Mitochondrial topoisomerase I (Top1mt) is ... - AACR Journals

Cancer Therapy: PreclinicalSee related commentary by Nitiss and Nitiss, p. 4737

Mitochondrial Topoisomerase I (Top1mt) Is a Novel LimitingFactor of Doxorubicin Cardiotoxicity

Salim Khiati1, Ilaria Dalla Rosa1, Carole Sourbier2, Xuefei Ma3, V. Ashutosh Rao4, Leonard M. Neckers2,Hongliang Zhang1, and Yves Pommier1

AbstractPurpose:Doxorubicin is one of the most effective chemotherapeutic agents. However, up to 30% of the

patients treated with doxorubicin suffer from congestive heart failure. The mechanism of doxorubicin

cardiotoxicity is likely multifactorial andmost importantly, the genetic factors predisposing to doxorubicin

cardiotoxicity are unknown. On the basis of the fact that mtDNA lesions and mitochondrial dysfunctions

have been found in human hearts exposed to doxorubicin and that mitochondrial topoisomerase 1

(Top1mt) specifically controls mtDNA homeostasis, we hypothesized that Top1mt knockout (KO) mice

might exhibit hypersensitivity to doxorubicin.

Experimental Design: Wild-type (WT) and KO Top1mt mice were treated once a week with 4 mg/kg

doxorubicin for 8 weeks. Heart tissues were analyzed one week after the last treatment.

Results: Genetic inactivation of Top1mt in mice accentuates mtDNA copy number loss and mtDNA

damage in heart tissue following doxorubicin treatment. Top1mt KOmice also fail to maintain respiratory

chain protein production and mitochondrial cristae ultrastructure organization. These mitochondrial

defects result in decreased O2 consumption, increased reactive oxygen species production, and enhanced

heartmuscle damage in animals treatedwith doxorubicin. Accordingly, Top1mtKOmice diewithin 45 days

after the last doxorubicin injection, whereas the WT mice survive.

Conclusions:Our results provide evidence that Top1mt, which is conserved across vertebrates, is critical

for cardiac tolerance to doxorubicin and adaptive response to doxorubicin cardiotoxicity. They also suggest

the potential of Top1mt single-nucleotide polymorphisms testing to investigate patient susceptibility to

doxorubicin-induced cardiotoxicity. Clin Cancer Res; 20(18); 4873–81. �2014 AACR.

IntroductionAnthracycline antibiotics, and especially doxorubicin,

are among the most widely used anticancer drugs (1).Their primary mechanism of action is by intercalationinto DNA (2) and by trapping topoisomerase II-DNAcleavage complexes (Top2cc; refs. 3, 4) as they bind at theTop2-DNA interface (5, 6). Top2cc, in turn selectivelykills cancer cells by blocking replication and transcrip-tion (4, 7–9).Despite the efficacy of doxorubicin in pediatric (10) and

adult cancers ranging from leukemia to lymphomas and

solid tumors such as breast cancers (11), the main adverseeffect of doxorubicin is cardiotoxicity, which can causecongestive heart failure in 30% of adults at high doses, anddelayed heart failure after terminating treatment in childrenonce they reach adulthood. The cardiotoxicity of doxoru-bicin appears separable from its therapeutic mechanismbecause cardiomyocytes are generally not replicative, andTop2a, the primary target of doxorubicin (7, 8), is notexpressed in quiescent cells and undetectable in heart tis-sues (12). On the other hand, Top2a is required for cellproliferation and its gene TOP2A is often amplifiedwith theHER-2 (ERBB2) oncogene in breast and other forms ofcancers (13).

The cardiotoxicity of doxorubicin remains difficult topredict and is often not detected until years after thecompletion of chemotherapy (14). Also, the genetic deter-minants of doxorubicin cardiotoxicity remain unknown, atleast in part, because doxorubicin cardiotoxicity is likelymultifactorial and complex (15). Free radical generation is aclassical mechanism by which doxorubicin injures themyocardium (16). The chemical structure of doxorubicinis prone to the generation of free radicals as doxorubicinreversibly oxidizes to a semiquinone, an unstable metabo-lite whose futile cycling within the mitochondria releasesreactive oxygen species (ROS; ref. 17). Unfortunately, free

1Developmental Therapeutics Branch and Laboratory of Molecular Phar-macology; 2Urologic Oncology Branch, Center for Cancer Research,National Cancer Institute; 3Laboratory of Molecular Cardiology, NationalHeart, Lung, and Blood Institute, NIH; and 4Center for Drug Evaluation andResearch, Food and Drug Administration, Bethesda, Maryland

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Yves Pommier, Developmental TherapeuticsBranch and Laboratory of Molecular Pharmacology, Center for CancerResearch, National Cancer Institute, NIH, Bethesda, MD 20892. Phone:301-496-5944; Fax: 301-402- 0752; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-3373

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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radical scavengers provide only limited heart tissue protec-tion (18–20). The heart is selectively sensitive to reactiveoxygen metabolites because of lowered antioxidant gluta-thione peroxidase, catalase, and superoxide dismutaselevels compared with other tissue (21). An additionalpossibility stems from the fact that doxorubicin not onlyinhibits Top2a, but also Top2b. A recent study showed thatgenetically engineered mice lacking Top2b in their heartavoidmyocardial injuries after doxorubicin treatment (22).A third possibility is the direct targeting of mitochondria bydoxorubicin (23). Doxorubicin being a cationic compoundreadily enters mitochondria, binds to cardiolipin, and inhi-bits the respiratory chain. Indeed, the electron–transportchain proteins require cardiolipin to function properly, andit has been proposed that because doxorubicin disrupts thecardiolipin–respiratory chainprotein interface,more super-oxide (O2

�) formation occurs (24–26). Finally, mtDNAcould be a direct target of doxorubicin (27), as mtDNAlesions and free radical-associated mitochondrial dysfunc-tion have been found in the hearts of patients treated withdoxorubicin (26).

Mitochondria are the only cellular organelles containingmetabolically active DNA outside the nucleus (28). DNAtopoisomerases are present in mitochondria to relievemtDNA topologic stress and entanglements generatedduring replication and transcription. To date, three topoi-somerases have been identified in vertebrate mitochon-dria: Top1mt (29), Top2b (30), and Top3a (31). Top3aand Top2b both function in mitochondria and the nucle-us, and the only specific mitochondrial topoisomerase invertebrates is Top1mt (29). Murine embryonic fibroblasts(MEF) from Top1mt knockout (KO) animals show amarked increase in ROS production, calcium signaling,and hyperpolarization of mitochondrial membranes (32).Top1mt activity in the regulatory region of mtDNA alsosuggests its importance in regulating mtDNA replication

(33). However, Top1mt-deficient mice (Top1mt�/�) areviable, fertile, normal in size, and do not display obviousbasal physical or behavioral abnormalities, indicatingcompensation by other topoisomerases and metabolicreprogramming. Indeed, Top1mt-deficient MEFs compen-sate their mitochondrial dysfunction by producing ATPthrough alternative metabolic pathways and increasingtheir antioxidant capacity (32).

On the basis of the fact that mtDNA lesions and radical-associated mitochondrial dysfunctions have been found inhuman hearts exposed to doxorubicin (26) and thatTop1mt specifically controls mtDNA homeostasis (32,33), we hypothesized that Top1mt KO mice might exhibitheart tissue sensitivity to doxorubicin.

Materials and MethodsMouse handling

Top1mtþ/þ (WT) and Top1mt�/� (Top1mt KO) micewere generated fromheterozygous (Top1mtþ/�) and pairedwithin the same litter. Each Top1mt KOmouse had at leastone brother WT as control. Starting at 7 weeks of age, micewere treated once a week with 4 mg/kg intraperitonealdoxorubicin or saline solution control for 8 weeks. Hearttissues were analyzed one week after the last treatment. Forsurvivals studies, mice were followed for up to 90 days afterthe last injection. Animal experiments were performed inaccordance with the guidelines of the Animal Care and UseCommittee of the NIH (Bethesda, MD).

Transmission electron microscopyMice were euthanized and heart tissues were immedi-

ately harvested and fixed in 4% formaldehyde, 2% glu-taraldehyde, and 0.1 mol/L Cacodylate (pH 7.4) for 2hours at room temperature. Small pieces of the fixed hearttissue were postfixed in 1% osmium tetroxide for 1 hourand stained in 0.5% uranyl acetate for another hour. Thesamples were then dehydrated in a graded series of 35%,50%, 70%, and 100% ethanol and exchanged to propyl-ene oxide. After infiltration at 1:1 propylene oxide andepoxy resin (Poly/Bed 812, Polysciences) overnight, sam-ples were embedded in 100% epoxy resin. Polymerizationof resin was performed for 3 days at 55�C. Thin sections of70 to 90 nm were cut with an ultramicrotome (Leica EMUC6, Leica Microsystems), stained with uranyl acetate andlead citrate, lightly carbon coated, and imaged in a Hitachi7650 or 7600 transmission electron microscope (HitachiHigh Technologies America). Images were taken with 2k� 2k AMT digital camera (Advanced Microscopy Techni-ques). Mitochondrial morphometric measurements wereperformed using ImageJ. A total of 250 mitochondria foreach condition were analyzed from 10 electron micro-graphs taken at �3,000 magnification in six differentsections. Two animals were used for each condition.

Mitochondria isolationMitochondria from hearts of mice were isolated follow-

ing the protocol for rats published by Rogers and colleagues

Translational RelevanceDoxorubicin is one of themost widely used anticancer

drugs. Yet, a significant number of patients treated withdoxorubicin develop cardiotoxicity. The exact mechan-isms of doxorubicin cardiotoxicity are likely multifacto-rial and complex, and identification of predicting factorsfor doxorubicin toxicity remains a clinical challenge.Here, we show that the mitochondrial topoisomerase1 (Top1mt) is critical to limit doxorubicin cardiotoxicity.Top1mt knockout (KO) mice show hypersensitivity todoxorubicin with significant mitochondrial dysfunc-tion, including mtDNA and cristae ultrastructure dam-age and respiratory chain proteins loss. Top1mtKOmiceshowheartmuscle defects with increased death rate aftertreatment. Our study demonstrates the importance ofmitochondrial DNA (mtDNA) regulation for doxorubi-cin cardiotoxicity. Deleterious genomic variants forTop1mt should be tested in patients hypersensitive todoxorubicin.

Khiati et al.

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(34). Briefly, 50 mg of heart tissue were trimmed to size of1 mm3 and resuspended in approximate 10 mL mitochon-dria isolation buffer (225 mmol/L mannitol, 75 mmol/Lsucrose, 10 mmol/L HEPES, 10mmol/L EDTA, 1 mg/mLbovine serum albumin; BSA). Tissues were homogenizedwith 40 strokes in a dounce homogenizer and centrifugedfor 10minutes at 1,000� g. Supernatant was centrifuged at12,000 � g for 10 minutes and crude mitochondria pelletswere washed twice with mitochondria isolation bufferwithout BSA. Proteins concentrations were quantified usingBio-Rad Protein Assay.

Mitochondrial membrane potential (Dcm)Dymwas determined in isolated mitochondria using JC-

1 according to the manufacturer’s protocol. Protein con-centration was used for normalization.

Reactive oxygen species production measured byglutathione assayROS production was measured quantifying reduced glu-

tathione (GSH) in heart tissue. GSH levels were assessed in50mg tissue lysates using the luminescence-basedGSH-GloGlutathione Assay (Promega) according to the manufac-turer’s protocol.

Mitochondrial Complex IV activityThe cytochrome C oxidase activity quantification in

isolated mitochondria was performed using the absor-bance-based assay Mitochondrial Complex IV (Mouse)Activity Assay Kit (Millipore) and following the manu-facturer’s protocol. The complex IV is immunocapturedwith the wells and its activity is determined by followingthe oxidation of reduced cytochrome c as an absorbancedecrease at 550 nm.

Quantification of mtDNA copy number and mtDNAdamageFor mtDNA quantification, total DNA was isolated from

30 mg of tissue using DNeasy Blood and tissues Kit (QIA-GEN). Quantitative PCRs were performed in triplicates in384-well reaction plates (Applied Biosystems). Each reac-tion (final volume 10 mL) contained 25 ng DNA, 5 mL ofPower SYBR-Green PCR Master Mix (Applied Biosystems),and 0.5 mmol/L of each forward and reverse primer. COX1gene was amplified and b2-microglobulin (b2m) was usedas normalizing control.MtDNA damage was quantified by long-range PCR

(35). A 10-kb fragment and a shorter region of mtDNAwere amplified. PCR reactions were limited to 18 cycles,to ensure that amplification process was still in theexponential phase. To compare mtDNA damage in eachsample, PCR products were quantified using PicoGreenand the quantity of the short-range PCR product (Q) wasnormalized to amount of the long-range PCR product (P)measured by analysis. The damage index is determined bythe ratio of Q/P.Primers sequences used for mtDNA analysis are listed in

supplementary table S1.

Western blottingFor detection of respiratory chain proteins, 50mgof heart

tissue were homogenized and lysed in radioimmunopreci-pitation assay buffer (RIPA) supplemented with 0.4 mol/LNaCl andprotease inhibitors (RocheApplied Science). After1 hour at 4�C, lysates were centrifuged for 10minutes at fullspeed and protein concentration in the supernatant wasmeasured (Bio-RadProteinAssay).Of note, 40mg of proteinwere subjected to SDS-PAGE and transferred onto nitrocel-lulose membranes (Bio-Rad). After 1 hour blocking with5% milk in PBST (PBS Tween 20, 0.1%), membranes wereincubated overnight with Anti-OxPhos Complex Kit anti-body (#457999, Invitrogen). After threewashes in PBST, themembrane was incubated with horseradish peroxidase-conjugated goat anti-mouse (1:5,000 dilution) antibody(Amersham Biosciences) for 1 hour and then washed threetimes. Immunoblot analyses were detected using enhancedchemiluminescence detection kit (Pierce).

For detection of Top1 and Top2b, 100 mg of heart tissuewere trimmed to size of 1 mm3, homogenized and lysedin RIPA buffer supplemented with protease inhibitors. After1 hour shaking at 4�C, lysates were centrifuged at full speedfor 10 minutes at 4�C. Supernatant was discarded and thepallet was lysed a second time for 1 hour in RIPA buffersupplementedwith 0.4mol/LNaCl andprotease inhibitors.After centrifugation, proteins in the supernatant were quan-tified and 40 mg were subjected to SDS-PAGE as describedabove. The primary antibodies used were: anti-Top1(#556597, BD Pharmingen), anti-Top2b (sc-25330, SantaCruz Biotechnology), and anti-a-tubulin (#05–829,Millipore).

Histologic analyses and immunofluorescenceHeart tissues were fixed in 10% phosphate buffered

formalin, pH 7.4, at room temperature for 2 hours. Fivemicrons sections from the paraffin-embedded hearts werestained with hematoxylin and eosin (H&E) for the analysisof nucleus hypertrophy. For cardiomyocyte cross-dimen-sions analysis, heart sections were deparaffined (3 times20 minutes in Xylene at room temperature) and fixed with4% formaldehyde in PBS for 1 hour. After PBS washes,sections were fixed and permeabilized with prechilled(�20�C) 70%ethanol for 20minutes and stained for 1 hourwith Wheat germ agglutinin coupled to Alexa Flour 488(1:200; Wheat Germ Agglutinin, Alexa Fluor 488 Conju-gate, Invitrogen). Tissues were then washed with PBS, andmounted using Vectashield mounting medium with 40, 60-diamidino-2-phenylindole to counterstain the DNA (Vec-tor Laboratories).

Slides after H&E staining were examined using high-resolution TV camera attached to a light microscope andthe magnification was calibrated with a stage micrometer(Zeiss). Slides stained with wheat germ agglutinin wereexamined using a laser scanning confocalmicroscope (ZeissLSM510). Images were collected and processed using theZeiss AIM software. Nucleus size and cardiomyocyte areaswere realized with ImageJ software. For each animal (n¼ 4for each condition), four to seven regions from sections of

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the right ventricular were counted and a mean value wasobtained.

ResultsLack of Top1mt increases doxorubicin-induced cardiacmitochondrial defects

To investigate the role of Top1mt in the adaptive responseto doxorubicin-induced cardiomyopathy, we treatedTop1mt KO (Top1mt�/�) and wild-type (WT; Top1mtþ/þ)mice born in similar litters from heterozygous (Top1mtþ/�)parents with doxorubicin. Figure 1A shows our treatmentscheme. Seven-week-oldmice were treated once a week withdoxorubicin at 4 mg/kg or with saline solution (control)given by injections for 8 consecutive weeks. One week afterthe last injection,heartswere analyzed.Additionalmicewerefollowed for survival for up to 90 days after the last injection(see below and Fig. 4).

Electron microscopy analysis of heart tissues showed noobvious difference inmitochondrial ultrastructure betweenWT and Top1mt KO mice treated with saline solution (Fig.1B, left). Accordingly, surface area analysis (SupplementaryFig. S1A) and mitochondria quantitation (SupplementaryFig. S1B) showed no significant difference betweenWT andTop1mt KO mice, and dense and regular cristae organiza-tions were observed in both tissues. After doxorubicintreatment, electron microscopy analyses showed Top1mtKO mice displaying more extensive mitochondrial damagecompared with WT mice. Mitochondria were swollen (Fig.1B and Supplementary Fig. S1A), and showed highly frag-

mented and degraded cristae (Fig. 1B). In addition, com-pared with WT mice, the Top1mt KO mice showed anattenuated upregulation of mitochondria number inresponse to doxorubicin (Supplementary Fig. S1B), indi-cating defective mitochondrion homeostasis in response todoxorubicin for the Top1mt KO mice.

Top1mt is required to maintain heart mitochondrialbiochemical functions and mtDNA integrity afterdoxorubicin treatment

To determine whether the ultrastructural defectsobserved by electron microscopy were accompanied bymitochondrial dysfunction, mitochondria isolated fromthe heart tissue were examined biochemically. Immuno-blotting showed that doxorubicin markedly decreasedthe steady-state levels of complexes I, III, and IV of therespiratory chain proteins in Top1mt KO mice (Fig. 2A,right panel showing a representative heart muscle exam-ple, and Supplementary Fig. S2 for quantitation).Although, it is well known that complexes I and III, andespecially complex IV, are depleted in heart tissue afterdoxorubicin treatment (36, 37), the decrease in thosecomplexes, which are both nuclear and mitochondrialencoded, was more dramatic in Top1mt KO comparedwith WT mice (Fig. 2A and Supplementary Fig. S2B). Onthe other hand, proteins of complexes II and V, which areassembled even in the complete absence of mitochondrialprotein synthesis, were unaffected (Fig. 2A). The effect ofdoxorubicin was specific for the heart muscle as the same

Figure 1. Increased mitochondrialdamage in Top1mt KO mice afterdoxorubicin (DOX) treatment. A,treatment scheme: 7-week-oldmice including paired Top1mt KOand WT mice from similar litterswere treated once a weekintraperitoneally with doxorubicinat 4 mg/kg or with saline solution(controls) for 8 weeks. One weekafter the last treatment, hearttissues were analyzed. For survivalstudy, animals were assessed upto 90 days after the last injection. B,representative ultrastructureimages of mitochondria obtainedby electron microscopy from WTand Top1mt KOmice heart tissues:left, after saline injection, and right,after doxorubicin treatment.

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respiratory chain proteins in skeletal muscle showed nodifference after doxorubicin treatment in both, WT andTop1mt KO mice (Supplementary Fig. S2A).Complex IV activity was analyzed further by measuring

cytochrome C oxidase activity in isolated heartmitochondria. Figure 2B shows that cytochrome C oxi-dase activity was decreased by 80% in Top1mt KO mice,whereas it decreased only by 20% in WT mice. As the final

electron acceptor in the electron transport chain is oxy-gen, we assessed mitochondrial respiration by measuringthe rate of oxygen consumption in isolated mitochondria.Oxygen consumption was decreased by about 50% inTop1mt KO compared with WT mice treated with doxo-rubicin (Fig. 2C). Likewise, the membrane potential inisolated mitochondria from heart tissue decreased by31% in Top1mt KO mice (Fig. 2D). As mitochondrial

Figure 2. Heart mitochondria and mtDNA alterations in Top1mt KO mice after doxorubicin (DOX) treatment. A, representative Western blot analyses ofrespiratory chain subunits in WT and Top1mt KO mice from same litters (left, control saline injections; right, after doxorubicin). Western blot analysisshows animals from the same litter. B, cytochromeC oxidase activity in heart tissue after saline injection or doxorubicin treatment inWT and Top1mt KOmice(n¼ 4 for each condition). C, oxygen consumption rates of isolatedmitochondria frommouse heart tissue after saline injection or doxorubicin treatment (n¼ 3for saline and n ¼ 5 for doxorubicin). D, mitochondrial membrane potential measured by staining isolated mitochondria from mouse heart tissue aftersaline injection or doxorubicin treatment with JC-1 (n¼ 3 for saline injection and n¼ 5 for doxorubicin treatment; �, P < 0.05; t test). E, drop in reduced GSH inmouse heart tissue lysate after saline injection or doxorubicin treatment (n¼ 5 for saline and n¼ 8 for doxorubicin; ��,P < 0.006; t test). F,mtDNA copy numberquantification in heart tissue after saline injection or doxorubicin treatment. mtDNA copy number was expressed relative toWT after saline injection, set as 1.Normalized intensity values are on a binary log scale (n¼ 6 for saline injection and n¼ 9 for doxorubicin treatments; ��,P < 0.006; t test). G, Left, representativeagarose gel images of mtDNA long fragment (Long-F) and mtDNA short fragment (Short-F) PCR products of heart tissue after saline injection or doxorubicintreatment. Top1mt KO andWT animal from the same litters were used. Right, ratio of long fragment to short fragment PCR products quantified by PicoGreen.Normalized intensity values are on a binary log scale (n ¼ 5 for saline injection and n ¼ 8 for doxorubicin treatments; ��, P < 0.006; t test).



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dysfunction generates ROS (38) that are quenched byGSH (39), we measured reduced GSH in Top1mt KOmice. Figure 2E shows that reduced GSH decreased by�80% in Top1mt KO mice, whereas this level decreasedby only �40% in WT mice following doxorubicin treat-ment (Fig. 2E).

Each mitochondrion contains several mtDNA copiesand prior observations point to the important contribu-tion of direct and/or indirect mtDNA damage in doxo-rubicin cardiotoxicity (40). Accordingly, we found thatdoxorubicin decreased mtDNA copy number both in WTand Top1mt KO mice (Fig. 2F). However, mtDNA deple-tion was significantly greater in the Top1mt KO mice (Fig.2F). Long-range PCR was also performed to evaluatemtDNA damage (41). Figure 2G shows doxorubicin-induced mtDNA damage both in WT and Top1mt KOmice. However, mtDNA damage was significantly greaterin the Top1mt KO mice. The effects of doxorubicin on the

mtDNA of WT mice are consistent with previous studies(24–27). However, we show here for first time that lack ofTop1mt accentuates mtDNA copy number loss andmtDNA damage.

Lack of Top1mt accentuates cardiomyocyte damageafter doxorubicin treatment

To further examine cardiomyocytes, cardiac sectionswere stained with fluorescein isothiocyanate-conjugatedwheat germ agglutinin, which delineate cardiomyocytedimensions by staining glycolipids and glycoproteinsenveloping individual cells (ref. 42; Fig. 3A). Doxorubicininduced hypertrophy of individual cardiomyocytes inboth WT (43) and Top1mt KO mice. However, the KOcardiomyocytes were significantly larger after doxorubicinthan those from WT mice (Fig. 3A and B). In addition,H&E staining showed an increased cardiomyocyte nuclearsize in Top1mt KO mice (Fig. 3C and D). These results

Figure 3. Doxorubicin (DOX)-induced cardiomyocytehypertrophy and fiber damage inTop1mt KOmice. A, representativeimages of cardiac right ventricularsections stained with fluoresceinisothiocyanate-conjugated wheatgerm agglutinin. B, quantitation ofmean mitochondrial area inTop1mt KO and WT hearts(�, P < 0.05; ��, P < 0.01 t test). C,representative H&E staining oflongitudinal sections of the rightventricular heart. Black arrowsindicate hypertrophic nuclei. D,quantification of nuclear sizesobtained by measuring the longerdiameter after H&E staining(��, P < 0.0001; t test). E,representative electronmicroscopy images of heartmuscle from WT and Top1mt KOmice after doxorubicin treatment.Left, red asterisks indicatedistances between individualcardiomyocytes. Right, redarrowheads indicate myofibrildefects.

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demonstrate that Top1mt activity prevents doxorubicin-induced cardiomyocytes hypertrophy.To address whether cardiomyocyte hypertrophy is

accompanied by defects in cardiac muscle at the ultrastruc-tural level, we analyzed heart tissue sections from Top1mtKO and WT mice by electron microscopy (Fig. 3E). Suchanalysis revealed prominent defects in the hearts of Top1mtKO mice, with marked structure alterations of individualmyofibrils after doxorubicin treatment. At the tissue level,the distance between individual cardiomyocytes was greaterin Top1mt KO than in WT mice (Fig. 3E, left, asterisks). Atthe intracellular level, several prominent defects in themyofibril structure could be observed. Top1mt KO miceexhibited a range ofmyofibril defects, including disintegrat-ing sarcomeres with unevenly spaced filaments "fraying"out of the myofibrils (Fig. 3E, right, arrowheads).

Top1mt inactivation increases the lethality ofdoxorubicinIn light of the accentuated heart abnormalities in the

Top1mtKO mice, we followed the survival of seven pairsof animals (Top1mt KO vs. WT) for 90 days followingthe last doxorubicin injection. None of the animalsreceiving saline died, whereas doxorubicin reduced thesurvival of both Top1mt KO and WT mice (Fig. 4).Notably, the Top1mt KO mice showed a markedly worsesurvival. All 7 Top1mt KO mice (100%) died within45 days, which is in contrast with the WT mice groupwhere only 1 of the 7 died at day 45, and 4 WT miceremained alive at day 90.

DiscussionCumulative evidence indicates the importance of mito-

chondrial dysfunction as a predisposing and potentiallycausal factor for the cardiotoxicity of doxorubicin. Ourstudy adds novel evidence for this concept, which wasrecently proposed for Parkin in a myocardial infarctionmodel (44). The difference is that Parkin is involved inmitochondrial recycling by mitophagy, whereas Top1mt isinvolved in mtDNA homeostasis (32).

Themechanismofmitochondrial toxicity of doxorubicinremains to be fully established. A recent study showingthe involvement of nuclear Top2b (22) questioned theprior notion that doxorubicin poisons mitochondria bygenerating ROS. Moreover, Top2b has been shown topresent in bovine heart mitochondria (30). However, wefound no evidence of Top2b overexpression to account forthe hypersensitivity of the Top1mt KO mice (Supplemen-tary Fig. S3A and S3B). Figure 5 outlines ourmodel explain-ing how Top2b and Top1mt exert opposite effect on doxo-rubicin-induced cardiotoxicity. Although doxorubicin trapsTop2b cleavage complexes, resulting inmitochondrialDNAdamage and dysfunction (45, 46), Top1mt protects mito-chondria (32) bymaintaining normalmtDNAhomeostasisand enabling damagedmtDNA to be replaced. Accordingly,tissue-specific mtDNA lesions, mtDNA copy loss, andabnormal arrangements of cristae have been found inhuman heart patients exposed to doxorubicin (26, 47).Mitochondrial protection is also supported as a cardiopro-tective strategy by recent evidence with mitochondriallytargeted redox active drugs in animal models (48–50).

Our study provides the first evidence that constitutivemtDNA alterations, exemplified by Top1mt deficiency,could help identify patients at risk of doxorubicin

Figure 4. Decreased survival of Top1mt-deficient mice after doxorubicin(DOX) treatment. Survival ofmice receiving doxorubicinwas assessed for90days after last treatment. Data are plotted asKaplan–Meier cumulativesurvival curves. P value was determined using the log-rank test. None ofthe control animals receiving saline died (n ¼ 7 for each condition).

Figure 5. Schematic representation of the mechanism by which Top1mtinfluences doxorubicin (DOX) cardiotoxicity. Poisoning of Top2b bydoxorubicin damages mtDNA, whereas Top1mt limits mtDNA damage.



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cardiotoxicity. Notably, we found that potentially dele-terious Top1mt variants exist in the normal population(Fig. 6).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerThe views expressed in this article are those of the authors and do not

necessarily reflect the official policy or position of the Department of HealthandHuman Services, nor doesmention of trade names, commercial products,or organizations imply endorsement by the United States Government.

Authors' ContributionsConception and design: S. Khiati, I.D. Rosa, X. Ma, V.A. Rao, L.M. Neckers,H. Zhang, Y. PommierDevelopment of methodology: V.A. Rao, H. Zhang, Y. PommierAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S. Khiati, I.D. Rosa, C. Sourbier, H. Zhang,Y. PommierAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S. Khiati, I.D. Rosa, C. Sourbier, X. Ma,V.A. Rao, L.M. Neckers, H. Zhang, Y. PommierWriting, review, and or revision of the manuscript: S. Khiati, X. Ma,V.A. Rao, L.M. Neckers, H. Zhang, Y. PommierAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Khiati, Y. PommierStudy supervision: H. Zhang, Y. Pommier

Grant SupportThis work was supported by the NIH through the Intramural Program of

the NCI, Center for Cancer Research (Z01 BC 006161).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 18, 2013; revised February 28, 2014; accepted March25, 2014; published OnlineFirst April 8, 2014.

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Figure 6. Distribution of potentially deleterious genomic amino acidchanging variants for Top1mt. Distribution of the mutations along theTop1mt polypeptide. Red: Deleteriousmissense variants. Blue: Essentialamino acids (aa) for Top1mt activity. Green: High frequent missensevariants.

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