in treatment of acute * Corresponding author. Niigata University of Pharmacy and Applied Life Sciences, Faculty of Pharmaceutical Sciences, 5-13-2, Kamishin-eicho, Niigata shi, Japan. Tel.: +81 25 268 1326; fax: +81 25 268 1230. E-mail address: [email protected] (K. Watanabe). 1 These authors contributed equally to the study. 0167-5273/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2005.08.061

myeloid leukemias [1]. However, its chronic administration can cause a cumulative, dose-dependent cardiomyopathy [2]. Several mechanisms have been suggested by which anthracyclines cause myocardial injury; which include free-radical formation, myocyte apoptosis, lipid peroxidation, mitochondrial impairment, alterations in calcium handling, and direct suppression of muscle-specific gene expression [3]. Anthracycline-induced cardiomyopathy is associated with increased cardiac angiotensin converting enzyme (ACE) activity, where ACE inhibition has been reported

M. Soga et al. / International Journal of Cardiology 110 (2006) 378 – 385

to significantly limit anthracycline toxicity in human [4,5] and animal studies [6– 11]. In addition, an angiotensin II type-1 receptor blocker (ARB) ‘‘RNH-6270’’ was shown to reduce doxorubicin-induced cardiomyopathy in mice [12]. Moreover, ARBs have been shown to improve sarcoplasmic reticulum function and calcium cycling in different animal models of heart failure [13 –15]. Herewith, we are the first to examine the effects of an ARB, candesartan, on DNR-induced cardiomyopathy (DNR-CM) in rats. In our investigation, we focused on studying the changes in left ventricular function, myocyte apoptosis, hypertrophy of residual myocytes, interstitial fibrosis, and altered gene expression of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) of DNR-CM rats.

2. Materials and methods 2.1. Animals and medication Eight-week-old male Sprague – Dawley rats were obtained from Charles River Japan Inc. (Kanagawa, Japan). On day 0, each animal received a single intravenous injection of daunorubicin hydrochloride at a dose of 3 mg/ kg (i.v.). The drug was administered in three equal injections over a period of 2 weeks for a cumulative dose of 9 mg/kg body weight. 28 days later, 36 daunorubicin-treated rats were randomly divided into two groups: group Cn (n = 16) received candesartan at a dose of 5 mg/kg/day (p.o.); and group D (n = 20) received vehicle alone (0.5% methylcellulose, p.o.). Age-matched normal rats were used as the normal control, group N (n = 10). Mortality of animals was observed starting from day 28 until the termination of treatment. Throughout the study, all animals were treated in accordance with the guidelines for animal experimentation of our institute. 2.2. Hemodynamic studies Rats were anesthetized with 2% halothane in O2 during the surgical procedures preceding hemodynamic measurements; this concentration was then reduced to 0.5% to minimize the anesthetic effect. The peak left ventricular pressure (LVP), the central venous pressure (CVP), the mean blood pressure (mBP), the left ventricular enddiastolic pressure (LVEDP), the rates of intraventricular pressure rise and decline (T dP/dt), and the heart rate (HR) were recorded as described previously [16]. 2.3. Echocardiographic measurements Two-dimensional echocardiography was performed for rats under 0.5% halothane anesthesia with an echocardiographic system (SSD-5500; Aloka, Tokyo, Japan) equipped with a 7.5-MHz linear scan probe. M-mode images were

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obtained from the epicardial surface of the right ventricle, and short-axis view of the left ventricle was recorded to determine the left ventricular dimension in diastole (LVDd), left ventricular dimension in systole (LVDs), and percentage fractional shortening (%FS); E/A ratio was measured using Doppler [16]. 2.4. Histological assessment of myocardial damage Paraffin sections were stained with hematoxylin-eosin and Azan – Mallory in order to visualize cell size and myocardial fibrosis, respectively. The area of fibrosis (blue color) was quantified with a color image analyzer (Mac Scope; Mitani Co., Fukui, Japan). 2.5. In situ terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay TUNEL assay was performed as specified in the in situ apoptosis detection kit (Takara Bio. Inc.; Shiga, Japan) using paraffin sections. For each animal, three sections were scored for apoptotic nuclei. Only nuclei that were clearly located in cardiac myocytes were considered. Data are expressed as the percentage of apoptotic cells relative to normal cells. 2.6. Ribonuclease protection assay Apical left ventricles from group N, group D, and group Cn were rapidly excised, frozen in acetone ice and stored at 80 -C. Anti-sense complementary RNA probes for SERCA2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were generated as described previously [16]. The ribonuclease protection assay for the quantification of SERCA2 mRNA levels was performed as described [16]. Each mRNA result was normalized to GAPDH mRNA. 2.7. Western blotting Proteins were extracted from freshly frozen hearts. Myocardial tissue was homogenized in a lysis buffer containing 50 mmol/L Tris – HCl (pH 7.4), 200 mmol/L NaCl, 20 mmol/L NaF, 1 mmol/L Na3VO4, and 1 mmol/L DTT with protease inhibitors. Samples were stored at 80 -C until use. Total protein concentration of each sample was measured using BCA (bicinchonic acid) protein assay kit (Pierce; Rockford, IL). Samples (each containing 20 Ag total protein) were electrophoretically separated on 7.5%, 10%, and 15% SDS-PAGE for the determination of SERCA2, GAPDH, and Fas ligand (Fas-L), respectively. Then each SDS-PAGE was transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat milk for 1 h at room temperature, then incubated with SERCA2 goat polyclonal antibody (1:5000), Fas-L rabbit polyclonal antibody (1:1000), or GAPDH goat polyclonal antibody (1:1000) overnight at 4 -C, washed and incubated with the suitable secondary antibody. The targeted bands were

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Table 1 Changes in the mortality rate, hemodynamic and echocardiographic parameters, ventricular weight-to-body weight ratio (VW/BW), and myocyte diameter/body weight ratio (MD/BW) in the three groups

Total number of animals/group Number of dead animals/group Mortality rate (%) HR (beats/min) CVP (mm Hg) mBP (mm Hg) LVP (mm Hg) LVEDP (mm Hg) +dP/dt (mm Hg/s) dP/dt (mm Hg/s) LVDd (mm) LVDs (mm) %FS E/A ratio VW/BW (g/kg) MD/BW (Am/kg)

Group N (n = 10)

Group D (n = 10)

Group Cn (n = 10)

10

20

16

0

10

3

0P 327 T 12 0.4 T 0.3 99 T 3 116 T 2 3.6 T 0.5 7534 T 212 8074 T 535 7.1 T 0.3 3.5 T 0.3 50.8 T 2.6 2.5 T 0.2 2.44 T 0.06 98 T 15

50P 359 T 26 1.2 T 0.6 125 T 8* 150 T 6** 11.0 T 2.0** 7173 T 200 7090 T 833 7.1 T 0.1 5.4 T 0.2** 37.7 T 2.0** 0.3 T 0.1** 3.73 T 0.06** 208 T 25*

19P 348 T 22 0.6 T 0.1 115 T 8# 120 T 7# 7.5 T 1.5# 6992 T 306 7809 T 625 6.8 T 0.3 4.1 T 0.2 41.2 T 2.1# 2.2 T 0.3## 3.20 T 0.11## 163 T 18##

**P < 0.01 and *P < 0.05 vs. group N; ##P < 0.01 and #P < 0.05 vs. group D; P P < 0.01 measured using chi-square test; (n) is the number of animals used to calculate the mean T S.E.M. of the presented data in each of group N (normal control rats), group D (rats that received daunorubicin followed by treatment with vehicle), and group Cn (rats that received daunorubicin followed by treatment with candesartan).

visualized using enhanced ECL-plus kit (Amersham, Piscataway, NJ). 2.8. Statistical analyses Data are presented as the mean T S.E.M. Statistical assessment was performed by the one-way analysis of variance followed by Tukey’s method; and chi-square test

was performed to measure the significance of the mortality rate result. Statistical significance was considered for P < 0.05.

3. Results 3.1. Mortality There were no deaths in the normal control group (0% mortality). In group D, mortality was elevated to 50% (10 rats died out of 20). Candesartan treatment reduced mortality to 19% (only 3 rats died out of 16) ( P < 0.01) (Table 1). 3.2. Blood pressure mBP was significantly elevated in group D ( P < 0.05 vs. group N), and it was significantly reduced by candesartan treatment ( P < 0.05 vs. group D). 3.3. Myocardial systolic and diastolic function Daunorubicin administration significantly increased LVP ( P < 0.01), LVEDP ( P < 0.01), and LVDs ( P < 0.01) relative to group N. While candesartan treatment significantly reduced LVP and LVEDP ( P < 0.05 vs. group D), the reduction in LVDs was not statistically significant. On the other hand, daunorubicin treatment significantly reduced % FS and E/A ratio ( P < 0.01 vs. group N), where both were significantly increased ( P < 0.05 and P < 0.01 vs. group D, respectively) to almost normal values by candesartan treatment (Table 1, Fig. 1), thus revealing an improvement in the systolic and diastolic functions of the myopathic heart by candesartan treatment.

Fig. 1. (A) Echocardiogram showing the left ventricular dimensions. IVS: intra-ventricular septum; LVDd: left ventricular dimension in diastole; LVDs: left ventricular dimension in systole. (B) Representative diagrams of Doppler E/A ratio [group N (normal control rats), group D (rats that received daunorubicin followed by treatment with vehicle), and group Cn (rats that received daunorubicin followed by treatment with candesartan)].

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Fig. 2. (A) Representative Azan – Mallory stained sections showing the fibrotic tissue (100 magnification). (B) Bar graph showing % fibrosis in each experimental group; the area of fibrosis (blue color) was quantified with a color image analyzer (**P < 0.01 vs. group N and ##P < 0.01 vs. group D) [group N (normal control rats), group D (rats that received daunorubicin followed by treatment with vehicle), and group Cn (rats that received daunorubicin followed by treatment with candesartan)].

3.4. Ventricular weight/body weight Ventricular weight/body weight (VW/BW) was significantly increased in group D ( P < 0.01 vs. group N) and was significantly reduced by candesartan treatment ( P < 0.01 vs. group D). 3.5. Histopathology Myocyte diameter/body weight ratio (MD/BW) was significantly increased in group D ( P < 0.05 vs. group N)

and reduced in group Cn ( P < 0.01 vs. group D). Group D showed higher % fibrosis ( P < 0.01 vs. group N); while in group Cn, fibrosis was reduced ( P < 0.01 vs. group D) (Table 1, Fig. 2). 3.6. Apoptotic Cells Compared with group N, % apoptosis was significantly increased in group D ( P < 0.01). However, it was significantly decreased in group Cn ( P < 0.01) vs. group D (Fig. 3).

Fig. 3. Effect of daunorubicin administration with and without candesartan treatment on myocytes apoptosis. (A) TUNEL stained sections showing the apoptotic nuclei (400 magnification). (B) Bar graph showing % apoptosis in each group, only nuclei that were clearly located in cardiac myocytes were considered. Data are expressed as the ratio relative to normal (**P < 0.01 vs. group N and ##P < 0.01 vs. group D) [group N (normal control rats), group D (rats that received daunorubicin followed by treatment with vehicle), and group Cn (rats that received daunorubicin followed by treatment with candesartan)].

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Fig. 4. Effect of daunorubicin administration with and without candesartan treatment on SERCA2 expression in the myocardium. (A) SERCA2 mRNA level, assayed by ribonuclease protection assay. (B) Western blots of SERCA2 and GAPDH. (C and D) Quantitative representation of SERCA2 mRNA and protein levels, respectively; the intensities of SERCA2 bands were normalized to those of GAPDH and then the percentage relative to normal was calculated (**P < 0.01 and *P < 0.05 vs. group N; ##P < 0.01 and #P < 0.05 vs. group D) [group N (normal control rats), group D (rats that received daunorubicin followed by treatment with vehicle), and group Cn (rats that received daunorubicin followed by treatment with candesartan)].

3.7. Expression of SERCA2 mRNA and protein The levels of left ventricular mRNA and protein expression of SERCA2 were significantly down-regulated in group D ( P < 0.05 and P < 0.01, respectively) relative to group N and were significantly up-regulated by candesartan treatment ( P < 0.05 and P < 0.01 relative to group D, respectively) (Fig. 4). 3.8. Expression of Fas-L protein Compared with group N, Fas-L protein was significantly over-expressed in the hearts of group D ( P < 0.01). However, its expression in group Cn was significantly reduced ( P < 0.05) relative to group D (Fig. 5).

4. Discussion We have previously reported that long-term treatment with the ARB candesartan improved the myocardial function in a rat model of post-myocarditis dilated cardiomyopathy [17]. In the present study, we examined

the effects of candesartan in a rat model of DNR-CM where it significantly improved the mortality, cardiac hypertrophy and dysfunction, myocardial fibrosis, apoptosis and Fas-L expression, as well as SERCA2 gene expression. 4.1. Clinical relevance Clinically, patients receiving anthracycline are continuously monitored by echocardiography during and after treatment, where % FS and E/A ratio are considered as important indicators of heart damage [18]. In this study, DNR treatment resulted in the deterioration of cardiac function as indicated by the worsened hemodynamic and echocardiographic parameters and high mortality rate (50%). Thus, it is important to note, in a clinical relevance, that candesartan treatment has significantly improved the worsened % FS and E/A ratio to almost normal values in cardiomyopathic rats. 4.2. Overall findings In cardiomyopathic rats, the mBP was significantly elevated; the direct involvement of Angiotensin II (Ang

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in different animal models [24,25], where an angiotensin converting enzyme inhibitor (ACEI) [26] and an ARB [12] have been studied in doxorubicin-induced cardiomyopathy and were successful in reducing myocytes apoptosis. On the other hand, candesartan has been used in different animal models where its effect on apoptosis has been controversial: Chen et al. have shown that candesartan increases myocyte apoptosis [27], while Moudgil et al. have shown that candesartan exerts no effect on myocyte apoptosis [28]. This discrepancy might be due to variation in the experimental model and/or candesartan dose. In DNR-CM, we are the first to find out that candesartan treatment significantly reduces DNR-induced myocyte apoptosis as revealed by the reduction in the number of apoptotic nuclei and Fas-L protein expression. 4.4. SERCA2

Fig. 5. Changes in Fas-L expression in the myocardium caused by daunorubicin administration with and without candesartan. (A) Fas-L and GAPDH Western blots. (B) Quantitative representation of Fas-L protein level; the intensities of Fas-L bands were normalized to those of GAPDH and then the percentage relative to normal was calculated (**P < 0.01 vs. group N and ##P < 0.01 vs. group D) [group N (normal control rats), group D (rats that received daunorubicin followed by treatment with vehicle), and group Cn (rats that received daunorubicin followed by treatment with candesartan)].

II) [12] and ACE [19] in anthracycline-induced cardiotoxicity has been shown; in our study, candesartan significantly normalized the mBP in DNR-CM rats, probably due to its ARB action. In addition, cardiac hypertrophy appeared 28 days after the initiation of DNR administration, as revealed by the increase in VW/BW and MD/BW ratios. On the microscopic level, % fibrosis was increased and apoptosis of the myocytes was obvious during the period of development of cardiomyopathy, with over-expression of apoptosis-related gene FasL. Finally, the expression of SERCA2 mRNA and protein was down-regulated. All these changes were statistically significant and were significantly improved by candesartan treatment where mortality rate was reduced to 19% only. 4.3. Apoptosis Apoptosis is an active process induced by a variety of stresses and plays a critical role in a variety of cardiovascular diseases [20]. Several recent studies have shown that anthracycline anti-tumor agents, such as doxorubicin and DNR, induce apoptosis [1,3,20 – 22]. The mechanism is supposed to be through the production of reactive oxygen species (ROS) [18]. Anthracyclineinduced cardiomyopathy also involves the expression of Fas-L which induces apoptosis [22,23], this was further confirmed in our study. Moreover, it is known that Ang II stimulation induces the apoptosis of ventricular myocytes

Another factor that appears to play a significant role in anthracycline cardiomyopathy [29 – 32] is sarcoplasmic reticulum (SR) dysfunction, since anthracycline-induced decrease in myocardial contractility is associated with impaired Ca2+ homeostasis [30,33 –36]. The Ca2+-ATPase of the SR is chiefly responsible for regulating intracellular Ca2+ concentration in cardiac myocytes during the excitation – contraction cycle [37]. Some authors [38,39] have suggested the genetic regulation of SERCA2 to be important in anthracycline cardiomyopathy [38]. Besides, it has been shown, in animal models of heart failure, that Ang II antagonism leads to the attenuation of the down-regulation of SERCA2 and to an improvement in intracellular Ca2+ handling [13 – 15]. We are the first to find out that candesartan treatment in DNR-CM significantly attenuates SERCA2 down-regulation in rat’s myocardium. 4.5. Conclusion In conclusion, this study demonstrates that long-term candesartan treatment improves the left ventricular function and reduces cardiomyocytes apoptosis, the hypertrophy of residual myocytes, and interstitial fibrosis, as well as SERCA2 down-regulation in DNR-CM. This suggests the potential application of candesartan in reducing the cardiomyopathic effect of anthracycline anti-tumor agents. 4.6. Study limitations However, further investigation is warranted to assess other aspects of candesartan treatment before clinical application can be initiated.

Acknowledgements This research was supported by grants from Yujin Memorial Grant, the Ministry of Education, Science, Sports

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and Culture of Japan, and the Promotion and Mutual Aid Corporation for Private Schools of Japan.

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