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1521-0103/355/3/516–527$25.00 http://dx.doi.org/10.1124/jpet.115.225375THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 355:516–527, December 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
Early Administration of Carvedilol Protected againstDoxorubicin-Induced Cardiomyopathy s
Yung-Lung Chen,2 Sheng-Ying Chung,2 Han-Tan Chai, Chih-Hung Chen, Chu-Feng Liu,Yi-Ling Chen, Tien-Hung Huang, Yen-Yi Zhen, Pei-Hsun Sung, Cheuk-Kwan Sun,Sarah Chua, Hung-I Lu, Fan-Yen Lee, Jiunn-Jye Sheu,1 and Hon-Kan Yip1
Division of Cardiology, Department of Internal Medicine (Y.-L.C., S.-Y.C., H.-T.C., Y.-L.C., T.-H.H., Y.-Y.Z., P.-H.S., S.C.,H.-K.Y.); Division of General Medicine, Department of Internal Medicine (C.-H.C.); Department of Emergency Medicine (C.-F.L.);Division of Thoracic and Cardiovascular Surgery, Department of Surgery (H.-I.L., F.-Y.L., J.-J.S.); Institute for TranslationalResearch in Biomedine (S.C., H.-K.Y.); and Center for Shockwave Medicine and Tissue Engineering (H.-K.Y.), Kaohsiung ChangGung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan; Department of EmergencyMedicine, E-Da Hospital, I-Shou University School of Medicine for International Students, Kaohsiung, Taiwan (C.-K.S.); andDepartment of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan (H.-K.Y.)
Received May 5, 2015; accepted October 7, 2015
ABSTRACTThis study tested for the benefits of early administration ofcarvedilol as protection against doxorubicin (DOX)-inducedcardiomyopathy. Thirty male, adult B6 mice were categorized intogroup 1 (untreated control), group 2 [DOX treatment (15 mg/everyother day for 2 weeks, i.p.], and group 3 [carvedilol (15 mg/kg/d,from day 7 after DOX treatment for 28 days)], and euthanized byday 35 after DOX treatment. By day 35, the left ventricularejection fraction (LVEF) was significantly lower in group 2 than ingroups 1 and 3, and significantly lower in group 3 than in group 1,whereas the left ventricular (LV) end-diastolic and LV end-systolic dimensions showed an opposite pattern to the LVEFamong the three groups. The protein expressions of fibrotic(Smad3, TGF-b), apoptotic (BAX, cleaved caspase 3, PARP),DNA damage (g-H2AX), oxidative stress (oxidized protein),mitochondrial damage (cytosolic cytochrome-C), heart failure(brain natriuretic peptide), and hypertrophic (b-MHC) biomarkers of
the LV myocardium showed an opposite pattern to the LVEFamong the three groups. The protein expressions of antifibrotic(BMP-2, Smad1/5), a-MHC, and phosphorylated-Akt showed anidentical pattern to the LVEF among the three groups. Themicroscopic findings of fibrotic and collagen-deposition areasand the numbers of g-H2AX1 and 53BP11 cells in the LVmyocardium exhibited an opposite pattern, whereas the num-bers of endothelial cell (CD311, vWF1) markers showed anidentical pattern to the LVEF among the three groups. Cardiacstem cell markers (C-kit1 and Sca-11 cells) were significantlyand progressively increased from group 1 to group 3. Addition-ally, the in vitro study showed carvedilol treatment signifi-cantly inhibited DOX-induced cardiomyoblast DNA (CD90/XRCC11, CD90/53BP11, and r-H2AX1 cells) damage. Earlycarvedilol therapy protected against DOX-induced DNA dam-age and cardiomyopathy.
IntroductionHypertrophic cardiomyopathy can be caused by any disease
that increases cardiac afterload and volume overload, somemyocardiotoxic drugs, and certain primary genetic disordersof themyocardium (Senni et al., 1998; Roura andBayes-Genis,2009; Masuda et al., 2012; Maron et al., 2014; Modesto andSengupta, 2014; Patel et al., 2015). Without appropriatetreatment, hypertrophic cardiomyopathy commonly developsinto dilated cardiomyopathy, and ultimately decompensated
heart failure (Senni et al., 1998; Roura andBayes-Genis, 2009;Masuda et al., 2012; Patel et al., 2015).Doxorubicin (DOX) is used to treat a variety of human neo-
plasms; however, its usage is limited because of its cardiotoxi-city (Blum and Carter, 1974; Von Hoff et al., 1979). Long-termtreatment with DOX can cause cardiomyopathy and congestiveheart failure (CHF) in a process that involves multiple factors,including the generation of free radicals that further damagecellular membranes (Rajagopalan et al., 1988; Keizer et al.,1990); disturbance of adrenergic function; alterations in intra-cellular Ca21 homeostasis (Kim et al., 1989); myocardial cellapoptosis/death (Arola et al., 2000;Wu et al., 2000); and selectiveinhibition of the expression of cardiac muscle-specific proteins(Jeyaseelan et al., 1997). DOX induced cardiac myocyteapoptosis/death through upregulated caspase 3 and downregu-lated kinase activities of PI 3-kinase and Akt (Negoro et al.,2001).
This study was supported by a program grant from Chang Gung MemorialHospital, Chang Gung University [Grant CMRPG8B0651; NMRPG8C0231]and Ministry of Science and Technology [Grant 102-2314-B-182A-106].
1Jiunn-Jye Sheu and Hon-Kan Yip are co-corresponding authors.2Yung-Lung Chen and Sheng-Ying Chung contributed equally to this work.dx.doi.org/10.1124/jpet.115.225375.s This article has supplemental material available at jpet.aspetjournals.org.
ABBREVIATIONS: BNP, brain natriuretic peptide; CHF, congestive heart failure; DOX, doxorubicin; ECL, enhanced chemiluminescence; HPF,high-power field; IF, immunofluorescent; LV, left ventricular; LVEF, left ventricular ejection fraction.
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Carvedilol, a cardioselective beta blocker/alpha-1 blocker, iswidely used to treat hypertension and CHF (Yue et al., 1994;Packer et al., 2002). It reduces morbidity and mortality inCHF (Packer et al., 2002), has antioxidant effects, inhibitslipid perioxidation, and reduces mitochondrial toxicity (Tadoliniand Franconi, 1998; Arozal et al., 2010, 2011; Pereira et al.,2011). Therefore, we used an animalmodel to test the hypothesisthat carvedilol would protect against myocardial damage causedby DOX and improve heart function through inhibiting DNAdamage, fibrosis, and apoptosis of the left ventricular (LV)myocardium in mice.
Materials and MethodsEthics. Eight-week-old male C57BL/6 mice were purchased
from Charles River Technology, BioLASCO Taiwan Co., Ltd.,Taiwan, and housed in our hospital in an animal facility approvedby the Association for Assessment and Accreditation of LaboratoryAnimal Care International (Frederick, MD) with controlled tem-perature (24°C), humidity (50% –70%), and light cycle (12/12) for 2weeks before administration of DOX. Additionally, animals weregiven ad libitum access to food (FWUSOW Industry Co., Ltd.,Taipei, Taiwan.) and autoclaved filtered reverse osmosis water(ELGAMEDICA ProWater System,West Midlands, United Kingdom).All experimental procedures were approved by the Institute of AnimalCare andUseCommittee of KaohsiungChangGungMemorial Hospitaland performed in accordance with the Guide for the Care and Use ofLaboratory Animals. The current investigations were restricted tomalemice only in order to avoid the possibility that gender specific–effectdifferences might increase experimental variation and confound statis-tical analyses.
Pilot Study of DOX-Induced Mouse Cardiomyopathy. Thedose dependency of DOX-induced myocardial damage (i.e., cardiomyop-athy) was determined in mice. Male, adult C57BL/6 (B6) mice (n 5 24),weighing 25–30mg (Charles River Technology, BioLASCO Taiwan Co.,Ltd.), were grouped to receive stepwise increases of DOXdoses i.p. [0mg(group A), 5 mg (group B), 10 mg (group C), and 15 mg (group D) mg]every other day for 2 weeks, for a total of 7 doses. The regimen and totalaccumulated dosage of DOX used in the present study was based onprevious reports (Olson et al., 2003; Miyagawa et al., 2010) with somemodification. Additionally, to minimize animal handling and possiblediscomfort associated with i.p. injections, DOX was administered everyother day for 2 weeks.
The mice were anesthetized with an inhaled anesthesia mixtureof isoflurane and oxygen 0.8–1 l/min and placed on a temperature-regulated table (25°C) to maintain body temperature. Isoflurane(2.0%) was vapored at a concentration of 32%–36% in a N2/O2
mixture for transthoracic echocardiographic examination. Monitor-ing of the depth of the anesthesia included testing the rear-footreflexes before skin incision. The respiratory pattern, mucousmembrane color, responsiveness to manipulations, and rear-footreflexes were closely surveyed throughout the procedure. Afterechocardiographic examination or DOX administration the micewere returned to the animal center before euthanasia. Themice wereeuthanized with an overdose of isoflurane (.5.0%, i.e., continueisoflurane exposure until 1 minute after the animal stopped breathing)by day 28 after DOX administration and each heart was harvested forindividual study.
The total heart weight to body weight ratio was significantly lowerin groupD than in the other groups and significantly lower in groups Aand B than in group C but it was not different between groups A and B(see Results). Additionally, the LV ejection fraction (LVEF) showed asimilar pattern of the heart weight to bodyweight ratio among the fourgroups (60% versus 54% versus 50% versus 47.3%, P , 0.001) (seeResults). Thus, DOX-induced cardiomyopathy was recreated in thepilot study.
Animal Grouping and Induction of Cardiomyopathy byDOX (15 mg/Every Other Day for 2 Weeks) and Rationale forthe Carvedilol Dose in the Current Study. To elucidate theimpact of carvedilol therapy on preserving the LVEF, four additionalanimals were used and categorized into receiving a higher dose(15 mg/kg/day) anda lower dose (5mg/kg/day) of carvedilol, respectively.The results of the pilot study showed that the LVEFwas better preservedin animals (n5 2) that received 15mg/kg/day of carvedilol than in animals(n 5 2) that received 5 mg/kg/day of carvedilol orally, i.e., by lavage(53.0% versus 49.0%; carvedilol was commenced from day 7 after DOXadministration and administered for 4 weeks). Additionally, a previousstudy has shown that the dosage of carvedilol up to 30 mg/kg daily forrodentswas still safewithout any side effects (Matsui et al., 1999). Thus,15 mg/kg/day dose of carvedilol was used in the present study.
In the present study, prior to DOX administration, themice (n5 30)were equally randomized into group 1 (untreated control, n 5 10),group 2 (DOX only, i.p.; n 5 10), and group 3 [DOX 1 carvedilol(15 mg/kg/d by lavage), commenced from day 7 after DOX administra-tion for 28 days, n5 10]. The present study did not provide a carvedilol-treated control group and was based on the spirit of the Guide for theCare and Use of Laboratory Animals, i.e., replace, reduce, and reduce,and the previous study, which had demonstrated that dosage ofcarvedilol up to 30 mg/kg/day for rodents was safe (Matsui et al., 1999).
In vitro Study for Identifying the Impact of Carvedilol onProtecting the Cardioblasts from DOX-Induced Toxicity. Forthe in vitro study, H9C2 cells (cardiomyoblast) were purchased fromthe American Type Culture Collection Manassas, VA. The cardiomyo-blast characteristics of H9C2 cells were verified by immunofluores-cent (IF) staining to detect the specific expressions of cardiac troponinT and cardiac sarcomeric a-actinin. Additionally, H9C2 cells werecultured in Dulbecco’s modified Eagle’s medium supplemented with10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml strepto-mycin in a T-75 culture flask at 37°C with 5% CO2. For passage,cells at 70% confluence were enzymatically dissociated with 0.25%trypsin/EDTA and subcultured to new flasks with fresh medium.
To determine the impact of carvedilol against the DOX-inducedDNA damage, H9C2 cells were first cultured in Dulbecco’s modi-fied Eagle’s medium culture, and then cocultured with 1) stepwiseincreases of DOX (0, 20, 100, and 500 nM) for 24 hours; 2) stepwiseincreases of carvedilol (0, 2, 5, and 10 mM) for 24 hours; or 3) DOX(100 nM) and carvedilol (10 mM) for 24 and 48 hours, respectively. Thecells were then collected for individual study.
Functional Assessment by Echocardiography. The procedureand protocol for transthoracic echocardiography was based on aprevious report (Chua et al., 2014). Transthoracic echocardiography(Vevo 2100, Visualsonics Toronto, Ontario, Canada.) was performedin each group prior to and on day 35 after DOX treatment by ananimal cardiologist blind to the experimental design. M-modestandard two-dimensional left parasternal/long axis echocardio-graphic examination was conducted. The LV internal dimensions[i.e., LV end-systolic diameter (LVESd) and LV end-diastolic di-ameter (LVEDd)] were measured at the mitral valve and papillarylevels of the left ventricle, according to the American Society ofEchocardiography (Morrisville, NC) leading-edge method using atleast three consecutive cardiac cycles. The LVEF was calculated asfollows: LVEF (%) 5 [(LVEDd3 2 LVESd3)/LVEDd3] ! 100%.
Specimen Collection. Mice in each groupwere euthanized by day35 after DOX treatment, and the heart in each mouse was rapidlyremoved and then immersed in cold saline. The LV tissues wereisolated and divided into three parts for 1) cryosections [embedded incompound (Tissue-Tek, Sakura, Netherlands]; 2) paraffin sections(fixed with 10% formalin); and (3) protein examination (stored at280°C refrigeration before using), respectively.
Western Blot Study. The procedure and protocol for western blotanalysiswere based on previous reports (Chen et al., 2014a,b). In brief,equal amounts (50 mg) of protein extracts were loaded and separatedby SDS-PAGE using acrylamide gradients. After electrophoresis,the separated proteins were transferred electrophoretically to a
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polyvinylidene difluoridemembrane (AmershamBiosciences, Freiburg,Germany.). Nonspecific sites were blocked by incubation of the mem-brane overnight in blocking buffer (5% nonfat dry milk in Tris-bufferedsaline containing 0.05% Tween 20). The membranes were incubatedfor 1 hour at room temperature with the indicated primary antibodies[Bax (1:1000, Abcam Cambridge, MA); cleaved poly (ADP-ribose)polymerase (PARP) (1:1000, Cell Signaling Danvers, MA); caspase 3(1:1000, Cell Signaling Danvers, MA); Smad3 (1:1000, Cell Signaling);transforming growth factor (TGF)-b (1:500, Abcam Cambridge, MA);Smad1/5 (1:1000, Cell Signaling); bone morphogenetic protein (BMP)-2(1:1000, Abcam); phosphorylation of histone H2AX (g-H2AX) (1:1000,Cell Signaling); a-MHC (1:300, Santa Cruz); b-MHC (1:1000, SantaCruz Santa Cruze, CA); brain natriuretic peptide (BNP) (1:800, Abcam);Akt (1:1000, Cell Signaling); Ku-70 (1:1000, Cell Signaling); andcytosolic (1:2000, BD San Jose, CA) and mitochondrial (1:2000, BDSan Jose, CA) cytochrome C]. Horseradish peroxidase–conjugatedanti-rabbit IgG (1:2000, Cell Signaling) was used as a secondary antibodyfor 1-hour incubation at room temperature. The washing procedurewas repeated eight times within 1 hour. Immunoreactive bands werevisualized by enhanced chemiluminescence (ECL) (Amersham Bio-sciences) and exposed to Biomax L film (Kodak Rochester, NY). Forthe purpose of quantification, ECL signals were digitized using theLabwork software (UVP Upland, CA).
In our western blot study for the assessment of specific proteinexpressions, we loaded the lysate in the same SDS/polyacrylamide geland then transferred it to the polyvinylidene difluoride membrane,followed by hybridization with different antibodies and with the use ofb-tubulin as the control for each lane in order to assess the expressionsof the proteins, which means the b-tubulin expression of each lane wasused to compare the expressions of different proteins on the same lane.
Oxidative Stress Measurement of LV Myocardium. The pro-cedure and protocol for assessing the protein expression of oxidativestress have been detailed in previous reports (Chen et al., 2014a,b).The Oxyblot Oxidized Protein Detection Kit was purchased fromChemicon (Billerica, MA). 2,4-dinitrophenylhydrazine (DNPH) deriva-tization was carried out in 6 mg protein for 15 minutes according to themanufacturer’s instructions. One-dimensional electrophoresis was car-ried out in 10% SDS/polyacrylamide gel after DNPH derivatization.Proteins were transferred to nitrocellulose membranes, which werethen incubated in the primary antibody solution (anti-dinitrophenyl[DNP] 1:150) for 2 hours, followed by incubation in secondary antibodysolution (1:300) for 1 hour at room temperature. The washing procedurewas repeated eight times within 40 minutes. Immunoreactive bandswere visualized by ECL (Amersham Biosciences), which was thenexposed to BiomaxL film (Kodak). For quantification, ECL signals weredigitized using the Labwork software (UVP). For oxyblot proteinanalysis, a standard control was loaded on each gel.
IF and Immunohistochemical Staining. IF staining was per-formed using the respective primary antibodies for examination ofCD311 (1:100, Abcam), g-H2AX1 (1:500, Abcam), Ku-70 (1:100,Abcam),CD901 (1:100, abcam), XRCC11 (1:200, Abcam), and 53BP11 (1:100,Abcam; 1:300, Novus Littleton, CO) cells and actinin-phalloidin (1:500,LuBio Science, Switzerland; 1:500, Life Technologies Carlsbad, CA) inLV myocardium based on recent studies (Rajagopalan et al., 1988; Kimet al., 1989; Arola et al., 2000;Wu et al., 2000). Moreover, immunohisto-chemical staining was performed for examinations of Sca-11 (1:300,BioLegend San Diego, CA) and C-kit1 (1:300, Santa Cruz) cells usingthe respective primary antibodies as described previously (Sung et al.,2009; Leu et al., 2011;Chen et al., 2014a,b; Chua et al., 2014). Irrelevantantibodies were used as controls in the current study.
Histologic Quantification of Myocardial Fibrosis and Col-lagen Deposition. The procedure and protocol have been describedin previous reports (Leu et al., 2011; Chua et al., 2014). Masson’strichrome stainingwas used to identify fibrosis of the LVmyocardium.Three serial sections of LV myocardium in each animal at the samelevels were prepared at 4 mm thickness by cryostat (Leica, Singapore).The integrated areas (mm2) of fibrosis in each section were calculatedusing the ImageTool 3 software, version 3.0 (University of Texas,
Health Science Center, San Antonio, TX). Three randomly selectedhigh-power fields (HPFs) (100!) were analyzed in each section. Afterdetermining the number of pixels in each infarct and fibrotic area perHPF, the numbers of pixels obtained from three HPFs were sum-mated. The procedure was repeated in two other sections for eachanimal. The mean pixel number per HPF for each animal was thendetermined by summating all pixel numbers and dividing by 9. Themean integrated area (mm2) of fibrosis in LV myocardium per HPFwas obtained using a conversion factor of 19.24 (where 1 mm2
represented 19.24 pixels).To analyze the extent of collagen synthesis and deposition, cardiac
paraffin sections (6 mm) were stained with Picrosirius Red (1% SiriusRed in saturated picric acid solution) for 1 hour at room temperatureusing standard methods. The sections were then washed twice with0.5% acetic acid. The water was physically removed from the slides byvigorous shaking. After dehydration three times in 100% ethanol, thesections were cleanedwith xylene andmounted in a resinousmedium.The HPFs (!100) of each section were used to identify the SiriusRed–positive area in each section. Analyses of collagen deposition areain LVmyocardiumwere identical to the description of the calculationsof the infarct and fibrotic areas.
Statistical Analysis. Quantitative data are expressed as means6S.D. Statistical analysis was performed by analysis of variance followedby theBonferronimultiple-comparisonpost hoc test. TheSAS statisticalsoftware forWindows, version 8.2 (SAS institute, Cary,NC)was used. Aprobability value ,0.05 was considered statistically significant.
ResultsIn Vitro Studies Identifying Protective Effect of
Carvedilol on DNA Damage Induced by DOX (Figs.1–4). To elucidate the impact of DOX therapy on DNAdamage, H9C2 cells (cardioblast cell line) were coculturedwith stepwise increases of DOX (0, 20, 100, and 500 nM) for24 hours, followed by collection of cells for western blot. Theresults showed that the protein expression ofg-H2AX, amarkerof DNAdamage, progressively increased,whereas that of Ku-70and phosporylated Akt, two indicators of DNA repair, pro-gressively decreased (Fig. 1).To assess whether carvedilol treatment would activate DNA
repair in H9C2 cells, the cells were cocultured with stepwiseincreases of carvedilol concentration (0, 2, 5, and 10 mM) for 24hours. The protein expressions of Ku-70 and phosporylatedAkt were progressively enhanced as the dose of carvedilol wasincreased (Fig. 1).To assess whether carvedilol could prevent DNA damage
induced by DOX, H9C2 cells were cocultured with DOX(100 nM) and carvedilol (10 mM) for 24 and 48 hours, re-spectively. The protein expressions of phosporylated Akt, Ku-70,and TNNI3K, three indicators of DNA repair, were preserved,whereas protein expression of g-H2AX was suppressedby carvedilol (Fig. 2). Additionally, the IF microscopicfindings demonstrated that the numbers of CD90/XRCC11,CD90/53BP11 (Fig. 3), and r-H2AX1 cells (Fig. 4), threemarkers of DNA damage, were significantly higher, whereasthe number of Ku701 cells (Fig. 4), an indicator of DNA repair,was significantly lower in the DOX-treated group comparedwith the control group and the group treated with DOX 1carvedilol.DOX Treatment of Dose-Dependent Myocardial Dam-
age and Alternation of Heart Weight and LV Functionin Living Animals (Fig. 5). By day 35 after DOX treatment,the protein expressions of Bax, cleaved caspase 3, and cleavedPARP, three indicators of apoptosis in LV myocardium,
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progressively increased as the DOX dose was increased (Fig. 5,A–C). Additionally, the lower dose of DOX (i.e., 10 mg) notablyinduced cardiac hypertrophy, whereas the higher dose of DOXcaused a significantly lower ratio of the heart weight to tibiallength (Fig. 5D) and total heart weight (Fig. 5E), an indirectindictor of loss of myocardium and cell death. Furthermore, theLVEF notably and progressively decreased as the DOX dosewas increased stepwise (Fig. 5F).The results of the immunohistochemical staining showed
that the fibrotic area (Fig. 5G) and collagen-deposition area in
LV myocardium (Fig. 5H) progressively increased as theDOX dose was increased (please see the illustrations of themicroscopic findings in the Supplemental Material). Theresults of the IF staining displayed that 53BP11 cells (Fig.5I) and g-H2AX1 cells (Fig. 5J), two markers of DNA damage,were identical to the expression of fibrosis in LV myocardium(please see the illustrations of microscopic findings in Supple-mental Material).Carvedilol Therapy Preserved LV Function and
Inhibited LV Remodeling by Day 35 after DOX Treat-ment (15 mg/Every Other Day for 2 Weeks) (Fig. 6). Byday 35 after DOX treatment, the LVEF was significantly lowerin group 2 (DOX) than in group 1 (untreated control) and group3 (DOX 1 carvedilol), and significantly lower in group 3 thanin group 1. Conversely, the LV end-diastolic and end-systolicdiameters showed a reversed pattern of LVEF among the threegroups. These findings suggest that carvedilol treatmentprotected against DOX-induced myocardial damage.Carvedilol Prevented Apoptosis, DNA, and Mito-
chondrial Damage Caused by DOX Treatment (15 mg/Every Other Day for 2 Weeks) in LV Myocardium (Fig.7). By day 35 after 15 mg of DOX therapy, the proteinexpressions of Bax, cleaved caspase 3 and PARP, g-H2AX,and cytosolic cytochorme C (i.e., an indicator of mitochondrialdamage) were significantly higher in group 2 than in groups 1and 3, and significantly higher in group 3 than in group 1. Onthe other hand, mitochondrial cytochrome C, an indicator ofmitochondrial integrity, revealed an opposite pattern toapoptosis among the three groups.Carvedilol Prevented Fibrosis and Myocardial Hy-
pertrophy Caused by DOX Treatment (15 mg/EveryOther Day for 2 Weeks) in LV Myocardium (Fig. 8). Byday 35 after DOX treatment, the protein expressions of Smad3and TGF-bwere significantly higher in group 2 than in groups1 and 3, and significantly higher in group 3 than in group 1.Conversely, the protein expressions of Smad1/5 and BMP-2,two antifibrotic biomarkers, exhibited an opposite pattern tofibrosis among the three groups. Additionally, the proteinexpressions of BNP and b-MHC, two indicators of pressureoverload/heart failure, showed an identical pattern of fibro-sis among the three groups. Conversely, protein expressionof a-MHC, a reverse myocardial hypertrophic biomarker,showed an opposite pattern to b-MHC among the threegroups.
Fig. 1. DOX-induced DNA damage andcarvedilol therapy upregulated the ca-pacity of DNA repair. (A) The proteinexpression of g-H2AX was progressivelyenhanced, whereas the protein expres-sions of Ku-70 and phosphorlyated (p)-Aktwere progressively attenuated, after step-wise increased DOX dosage (i.e., 0, 20,100, and 500 nM). (B) The protein expres-sions of Ku-70 and phosporylated Akt(p-Akt) (i.e., two indicators of DNA repair)were progressively enhanced as the doseof carvedilol increased (i.e., 0, 2, 5, and10 mM).
Fig. 2. In vitro study of the protective effect of carvedilol on DNA damageinduced by DOX. (A) Demonstrates the dosage and timing of DOX andcarvedilol to be used in coculture with H9C2 cells. (B) The proteinexpressions of Ku-70, phosporylated Akt (p-Akt), and cardiac troponinI-interacting kinase (TNNI3K). Three indicators of DNA repair werenotably and progressively downregulated at two time interval points (i.e.,at 24 hours or extended to 48 hours) in DOX (100 nM) treatment, whereasthese three parameters were stepwise upregulated in carvedilol (10 mM)treatment at two time interval points.
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Carvedilol Prevented Microscopic Findings of Fibro-sis and Collagen Deposition caused by DOX Treatment(15 mg/Every Other Day for 2 Weeks) in LV Myocar-dium (Fig. 9). By day 35 after DOX treatment, Mason’strichrome staining showed that the fibrotic areawas significantly
higher in group 2 than in groups 1 and 3, and significantlyhigher in group 3 than in group 1. Additionally, the results ofthe Sirius Red staining exhibited that the collagen depositionarea displayed an identical pattern of fibrosis among thethree groups.
Fig. 3. IF stain for identifying DOX-induced DNA damage that was reversed by carvedilol therapy. (A–D) The IF microscopic finding (400!) illustratesthe CD90/XRCC1+ cells. The red color indicates positively stained XRCC1, the green color indicates positively stained CD90, and the positively doublestain (i.e., red and green colors) indicates CD90/XRCC1+ cells. (E) Analytical result of CD90/XRCC1+ cells (*) versus other groups with different symbols(*, †, ‡), P , 0.0001. (F–I) The IF microscopic finding (400!) illustrates the CD90/53BP1+ cells. The red color indicates positively stained 53BP1, thegreen color indicates positively stained CD90, and the positively double stain (i.e., red and green colors) indicates CD90/53BP1+ cells. (J) Analyticalresult of CD90/53BP1+ cells (*) versus other groups with different symbols (*, †, ‡), P , 0.0001. Scale bars in right, lower corner represent 20 mm. Allstatistical analyses were performed by one-way analysis of variance, followed by the Bonferroni multiple comparison post hoc test (n = 6 for each group).Symbols (*, †, ‡) indicate significance (at the 0.05 level). Dox = doxorubicin; CAR = carvedilol.
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Cardvedilol Prevented the Expressions of g-H2AX1
and 53BP11 Cells Caused by DOX (15 mg/Every OtherDay for 2 Weeks) Expressions and Upregualtion ofKu-701 Cell Expression in LVMyocardium (Fig. 10). Byday 35 after DOX treatment, the numbers of g-H2AX1 and53BP11 cells, two indices of DNA damage markers, weresignificantly increased in group 2 compared with groups 1 and
3, and significantly higher in group 3 than in group 1. On theother hand, the cellular expression of Ku-70, a DNA repairbiomarker, exhibited an opposite pattern of DNA damagemarkers among the three groups.Carvedilol Therapy Enhanced the Expressions of
Cardiac Stem Cells and Endothelial Cells in LVMyocardium (Fig. 11). By day 35 after DOX treatment
Fig. 4. IF stain for identifying DOX-augmented DNA damage and suppressed DNA repair that were reversed by carvedilol therapy. (A–D) The IF microscopicfinding (400!) illustrates the g-H2AX+ cells (red color). (E) Analytical result of g-H2AX+ cells (*) versus other groups with different symbols (*, †, ‡), P, 0.0001.(F–I) The IFmicroscopic finding (400!) illustrates theKu-70+ cells (green color). (J) Analytical result of Ku-70+ cells (*) versus other groupswith different symbols(*, †, ‡), P, 0.0001. Scale bars in right, lower corner represent 20 mm. All statistical analyses were performed by one-way analysis of variance, followed by theBonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance (at the 0.05 level). Dox = doxorubicin; CAR = carvedilol.
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(15 mg/every other day for 2 weeks), the numbers of Sca-11
and C-kit1 cells, two indicators of cardiac stem cells, weresignificantly higher in group 2 and significantly increased ingroup 3 compared with group 1.We suggest that the increasednumbers of cardiac stem cells after DOX treatment could be
an intrinsic response to cardiotoxicity and DNA damage. Addi-tionally, carvedilol therapy had an extrinsic capacity toenhance the increase of cardiac stem cells for myocardialrepair. Conversely, the number of CD311 cells, an indicator ofendothelial cells, was significantly lower in group 2 than in
Fig. 5. DOX treatment of dose-dependent augmentation of myocardial damage and alternation of heart weight and LV function in living animals. (A)Protein expression of Bax; * versus other groups with different symbols (*, †, ‡), P , 0.0001. (B) Protein expression of cleaved caspase 3 (c-Casp 3);* versus other groups with different symbols (*, †, ‡, x), P , 0.0001. (C) Protein expression of cleaved Poly (ADP-ribose) polymerase (c-PARP); * versusother groups with different symbols (*, †, ‡),P, 0.0001. (D) The ratio of heart weight to tibial length; * versus other groups with different symbols (*, †, ‡),P, 0.0001. (E) The analytical results of total heart weight; * versus other groups with different symbols (*, †, ‡), P, 0.001. (F) The analytical resultsof the LVEF; * versus other groups with different symbols (*, †, ‡, x), P , 0.0001. (G) Analytic result of the fibrosis area; * versus other groups withdifferent symbols (*, †, ‡, x), P, 0.0001. (H) Analytic result of the collagen deposition; * versus other groups with different symbols (*, †, ‡, x), P, 0.0001.(I) Analytical result of 53BP1+ cells (*) versus other groups with different symbols (*, †, ‡), P , 0.001. (J) Analytical result of g-H2AX+ cells (*) versusother groups with different symbols (*, †, ‡), P , 0.0001. The lysate was loaded in the same SDS/polyacrylamide gel and then transferred to thepolyvinylidene difluoride membrane, followed by hybridization with different antibodies and with the use of b-tubulin as the control for each lane toassess the protein expressions of Bax, c-caspase 3, and c-PARP. All statistical analyses were performed by one-way analysis of variance, followed by theBonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance (at the 0.05 level). Dox = doxorubicin.
Fig. 6. Carvedilol therapy against DOX-induced deterioration of heart function and enhancement of LV remodeling by day 35 after DOX treatment(15mg/every other day for 2 weeks). (A) Analytical results of the LVEF; * versus other groups with different symbols (*, †, ‡),P, 0.001. (B) Analytical resultsof the LV end-diastolic dimension (LVEDd); * versus other groups with different symbols (*, †, ‡), P, 0.001. (C) Analytical results of the LV end-systolicdimension (LVESd); * versus other groups with different symbols (*, †, ‡), P , 0.001. All statistical analyses were performed by one-way analysis ofvariance, followed by the Bonferroni multiple comparison post hoc test (n = 10 for each group). Symbols (*, †, ‡) indicate significance (at the 0.05 level).Dox = doxorubicin; CAR = carvedilol.
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groups 1 and 3, and significantly lower in group 3 than ingroup 1.
DiscussionThis study, which investigated the protective effect of
carvedilol therapy on myocardium against DOX damage,yielded several striking implications. First, DOX causedsignificant LV dysfunction and increased LV remodeling.Second, DOX-induced LV dysfunction and remodeling were
shown to be mainly through fibrosis, apoptosis, DNA damage,mitochondrial dysfunction, and oxidative stress. Third, DOXtherapy was associated with cumulative and dose-dependentcardiomyopathy. Fourth, these molecular-cellular and func-tional perturbations caused by DOX were significantly re-versed by carvedilol, suggesting that such therapy could beeffective for patients with neoplasms and DOX-inducedcardiotoxicity/cardiomyopathy.Undoubtedly, accumulating doses of DOX therapy are
associated with irreversible dilated cardiomyopathy. Once
Fig. 7. Protein expressions of apoptotic, NDA-damaged, and mitochondria-damaged markers in LV myocardium by day 35 after DOX treatment (15 mg/every other day for 2 weeks). (A) Protein expression of Bax; * versus other groups with different symbols (*, †, ‡), P, 0.001. (B) Protein expression of cleavedcaspase 3 (c-Csp 3);, * versus other groups with different symbols (*, †, ‡), P , 0.0001. (C) Protein expression of cleaved poly(ADP-ribose) polymerase(c-PARP); * versus other groups with different symbols (*, †, ‡), P, 0.0001. (D) Protein expression of g-H2AX; * versus other groups with different symbols(*, †, ‡), P, 0.001. (E) Protein expression of cytosolic cytochrome C (cyt-Cyt C); * versus other groups with different symbols (*, †, ‡), P, 0.0001. (F) Proteinexpression of mitochondrial cytochrome C (mit-Cyt C); * versus other groups with different symbols (*, †, ‡), P , 0.001. The lysate was loaded in the sameSDS/polyacrylamide gel and then transferred to the polyvinylidene difluoride membrane, followed by hybridization with different antibodies and with theuse of b-tubulin as the control for each lane to assess the protein expressions of Bax, c-caspase 3, and c-PARP, and for each lane to assess the proteinexpressions of g-H2AX and cyto-Cyt C, respectively. All statistical analyses were performed by one-way analysis of variance, followed by the Bonferronimultiple comparison post hoc test (n = 10 for each group). Symbols (*, †, ‡) indicate significance (at the 0.05 level). Dox = doxorubicin; CAR = carvedilol.
Fig. 8. Protein expressions of fibrotic, antifibrotic, and hypertrophic biomarkers in LV myocardium by day 35 after DOX treatment (15 mg/every otherday for 2 weeks). (A) Protein expression of Samd3; * versus other groups with different symbols (*, †, ‡), P , 0.0001. (B) Protein expression oftransforming growth factor (TGF)-b; * versus other groups with different symbols (*, †, ‡), P , 0.001. (C) Protein expression of Sam1/5; * versus othergroups with different symbols (*, †, ‡), P , 0.0001. (D) Protein expression of bone morphogenetic protein (BMP)-2; * versus other groups with differentsymbols (*, †, ‡), P , 0.0001. (E) Protein expression of BNP; * versus other groups with different symbols (*, †, ‡), P , 0.0001. (F) Protein expression ofb-MHC; * versus other groups with different symbols (*, †, ‡), P, 0.0001. (G) Protein expression of a-MHC; * versus other groups with different symbols(*, †, ‡), P, 0.001. The lysate was loaded in the same SDS/polyacrylamide gel and then transferred to the polyvinylidene difluoride membrane, followedby hybridization with different antibodies and with the use of b-tubulin as the control for each lane to assess the protein expressions of p-Smad3,Smad1/5, and BMP-2. All statistical analyses were performed by one-way analysis of variance, followed by the Bonferroni multiple comparison post hoctest (n = 10 for each group). Symbols (*, †, ‡) indicate significance (at the 0.05 level). Dox = doxorubicin; CAR = carvedilol.
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Fig. 9. Fibrosis and collagen deposition in LV myocardium by day 35 after DOX treatment (15 mg/every other day for 2 weeks). (A–C) The microscopicfindings (100!) of Masson’s Trichrome staining illustrate the fibrosis in LV myocardium among the three groups. (D) Analytic result of the fibrosis area;* versus other groups with different symbols (*, †, ‡), P , 0.0001. (E–G) The microscopic findings (100!) of Sirius Red staining illustrate the collagendeposition in LVmyocardium among the three groups. (H) Analytic result of the collagen deposition; * versus other groups with different symbols (*, †, ‡, x),P , 0.0001. Scale bars in right, lower corner represent 100 mm. All statistical analyses were performed by one-way analysis of variance, followed by theBonferroni multiple comparison post hoc test (n = 10 for each group). Symbols (*, †, ‡) indicate significance (at the 0.05 level). Dox = doxorubicin; CAR =carvedilol.
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established, medical therapy is mostly ineffective. Therefore,prevention of cardiotoxicity has great clinical importance.One important finding in the present study was that cardio-
toxicity was strongly associated with stepwise increases inDOX dosage. Additionally, cardiotoxicity/cardiomyopathyoccurred at an early stage of DOX therapy (by day 28). In-triguingly, one clinical observational study previously revealedthat subclinical systolic dysfunction occurs in almost 50% ofpatients early after anthracycline (DOX is one kind ofanthracycline) therapy (Lotrionte et al., 2007). Similarly,another observational study showed that changes in LVlongitudinal peak systolic strain and LV remodeling wereobserved early in patients receiving anthracycline chemo-therapy (Poterucha et al., 2012). Accordingly, the results ofour experimental study (i.e., preclinical study) support thefindings of those previous studies (Lotrionte et al., 2007;Poterucha et al., 2012) (i.e., clinical observational study)that cardiotoxicity quite often developed at an early stage ofDOX therapy.The benefit of carvedilol on preserving heart function has
been extensively discussed by numerous clinical observationalstudies (Kalay et al., 2006; Elitok et al., 2014). The mostimportant finding in the present study was that earlyadministration of carvedilol significantly preserved LV func-tion and inhibited LV remodeling. Our findings strengthenprevious work (Kalay et al., 2006; Elitok et al., 2014) andhighlight that early administration is essential for protectingagainst DOX-induced cardiomyopathy. This finding has im-portant preclinical relevance that can be extrapolated to theclinical setting: early provision of carvedilol for chemotherapypatients may be crucial to prevent cardiotoxicity, which inturn preserves heart function and inhibits LV remodeling.The cardiotoxicity/cardiomyopathy caused by DOX is due
to multiple mechanisms, including generation of free oxy-gen radicals (Olson and Mushlin, 1990), apoptosis/cell death(Kalyanaraman et al., 2002), mitochondrial dysfunction(Olson and Mushlin, 1990; Wallace et al., 1997), activation ofmatrix metalloproteinase (Bai et al., 2004), DNA damage/abnormal protein processing (Shi et al., 2011), and decreasedvasculogenesis (Shi et al., 2011). Another important finding inthe present study was that the fibrotic and collagen depositionareas were remarkably higher in the DOX-treated group thanin the untreated control group. Additionally, both in vitro andin vivo studies exhibited that the DNA damage markers,apoptotic and fibrotic biomarkers, as well as the oxidativestress and mitochondrial dysfunction markers (cytosoliccytochrome C increased and mitochondrial cytochrome C de-creased) were substantially increased in DOX-treated ani-mals compared with those of untreated control animals.Accordingly, our findings corroborate the findings of thoseprevious studies (Olson and Mushlin, 1990; Kalyanaramanet al., 2002; Shi et al., 2011). However, all of these perturba-tion parameters were markedly reversed and those of theantifibrotic biomarkers were notably increased after carvediloltherapy. Therefore, our findings, in addition to supportingthe findings of previous studies (Olson and Mushlin, 1990;
Fig. 10. The expressions of DNA-damaged and DNA-repaired cells in LVmyocardium by day 35 after DOX treatment (15 mg/every other day for 2weeks). (A–C) The IF microscopic findings (400!) illustrate the expressionof g-H2AX+ cells in LV myocardium (pink color). (D) Analytical result ofg-H2AX+ cells (*) versus other groups with different symbols (*, †, ‡), P ,0.0001. Scale bars in right, lower corner represent 20 mm. (E–G) The IFmicroscopic findings (200!) illustrate the expression of 53BP1+ cells in LVmyocardium (red color). (H) Analytical result of 53BP1+ cells * versusother groups with different symbols (*, †, ‡), P , 0.0001. Scale bars inright, lower corner represent 50 mm. (I–K) The IF microscopic findings(400!) illustrate the expression of Ku-70+ cells in LV myocardium (greencolor). (L) Analytical result of Ku-70+ cells (*) versus other groups withdifferent symbols (*, †, ‡), P , 0.0001. Scale bars in right, lower cornerrepresent 20 mm. All statistical analyses were performed by one-way
analysis of variance, followed by the Bonferroni multiple comparison posthoc test (n = 10 for each group). Symbols (*, †, ‡) indicate significance (atthe 0.05 level). Dox = doxorubicin; CAR = carvedilol.
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Kalyanaraman et al., 2002; Shi et al., 2011), could lend partialexplanation to why LV function was preserved and LVremodeling was inhibited in DOX-treated animals after re-ceiving carvedilol therapy. In this way, the results of ourstudies encourage the early use of carvedilol for patients withthe requirement of DOX treatment.Interestingly, in the present study we found that DOX-
induced cardiotoxicity enhanced the generation of cardiacstem cells in myocardium. We suggest that such progenitorcell renewal could be an intrinsic response to myocardialdamage for regeneration of myocardium. Of importance wasthat carvedilol therapy further enhanced this phenomenon ofprogenitor cell renewal. This finding could, at least in part,explain why carvedilol therapy preserved heart function andabrogated LV remodeling in DOX-treated animals.An essential finding in the present study was that the
number of CD31 cells was significantly reduced in DOX-treated animals than in those of the untreated control group,indicating that DOX therapy not only induced cardiotoxicity
but also destroyed endothelial cells/endothelial function andangiogenesis. Importantly, this endothelial cell was signifi-cantly reversed (i.e., from 0.5% to 2.5% with an increment of5.0 times) after carvedilol treatment. This increment inCD311 cells may indicate that the microcirculation and bloodsupply in myocardium was preserved after carvedilol treat-ment; therefore, protecting cardiomyocytes against ischemiaand death. In this way, our findings could also once againexplain why carvedilol treatment preserved LV function andameliorated LV remodeling.A principal finding in the present study was that the protein
expressions of BNP and b-MHC were significantly enhanced,whereas the protein expression of a-MHC was notably re-duced in the DOX group compared with the untreatedcontrols. In fact, numerous studies have shown a correlationbetween an increase in circulating levels of BNP and CHF/pressure overload and prognostic outcome in patients afterischemic myocardial infarction (Wu et al., 2006). Additionally,cardiac hypertrophy is characterized by a switch from a- to
Fig. 11. Expressions of cardiac stem cells and endothelial cells in LV myocardium by day 35 after DOX treatment (15 mg/every other day for 2 weeks).(A–C) The microscopic findings (400!) of immunohistochemical (IHC) staining identify the expression of Sca-1+ cells in LVmyocardium (red arrows). (D)Analytical result of Sca-1+ cells (*) versus other groups with different symbols (*, †, ‡), P, 0.001. Scale bars in right, lower corner represent 20 mm. (E–G)The microscopic findings (400!) of IHC staining identify the expression of Sca-1+ cells in LV myocardium (red arrows). (H) Analytical result of Sca-1+cells (*) versus other groups with different symbols (*, †, ‡), P , 0.001. Scale bars in right, lower corner represent 20 mm. (I–K) The IF microscopicfindings (200!) illustrate the expression of CD31+ cells in LV myocardium (yellow arrows). Scale bars in right, lower corner represent 50 mm. Allstatistical analyses were performed by one-way analysis of variance, followed by the Bonferroni multiple comparison post hoc test (n = 10 for each group).Symbols (*, †, ‡) indicate significance (at the 0.05 level). Dox = doxorubicin; CAR = carvedilol.
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b-MHC mRNA expression (i.e., reactivation of fetal geneprogram) (Sun et al., 2014). Intriguingly, these biomarkerswere remarkably reversed after carvedilol therapy. In thisway, our findings, in addition to being consistent with that ofprevious studies (Wu et al., 2006; Sun et al., 2014), once moreexplain why carvedilol therapy preserves LV function andameliorates LV remodeling.
Acknowledgments
The authors thank the molecular imaging core of the Center forTranslational Research in Biomedical Sciences, Kaohsiung ChangGung Memorial Hospital, Kaohsiung, Taiwan, for technical andfacility support on Echo Vevo 2100.
Authorship Contributions
Participated in research design: Yung-Lung Chen, Chung, Shue,Yip.
Conducted experiments: Sung, Lu, Yi-Ling Chen, Huang, Zhen.Contributed new reagents or analytic tools: Chai, C.-H. Chen, Liu,
Chua.Performed data analysis: Yung-Lung Chen, Chung, Shue, Yip.Wrote or contributed to the writing of the manuscript: Sun, Lee,
Yip.
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Address correspondence to: Dr. Hon-Kan Yip, Division of Cardiology,Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital,123 Dapi Road, Niaosung District, Kaohsiung City, 83301, Taiwan, R.O.C.E-mail: [email protected], or Dr. Jiunn-Jye Sheu, Division of Thoracicand Cardiovascular Surgery, Department of Surgery, Kaohsiung Chang GungMemorial Hospital, 123, Dapi Road, Niaosung Dist., Kaohsiung city, 83301,Taiwan, R.O.C. E-mail: [email protected].
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