Overexpression of CYP2J2 provides protection against doxorubicin-induced cardiotoxicity

Overexpression of CYP2J2 provides protection against doxorubicin-inducedcardiotoxicity

Yunfang Zhang,1 Haitham El-Sikhry,1 Ketul R. Chaudhary,1 Sri Nagarjun Batchu,1

Anooshirvan Shayeganpour,1 Taibeh Orujy Jukar,1 J. Alyce Bradbury,2 Joan P. Graves,2

Laura M. DeGraff,2 Page Myers,2 Douglas C. Rouse,2,4 Julie Foley,2 Abraham Nyska,2,3 Darryl C. Zeldin,2

and John M. Seubert1

1Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada; 2Division ofIntramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research TrianglePark, North Carolina; 3Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; and 4Duke University MedicalCenter, Durham, North Carolina

Submitted 9 September 2008; accepted in final form 29 April 2009

Zhang Y, El-Sikhry H, Chaudhary KR, Batchu SN, Shayegan-pour A, Jukar TO, Bradbury JA, Graves JP, DeGraff LM, Myers P,Rouse DC, Foley J, Nyska A, Zeldin DC, Seubert JM. Overexpressionof CYP2J2 provides protection against doxorubicin-induced cardiotoxic-ity. Am J Physiol Heart Circ Physiol 297: H37–H46, 2009. First pub-lished May 8, 2009; doi:10.1152/ajpheart.00983.2008.—Human cyto-chrome P-450 (CYP)2J2 is abundant in heart and active in bio-synthesis of epoxyeicosatrienoic acids (EETs). Recently, wedemonstrated that these eicosanoid products protect myocardiumfrom ischemia-reperfusion injury. The present study utilized trans-genic (Tr) mice with cardiomyocyte-specific overexpression of humanCYP2J2 to investigate protection toward toxicity resulting from acute(0, 5, or 15 mg/kg daily for 3 days, followed by 24-h recovery) orchronic (0, 1.5, or 3.0 mg/kg biweekly for 5 wk, followed by 2-wkrecovery) doxorubicin (Dox) administration. Acute treatment re-sulted in marked elevations of serum lactate dehydrogenase andcreatine kinase levels that were significantly greater in wild-type(WT) than CYP2J2 Tr mice. Acute treatment also resulted in lessactivation of stress response enzymes in CYP2J2 Tr mice (catalase750% vs. 300% of baseline, caspase-3 235% vs. 165% of baselinein WT vs. CYP2J2 Tr mice). Moreover, CYP2J2 Tr hearts exhib-ited less Dox-induced cardiomyocytes apoptosis (measured byTUNEL) compared with WT hearts. After chronic treatment,comparable decreases in body weight were observed in WT andCYP2J2 Tr mice. However, cardiac function, assessed by measure-ment of fractional shortening with M-mode transthoracic echocar-diography, was significantly higher in CYP2J2 Tr than WT heartsafter chronic Dox treatment (WT 37 � 2%, CYP2J2 Tr 47 � 1%).WT mice also had larger increases in �-myosin heavy chain andcardiac ankryin repeat protein compared with CYP2J2 Tr mice.CYP2J2 Tr hearts had a significantly higher rate of Dox metabo-lism than WT hearts (2.2 � 0.25 vs. 1.6 � 0.50 ng �min�1 �100 �gprotein�1). In vitro data from H9c2 cells demonstrated that EETsattenuated Dox-induced mitochondrial damage. Together, thesedata suggest that cardiac-specific overexpression of CYP2J2 lim-ited Dox-induced toxicity.

cytochrome P-450 2J2; heart; function

CYTOCHROME P-450 (CYP) epoxygenases are predominant en-zymes responsible for the epoxidation of endogenous arachi-donic acid (AA) to four regioisomeric epoxyeicosatrienoicacids (5,6-, 8,9-, 11,12-, and 14,15-EETs). These epoxy fatty

acid products have an important role in cellular signaling andpossess numerous biological activities in the cardiovascularsystem (54). The actions of EETs are terminated by conversionto the corresponding and less biologically active dihydroxyei-cosatrienoic acids (DHETs) by epoxide hydrolases (41). Therole of EETs in cardiovascular homeostasis and protectionagainst ischemia-reperfusion injury has been demonstrated inboth rodent and dog (19, 48). While a protective role inischemia-reperfusion injury is known, it remains to be inves-tigated whether EETs mediate myocardial protection againstother pathological stressors.

Doxorubicin (Dox, Adriamycin) is a quinone-containinganthracycline antibiotic commonly used in treatment of avariety of human neoplastic diseases and solid cancers. Itsclinical use is limited by irreversible cardiomyopathy andheart failure (11, 39). Dox-induced cardiotoxicity can becharacterized by acute myocardial injury, which occursimmediately after an initial treatment. Alternatively, chroniccardiotoxicity, which can lead to conditions such as conges-tive heart failure, ventricular dysfunction, and arrhythmia,may occur years to decades after treatment (36, 47, 55). It iswell established that the adverse effect of Dox treatment ismediated by mechanisms distinct from its therapeutic modeof action, which involves interacting with DNA by interca-lation and thereby inhibiting macromolecular biosynthesis(18). Dox-induced cardiotoxicity results from a series ofadverse reactions involving increased oxidative stress, al-teration in cellular energetics, and initiation of cell deathpathways (39, 57, 59). Numerous reports have demonstratedmoderate protection against Dox-induced cardiotoxicity, yetthe exact mechanism(s) and optimal therapy remain un-known (3, 6, 7, 9, 31).

Recently, we reported (48, 50) that enhanced postischemicrecovery of left ventricular (LV) function and reduced cardiacinjury are found in transgenic (Tr) mice that overexpresshuman CYP2J2 and in mice with targeted deletion of solubleepoxide hydrolase. Whether the overexpression of CYP2J2 canprotect against Dox-induced cardiotoxicity has not been inves-tigated. To examine the role of human CYP2J2 isoform in amodel of Dox-induced toxicity we utilized Tr mice withcardiac-specific overexpression of CYP2J2. Results of thesestudies indicate that CYP2J2 overexpression provided signifi-cant protection in both an acute and a chronic model ofDox-induced injury.

Address for reprint requests and other correspondence: J. M. Seubert, 3126Dentistry/Pharmacy Centre, Faculty of Pharmacy and Pharmaceutical Sci-ences, Univ. of Alberta, Edmonton, AB, Canada T6G 2N8 (e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 297: H37–H46, 2009.First published May 8, 2009; doi:10.1152/ajpheart.00983.2008.

http://www.ajpheart.org H37

MATERIALS AND METHODS

Animals. Mice with cardiomyocyte-specific overexpression of hu-man CYP2J2 and their wild-type (WT) littermate controls wereutilized (48). All experiments used male and female mice aged 3–5mo and weighing 25–35 g and were approved by the National Instituteof Environmental Health Sciences/National Institutes of Health andUniversity of Alberta Animal Care and Use Committees.

Treatment protocols. In the acute protocol, mice were randomlydivided into three groups and received 0, 5, or 15 mg/kg Dox byintraperitoneal injections (Fig. 1A). Mice were treated with a singledose each day for 3 days (0, 24, and 48 h) and were killed by CO2

asphyxiation on the fourth day (72 h). Hearts were either isolated andperfused in the Langendorff mode to assess heart function (see below)or collected for histological and biochemical analyses.

In the chronic protocol, mice were randomly divided into threegroups and received 0, 1.5, or 3.0 mg/kg Dox by intraperitonealinjections (Fig. 1B). Dox was administered twice a week for 5 wk fora total of 10 treatments. A 2-wk “washout” period was allowed afterthe last treatment, at which point cardiac function was assessed byechocardiography. Mice were then killed by CO2 asphyxiation, andcardiac specimens were analyzed. All studies were conducted byinvestigators who were blinded to treatment group assignments.

Biochemical analyses. At the end of each protocol, blood wasdrawn from the inferior vena cava to assess levels of lactate dehydro-genase (LDH) and creatine kinase (CK). Serum was collected within2 h from clotted blood by centrifugation and analyzed with end pointassay kits (Sigma Diagnostics, St. Louis, MO). LDH and CK activitieswere expressed as units per liter. Subcellular fractions were preparedby differential centrifugation from frozen hearts as described previ-ously (15). Catalase activity was measured with a spectrophotometricassay that monitored H2O2 disappearance at 240 nm and used themolar extinction coefficient 43.6 M�1cm�1 (2, 15). Caspase-3 activ-ity was assessed with a spectrofluorometric assay as previouslydescribed (49). Briefly, caspase-3 activity was determined in cytosolicfractions by monitoring the release of 7-amino-4-methylcoumarin(AMC) by proteolytic cleavage of the peptide Ac-DEVD-AMC (20

�M) (Sigma-Aldrich, Oakville, ON, Canada). Fluorescence was mon-itored at wavelengths of 380 nm (excitation) and 460 nm (emission).Specific activities were determined to be within the linear range of astandard curve established with AMC. Protein quantities were deter-mined with a Bradford protein assay kit (Bio-Rad Laboratories).

Isolated, perfused hearts. Hearts were perfused in the Langendorffmode as described previously (48, 50). Hearts from CYP2J2 Tr andage-/sex-matched WT littermate control animals were perfused in aretrograde fashion at constant pressure (90 cmH2O) with continuouslyaerated (95% O2-5% CO2) Krebs-Henseleit buffer at 37°C. Heartswere perfused for 40 min (stabilization) and then subjected to 20 minof global no-flow ischemia, followed by 40 min of reperfusion.Recovery of contractile function was taken as LV developed pressure(LVDP) at the end of reperfusion expressed as a percentage ofpreischemic LVDP.

Transthoracic echocardiography. Two-dimensional M-mode echo-cardiography was performed with a Vevo 770 high-resolution imag-ing system (VisualSonics) as described previously (48, 50). LVend-diastolic dimension (LVDd, mm) and LV end-systolic dimension(LVDs, mm) were assessed. Fractional shortening (FS) of the LV wasexpressed as %FS � [(LVDd � LVDs)/LVDd] � 100.

Histology and gene expression. Histological analyses were per-formed on hearts from both CYP2J2 Tr and WT mice as previouslydescribed (48). Briefly, hearts were removed, dissected, fixed in 10%neutral buffered formalin, embedded in paraffin, and sectioned forexamination. Sections were used for a terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) procedure fordetecting apoptotic cardiomyocytes as previously described (16). Thepercentage of TUNEL-positive nuclei was determined by counting 10random fields. All tissues were stored at �80°C until preparation ofRNA. Total RNA was isolated with an RNeasy Midi kit (Qiagen,Valencia, CA) and concentrated with a Microcon YM-30 column(Millipore, Billerica, MA). A formaldehyde agarose gel containingethidium bromide was used to assess the quality of the RNA. Semi-quantitative PCR analysis was performed for alterations in geneexpression. PCR primers for �-myosin heavy chain (�-MHC) were5�-GGA AGA GTG AGC GGC GCA TCA AGG-3� (forward) and5�-CTG CTG GAG AGG TTA TTC CTC G-3� (reverse); for �-myosin heavy chain (�-MHC) were 5�-GCC AAC ACC AAC CTGTCC AAG TTC-3� (forward) and 5�-TGC AAA GGC TCC AGGTCT GAG GGC-3� (reverse); for cardiac ankyrin repeat protein(CARP) were 5�-TGC GAT GAG TAT AAA CGG ACG-3� (forward)and 5�-GTG GAT TCA AGC ATA TCT CGG AA-3� (reverse); andfor glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5�-ACC ACA GTC CAT GCC ATC AC-3� (forward) and 5�-TCC ACCACC CTG TTG CTG TA-3� (reverse). Amplification was as follows:1) 50°C for 2 min; 2) 95°C for 5 min; 3) 30 cycles of 94°C for 30 s,55°C for 30 s, 72°C for 2 min; 4) 72°C for 10 min. The products werefractionated on a 2% agarose gel and visualized by ethidium bromidestaining. The ratio of �-MHC to �-MHC and CARP expression werequantitated by assessing relative cDNA levels of the genes comparedwith GAPDH expression from the same sample. Immunoblots wereprepared with S9 fractions (50 �g protein) isolated from hearts andprobed with antibodies to anti-CYP2J2pep1 (1:1,000) and GAPDH(1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA) (32, 48, 50).

Measurement of Dox metabolism. The ability of WT and CYP2J2Tr mice to metabolize Dox was evaluated in an HPLC assay. Briefly,microsomal proteins from WT and CYP2J2 Tr hearts (1 mg protein/ml) were incubated with Dox (500 nM) at 37°C for 60 min in a buffercontaining 50 mM potassium phosphate, 1.15% KCl, pH 7, and 1 mMNADPH (17, 34). Control experiments were performed with theselective P-450 epoxygenase inhibitor N-methylsulfonyl-6-(propargyl-oxyphenyl)hexanamide (MS-PPOH) (50 �M; generously provided byDr. J. Falck, University of Texas, Dallas, TX). The reactions werestopped by the addition of 300 �l of acetonitrile, and Dox wasextracted with a chloroform and 2-propanol (1:1 vol/vol) procedure,dried, and redissolved in 120 �l of methanol. Samples were injected

Fig. 1. A: acute doxorubicin (Dox) treatment protocol. B: chronic Dox treat-ment protocol.

H38 CYP2J2-MEDIATED CARDIAC PROTECTION

AJP-Heart Circ Physiol • VOL 297 • JULY 2009 • www.ajpheart.org

into a Waters 712 WISP HPLC equipped with a Schima D RF-10AXLfluorescence detector (17, 34, 38). A C18 10-�m Bondapak columnwas utilized with a formic acid (0.05%):acetonitrile gradient mobilephase in reverse mode. All products were identified based on coelu-tion with authentic standards. Standard curves prepared with Dox(0–1,000 ng/ml) were used to determine concentration differences andspecific activity (excitation 470 nm, emission 560 nm).

Mitochondrial assessment. H9c2 cells (American Type CultureCollection, Manassas, VA) were cultured and grown in DMEMsupplemented with 10% bovine serum albumin and antibiotics such aspenicillin and streptomycin at 37°C in an atmosphere of 5% CO2-95%air. Cells were loaded with 150 nM tetramethylrhodamine ethyl ester(TMRE) (Invitrogen) for 30 min and then subjected to time-lapseimaging for 60 min at 37°C and 5% CO2. A Zeiss Axio Observer Z1inverted epifluorescence microscope was used to take z-stack imagesevery minute with 200-ms exposure time. Cells were observed undera �40 objective, fluorescence was excited at the 555-nm line, andemission was recorded with a band-pass filter of 575–640 nm.Changes in fluorescence were recorded in cells treated with vehicle(0.5% ethanol in PBS), Dox (10 �M), 11,12-EET (1 or 10 �M) or14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) (1 �M; generouslyprovided by Dr. J. Falck, University of Texas). Mitochondrial mor-phology changes were visualized over the 60-min exposure period,and individual mitochondria were assessed for alterations to theelongated and filamentous appearance found in control cells. The term“punctate mitochondria” was used to describe both condensed andfragmented mitochondrial morphology as described elsewhere (35).Measurements were taken from four or five individual experiments,and intensities were quantified relative to background levels. Individ-ual mitochondria were quantified in multiple images taken at similarmagnifications with AxioVision Software (Carl Zeiss Imaging Solu-tions). Changes in fluorescence were expressed as percent changerelative to baseline levels.

Statistical analysis. Values are expressed as means � SE. Statis-tical significance was determined by the unpaired Student’s t-test andone-way ANOVA followed by a Duncan’s test to assess multiplegroup comparisons. Values were considered significant if P 0.05.

RESULTS

Effects of acute Dox administration. Serum CK and LDHactivities increased in a dose-dependent manner after threeconsecutive daily administrations of Dox in both WT andCYP2J2 Tr mice (Fig. 2, A and B). However, overexpression ofCYP2J2 resulted in lower serum CK and LDH activities afterDox treatment, indicative of reduced myocardial injury. Dox-mediated cardiotoxicity has been shown to involve productionof reactive oxygen species (ROS) (1, 8). In turn, increasedintracellular ROS activate endogenous antioxidant enzymes,such as catalase. Catalase is an important antioxidant enzymethat catalyzes the decomposition of hydrogen peroxide to waterand oxygen. Acute Dox treatment resulted in significant in-creases in cardiac catalase activities in both WT and CYP2J2Tr mice, although this increase was less pronounced in thelatter group (Fig. 2C).

Postischemic cardiac performance was decreased in Dox-treated WT mice compared with vehicle-treated mice as mea-sured by LVDP (21.8 � 5% vs. 12.3 � 3% for the 0 and 15mg/kg groups, respectively; P 0.05) (Fig. 2D). CYP2J2 Trmice exhibited improved postischemic cardiac function com-pared with WT mice, consistent with previously publishedresults (48). Importantly, no decrease in LVDP was observedin CYP2J2 Tr hearts after Dox treatment (38 � 3% vs. 36 �11% for the 0 and 15 mg/kg groups, respectively) (Fig. 2D).

Fig. 2. Acute exposure to Dox. A and B: serum creatinekinase (CK; A) and lactate dehydrogenase (LDH; B)levels after 3 consecutive daily administrations of Dox(0, 5, or 15 mg/kg); n � 6–8 per group. *P 0.05treated vs. control of the same genotype; ^P 0.05 vs.wild type (WT). CYP2J2, cytochrome P-450 2J2; Tr,transgenic. C: catalase activity in hearts from mice after3 consecutive daily administrations of Dox (0 or 15mg/kg); n � 5 per group. *P 0.05 treated vs. controlof the same genotype; ^P 0.05 vs. WT. D: postische-mic left ventricular (LV) developed pressure (LVDP)recovery at 40 min of reperfusion (R40) expressed as %of baseline LVDP in hearts from mice after 3 consecutivedaily administrations of Dox (0 or 15 mg/kg); n � 5 pergroup. *P 0.05 vs. control of the same genotype; ^P 0.05 vs. WT.

H39CYP2J2-MEDIATED CARDIAC PROTECTION


Evidence suggests that cellular apoptotic responses may betriggered subsequent to Dox exposure. Therefore, we furtherassessed cardiac injury by analyzing cytosolic fractions forcaspase-3 activity. Compared with mice treated with vehicle,caspase-3 activity was significantly higher in both WT andCYP2J2 Tr mice treated with Dox (15 mg/kg) (Fig. 3A).Importantly, caspase-3 activity was significantly higher in WThearts than in CYP2J2 Tr hearts after Dox treatment (84 � 8vs. 55 � 9 pmol �min�1 �mg protein�1, respectively; P 0.05)(Fig. 3A). Consistent with these results, significant increases inTUNEL-positive nuclei were observed in WT hearts after acutetreatment with Dox, whereas no significant changes wereobserved in CYP2J2 Tr hearts (Fig. 3B). However, similar to arecent report by Hiraumi et al. (21), we did not observe anysignificant histological changes with hematoxylin and eosinstaining on repeated blinded analyses.

Effects of chronic Dox administration. Chronic treatmentwith Dox resulted in significant dose-dependent decreases inbody weight in both WT and CYP2J2 Tr mice (Fig. 4, A andB). After Dox treatment was stopped and during the “washout”period, body weight stabilized and began to recover in allDox-treated animals. Mice were killed at the end of the

recovery period, after which heart weights were measured andcompared with changes in body weight. As shown in Fig. 4C,heart weight-to-body weight ratios increased in a dose-depen-dent manner after Dox treatment. However, the magnitude ofincrease was significantly larger in WT mice than in CYP2J2Tr mice at the highest Dox dose, suggestive of cardiac hyper-trophy.

In contrast to the acute protocol, serum CK and LDHactivities were not different in Dox-treated mice comparedwith vehicle control animals at the end of the chronic protocol(see Supplemental Data for this article).1 Additionally, nosignificant difference in caspase-3 activity was observed be-tween Dox-treated and vehicle-treated mice in the chronicstudy (see Supplemental Data). To determine whether chronicDox treatment resulted in significant changes to the cardiacstructure, we examined expression levels of MHC and CARPin WT and CYP2J2 Tr mice. Analysis for MHC gene expres-sion following chronic Dox treatment revealed an increased�-MHC-to-�-MHC ratio in WT mice but not in CYP2J2 Trmice (Fig. 4D). CARP is a transcriptional cofactor and struc-tural component of the sarcomere involved in cardiogenesisand muscle injury (46). Studies have demonstrated that Doxtreatment can induce CARP in vivo but repress CARP expres-sion in cell culture systems (5, 60). As shown in Fig. 4E,chronic Dox treatment increased CARP expression only in WTmice. Together the data indicate that CYP2J2 Tr mice had lesscardiac injury than WT mice after chronic Dox treatment.

To examine whether CYP2J2 can metabolize Dox, we in-cubated microsomes from CYP2J2 Tr and WT mouse heartsand measured Dox turnover. As shown in Fig. 4F, CYP2J2 Trmice demonstrated a significantly higher rate of Dox metabo-lism (2.2 � 0.25 ng �min�1 �100 �g protein�1) compared withWT mice (1.6 � 0.50 ng �min�1 �100 �g protein�1). Impor-tantly, the selective epoxygenase inhibitor MS-PPOH abol-ished the improved metabolism observed in CYP2J2 Tr mice(Fig. 4F). Thus the enzymatic activity was comparable in thetwo genotypes after treatment with MS-PPOH. Immunoblotanalysis demonstrated that Dox treatment did reduce the ex-pression level of CYP2J2 protein in hearts after chronic ad-ministration (Supplemental Data).

Cardiac function after chronic administration of Dox. Todetermine whether contractile function was affected by chronicDox administration, LVDd and LVDs were assessed by trans-thoracic echocardiography at the end of the recovery periodand LVFS was calculated. As shown in Fig. 5, there was nosignificant difference in these parameters between WT miceand CYP2J2 Tr mice in the vehicle control groups, indicatingthat CYP2J2 Tr mice had normal chamber dimensions andbasal contractile function. Dox treatment caused significantdose-dependent increases in both LVDd and LVDs in WT mice(Fig. 5, A and B). In contrast, there were no significant changesin LVDd or LVDs in CYP2J2 Tr mice after Dox treatment.DOX caused a reduction in %FS in WT and CYP2J2 Tr mice,although the decrease in %FS was significantly less in CYP2J2Tr mice (Fig. 5C). These data suggest that CYP2J2 Tr heartshad significantly better cardiac function than WT hearts afterchronic Dox treatment. There were no significant changes inheart rate between the groups (Fig. 5D).

1 The online version of this article contains supplemental material.

Fig. 3. Apoptotic response following acute Dox. A: cardiac caspase-3 activity.Cytosolic fractions were prepared from hearts after acute exposure to Dox (0or 15 mg/kg) and analyzed for caspase-3 activity with Ac-DEVD-AMC assubstrate; n � 5. *P 0.05 Dox vs. control of the same genotype; ^P 0.05CYP2J2 Tr vs. respective WT mice. B: terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-positive cells. Myocardium apop-tosis was assessed by TUNEL assay in heart slices after acute exposure to Dox(0 or 15 mg/kg). Values represent % increase in TUNEL-positive nuclei abovecontrol groups from the same genotype; n � 10–15 hearts per group. *P 0.05 Dox vs. control of the same genotype.



EETs limit mitochondrial damage caused by Dox in H9C2cells. Recent reports indicate that mitochondria undergo dra-matic fragmentation and dysfunction in response to Dox-induced toxicity (13, 21, 23, 43). To determine whether EETscan limit Dox injury, we investigated the mitochondrial mor-phology and membrane potential in H9c2 cells by real-timeimaging. Mitochondria, which exhibit elongated and filamen-tous morphology in healthy control cells, became dramaticallyshorter and round after Dox exposure in H9c2 cells loaded withTMRE (Fig. 6 and Fig. 7A). Dox exposure resulted in thedissipation of fluorescence from the cells within 30 min (Fig.7B), indicating changes in mitochondrial membrane potential.Pretreatment of H9c2 cells with 11,12-EET significantly atten-uated the fragmentation and conversion of tubular mitochon-dria to punctate mitochondria (Fig. 6 and Fig. 7A). Blindedquantitative analysis revealed significantly higher relative fluo-rescence intensity in cells cotreated with 11,12-EET and Doxcompared with Dox alone (Fig. 7B). Together these datasuggest that EETs attenuated mitochondrial fission andslowed the collapse of the membrane potential. To confirm

that the effect was mediated by EETs, we conducted exper-iments in the presence of 14,15-EEZE. This putative pan-EET receptor antagonist had no effect on mitochondriawhen administered alone and abrogated the effect of 11,12-EET in H9c2 cells (Figs. 6 and 7). In further analysis ofdownstream effects of mitochondrial dysfunction, Dox-induced caspase-3 activity was partially attenuated by co-treatment with EETs (Fig. 7C).

DISCUSSION

Several hypotheses have been put forth to explain the car-diotoxicity that limits the therapeutic use of Dox (37, 52, 53),including generation of free radicals in cardiomyocyte mito-chondria. There is an increasing amount of literature reportingthe functional significance of CYP monooxygenase enzymes inthe heart. This is particularly true for CYP2J2, a primarilycardiac P-450 active in the epoxidation of AA to EETs (61). Inthe present study, we demonstrate that cardiac overexpressionof the human CYP2J2 cDNA limits Dox-induced toxicity in

Fig. 4. Chronic exposure to Dox. A and B: bodyweight change in WT (A) and CYP2J2 Tr (B) mice.Animal weights were taken during the chronic Doxprotocol; n � 12–15. C: analysis of heart weight-to-body weight ratios (HW:BW) in WT andCYP2J2 Tr mice after chronic Dox treatment (0,1.5, or 3.0 mg/kg); n � 10–15. *P 0.05 DOX vs.control of the same genotype; ^P 0.05 CYP2J2Tr vs. respective WT. D: semiquantitative PCRanalysis of myosin heavy chain (MHC) gene ex-pression. �-MHC:�-MHC expression was quanti-tated by assessing relative cDNA of both genesfrom the same individual samples; n � 3. *P 0.05Dox vs. control. E: semiquantitative PCR analysisof cardiac ankyrin repeat protein (CARP) geneexpression. CARP expression was quantitated byassessing relative cDNA to GAPDH expressionfrom the same individual samples; n � 3. *P 0.05Dox vs. control. F: Dox metabolism is increased inCYP2J2 Tr compared with WT hearts and attenu-ated by the P-450 epoxygenase inhibitor N-methyl-sulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH, 50 �M). Values are means � SE; n � 4.*P 0.05 vs. WT.



mice by maintaining LV function and increased Dox metabo-lism. Moreover, our in vitro data demonstrate direct protectiveeffects of CYP2J2-derived metabolites, EETs, toward Dox-mediated mitochondrial damage.

Dox-induced injury can be indirectly monitored by therelease of CK and LDH into the serum. No differences inbaseline CK and LDH were observed between CYP2J2 Tr andWT mice, but acute Dox treatment resulted in significant

Fig. 5. Assessment of cardiac function afterchronic Dox treatment. A and B: LV end-dia-stolic dimension (LVDd, mm; A) and LV end-systolic dimension (LVDs, mm; B) after chronicDox treatment (0, 1.5, or 3.0 mg/kg); n �12–17. *P 0.05 Dox vs. control of the samegenotype; ^P 0.05 CYP2J2 Tr vs. respectiveWT. C: fractional shortening (FS) after chronicDox treatment (0, 1.5, or 3.0 mg/kg). FS of theLV is expressed as %FS � (LVDd � LVDs)/LVDd � 100; n � 12–17. *P 0.05 Dox vs.control of the same genotype; ^P 0.05CYP2J2 Tr vs. respective WT. D: heart rate(HR) after chronic DOX treatment (0, 1.5, or 3.0mg/kg); n � 12–17. bpm, beats/min.

Fig. 6. Assessment of mitochondrial mor-phology in H9c2 cells. Representativeframes from time-lapse series show H9c2cells treated with vehicle (0.5% EtOH inPBS), 14,15-epoxyeicosa-5(Z)-enoic acid(14,15-EEZE, 1 �M), Dox (10 �M), Dox(10 �M) 11,12-epoxyeicosatrienoic acid(11,12-EET, 10 �M), or Dox (10 �M) 11,12-EET (10 �M) 14,15-EEZE (1 �M)at 0, 30, and 60 min. Mitochondrial mor-phology, filamentous and tubular shape, ofthe control cells remains unaltered duringthis time period. In contrast, Dox-treatedcells exhibit significant punctate and frag-mented mitochondrial morphology, markedby arrows, which is attenuated in EET-treated cells.



increases in serum LDH and CK levels suggestive of tissuedamage, consistent with other reports (56). Importantly, theselevels were significantly lower in mice overexpressing CYP2J2than in WT mice. Although Dox toxicity occurs more selec-tively in heart than in other tissues, serum LDH and CK canarise from multiple organs. However, as CYP2J2 was only

overexpressed in cardiomyocytes of CYP2J2 Tr mice andcirculating EETs levels are similar between WT and CYP2J2Tr mice (48), the data presented here infer a reduction incardiac-specific CK and LDH as a result of CYP2J2 overex-pression.

Evidence indicates that cardiomyocyte apoptosis plays asignificant role in cardiac dysfunction in Dox-induced cardio-myopathy (39, 59). Here we observed that hearts from CYP2J2Tr mice had reduced activation of caspase-3 and reducedTUNEL-positive cells after acute Dox administration, consis-tent with reports demonstrating the antiapoptotic effects ofCYP2J2-derived EETs in other cell types (12, 26, 63). It isplausible that the increased level of EETs in the hearts ofCYP2J2 Tr mice mediated this response or that CYP2J2 wasinvolved in the metabolism of Dox. Although the exact antiapop-totic mechanism(s) of EETs is not known, it appears to involvep42/p44-MAPK and phosphatidylinositol 3-kinase/Akt path-ways (12, 26, 63). Manifestation of acute injury results infunctional decline in cardiac performance following Dox tox-icity. In this regard, cardiac dysfunction was marginally evi-dent during baseline perfusions as evidenced by decline in bothinotropy and lusitropy before ischemic insult, consistent withother reports (58). However, the cardioprotective effect ofCYP2J2 was prominent after ischemia-reperfusion, when theLVDP of Dox-treated CYP2J2 Tr mice did not differ signifi-cantly from that of vehicle-treated mice whereas Dox-treatedWT mice had a significant decline in LVDP compared withvehicle-treated mice. These data are strongly suggestive of aprotective effect of CYP2J2 in maintaining cardiac functionafter Dox administration.

To assess the influence of CYP2J2 on late events in Dox-mediated cardiotoxicity, we used a chronic Dox administrationprotocol. Our results revealed a general toxicity that occurredin both WT and CYP2J2 Tr mice and manifested as a signif-icant decrease in body weight, most likely stemming fromsevere anorexia, poor oral intake, and dehydration. Interest-ingly, Dox-induced increases in heart weight-to-body weightratios, �-MHC-to-�-MHC ratios, and CARP expression weregreater in WT mice than in CYP2J2 Tr mice. While thefunctional role of CARP is not fully understood, evidencesuggests its involvement in mechanical or stress responses,where it contributes to tissue repair (46). CARP appears tohave a role in structural organization of sarcomeres as well asin the transcriptional machinery of cardiomyocytes, striatedmuscles, and vasculature (51). Increased expression of CARPhas been observed in several cardiovascular injuries, such asLV dilated cardiomyopathy and pressure-overload hypertrophy(4, 46). Interestingly, opposing data are available regardingDox-mediated regulation of CARP expression, with increasedexpression reported in vivo (60) and repression reported invitro; these differences have been attributed to differences intiming and models utilized (46). Data presented here indicatethat cardiac overexpression of CYP2J2 resulted in maintenanceof control levels of CARP expression after chronic Dox ad-ministration, although the significance of this finding for themaintenance of overall cardiac function is unclear. Consistentwith adverse effects, there was a decrease in cardiac function inWT mice that was either not apparent (LVDd and LVDs) or notas severe (LVFS) in CYP2J2 Tr mice. As such changes havebeen well documented in various models of DOX-inducedheart failure (28, 29, 42, 44), the results found here are

Fig. 7. Assessment of mitochondrial morphology and membrane potential(��m) in H9c2 cells. A: mitochondrial morphology. Histograms represent %of punctate mitochondria present in individual cells relative to total mitochon-dria. Cells were treated with vehicle (0.5% EtOH in PBS), Dox (10 �M),14,15-EEZE (1 �M), Dox (10 �M) 11,12-EET (10 �M), or Dox (10 �M) 11,12-EET (10 �M) 14,15-EEZE (1 �M). Values represent means � SE;n � 4 or 5. *P 0.05 vs. vehicle control; ^P 0.05 vs. DoxEET treated;�P 0.05 vs. DoxEETEEZE treated. B: ��m. Histograms representing %of tetramethylrhodamine ethyl ester (TMRE) fluorescence lost in H9c2 cellsafter collapse of ��m. Cells were treated with vehicle (EtOH), Dox (10 �M),14,15-EEZE (1 �M), Dox (10 �M) 11,12-EET (10 �M), or Dox (10 �M) 11,12-EET (10 �M) 14,15-EEZE (1 �M). Values are mean � SE % changein relative fluorescence from baseline; n � 4 or 5. *P 0.05 vs. vehiclecontrol; ^P 0.05 vs. DoxEET treated. C: caspase-3 activity in H9c2 cellsusing Ac-DEVD-AMC as substrate. Cells were treated with vehicle (EtOH),Dox (10 �M), 11,12-EET (10 �M), or Dox (10 �M) 11,12-EET (10 �M)for 24 h. Values represent means � SE; n � 4 or 5. *P 0.05 vs. vehiclecontrol; ^P 0.05 vs. DoxEET treated.



indicative of a significant protection against Dox-induced car-diac dysfunction by CYP2J2 overexpression.

Evidence demonstrates that CYP enzymes are expressed inthe heart, where they may participate in the metabolism oftherapeutic agents, environmental toxicants, and endogenouscompounds (10, 14, 48). Currently limited information isavailable regarding the regulation and role CYP enzymes playin the pathophysiology of heart diseases and cardiac drugmetabolism. Our present results demonstrate that CYP2J2 Trmice can limit the Dox-induced injury. Recent studies docu-menting that P-450-derived eicosanoids can affect cardiomyo-cyte function in vitro (25, 27, 32, 33, 40, 45, 62) and protectagainst ischemia-reoxygenation injury (19, 20, 48, 50) have ledto the hypothesis that these metabolites may have importantendogenous functions in the heart. However, the reduced injuryobserved in our CYP2J2 Tr mice may be partially attributed tothe increased Dox metabolism compared with WT mice. In-teresting data from H9c2 cell culture experiments show thatDox can induce CYP enzymes, notably CYP2J2 isoforms (65).The increased turnover of Dox in CYP2J2 Tr mice suggests apotential mechanism for the reduced toxicity observed in theseanimals. Considering that CYP enzymes can produce bioactivemetabolites and metabolize foreign compounds, many impor-tant questions remain regarding the role of cardiac CYP.

Cellular excitation-contraction and mitochondrial energeticsare tightly regulated in cardiac cells to meet the high energeticflux during cardiac work. Importantly, cellular stress condi-tions can result in distinct morphological changes that reducemitochondrial dynamics influencing the energetic state of thecell (24). We recently demonstrated that a marked disorgani-zation of cardiomyocyte ultrastructure following ischemia-reperfusion was significantly reduced in CYP2J2 Tr mice;moreover, EETs can minimize adverse effects of stress onmitochondrial function (30). Mitochondria are dynamic or-ganelles that migrate through the cell and undergo continuousfusion or fission processes to maintain proper function andmeet cellular demands (22). Significant decreases in fusion orincreases in fission resulting from disease or toxicity can leadto punctate, fragmented mitochondria, which are thought toplay a critical role in cellular dysfunction and death (22, 35,64). Dox-induced toxicity can result in mitochondrial swellingand ultrastructural changes and alter function. Recently,Hiraumi et al. (21) demonstrated that Dox-induced mitochon-drial damage can begin to occur at an early phase in cardiacinjury before apoptotic changes. In the present study using anin vitro model, we demonstrate that Dox-increased mitochon-drial fragmentation and membrane depolarization occur within1 h before caspase-3 activation at 24 h in H9c2 cells. Consis-tent with our previous data, our present study demonstrates thatEETs minimize the adverse effects of Dox on mitochondrialfunction. While these results are limited to our in vitro model,the implication that EETs can attenuate formation of punctatemitochondria is of particular significance. Indeed, the factEETs can inhibit apoptotic events and maintain mitochondrialfunction highlights an interesting dichotomy, where elevatedEETs may provide benefit to reduction in cellular injury butcan also be detrimental for cancer therapy. Further studies areneeded to investigate how EETs might influence mitochondrialfission and fusion and, moreover, how this might affect in vivocardiac function and cardioprotection and what implicationsthere are for oncogenesis.

In summary, the results obtained here illustrate that cardiacCYP2J2 overexpression limits the progression of cardiac injuryand preserves cardiac function in mice after treatment withDox under two different administration protocols. Indeed, asassessed by various biochemical and functional end points, amore advanced progression toward development of cardiacinjury was observed in WT mice compared with CYP2J2 Trmice after treatment with Dox. These studies are the first todocument a protective effect of CYP2J2 in Dox-induced car-diotoxicity and may have implications for treatment of cardiacinjury.

GRANTS

This work was supported by a Canadian Institutes of Health Research Grant(MOP79465, J. M. Seubert) and by the Intramural Research Program of theNational Institute of Environmental Health Sciences (Z01-ES-025034). J. M.Seubert is the recipient of a New Investigator Award from the Heart and StrokeFoundation of Canada and a Health Scholar Award from the Alberta HeritageFoundation for Medical Research.

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Overexpression of CYP2J2 provides protection against doxorubicin-induced cardiotoxicity

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