persistent effects of doxorubicin on cardiac gene expression

J Mol Cell Cardiol 31, 1435–1446 (1999)

Article No. mc980972, available online at http://www.idealibrary.com on

Persistent Effects of Doxorubicin onCardiac Gene ExpressionRobert J. Boucek, Jr.∗1, Ann Miracle1, Michelle Anderson4,Robert Engelman1, James Atkinson3 and Debra A. Dodd2

1Division of Pediatric Cardiology, Department of Pediatrics, University of South Florida College ofMedicine, Tampa, FL., USA; Departments of 2Pediatrics and 3Pathology, Vanderbilt University Schoolof Medicine, Nashville, TN, USA and 4Division of Biology, California Institute of Technology, PasadenaCA 91125, USA

(Received 13 March 1998, accepted in revised form 23 April 1999)

R. J. B, J., A. M, M. A, R. E, J. A D. A. D. Persistent Effects ofDoxorubicin on Cardiac Gene Expression. Journal of Molecular and Cellular Cardiology (1999) 31, 1435–1446.During administration of the anthracycline antitumour agents, their cardiotoxicity can progress from cardiacdysfunction to heart failure. Cardiomyopathy can also develop years after receiving anthracyclines. To determineif persistent and/or progressive anthracycline effect(s) are referable to anthracycline effects on cardiac geneexpression, steady-state mRNA levels were determined 4 days (n=8), 4 weeks (n=7) and 10 weeks (n=7) afterdoxorubicin (DOX; 2 mg/kg IV) in a well-characterized rabbit model. Levels of mRNA for a-actin, b-myosin heavychain and the calcium pump of the sarcoplasmic reticulum (SERCA2a) in the left ventricle (LV) were determinedby Northern blot hybridization and expressed relative to an 18S constitutive marker. The mRNA levels for thehigh molecular weight subunit (cardiac isoform) of the ryanodine receptor (RyR2), sarcolemmal calcium channel(dihydropyridine receptor; DHPR), angiotensin-converting enzyme (ACE), angiotensin II receptor (ATR) and atrialnaturetic peptide prohormone (ANP) were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis, and expressed relative to GAPDH, a constitutive marker. Histopathologicevidence for anthracycline-induced myocardial cell injury was absent (score <1) in all hearts examined exceptone (score=1.1; 4 weeks post-DOX), which was considered separately. Relative mRNA levels for b-myosin heavychain 4 days after DOX increased 1.9-fold compared to the vehicle-treated group, but by 4 weeks levels hadreturned to baseline. Relative mRNA levels for DHPR were increased 1.2-fold 4 days after DOX and werepersistently increased 1.9- and 2.2-fold 4 and 10 weeks after DOX, respectively. The mRNA levels for ANP werefirst decreased (4.5-fold) 4 days after DOX. Four weeks after DOX, ANP message levels approached Control inseven out of eight rabbits. The one rabbit with early LV histopathology 4 weeks post-DOX had increased mRNAfor DHPR (2.7-fold) and ANP (80-fold). Between 4 and 10 weeks after DOX, mRNA levels for ANP increased>16-fold: evidence for late progression. In situ hybridization with specific riboprobes localized the persistentincrease in DHPR and the progressive increase in ANP to myocytes. Thus, DOX alters steady-state mRNA levelsin LV that are referable to both persistent and progressive anthracycline effects on myocellular gene expression.

1999 Academic Press

K W: Anthracyclines; Doxorubicin; Cardiomyopathy; Ryanodine receptor; Cardiac Gene expression;Atrial Naturetic Peptide; Dihydropyridine receptor.

tration (Singal and Iliskovic, 1998). AnthracyclineIntroductioncardiotoxicity can be either acute or cumulativedose-dependent, the latter can progress to overtThe cardiotoxicity of the anthracycline antitumour

agents is a major complication of their adminis- contractile failure (vonHoff et al., 1979; Goorin et

∗Please address all correspondence to: Robert J. Boucek, Jr., Professor and Chief, Division of Pediatric Cardiology, University of SouthFlorida College of Medicine, 880 Sixth St. So., St Petersburg, FL 33701-4827, USA.

0022–2828/99/081435+12 $30.00/0 1999 Academic Press

R. J. Boucek, Jr et al.1436

al., 1981; Schwartz et al., 1987). Why the toxicity 85-23 revised 1985. All animal protocols wereapproved by the Institutional Animal Care and Useof the anthracyclines is targeted to the heart is not

understood (Olson and Mushlin, 1990). Recently, Committee of the University of South Florida. Adultfemale New Zealand white rabbits (Oryctolagus cu-a “late”, or delayed onset cardiomyopathy has been

described in children years after receiving an- niculus; Marvin Cummings, Lutz, Florida) rangingin weight from >2.5 to 4 kg were fed weighedthracyclines (Steinherz et al., 1991; Steinherz et al.,

1995; Lipshultz et al., 1991). portions of standard rabbit chow (Purina RabbitChow) per day and water ad libitum. The bodyRecent evidence suggests the mechanistic hypo-

thesis that the late progression of anthracycline’s weight of the rabbits and the amount of chowintake were recorded daily.cardiotoxicity could be due to anthracycline effects

on cardiac gene expression. Anthracylines decrease Rabbits were randomly assigned to receive intra-venous doxorubicin or a saline vehicle. All intra-cardiac-specific transcription of myocellular pro-

teins, most notably a-actin (Cappelli et al., 1989; venous procedures used aseptic techniques, steriledisposable needles and syringes and sterile so-Lewis and Gonzales, 1987; Lewis et al., 1983).

Anthracyclines decrease mRNA for muscle-specific lutions. All intravenous infusions were through amarginal ear vein over a period of 30 min at a ratea-actin while not affecting mRNA for non-muscle

specific pyruvate kinase (Ito et al., 1990; Akimoto of 1 ml/min with a syringe pump (KD Scientific).Prior to infusion, doxorubicin (Adriamycin; Adriaet al., 1993). In vitro, anthracyclines intercalate

with DNA (Leonard et al., 1992), interact with a Laboratories) was diluted to a 2 mg/kg solutionwith sterile normal saline (0.9% NaCl) and shieldedunique consensus sequence located in upstream

regulatory sequence(s) (Eliopoulos et al., 1991; Ku- from light. DOX-treated rabbits were given DOX(1 mg/kg twice in one week). DOX-treated rabbitsrabayashi et al., 1992; Kurabayashi et al., 1993)

and interact with topoisomerase (Foglesong et al., were paired with Control group rabbits that receivedintravenous saline infusion alone (30 ml twice in1992), effects which could affect cardiac gene ex-

pression. The significance of these observations to one week). The cumulative DOX dose, 2 mg/kg, iswell below the threshold of expression for the acutethe late cardiomyopathy in man is uncertain, in

part because the dosing was high (>10 mg/kg), cardiotoxicity, while the dosing protocol duplicatesthe protocol which produces the cumulative-doseapproaching lethal single doses.

Recently, and in a clinically-relevant model with cardiotoxicity (Dodd et al., 1993).more clinically-relevant doses, we have reportedpreliminarily that doxorubicin alters cardiac mRNAlevels (Dodd et al., 1995; Dodd et al., 1996). Thepurpose of this study is to determine whether cumu-lative dosing with anthracyclines leads to effects on Euthanasia and tissue distributioncardiac gene expression, whether there is a patternof effects similar to other injury responses, and Groups were euthanized 4 days (early group), 4

weeks (intermediate group) or 10 weeks (late group)whether these effects are persistent and/or pro-gressive. Specifically, this study determined the ef- after the last doxorubicin dose. Rabbits were

weighed, anticoagulated with heparin (100 units/fects of a cumulative dose of 2 mg/kg of doxorubicin(DOX) on steady-state mRNA abundance in a well- kg) and euthanized with pentobarbital (50 mg/kg

i.v.).characterized experimental rabbit model of thecardiomyopathy. The early (4 days post-DOX ex- The heart was rapidly removed through a median

sternotomy and prepared for examination as fol-posure) effects on mRNA abundance were com-pared with effects 4 and 10 weeks post-DOX lows. All of the major vessels and fatty deposits

were removed from the heart, which was thenexposure.blotted dry and weighed. The ventricles were dis-sected, blotted and weighed. Three 10 mg(5×5 mm) pieces of the left ventricular (LV) pap-illary muscle were placed in a 2.5% glutaraldehydeMaterials and Methodssolution in cacodylate buffer for later histo-pathologic evaluation. The other papillary muscleRabbit modelwas placed in 10% buffered formalin for in situhybridization studies. The LV free wall and theThe experimental design of this investigation con-

forms with the Guide for the care and use of laboratory intraventricular septum were then removed, blot-ted, weighed, and minced. The intraventricularanimals published by the US NIH publication No.

Doxorubicin and Cardiac Gene Expression 1437

septum was immediately placed in a homo- Dodd et al., 1996), an observation confirmed byArai et al., (1998). Doxorubicin has been reportedgenization buffer (imidazole-HCL, sodium azide, suc-

rose, DTT, Na2EDTA, PMSF), homogenized using a to decrease mRNA for acardiac-actin and b-myosinheavy chain (b-MHC; Ito et al., 1990; Akimoto et al.,Polytron homogenizer and frozen for later protein

analysis. The LV free wall was used for RNA isol- 1993). Other gene products were selected because ofreports that their expression was affected in otheration and subsequent analysis. Initially the minced

LV free wall was placed in TRI-Reagent (Molecular forms of myocardial injury or stress. Ventricularexpression of atrial naturetic peptide (ANP;Research Center, Inc. Cincinnati, OH, USA) and

quickly homogenized to prevent degradation. In Schwartz et al., 1992), a marker of the so-calledembryonic repertoire, b-MHC (Cao et al., 1995;recent experiments the LV free wall was snap-frozen

in liquid nitrogen and stored at −80°C prior to Crow et al., 1996) and SERCA2a, as well as RyR2,(Schwartz et al., 1992) are reported to be alteredhomogenization, which improved recovery of total

RNA by approximately 30%. in experimental pressure overload-induced injury/hypertrophy models. Gene expression for the sar-colemmal calcium channel (dihydropyridine re-ceptor; DHPR) was also evaluated, as it is relatedHistopathological grading of the cardiomyopathyfunctionally to the sarcolemmal reticulum (SR) cal-cium release channel.The LV papillary muscle samples (in glutaraldehyde)

were sent to one collaborator (JA) for analysis Plasmids for Northern blots containing RyR2were provided through collaboration with Drsblinded to the grouping of the samples. The severity

of doxorubicin-induced cardiomyopathy was in- Sidney Fleischer and Debra Dodd (Vanderbilt Uni-versity). Plasmids containing cDNA to SERCA2adexed by histopathologic grading by electron mi-

crograph (EM). The histopathological scoring and pBS-18S were obtained from Dr David Fisher(Dept of Pediatrics, University of Cincinnati Schoolsystem of Billingham (1991) was used.of Medicine). Each insert was excised from theirrespective plasmids and digested with respectiverestriction enzymes to generate double-strandedRNA isolationDNA probes for Northern blots.

The 726 base pair cDNA probe for a-actin wasMinced LV free wall was homogenized and the totalRNA isolated using modifications of the method of constructed with primers for base pairs 214–939

(Putney et al., 1993) with the addition of restrictionChomczynski and Sacchi (1987). TRI-Reagent RNAIsolation reagent (Molecular Research Center, Inc.; sites Sst I (5′) and Hind III (3′). Primers spanning

base pairs 647 to 1163 were constructed based onCincinnati, OH, USA) was added prior to homo-genization followed with a chloroform extraction, mRNA sequence of b-MHC (Kavinsky et al., 1984)

with restriction endonuclease sites Sst I (5′) andisopropanol precipitation, and ethanol wash(Chomczynski and Sacchi, 1993). More recently, Hind III (3′) added for cloning of the 517 base

pair fragment. The pulmonary isoform of ACE wasRNA has been isolated with the Totally RNA Isol-ation Kit (Ambion; Austin, TX, USA) according isolated using primers based on an mRNA sequence

from Thekkumkara et al. (1992). Restriction sitesto the manufacturer’s protocol. RNA pellets weresolubilized with 0.1 m EDTA prepared in RNase- Sst I (5′) and Hind III (3′) were built in to the

primers that spanned base pairs 384 through 812,free dH2O and quantified by UV spectrophotometry.Aliquots were stored frozen at −80°C. resulting in a 429 base pair fragment. Using ANP

mRNA sequence identified by Oikawa et al. (1985),primers were constructed to span base pairs 215to 538 resulting in a 324 base pair fragment.cDNA probesPrimers were constructed to include Hind III (5′)and Sst I (3′) restriction endonuclease sites forThe rationale for the selections of the various gene

expression products is as follows. Firstly, we had cloning of ANP fragment into a transcription vector.A 572 base pair fragment of the angiotensin IIpreviously reported that loss of high affinity ry-

anodine binding and reduced immunoreactive ry- receptor type 1 (ATR) was isolated using mRNAsequence of rabbit kidney cortex (Burns et al.,anodine receptor (RyR2) correlates with contractile

failure following cumulative DOX exposures (Dodd 1993). Enzyme sites Hind III (5′) and Sst I (3′)were designed into these primers for future cloning.et al., 1993; Boucek et al., 1997). Recently, we

have reported preliminarily that doxorubicin re- DHPR primers were constructed to span 678 basepairs that included the first five exons of the receptorduces cardiac RyR2 mRNA levels (Dodd et al., 1995;


Table 1 Probe and primer construction for messenger RNA fragments resolved or amplifiedfrom rabbit left ventricle. a-ACT, rabbit cardiac alpha-actin; ACE, rabbit pulmonary isoform ofangiotensin-converting enzyme; ANP, rabbit atrial natriuretic peptide; ATR, rabbit angiotensinreceptor II type I; b-MHC, rabbit cardiac beta-myosin heavy chain; CPP, calcium pump protein;DHPR, rabbit dihydropyridine receptor; GAPDH, rat glyceraldehyde-3-phosphate de-hydrogenase; H-CRC, heart calcium release channel (ryanodine receptor); RyR, rabbit ryanodinereceptor; 18S, ribosomal subunit 18s. Fragment lengths are given in base pairs and do notinclude restriction sites that were added to primer constructs where noted. Primer sequencewas taken from each corresponding source.

Message Fragment Size Primer Restriction Sites Source/Reference(bp) (5′/3′)

a-ACT 726 Sst I/Hind III Putney et al., 1983bp 214–939 (cardiac)

ACE 429 Hind III/Sst I Thekkumkara et al., 1992bp 384–812

ANP 324 Hind III/Sst I Oikawa et al., 1985bp 215–538

ATR 572 Hind III/Sst I Burns et al., 1993bp 235–806

b-MHC 517 Sst I/Hind III Kavinsky et al., 1984bp 647–1163

CPP 719 BamH I/EcoR I DoddDHPR 678 None Mikami et al., 1989

bp 522–1199GAPDH 349 None Makino et al., 1996H-CRC 630 BamH I/EcoR I Dodd/FleischerRyR 658 None Otsu et al., 1990

bp 2040–269718S 450 BamH I/Pst I Dodd

(bp 522–1199), as described in Mikami et al. (1989). Analysis for the expression of specific gene productsPrimers for GAPDH were modified from Makino et

Two methods were employed to estimate the relativeal. (1996). GAPDH mRN was used as an internalabundances of selected mRNAs, a Northern blottingcontrol for differences in extraction and loading.analysis and a method using RT-PCR followed byAll probes were sequenced to confirm the fidelitySouthern hybridization of the PCR-amplified mes-of their amplification.sage. PCR reagents were purchased from PromegaAlthough RyR2 was initially detected by North-(Madison, WI, USA).ern blot, mRNA was not detectable in all samples.

Therefore, new primers were constructed to spananother area of the ryanodine receptor mRNA (RyR)for detection by reverse transcription-polymerase Northern analysischain reaction (RT-PCR), and to test the differencein expression between treatment pairs by Northern Ten micrograms of RNA were added to a loadingand RT-PCR assays. Primers were based on base mix (formamide, formaldehyde, 10×MOPS/EDTA,pair sequence 2040 to 2697 in Otsu et al. (1990) blue loading dye) containing ethidium bromide andand yielded a 658 base pair fragment. loaded onto a 1% denaturing formaldehyde gel (1%

Primers used for PCR are listed in Table 1. Poly- agarose, 8% formaldehyde, 11×14 cm, 100 ml),merase chain reactions of each primer pair on rabbit flanked with a RNA ladder (Gibco, Bethesda, MD,left ventricle cDNA were performed to isolate each USA). The gels were electrophoresed in 1×MOPS/message. Amplified cDNA fragments were then cut EDTA overnight at 20V. The gels were photographedwith restriction endonucleases at sites in the and prepared for Northern blotting by sequentialprimers, cloned into pBluescript SK II (Stratagene, rinsing with dH2O (three times for 10 min), aLa Jolla, CA, USA) and digested at similar enzyme denaturing solution (0.5 NaOH/1.5 NaCl) forsites. The cDNA was sequenced to verify amp- 5 min, and then in a neutralization solution (1.0

TrisHCL/1.5 NaCl, pH 6.8) for 30 min. RNAlification.


was transferred from the gel to a Zeta-probe GT extension for 10 min. The number of cycles wasoptimized for each message to ensure linearity be-Nitrocellulose membrane (Biorad; Hercules, CA,

USA) using a TurboBlotter Rapid Downward Trans- tween PCR amplified product and message abund-ance. Twenty cycles were used for amplification offer System (Schleicher and Schuell; Keene, NH,

USA), for 5 h in 20×SSC (NaCl, Na citrate). The GAPDH and ANP and yielded 330 bp and 460 bpfragments, respectively. Thirty cycles were usedmembrane was rinsed in a 3×SSC buffer, air dried

and vacuum baked at 80°C for 30 min. Membranes for amplification of RyR and DHPR (amplificationsyielded 658 and 678 bp fragments, respectively)were subsequently hybridized with randomly

labeled probes in 1×SET buffer (0.6 NaCl, 20 m and ACE and ATR (amplifications yielded 450 bpand 590 bp fragments, respectively).EDTA, 0.2 Tris, 0.5% SDS, 0.1% sodium pyro-

phosphate) at 65°C overnight, washed, dried and Southern blots were used to determine differencesin mRNA abundance within the different groups.exposed to X-ray film. Probes were randomly labeled

using 32P-dCTP (Sambrook et al., 1989). Ap- Ten microlitres of each PCR reaction were elec-trophoresed on a 1.2% agarose gel and run forproximately 105 cpm of a single labeled probe were

hybridized at 65°C overnight to each Northern blot 30–45 min in 1×TBE buffer at 100 V. Gels werestained with ethidium bromide, photographed, andfilter in 10 ml of 1×SET hybridization buffer. Filters

were washed three times for 15–30 min each in standard references were marked before proceedingwith Southern transfer. Gels were denatured in300 ml of blot wash 1×SSC, 0.5% sodium pyro-

phosphate, 0.1% SDS) at 42°C, air-dried, and ex- 0.5 NaOH/1.0 NaCl for 30 min followed byneutralization in 1.5 NaCl/0.5 Tris-HCl, pH=posed to autoradiography film. After sufficient film

exposures were obtained, filters were stripped of 7.5, for 30 min. Gels were soaked in 20×SSC for15 min just prior to blotting and transferred ontoprobe by being placed in boiling 0.1% SDS and

allowed to cool to room temperature over a 30- nytran membranes with the TurboBlotter System(Schleicher & Schuell, Keene, NH, USA) 3 h in 20×min period. Filters were then probed again using a

different probe by the method just described. An SSC buffer. Membranes were rinsed in 2×SSC anduv crosslinked. Membranes were hybridized,internal RNA control probe specific for 18S ri-

bosomal RNA was applied to filters after other washed and exposed as described for Northern blots.probing was completed due to high specificity andbinding of probe to the filter.

In situ hybridizationRT-PCR and Southern blotting

In order to determine if expression was myocytespecific, in situ hybridization was used to determineReverse transcription of messenger RNA followed

by amplification using PCR was used to resolve the origin of expression for significant differencesbetween DOX-treated and Control animals. LV pap-mRNA not detectable by Northern blot assays. One

microgram of left ventricle total RNA from each illary muscle was embedded in paraffin and slicedto a five-micron thickness onto glass slides. Severalsample was added to 200 ng oligo-dT and heated

to 65°C for 15 min followed by chilling on ice. xylene washes, followed by hydration to deionizedwater, freed the sections from paraffin prior toSamples were then reverse transcribed with the

addition of 1.0 U RNase inhibitor, 2 ll pepsin digestion for 30 min at room temperature.After washing with DepC water (Research Genetics,10×transcription buffer with 10 m each dNTP,

and 200 U superscript reverse transcriptase (BRL, CA, USA), riboprobes (100 ng) were added to in-dividual sections, heated to 95°C and cooled toBethesda, MD, USA), heated to 42°C for 1 h, 52°C

for 30 min, and 99°C for 5 min followed by chilling 45°C for overnight hybridization. Excess probe wasremoved by washing, and sections were conditionedon ice. To each newly transcribed cDNA sample

a PCR reaction mixture was added consisting of (Tissue Conditioner, Biomeda, CA, USA) to preventendogenous alkaline phosphatase reactions. Anti-200 m each dNTP, 5 ll 10× PCR buffer, 300 ng

of each primer, 0.5 U Taq polymerase, and deionized digoxigenin conjugated to alkaline phosphatase wasadded to bind with bound riboprobe on the sectionswater for a total volume of 50 ll. DNA con-

tamination of the RNA samples was determined at 37°C and color reactions were detected by useof BCIP/NBT chromogen (Boehringer Mannheim,by inclusion of controls with no added reverse

transcriptase. Cycling conditions were as follows: IN, USA). Reactions were stopped by dehydratingtissue sections which were mounted in xylene with96°C denaturation for 1 min, 60°C annealing for

1 min, 72°C extension for 1.5 min, 72°C final an aqueous mount. Sections were examined for


pattern and cell specificity of staining for anti-sense riboprobes v sense riboprobes and lack of anyriboprobe.

Riboprobes were generated by transcribing anti-sense and sense RNA from linearized plasmids con-taining the probe of choice (Table 1) along withthe incorporation of digoxigenin-labeled UTP(Boehringer Mannheim, IN, USA). Riboprobes weretreated with DNase to remove template and werecolumn-purified (STE Select-D(RF); 5 Prime, 3Prime, CO), diluted to a concentration of 50 ng/ll,and stored at −20°C.

Data analysis

The two methods used to estimate the relativemyocardial abundances of selected mRNAs wereNorthern blotting and, for less abundant messages, Figure 1 A representative Southern blot with cDNA

specific for ANP and GAPDH is shown. Aliquots of RNART-PCR followed by Southern hybridization of thewere treated with reverse transcriptase and PCR amp-PCR-amplified message. In both methods the rel-lified. The product DNA was added to the lanes asative abundances of mRNA were determined asindicated, agarose electrophoresed, blotted and hybridized

follows. Autoradiographs of the hybridized transfer with radiolabeled probe. The autoradiogram of one blotmembranes were scanned using a Truvel den- is shown to illustrate paired comparisons of LV RNA from

DOX-treated (lanes labeled D) and Control (lanes labeledsitometer with a BioImage Visage 2000 image ana-C) at 4 days (lanes D and C), 4 weeks (lanes D and C)lysing system (Millipore, Bedford, MA, USA). Forand 10 weeks (lanes D and C) after either DOX (2 mg/each lane the optical density was determined forkg) or saline intravenous infusions.

the band of interest and for the internal standardband, either 18S (Northern analysis) or GAPDH(RT-PCR). The abundance ratios of the band of (n=8; intermediate). The median histopathologic

score for the DOX-treated group 4 weeks after DOXinterest to the internal standard (either 18S orGAPDH) were calculated to correct for differences exposure was 0.5 and ranged from 0 to 1.1 (n=

8; intermediate). One of eight DOX-treated rabbitin loading and transfer efficiency. Relative messagelevels for the DOX-treated group and the paired hearts in the intermediate subgroup had a histo-

pathological score greater than 1 (1.1) and wasControl group were determined from of the ab-sorbance ratios of the DOX-treated lane to the paired considered separately (dai110395). No evidence for

histopathology was observed in DOX-treated groupControl lane on the same gel (Fig. 2). The effectsof DOX were expressed as the fold change in the 10 weeks after DOX exposure (n=7; late).

The mRNA levels for ANP were decreased 4.5-paired DOX-treated absorbance ratio compared tothe Control absorbance ratio. The effects of DOX fold 4 days after DOX infusion (P<0.05; Figures 1

and 2C). ANP message returned to Control levelswere analysed for significance as continuous non-parametric paired data using the Wilcoxon Sign by 4 weeks in all but the one rabbit heart with the

histopathologic score of 1.1 in which the ANPRank test, which does not assume normal or equalvariances. The null hypothesis was rejected for message had increased nearly 80-fold (P<0.05; Fig.

2C). ANP message levels were then increased>16-PΖ0.05. Unless otherwise specified, all results arepresented as mean±.. fold 10 weeks after DOX (P<0.05; Fig. 1 and 2C).

The mRNA levels for b-myosin heavy chain byNorthern blot analysis were 2.9-fold increased com-pared to Control (Fig. 2A). b-myosin heavy chainResultsmRNA levels had returned to baseline 4 and 10weeks after the second DOX dose (Fig. 2A). a-actinHistopathologic evaluation of the LV papillary

muscle was performed in all of the intermediate (4 mRNA was not significantly affected 4 days, 4 or10 weeks after DOX (Fig. 2A).weeks after DOX) and late (10 weeks after DOX)

subgroups. The median histopathologic score for The relative levels of mRNA for the high mo-lecular weight subunit (cardiac isoform) of RyR2the Control group was 0.2, and ranged from 0 to 0.5


100.00

0.10mRNA:

Fol

d ch

ange

10.00

1.00

ACE/gapdh ATR/gapdh ANP/gapdh

10.00

0.10mRNA:

Fol

d ch

ange

1.00

CPP/18S RyR/gapdh DHPR/gapdh

0.10mRNA:

Fol

d ch

ange 10.00

1.00

a-Act/18S b-MHC/18S 18S

**

*

*

(A)

(B)

(C)

Figure 2 Panels A, B and C illustrate group mean (±..) changes in steady-state LV mRNA levels 4 days (early; n=8), 4 weeks (int.; n=7) and 10 weeks (late; n=7) after DOX (2 mg/kg i.v.) expressed as a fold change compared to apaired control group that received saline intravenous infusion. Also presented are the LV mRNA levels for a DOX-treated and control pair evaluated 4 weeks after DOX that was found to have significant histopathology (dai 110395).Each message is normalized to either 18S or GAPDH, as indicated. Messenger RNA fragments amplified from rabbitheart were: a-Act or a-Actin; angiotensin converting enzyme (ACE); atrial natriuretic peptide (ANP); angiotensin IIreceptor type I (ATR); cardiac beta-myosin heavy chain (b-MHC) calcium pump protein SERCA 2a (CPP); dihydropyridinereceptor (DHPR); the cardiac isoform of the ryanodine receptor (RyR). Panel A presents mRNA levels for selectedcontractile proteins and for the constitutive marker designated 18S. Panel B presents mRNA levels for selected proteinsinvolved in calcium-mediated excitation-contraction coupling. Panel C presents mRNA levels for selected proteinsrelevant to the neurohumoral response mechanism of heart. (D) Early, n=8; (P) Int., n=7; (C) Late, n=7; (Q) dai110395. ∗P<0.05.

were determined both by Northern blotting and RT- and 2.2-fold 4 and 10 weeks after DOX (Fig. 2B).Activation of the cardiac renin–angiotensin sys-PCR. Since the results were comparable, only the

RT-PCR results are presented. The mRNA levels for tem by DOX was not detected at the level of geneexpression. The levels of mRNA for ACE, determinedRyR2, determined by RT-PCR, were increased 1.3-

fold at 4 days after DOX, and were decreased 1.2-fold by RT-PCR, were increased (1.5-fold) 4 days afterDOX and fell to below Control group levels (1.8-and 1.8-fold 4 and 10 weeks after DOX, respectively

(Fig. 2B). The levels of mRNA for the calcium fold; Fig. 2) at 4 weeks and at Control group levelsat 10 weeks (Fig. 2). An exception was that ACEpump protein (SERCA2a), determined by Northern

blotting, were increased 1.4-fold at 4 days after message levels were increased (3.3-fold) in onerabbit with a histopathology score >1 (Fig. 2C).DOX, and then were decreased 1.8-fold and 2.3-

fold 4 and 10 weeks after DOX, respectively (Fig. Message levels for ATR in the DOX-treated groupwere not significantly different than Control group2B). The levels of mRNA for DHPR were increased

1.2-fold at 4 days after DOX. Of particular note, at any time point (Fig. 2C).Message expression, as determined by in situthe DOX-treated to Control ratio of mRNA for DHPR

and was persistently increased 1.9-fold (P<0.05) hybridization, was used to determine specific cell


Figure 3 Representative photomicrographs of an in situ hybridization for DHPR are shown. Paraffin embedded slicesfrom the papillary muscle from the LV of a DOX-treated (2 mg/kg) rabbit were hybridized with anti-sense (Panel a) orsense (Panel b) riboprobe specific for DHPR. Note the localization of mRNA for DHPR to the cytoplasm of cardiomyocytes(Panel a) with only nuclear localization with the sense control probe.

expression. Antisense probes for DHPR (Fig. 3) and DiscussionANP (not shown) indicated predominant pro-duction of mRNA localized to cardiomyocytes, as This study demonstrated that anthracyclines, when

given in a clinically-relevant dosing schedule, haveopposed to other cell types found in the heart. Incontrast, sense probes showed specificity only to both immediate (“early”) and persistent (“late”)

effects on cardiac gene expression. This study hasthe nucleus, probably due to binding of the senseRNA to the complimentary antisense DNA strand. identified DOX-induced changes in gene expression


4 days after DOX exposure. These immediate effects different patterns of gene expression have beenreported in heart failure models, stress-inducedwere to increase b-myosin heavy chain and de-

crease ANP mRNA abundances (Fig. 2). This study hypertrophy models and cardiomyopathy models.Alternatively, differences in the cDNA probes be-also has identified persistent and progressive DOX-

induced effects on myocardial gene expression. Four tween the studies could also account for the ob-served difference in results. In specific, Ito et al.and 10 weeks after DOX exposure persistent effects

on message levels were observed, the largest change selected a probe for the 3′-untranslated region ofthe codon presumably to be specific for the cardiacfrom baseline observed was an increase in DHPR

(Figs 2 and 3). DOX resulted in effects that were isoform. The cDNA probe used in this study (Table1) was from the coding sequence and detects bothprogressive; the largest changes over the interval

were in ANP mRNA (Figs 1 and 2). These effects the cardiac and skeletal isoforms.Possible mechanisms for anthracycline effects onare largely myocellular based on in situ hy-

bridization (Fig. 3). Thus, this study has dem- cardiac gene expression include direct an-thracycline effects on the determinants of mRNAonstrated anthracycline effects on myocellular gene

expression that are both rapid in onset, relatively production and/or a response secondary to an-thracycline-induced myocellular injury. Earlierpersistent (up to 10 weeks) and also progressive

after brief anthracycline exposure. studies had suggested that anthracyclines effect onmyocellular transcription (Zahringer et al., 1981)Message levels for RyR2 and SERCA2a followed

a downward trend over time post-DOX, though by intercalation with DNA (Leonard et al., 1992),and/or by an interaction with a unique consensusthese differnces could be by chance. This reduction

in mRNA for RyR2 (Fig. 2) is consistent with sequence located in upstream regulatory se-quence(s) (Eliopoulos et al., 1991; Kurabayashi etreduced transcription, and suggests that a tran-

scriptional mechanism is possible for the loss of al., 1992; Kurabayashi et al., 1993), and/or by aninteraction with topoisomerase (Foglesong et al.,high affinity ryanodine binding and immuno-

reactive RyR2 reported with the contractile failure 1992). Further studies will be necessary to deter-mine if the effects of DOX on cardiac gene expressionof the cardiomyopathy due to cumulative DOX

exposures (Dodd et al., 1993; Boucek et al., 1997). reported in this study are direct effects of DOX oncardiac transcriptional events. Alternatively, theIt is possible that the small changes in RyR2 mRNA

with doxorubicin (Fig. 2) reflect the minimal histo- effects of DOX on cardiac gene expression reportedherein could be responses secondary to myocellularpathology, as earlier reports have shown that the

doxorubicin-induced loss of ryanodine binding cor- injury and remodeling. Cardiac hypertrophy canbe an early response to certain types of myocardialrelated with histopathology and contractile failure

(Dodd et al., 1993; Boucek et al., 1997). Since injury. The response is initially adaptive and isassociated with reversion to a fetal pattern of cardiacsubmission of this manuscript, Arai et al. (1998)

reported the downregulation of RyR2 and SERCA2a gene expression (Francis and McDonald, 1992).Since cardiac mass is increased early in the de-in rabbit heart following DOX treatments. It is

important to note that the cumulative DOX dose velopment of the cumulative doxorubicin-inducedcardiomyopathy (Dodd et al., 1995), the pattern ofused in this study was 20 mg/kg, 10-fold greater

than used in the study reported herein. responses to DOX in this study was compared tothe responses of cardiac muscle gene expressionPrevious studies have demonstrated an-

thracycline effects on cardiac gene expression previously reported in hypertrophy models (Table3). Qualitatively, the pattern of DOX effects onwithin 24 h after a high dose. Exposure of cultured

neonatal rat cardiomyocytes to DOX (0.5 l) has cardiac gene expression are not similar to the pat-tern activated with cardiac remodeling and growth/been reported to reduce acardiac-actin mRNA levels

(Northern blot analysis)s while not affecting mRNA hypertrophy. However, comparisons are prob-lematic: firstly, no significant increase in LV massfor a non-muscle specific gene, pyruvate kinase

(Akimoto et al., 1993). The mRNA levels for a- was noted in this study (Table 2); and secondly, thetime dependency of mRNA response in the injury-actin and b-myosin heavy chain were reduced 24 h

after the intraperitoneal injection of a single dose hypertrophy models must also be defined and com-pared.(2–40 mg/kg) of DOX, while not affecting the ex-

pression of pyruvate kinase in rat hearts (Ito et al., The time-dependent increase in ventricular ANPmRNa is consistent with progressive activation of1990). The effects on a-actin on gene expression

are qualitatively different from these reports (Ito et ANP expression, possibly due to the re-inductionof the so-called fetal program of gene expressional., 1990; Akimoto et al., 1993), possibly due to

the differences between the models. In this context, (Feldman et al., 1993). The increase in ventricular


Table 2 Comparison of mRNA responses following hypertrophy-inducing stress andfollowing DOX (2mg/kg).

Hypertrophy Doxorubicin-inducedMessage Response Early Intermediate Late

b-MHC ↑ ↑ ↔ ↔SERCA 2a Usually decreased ± ↔ ↓

Usually decreased ↔ ?↓RyR2 ↔ANP ↑ ↓ ± ↑↑ACE ↑ ↔ ↔ ↔DHPR ↔ ↑ ↑ATR ↔ ↔ ↔ ↔

↑=Increase; ↓=decrease; ↔=no change; ±=equivocal.

Table 3 Characteristics of the early, intermediate and late Control and DOX-treated groups are tabulated. No significantdifferences were noted between Control and DOX-treated groups (unpaired T-test).

Early Intermediate Late

AE group parameters Control DOX-treated Control DOX-treated Control DOX-treated(mean±SD) (n=6) (n=8) (n=5) (n=8) (n=4) (n=7)Body wt (kg) 3.2±0.6 3.2±0.9 3.0±0.3 3.0±0.7 2.9±0.4 3.0±0.4DOX dose (mg/kg) 0 2 0 2 0 2Final body wt (kg) — — 4.0±0.6 3.7±0.5 4.0±0.4 4.1±0.4Heart wt/body wt 1.5±0.7 2.1±0.7 1.7±0.2 1.8±0.2 1.6±0.1 1.7±0.1RNA mg/gr 0.88±0.4 0.85±0.2 1.7±1.2 1.4±0.6 1.3±0.6 1.2±0.2A260/A280 1.9±0.1 1.6±0.7 1.8±0.1 1.8±0.1 1.9±0.0 1.9±0.0

ANP mRNA is consistent with the activation of injury, activation of transcriptional responses andprogression of this cardiomyopathy to failure. Fur-ANP expression noted by 8 weeks after 20 mg/kg

DOX (Arai et al., 1998). In addition, circulating or ther studies are necessary to determine how longthese responses persist.plasma levels of ANP are elevated in rats (Bernardini

et al., 1992) and patients (Bauch et al., 1992) Recently, a “late”, or delayed onset cardio-myopathy has been described in children years aftertreated with anthracyclines. The early decrease in

ventricular ANP mRNA is consistent with transient receiving anthracyclines (Steinherz et al., 1991;Steinherz et al., 1995; Lipshultz et al., 1991). Therepression of ventricular gene expression for ANP,

which has also been reported during cardiac on- pathogenesis of this late onset anthracycline-relatedcardiomyopathy has not been elucidated, but atogeny (Bloch et al., 1986).

The significance of anthracycline-induced effects persistent and/or progressive anthracyline effect(s)on cardiac gene expression is now a plausible mech-on cardiac gene expression and the persistent ac-

tivation of ventricular ANP expression is not known anism to consider based on the results of this study.at this time. In general, persistent “injury” can leadto a maladaptive growth response contributing tothe progression of cardiomyopathies to failure (Katz, Acknowledgements1994). One interpretation of the observation re-ported herein is that the effect of doxorubicin on The authors were supported by AHA FL Affiliategene expression for DHPR is related to effects on Grant in Aid #9401217 and the Suncoast Cardio-excitation-contraction coupling related to heart fail- vascular Research and Education Foundation.ure (Arai et al., 1996; Rannou et al., 1996; Gomez etal., 1997). In the development of the anthracycline-induced cardiomyopathy, myocardial morphologic Referenceslesions do continue to develop for weeks after thelast dose (Jaenke, 1976). Thus, the continued ex- A H, B NA, S DL, B ME, T

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