Glutathione peroxidase 1-deficient mice are more susceptible todoxorubicin-induced cardiotoxicity

Jinping Gao1, Ye Xiong2,3, Ye-Shih Ho2,3, Xuwan Liu1, Chu Chang Chua1, Xingshun Xu1,Hong Wang1, Ronald Hamdy1, and Balvin H.L. Chua1

1Cecile Cox Quillen Laboratory of Geriatric Research, James Quillen College of Medicine, East TennesseeState University, James H. Quillen Veterans Affairs Medical Center, Johnson City, TN 37614

2Institute of Environmental Health Sciences, Wayne State University, Detroit, MI 48201

3Department of Biochemistry and Molecular Biology, Wayne State University, Detroit, MI 48201

AbstractDoxorubicin (DOX)-induced cardiotoxicity is thought to be mediated by the generation of superoxideanion radicals (superoxide) from redox cycling of DOX in cardiomyocyte mitochondria. Reductionof superoxide generates H2O2, which diffuses throughout the cell and potentially contributes tooxidant-mediated cardiac injury. The mitochondrial and cytosolic glutathione peroxidase 1 (Gpx1)primarily functions to eradicate H2O2. In this study, we hypothesize that Gpx1 plays a pivotal rolein the clearance of H2O2 generated by DOX. To test this hypothesis, we compared DOX-inducedcardiac dysfunction, mitochondrial injury, protein nitration, and apoptosis in Gpx1-deficient and wildtype mouse hearts. The Gpx1-deficient hearts showed increased susceptibility to DOX-induced acutefunctional derangements than wild type hearts, including impaired contractility and diastolicproperties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impairedthe mitochondrial function of Gpx1-deficient hearts. Specifically, Gpx1-deficient hearts treated withDOX demonstrated an increased rate of NAD-linked state 4 respiration and a decline in the P/O ratiorelative to wild type hearts, suggesting that DOX uncouples the electron transfer chain and oxidativephosphorylation in Gpx1-deficient hearts. Finally, apoptosis and protein nitration were significantlyincreased in Gpx1-deficient mouse hearts compared to wild type hearts. These studies suggest thatGpx1 plays significant roles in protecting DOX-induced mitochondrial impairment and cardiacdysfunction in the acute phase.

Keywordsdoxorubicin; glutathione peroxidase deficiency; mitochondrial function; cardiac function; apoptosis;protein nitration

1. IntroductionDoxorubicin (DOX) is an anthracycline antibiotic that is widely used as a chemotherapeuticagent. However, the administration of DOX is known to induce numerous cardiotoxic effects,including transient arrhythmias, nonspecific electrocardiographic abnormalities, pericarditis,

Correspondence and requests for materials should be addressed to B.H.L. Chua (chua@mail.etsu.edu).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

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Published in final edited form as:Biochim Biophys Acta. 2008 October ; 1783(10): 2020–2029. doi:10.1016/j.bbamcr.2008.05.027.

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and acute heart failure (1,2). DOX can also cause congestive heart failure months or years aftertreatment. The mechanism of DOX-induced cardiac injury has been actively investigated, andseveral hypotheses have been suggested to explain the acute and chronic cardiotoxicity ofDOX. These include the formation of free radicals, inhibition of enzymes and proteins, changesin cardiac muscle gene expression, alterations of mitochondrial membrane function,sensitization of Ca2+ release from sarcoplasmic reticulum channels, mitochondrial DNAdamage and dysfunction (3), and induction of apoptosis (4,5).

The free radical hypothesis of DOX-induced cardiotoxicity has gained significant support (6,7). DOX is known to generate free radicals either by redox cycling between a semi-quinoneform and a quinone form or through the formation of a DOX-Fe3+ complex (8). Molecularoxygen is reduced to superoxide anion (O2

−), which is converted to other forms of reactiveoxygen species such as hydrogen peroxide (H2O2) and hydroxyl radical (OH·). It ishypothesized that free radicals induce damage to the heart, an organ that is known to have arelatively low level of antioxidant enzymes such as superoxide dismutase (SOD) and catalase(9). In support of this hypothesis, DOX-induced cardiotoxicity can be inhibited by theoverexpression of antioxidant enzymes MnSOD and catalase (10,11).

MnSOD, which is located in mitochondria, specifically reduces superoxide to H2O2. In turn,H2O2 is decomposed by the enzymes catalase, glutathione peroxidase, and peroxiredoxin inmammalian cells (12,13). Catalase is located in the peroxisomes of cells (14), so diffusion ofH2O2 from other subcellular sites is necessary. The major isoform of glutathione peroxidase,glutathione peroxidase 1 (Gpx1), is present in both mitochondria and the cytosol (15). Thelocation and low Km of Gpx1 for H2O2 may facilitate its ability to detoxify the H2O2 that isgenerated in the mitochondria of heart following DOX treatment (16,17). It has been shownthat an early and persistent decrease in Gpx1 after DOX treatment may contribute to DOX-induced cardiotoxicity (18,19). Treatment with probucol increases cardiac Gpx1 activity,decreases lipid peroxidation, and improves mortality (18). All of these lines of evidence suggestthat there Gpx1 may play a role in minimizing the cardiotoxicity of DOX.

If Gpx1 indeed does minimize DOX-induced cardiotoxicity, then it follows that Gpx1deficiency would render hearts more susceptible to DOX-induced cardiotoxicity. To explorethis hypothesis, cardiac performance was measured in a DOX-treated isolated mouse heartpreparation as well as DOX-treated mouse hearts in vivo. In addition, this study measured themitochondrial function, amount of apoptosis, and level of protein nitration in the hearts ofDOX-treated Gpx1-dificient mice and their wild type littermates.

2. Materials and Methods2.1. Materials

DOX HCl (2 mg/ml) was purchased from Bedford Laboratories (Bedford, OH). An ApopTagIn Situ Oligo Ligation kit was obtained from Chemicon International (Temecula, CA). All otherreagents were of the highest grades commercially available.

2.2. Animal studiesGpx1 knockout mice (Gpx1−/−) were generated as described previously (20). Gpx1 is thepredominant form of the Gpx isozymes, and little to no Gpx activity was detected in tissues ofhomozygous knockout mice. In the heart, Gpx activity in knockout mice is less than 10% ofthe total Gpx activity in wild type mice (20). Mice were maintained in AAALAC-accredited,climate-controlled facilities. Wild type Gpx1+/+ and Gpx1−/− mice (4–5 month old, in a C57BL/6 and 129SV mixed genetic background) were randomly assigned to four groups: Gpx1+/+,Gpx1+/+ + DOX, Gpx1−/− mice, or Gpx1−/− mice + DOX. A single dose of DOX (22.5 mg/kg,

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i.p.) or an equivalent volume of saline was injected. These animals were free of pathogensduring the entire course of experiments. All the animal protocols were approved by the EastTennessee State University Animal Committee.

2.3. Isolation of cardiac mitochondria and measurement of mitochondrial respiratory andphosphorylating activities

Mouse cardiac mitochondria were isolated, and their respiratory and phosphorylating acivitiesmeasured according to the methods described previously (21). Respiratory control index (RCI)is defined as the ratio of the state 3 respiratory rate to the state 4 respiratory rate. ADP/O ratio(P/O ratio) was determined by dividing the amount (nmoles) of ADP added by the amount(natoms=nanoatoms) of oxygen consumed during state 3 respiration (22).

2.4. In vitro cardiac function measurementIn vitro mouse cardiac function was analyzed in a modified Langendorff perfusion model aspreviously described (23). Briefly, age- and gender-matched Gpx1+/+ and Gpx1−/− mice wereinjected with anticoagulant sodium heparin (500 units/kg, i.p.) 20 min prior to anesthetizationwith sodium pentobarbital (120 mg/kg, i.p.). Following anesthetization, mouse hearts wererapidly removed, cannulated, and perfused retrogradely at 37°C with a Krebs-Henseleitbicarbonate buffer. After 30 min perfusion to stabilize the cardiac function, mouse hearts wereperfused with the buffer in the presence or absence of DOX for another 60 min. The leftventricular pressure trace was continuously recorded through a pressure transducer connectedto a computerized data acquisition system (DATAQ Instruments, Inc., Akron, OH). Thecoronary effluent was collected and measured every 15 min. Cardiac function parameters,including heart rate (HR), left ventricular develop pressure (LVDP), and maximumdevelopment of pressure over time (±dP/dt), were analyzed from the recorded data with asoftware.

2.5. Analysis of in vivo cardiac functionA single dose of DOX (22.5 mg/kg, i.p.) or an equivalent volume of saline was injected. Fivedays after DOX or saline administration, mice were injected with heparin (500 units/kg, i.p.)and anesthetized with 2% isoflurane. Each mouse was intubated with a 22-gauge soft catheterand ventilated with a rodent ventilator (Columbus Instruments International Corp., Columbus,OH) at a tidal volume of 0.3–0.5 ml and a respiratory rate of 120 breaths/min. After leftthoracotomy, the pericardium was dissected to expose the heart. A microtip pressure–volumecatheter (SPR-839; Millar Instruments, Houston, TX) was inserted through a 25-gauge apicalstab into the left ventricle to measure steady state cardiac function. To change the cardiacpreload, the inferior vena cava was occluded for 1 second. At the completion of the study, 10µl of hypertonic saline (15%) was injected into the right atrium to calibrate Vp, the parallelvolume. The signals were continuously recorded at a sampling rate of 1000 s−1 using an ARIApressure-volume conductance system (Millar Instruments) coupled to a Powerlab/4SP A/Dconverter (AD Instruments, Mountain View, CA). All pressure–volume loop data wereanalyzed with a cardiac pressure–volume analysis program (PVAN3.4; Millar Instruments).At the end of function analysis, animals were anesthetized with tribromoethanol (270 mg/kg,i.p.). Hearts were removed and perfused for 2 min as Langendorff preparations to remove theremaining blood. Portions of the mid-ventricle were fixed in 4% buffered formaldehyde for 24h, dehydrated, and embedded for immunological and apoptosis studies.

2.6. ISOL analysisA 2-mm section of mid-ventricles was sliced, fixed in 4% buffered formaldehyde for 24 h,dehydrated, and embedded. Myocardial sections (5 µm) were mounted on siliconized slidesand dried at 37°C overnight. In situ staining of DNA strand breaks in the serial section of each

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specimen was detected by the ApopTag ISOL (In Situ Oligo Ligation) kit using oligo A aspreviously described (24). The percentage of ISOL-positive myocytes was determined bycounting 10 random fields per section under an Olympus BX40 microscope.

2.7. ImmunohistochemistryImmunostaining of nitrotyrosine in mouse hearts was performed using monoclonal anti-nitrotyrosine antibody at a dilution of 1:200 (Cayman, Ann Arbor, MI) at 4°C overnight.Antigen-antibody complexes were detected by the Super-sensitive alkaline phosphatase kit(BioGenex; San Ramon, CA) using fast red as a chromogen. Hematoxylin was used as acounterstain. Images were captured using an SPOT camera and transferred into research-baseddigital image analysis software (Image Pro Plus; Media Cybernetics, Silver Spring, MD). Theextent of immunoreactivity in the LV was determined in the tissues by applying intensitythresholding analysis (25). The sum density of total LV image pixels in the 80-to-155 rangewas used as a semi-quantitative measure of relative immunoreactivity.

2.8. Statistical AnalysisAll values were expressed as mean ± SEM. One-way ANOVA was used followed by Tukey’smultiple comparison tests if there were significant differences between groups. Significanceof differences between two groups was established by Student’s t-test. Significance wasindicated if P<0.05.

3. ResultsMouse hearts deficient in Gpx1 are more susceptible to DOX-induced cardiac dysfunction

In vitro cardiac function was analyzed in a mouse heart perfusion model to determine the roleof Gpx1 in protecting hearts against DOX-induced cardiotoxicity. Our study found that DOXcaused significantly more cardiac dysfunction in Gpx1-deficient (Gpx1−/−) mouse hearts thanin wild type or Gpx−/− mice. However, as shown in Table 1, there was no difference in thebasal levels of hemodynamic indices of cardiac function between the wild type mice andGpx1−/− mice; however, perfusion with 5 µM DOX for 60 min resulted in both inotropic andchronotropic dysfunction. As shown in Fig. 1, hearts from Gpx1−/− mice showed a 44%suppression of +dP/dt (maximum rate of pressure development during systole), a 47%suppression of −dP/dt (maximum rate of pressure development during diastole), and a 34%suppression of LVDP (left ventricular developing pressure) following exposure to DOX for60 min, compared to a 14% suppression of +dP/dt, a 12% suppression of −dP/dt, and a 10%reduction of LVDP from Gpx1+/+ mice. The chronotropic index of cardiac function HRdecreased 9% in Gpx1+/+ hearts and 34% in Gpx1−/− mouse hearts following exposure to DOXfor 60 min (P< 0.05 compared to that in the Gpx1+/+ mice). With HR×LVDP, which measuresboth inotropic and chronotropic changes, homozygous Gpx1−/−mouse hearts displayed a 49%reduction vs. a 14% reduction in Gpx1+/+ mice.

The above data indicate that DOX induces significantly greater cardiac dysfunction inGpx1−/− mice. In addition, the coronary flow rate during DOX perfusion was reduced from43% in Gpx1−/− to 13% in the Gpx1+/+ mice, suggesting that the decrease of cardiac perfusionis either due to vascular constriction or endothelial damage following exposure to DOX.Overall, the in vitro cardiac function study suggests that knockout of the Gpx1 gene leads tosignificantly compromised cardiac systolic and diastolic function following exposure to DOX.

To further validate our in vitro study findings, we investigated cardiac function in DOX-treatedGpx1−/− mice and their wild type littermates with a sophisticated in vivo functional study. Assummarized in Table 2, there was no difference between the initial body weight and cardiacfunction of Gpx1+/+ mice and Gpx1−/− mice. DOX-treated mice showed numerous changes

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after 5 days of treatment. First, DOX treatment caused weight loss in both Gpx1−/− mice andGpx1+/+ mice, but the weight loss was significantly greater in Gpx1−/− mice. Second, DOXtreatment led to profound suppression of systolic cardiac function in both Gpx1−/− miceGpx1+/+ mice, but the suppression was significantly higher in Gpx1−/− mice. Indeed, ejectionfraction was significantly lower in Gpx1−/− mice compared to Gpx1+/+ mice (41.8 ± 1.71%vs. 51.2 ± 1.01%, P<0.05), which was the result of a greater suppression of end-systolicventricular pressure (60.5 ± 2.68 mmHg vs. 67.1 ± 1.69 mmHg, P<0.05) and a greater elevationof end-systolic volume (9.19 ± 0.18 µl vs. 7.83 ± 0.26 µl, P<0.05). The pressure developmentrate of the left ventricle (+dP/dt) was significantly reduced in Gpx1−/− mice compared to theGpx1+/+ group (4406 ± 331 mmHg/s vs. 5,566 ± 111 mmHg/s, P<0.05), which in returnaccounted for the greater suppression in preload recruitable stroke work (PRSW) in Gpx1−/−

mice (44.5 ± 3.26 vs. 62.2 ± 2.96 mm Hg).

Finally, Gpx1−/− mice showed significantly worse diastolic function following DOX treatmentthan Gpx1+/+ mice. The end-diastolic volume was more reduced in Gpx1−/− mice comparedto Gpx1+/+ mice (14.87 ± 0.50 µl vs. 16.02 ± 0.25 µl, P<0.05). LV diastolic dysfunction wasalso more impaired in Gpx1−/− mice, as evidenced by the suppressed maximal rate of LVpressure decreasing (5,599 ± 142 mmHg/s vs. 3504 ± 336 mmHg/s, P<0.05) and elevated timeconstant of isovolumic relaxation (7.96 ± 0.26 ms vs. 10.26 ± 0.64 ms). It is worthwhile tomention that although the slope of end-diastolic pressure-volume relationship (β) is lower inboth Gpx1−/− and Gpx1+/+ mice following DOX treatment, we did not find a significantdifference between the DOX-treated Gpx1−/− and Gpx1+/+ and saline-treated controls. DOXhad a significant effect on peripheral vascular resistance (Ea) in Gpx1−/− mice, but not inGpx1+/+ mice.

In summary, DOX treatment produced a profound impairment on cardiac performance in bothGpx1−/− and Gpx1+/+ mice, but the impairments were more pronounced in Gpx1−/− mice.

Mouse hearts deficient in Gpx1 are more susceptible to DOX-induced mitochondrialdysfunction

Mitochondria are the primary subcellular organelle in which DOX-induced generation of ROSis observed. Therefore, mitochondria are a direct target of oxidative stress. We measured theefficiency of mitochondrial respiration in mouse hearts one day after DOX administration toelucidate the biochemical abnormalities that occur in cardiac mitochondria in response to DOXtreatment. We utilized NAD-linked substrates (glutamate and malate) in the measurement ofmitochondrial respiration. DOX treatment had no effect on state 3 respiration in cardiacmitochondria of either wild type or Gpx1−/− (Fig. 2A). However, DOX caused a 64% increasein the rate of state 4 respiration in cardiac mitochondria of Gpx1−/− mice as compared to saline-treated counterparts. However, this was not observed in cardiac mitochondria of wild type mice(Fig. 2B).

RCI is derived from the ratio of the rate of state 3 respiration to the rate of state 4 respiration.Thus, a change in the state 4 respiratory rate would lead to an altered RCI. RCI of cardiacmitochondria from Gpx1−/− mice treated with DOX was 56% of the RCI of saline-treated mice(Fig. 2C). Although the rates of state 3 and state 4 respiration in cardiac mitochondria of wildtype mice were statistically unaffected by DOX treatment, a relatively modest decrease (26%)of RCI was observed in these samples.

The decrease in RCI of Gpx1−/− mitochondria in response to DOX treatment was greater thanwild type mitochondria (p<0.001). DOX treatment also decreased the P/O ratio in wild typeor Gpx1−/− mice. The P/O ratio of the latter samples was lower than the former (p < 0.001)(Fig. 2D).

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The effect of DOX on mitochondrial respiration with FAD-linked substrate (succinate) wasnot as dramatic as the effect with NAD-linked substrates. DOX treatment did not alter the ratesof state 3 and state 4 respiration and RCI in wild type and Gpx1−/− mice (Fig. 3A–C). The P/O ratio of Gpx1−/− cardiac mitochondria, but not wild type mitochondria, was decreased by20%, as compared to mitochondria isolated from saline-treated counterparts (Fig. 3D).

These studies demonstrate that DOX treatment impairs mitochondrial respiration in hearts ofwild type and Gpx1−/− mice. However, the extent of functional decline in cardiac mitochondriaof Gpx1 knockout mice is significantly increased.

Gpx1-deficient hearts undergo enhanced apoptosisApoptosis is observed in cultured cardiomyocytes and hearts of DOX-treated animals (4,5).The ISOL method, which is more specific for double-stranded DNA breaks during apoptosis,was used to identify apoptotic myocytes in DOX-treated mouse hearts. The results showed that9 ± 0.8% of cardiomyocytes in DOX-treated Gpx1+/+ mice were ISOL-positive (Fig. 4). Incontrast, there were 15 ± 0.64% ISOL-positive cardiomyocytes in DOX-treated mouseGpx1−/− hearts.

Mouse hearts deficient in Gpx1 display increased protein nitrationProtein nitration was investigated by the immunostaining of 3-nitrotyrosine (3-NT) in heartsections of DOX-treated Gpx1+/+ and Gpx1−/− mice and controls. Diffused cytoplasmicstaining was observed throughout the heart sections. The immunostaining density of 3-NT wasanalyzed using a digital image system. The background of 3-NT staining was negligible inboth Gpx1+/+ and Gpx1−/− control mouse hearts (data not shown). DOX caused elevatedimmunostaining of 3-NT in mouse hearts; and the density was three-fold higher inGpx1−/−hearts relative to Gpx1+/+ hearts (Fig. 5B, 5C). Little staining was observed in theabsence of primary nitrotyrosine antibody (Fig. 5A)

4. DiscussionFree radicals mediate DOX-induced cardiotoxicity. However, a minimal set of studies haveinvestigated the role of the antioxidant enzyme Gpx1 in DOX-induced cardiotoxicity. Wehypothesized that if Gpx1 plays a critical role in preventing DOX-induced cardiotoxicity, thenmouse hearts overexpressing this enzyme will exhibit a resistant phenotype. Conversely, wehypothesized that mouse hearts deficient in this enzyme will exhibit a susceptible phenotype.We previously demonstrated that overexpression of Gpx1 in the heart attenuated cardiacdysfunction and improved mitochondrial complex respiration activity (21). Our current studydemonstrates that Gpx1 deficiency dramatically exacerbates DOX-induced in vivo cardiacdysfunction. We also show that DOX-induced impairment in mitochondrial respiratory activitywas more profound in mice lacking Gpx1. It may be possible that impaired mitochondrialactivity leads to increased H2O2, increased protein nitration, and ultimately cardiomyocyteapoptosis.

Our in vitro studies demonstrated that Gpx1 partially reversed the DOX-induced deficits,including impaired contractility and diastolic function, decreased coronary flow rate, andreduced HR (Fig. 1). The toxic effects of DOX have been observed on perfused rat hearts inother studies (26,27). Although the mechanism responsible for these alterations of cardiacfunction is uncertain, it has been previously shown that OH· formation in DOX-perfused rathearts is involved in the toxic response (7). Free radicals are known to have a detrimental effecton endogenous vascular regulators, such as nitric oxide (NO), a strong vascular dilator.Coronary vascular constriction or endothelial damage could play a role as well. Pelikan et al.has found that DOX elevates coronary resistance in DOX-perfused rat heart (27), which may

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explain why the coronary flow decreased in DOX-perfused Gpx1−/− mouse hearts but not inwild type mouse hearts in our study. It is likely that the cardioprotective effects of Gpx1 canbe primarily attributed to its antioxidant activity. However, it should be noted that heart ratewas significantly decreased following exposure to DOX in our in vitro cardiac function study,which was done in the absence of pacing. Reduction of heart rate likely has a negative impacton cardiac function and contractility due to underperfusion of cardiac muscle. The exactmechanism of DOX cardiotoxicity would ideally be explored in an isolated and paced workingheart, although to date, this has never been technically achieved in mouse hearts.

Our in vivo cardiac function study shows that DOX significantly decreased CO, EF, and PRSW,indicating heart failure in these mice. DOX treatment induced both systolic and diastolicdysfunction, which was magnified in Gpx1−/− mice. DOX–induced decrease in dP/dtmax-Vedsuggests an effect on the calcium cycling proteins during isovolumic contraction, while DOX–induced decrease in τ suggests an effect on SERCA2a and/or phospholamban duringisovolumic relaxation. An increase in Ea suggests that arterial vascular resistance is affectedby DOX in Gpx1−/− mice. There was no change in β in DOX-treated mice, suggesting thatthere is no effect on the cardiac extracellular matrix during the passive filling phase.

Mitochondria are the major intracellular ATP generation sites; their swelling and destructionin human and animal hearts following exposure to DOX are well documented in many studies(28,29). To further explore the possible role of Gpx1 in mitochondrial damage, we assessedmitochondrial respiration function from DOX- or saline-treated mouse hearts. Our studiesshowed that DOX treatment significantly compromises the efficiency of mitochondrialrespiration in hearts of both wild type and Gpx1 knockout mice. However, the functionaldecline of cardiac mitochondria in knockout mice was significantly more severe, suggestingthat Gpx1 mediates mitochondrial protection subsequent to DOX-induced damage.

The effect of DOX on cardiac mitochondrial respiration has been previously reported in anumber of reports. The size of the effect varies according to differences in the dosage of DOXtreatment, the genetic background of the mice, and the species (mice vs. rats) used in the studies(30–34). In our study, DOX at 22.5 mg/kg had no effect on NAD- or FAD-linked state 3respiration in mitochondria of either wild type or Gpx1 knockout mice, suggesting that DOXdoes not affect the rate of ATP synthesis. Nonetheless, DOX treatment caused a more profounduncoupling of oxidative phosphorylation from the electron transport chain in NAD-linkedrespiration in mitochondria of Gpx1-deficient hearts compared to those of normal hearts, assuggested by the decreased P/O ratios of these samples. The uncoupling was also observed inFAD-linked respiration in mitochondria of Gpx1-deficient hearts but not wild type hearts.Finally, it should be noted that the rate of NAD-linked state 4 respiration was significantlyincreased upon DOX administration in cardiac mitochondria of Gpx1 knockout mice, but notwild type mice. This increased state 4 respiratory rate suggests that mitochondria cannotmaintain a proper chemiosmotic gradient, possibly because DOX treatment significantlydamages the mitochondrial inner membrane. On the other hand, FAD-linked state 4 respirationwas not affected in Gpx1-deficient cardiac mitochondria, indicating that the DOX-inducedincrease in the rate of state 4 respiration is substrate-specific. The mechanisms by whichsubstrates affect state 4 respiration remain to be determined.

In our study, we showed increased protein nitration in Gpx1-deficient mice, suggesting thatDOX-induced ROS exert their deleterious effects partly by increasing protein nitration.Previous studies have shown that DOX treatment of the heart raises nitric oxide levels byincreasing iNOS expression (35). NO and superoxide anion react to generate peroxynitrite, apotent oxidant that partially mediates DOX-induced cardiotoxicity by causing nitrosative stresson proteins, lipids, and nucleic acids (36). Mitochondria are particularly sensitive targets forperoxynitrite-induced toxicity, which may lead to their impaired function resulting in further

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production of superoxide and H202. Pacher et al. established a pivotal role of peroxynitrite inDOX-induced cardiac dysfunction by demonstrating that FP-15, a peroxynitrite decompositioncatalyst, protected against protein nitration and cardiac dysfunction (37). Furthermore, bothH202 and peroxynitrite can induce DNA damage and lead to the overactivation of the nuclearenzyme PARP-1, which may also contribute to DOX-induced cardiotoxicity (38).

DOX-induced reactive oxygen species and reactive nitrogen species also activate matrixmetalloproteinases, such as MMP-2 and MMP-9 (39,40). These matrix degrading enzymeshave been associated with cardiac failure. Whether DOX treatment leads to higher MMP-2 orMMP-9 levels in Gpx1−/− mouse hearts warrants further investigation.

Several groups have reported that cardiomyocyte apoptosis is involved in cardiac injuryinduced by anthracycline antibiotics (4,5,41–43). Peroxynitrite has been found to causeapoptosis in cardiomyocytes (44). As shown in Fig. 4, Gpx1 hearts displayed a significantincrease in ISOL-positive apoptosis. These results support a recent study that showed thatoxidative stress is involved in DOX-induced apoptosis and that this stress can be inhibited byGpx1.

In summary, our study demonstrates that Gpx1 deficiency renders hearts more susceptible toDOX-induced cardiotoxicity. This cardiotoxic effect involves mitochondrial dysfunction, anincrease in apoptosis, and an increase in protein nitration. These findings suggest a therapeuticrole for agents such as probucol (18) and melatonin (45,46), both of which increase Gpx1activity in the heart. This possibility warrants further study.

AcknowledgementsWe are grateful for Dr. Douglas F. Larson, University of Arizona, for his technical advice on the LV loop analysis.This study was supported by NIH grant HL087271, a grant from the Department of Veterans Affairs Merit Reviewand a grant-in-aid from the American Heart Association Southeast Affiliate to B.H.L.C.; and by American HeartAssociation Midwest Affiliate grants 0455876Z and 0655631Z to Y.-S. H. The use of equipment in the Imagine andCytometry Facility Core was supported by a Center grant P30 ES06639.

References1. Billingham ME, Mason JW, Bristow MR, Daniels JR. Anthracycline cardiomyopathy monitored by

morphologic changes. Cancer Treat. Rep 1978;62:865–872. [PubMed: 667860]2. Bristow MR, Thompson PD, Martin RP, Mason JW, Billingham ME, Harrison DC. Early anthracycline

cardiotoxicity. Am. J. Med 1978;65:823–832. [PubMed: 707541]3. Olson RD, Mushlin PS. Doxorubicin cardiotoxicity: analysis of prevailing hypotheses. FASEB J

1990;4:3076–3086. [PubMed: 2210154]4. Arola OJ, Saraste A, Pulkki K, Kallajoki M, Parvinen M, Voipio-Pulkki LM. Acute doxorubicin

cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res 2000;60:1789–1792. [PubMed:10766158]

5. Kumar D, Kirshenbaum L, Li T, Danelisen I, Singal P. Apoptosis in isolated adult cardiomyocytesexposed to adriamycin. Ann. N. Y. Acad. Sci 1999;874:156–168. [PubMed: 10415529]

6. Monti E, Prosperi E, Supino R, Bottiroli G. Free radical-dependent DNA lesions are involved in thedelayed cardiotoxicity induced by adriamycin in the rat. Anticancer Res 1995;15:193–197. [PubMed:7733633]

7. Rajagopalan S, Politi PM, Sinha BK, Myers CE. Adriamycin-induced free radical formation in theperfused rat heart: implications for cardiotoxicity. Cancer Res 1988;48:4766–4769. [PubMed:2842038]

8. Davies KJ, Doroshow JH. Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracyclineradical formation by NADH dehydrogenase. J. Biol. Chem 1986;261:3060–3067. [PubMed: 3456345]

Gao et al. Page 8

Biochim Biophys Acta. Author manuscript; available in PMC 2009 October 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

9. Doroshow JH, Locker GY, Myers CE. Enzymatic defenses of the mouse heart against reactive oxygenmetabolites: alterations produced by doxorubicin. J. Clin. Invest 1980;65:128–135. [PubMed:7350193]

10. Kang YJ, Chen Y, Epstein PN. Suppression of doxorubicin cardiotoxicity by overexpression ofcatalase in the heart of transgenic mice. J. Biol. Chem 1996;271:12610–12616. [PubMed: 8647872]

11. Yen HC, Oberley TD, Vichitbandha S, Ho Y-S, St Clair DK. The protective role of manganesesuperoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J. Clin.Invest 1996;98:1253–1260. [PubMed: 8787689]

12. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol. Rev1979;59:527–605. [PubMed: 37532]

13. Rhee SG, Chae HZ, Kim K. Peroxiredoxins: a historical overview and speculative preview of novelmechanisms and emerging concepts in cell signaling. Free Radic. Biol. Med 2005;38:1543–1552.[PubMed: 15917183]

14. van der Bosch H, Schutgens RB, Wanders RJ, Tager JM. Biochemistry of peroxisomes. Annu. Rev.Biochem 1992;61:157–197. [PubMed: 1353950]

15. Esworthy RS, Ho Y-S, Chu F-F. The Gpx1 gene encodes mitochondrial glutathione peroxidase in themouse liver. Arch. Biochem. Biophys 1997;340:59–63. [PubMed: 9126277]

16. Flohé L, Loschen G, Gunzler WA, Eichele E, Glutathione peroxidase V. The kinetic mechanism.Hoppe Seylers Z. Physiol. Chem 1972;353:987–999.

17. Makino N, Mochizuki Y, Bannai S, Sugita Y. Kinetic studies on the removal of extracellular hydrogenperoxide by cultured fibroblasts. J. Biol. Chem 1994;269:1020–1025. [PubMed: 8288557]

18. Li T, Singal PK. Adriamycin-induced early changes in myocardial antioxidant enzymes and theirmodulation by probucol. Circulation 2000;102:2105–2110. [PubMed: 11044428]

19. Sazuka Y, Tanizawa H, Takino Y. Effect of adriamycin on the activities of superoxide dismutase,glutathione peroxidase and catalase in tissues of mice. Jpn. J. Cancer Res 1989;80:89–94. [PubMed:2496064]

20. Ho Y-S, Magnenat JL, Bronson RT, Cao J, Gargano M, Sugawara M, Funk CD. Mice deficient incellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia. J.Biol. Chem 1997;272:16644–16651. [PubMed: 9195979]

21. Xiong Y, Liu X, Lee C-P, Chua BHL, Ho Y-S. Attenuation of doxorubicin-induced contractile andmitochondrial dysfunction in mouse heart by cellular glutathione peroxidase. Free Radic. Biol Med2006;41:46–55. [PubMed: 16781452]

22. Lee CP, Sciamanna M, Peterson PL. Intact rat brain mitochondria from a single animal: Preparationand properties. Meth. Toxicol 1993;2:41–49.

23. Liu X, Chen Z, Chua CC, Ma YS, Youngberg GA, Hamdy R, Chua BH. Melatonin as an effectiveprotector against doxorubicin-induced cardiotoxicity. Am. J Physiol. Heart Circ. Physiol2002;283:H254–H263. [PubMed: 12063298]

24. Chen Z, Chua CC, Ho Y-S, Hamdy RC, Chua BH. Overexpression of Bcl-2 attenuates apoptosis andprotects against myocardial I/R injury in transgenic mice. Am. J. Physiol. Heart Circ. Physiol2001;280:H2313–H2320. [PubMed: 11299236]

25. Mihm MJ, Coyle CM, Jing L, Bauer JA. Vascular peroxynitrite formation during organic nitratetolerance. J. Pharmacol. Exp. Ther 1999;291:194–198. [PubMed: 10490904]

26. Ganey PE, Carter LS, Mueller RA, Thurman RG. Doxorubicin toxicity in perfused rat heart.Decreased cell death at low oxygen tension. Cir. Res 1991;68:1610–1613.

27. Pelikan PC, Weisfeldt ML, Jacobus WE, Miceli MV, Bulkley BH, Gerstenblith G. Acute doxorubicincardiotoxicity: functional, metabolic, and morphologic alterations in the isolated, perfused rat heart.J. Cardiovasc. Pharmacol 1986;8:1058–1066. [PubMed: 2429080]

28. Lambertenghi-Deliliers G, Zanon PL, Pozzoli EF, Bellini O. Myocardial injury induced by a singledose of adriamycin: an electron microscopic study. Tumori 1976;62:517–528. [PubMed: 1020054]

29. Lefrak EA, Rosenheim JS, Gottlieb JA. A clinicopathologic analysis of adriamycin cardiotoxicity.Cancer 1973;32:302–314. [PubMed: 4353012]

30. Childs AC, Phaneuf SL, Dirks AJ, Philips T, Leewenburgh C. Doxorubicin treatment in vivo causescytochrome C release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency,

Gao et al. Page 9

Biochim Biophys Acta. Author manuscript; available in PMC 2009 October 1.

NIH

-PA Author Manuscript

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-PA Author Manuscript

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-PA Author Manuscript

superoxide dismutase activity, and Bcl-2:Bax ratio. Cancer Res 2002;62:4592–4598. [PubMed:12183413]

31. Kawasaki S, Akiyama S, Kurokawa T, Kataoka M, Dohmitsu K, Kondoh K, Yamauchi M, Ito K,Watanabe T, Sugiyama S, Ozawa T, Matsuyama M, Takagi H. Polyoxyethylene-modified superoxidedismutase reduces side effects of adriamycin and mitomycin C. Jpn. J. Cancer Res 1992;83:899–906.[PubMed: 1399827]

32. Matsumura M, Nishioka K, Fuijii T, Yoshibayashi M, Nozaki K, Nakata Y, Temma S, Ueda T,Mikawa H. Age-related acute adriamycin cardiotoxicity in mice. J. Mol. Cell. Cardiol 1994;26:899–905. [PubMed: 7966358]

33. Praet M, Pollakis G, Goormaghtigh E, Ruysschaert JM. Damages of the mitochondrial membrane inadriamycin treated mice. Cancer Lett 1984;25:89–96. [PubMed: 6097353]

34. Yen H-C, Oberley TD, Gairola CG, Szweda LI, St Clair DK. Manganese superoxide dismutaseprotects mitochondrial complex I against adriamycin-induced cardiomyopathy in transgenic mice.Arch. Biochem. Biophys 1999;362:59–66. [PubMed: 9917329]

35. Weinstein DM, Mihm MJ, Bauer JA. Cardiac peroxynitrite formation and left ventricular dysfunctionfollowing doxorubicin treatment in mice. J. Pharmacol. Exp. Ther 2000;294:396–401. [PubMed:10871338]

36. Pacher P, Bechkman JS, Liaudet L. Nitric Oxide and peroxynitrite in health and disease. Physiol. Rev2007;87:315–424. [PubMed: 17237348]

37. Pacher P, Liaudet L, Bai P, Mabley JG, Kaminski PM, Virág L, Deb A, Szabó E, Ungvári Z, WolinMS, Groves JT, Szabó C. Potent metalloporphyrin peroxynitrite decomposition catalyst protectsagainst the development of doxorubicin-induced cardiac dysfunction. Basic Sci. Rep 2003;107:896–904.

38. Pacher P, Liaudet L, Bai P, Virag L, Mabley JG, Haskó G, Szabó C. Activation of poly(ADP-ribose)polymerase contributes to development of doxorubicin-induced heart failure. J. Pharmacol. Exp. Ther2002;300:862–867. [PubMed: 11861791]

39. Bai P, Mabley JG, Liaudet L, Virag L, Szabó C, Pacher P. Matrix metalloproteinase activation is anearly event in doxorubicin-induced cardiotoxicity. Oncol. Rep 2004;11:505–508. [PubMed:14719091]

40. Kizaki K, Ito R, Okada M, Yoshioka K, Uchide T, Temma K, Mutoh K, Uechi M, Hara Y. Enhancedgene expression of myocardial matrix metalloproteinases 2 and 9 after acute treatment withdoxorubicin in mice. Pharm. Res 2006;53:341–346.

41. Wang GW, Klein JB, Kang YJ. Metallothionein inhibits doxorubicin-induced mitochondrialcytochrome c release and caspase-3 activation in cardiomyocytes. J. Pharmacol. Exp. Ther2001;298:461–468. [PubMed: 11454906]

42. Zhu W, Zou Y, Aikawa R, Harada K, Kudoh S, Uozumi H, Hayashi D, Gu Y, Yamazaki T, Nagai R,Yazaki Y, KomuroI I. MAPK superfamily plays an important role in daunomycin-induced apoptosisof cardiac myocytes. Circulation 1999;100:2100–2107. [PubMed: 10562267]

43. Mukhopadhyay P, Bátkai S, Rajesh M, Czifra N, Harvey-White J, Haskó G, Zsengeller Z, GerardNP, Liaudet L, Kunos G, Pacher P. Pharmacological inhibition of CB1 cannabinoid receptor protectsagainst doxorubicin-induced cardiotoxicity. J. Am. Coll. Cardiol 2007;50:528–536. [PubMed:17678736]

44. Levrand S, Vannay-Bouchiche C, Pesse B, Pacher P, Fiehl F, Waeber B, Liaudet L. Peroxynitrite isa major trigger of cardiomyocyte apoptosis in vitro and in vivo. Free Rad. Biol. Med 2006;41:886–895. [PubMed: 16934671]

45. Dziegiel P, Murawska-Cialowicz E, Jethon Z, Januszewska L, Podhorska-Okolow M, Surowiak P,Zawadzki M, Rabczynski J, Zabel M. Melatonin stimulates the activity of protective antioxidativeenzymes in myocardial cells of rats in the course of doxorubicin intoxication. J Pineal Res2003;35:183–187. [PubMed: 12932202]

46. Pablos MI, Agapito MT, Gutierrez R, Recio JM, Reiter RJ, Barlow-Walden L, Acuna-Castroviejo D,Menendez-Pelaez A. Melatonin stimulates the activity of the detoxifying enzyme glutathioneperoxidase in several tissues of chicks. J Pineal Res 1995;9:111–115. [PubMed: 8750343]

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Fig. 1.Effect of DOX on the cardiac function and coronary flow rate of hearts from Gpx1−/− and wildtype mice. Mouse hearts were perfused in vitro with Krebs-Henseleit buffer as described inMaterials and Methods. Cardiac function parameters are expressed as the recovery percentageafter exposure to buffer or DOX compared to the basal levels measured in the first 30-minperfusion. Open and filled bars represent mouse hearts perfused with Krebs-Henseleit bufferonly (as control) and Krebs-Henseleit buffer containing 5 µM DOX, respectively. Values aremean ± SEM of six hearts. Empty bars, hearts perfused with buffer only; filled bars, heartsperfused with buffer containing DOX. a, P < 0.01 vs. the same measurement of mouse heartsperfused with the buffer, or the Gpx1+/+ mouse hearts perfused with DOX.

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Fig. 2.Gpx1-deficient mouse hearts are more susceptible to DOX-induced declines in NAD-linkedelectron transfer and energy coupling capacities in mitochondria. (A) DOX treatment had noeffect on NAD-linked state 3 respiration in cardiac mitochondria of either wild type or Gpx1knockout mice. (B) However, the same treatment caused an increase in the rate of state 4respiration in cardiac mitochondria of Gpx1 knockout mice, but not wild type mice. a, p <0.001 vs. saline-treated Gpx1 knockout mice and saline- or DOX-treated wild type mice. Theefficiency of NAD-linked oxidative phosphorylation in cardiac mitochondria, as determinedby RCI (C) and P/O ratio (D), was more severely affected in by DOX treatment in Gpx1knockout mice than in wild type mice. In (C), b indicates p < 0.001 vs. saline-treated wild typemice. In (D), c indicates p < 0.01 vs. saline-treated wild type mice. In (C) and (D), a indicatesp < 0.001 vs. saline-treated Gpx1 knockout and saline- or DOX-treated wild type mice. Valuesare mean ± SEM for all figures. N ≥ 4 mice for each experiment.

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Fig. 3.Effect of Gpx1 deficiency on FAD-linked electron transfer and energy coupling capacities incardiac mitochondria of wild type and Gpx1 knockout mice in response to DOX treatment.Gpx1 deficiency had no effect on FAD-linked state 3 respiration (A), state 4 respiration (B),and RCI (C) of cardiac mitochondria from either wild type or Gpx1 knockout mice. The P/Oratio of cardiac mitochondria from Gpx1 knockout mice, but not wild type mice, decreasedafter DOX treatment (D). In (D), a indicates p < 0.001 vs. saline-treated Gpx1 knockout andsaline- or DOX-treated wild type mice. Values are mean ± SEM for all figures. N ≥ 4 mice foreach experiment.

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Fig. 4.Effect of DOX on apoptosis in hearts from Gpx1−/− mice. ISOL procedure was performed inparaffin-embedded myocardial sections as described in Materials and Methods. Representativestaining pattern of hearts from Gpx1+/+ (A), DOX + Gpx1+/+ (B), Gpx1−/− (C), and DOX +Gpx1−/− were shown. Arrows indicate ISOL-positive myocytes. Immunolabeled nuclei ofmyocytes were determined by random counting of 10 fields per section. Each bar representsmean ± SEM of six hearts. a=P<0.05, DOX vs. respective control mice, b=P<0.05, DOX +Gpx1−/− mice vs. DOX + Gpx1+/+ mice.

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Fig. 5.Immunohistochemical staining of mouse hearts with anti-nitrotyrosine antibody. Sections fromDOX + Gpx1+/+ (panel B) or DOX + Gpx1−/− mouse hearts (panel C) were stained with anti-tyrosine antibody. Staining pattern of DOX + Gpx1−/− heart in the absence of specific antibodywas shown in Panel A. Semi-quantitative analysis was performed using Image Pro Plus. Eachbar represents mean ± SEM of 4 hearts. a=P<0.05, Gpx1−/− mice + DOX vs. Gpx1+/+ mice +DOX.

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Table 1Basal levels of cardiac parameters of perfused mouse hearts

Gpx1+/+ Gpx1+/++DOX Gpx1−/− Gpx1−/−+DOXHR (beats/min) 278±10 278±17 270±10 287±14LVDP (mmHg) 75.3±4.5 82.4±2.2 80.4±1.5 76.8±2.3

+dP/dt (mmHg/sec) 2882±271 2580±100 2527±208 2617±99−dP/dt (mmHg/sec) 1665±176 1612±72 1644±136 1773±93

HR×LVDP 22943±1377 23612±903 21769±1418 23471±587CFR (ml/min) 1.38±0.10 1.28±0.05 1.29±0.07 1.27±0.09

Values represent the cardiac functional parameters before exposure to buffer only or buffer containing DOX and are expressed as mean ± SEM of sixmouse hearts. Measurements of the maximum rates of pressure development (± dP/dt), left ventricular developed pressures (LVDP), heart rates (HR), andcoronary flow rate (CFR) were made after 30 min of preliminary perfusion. There is no significant difference among all groups.

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Gao et al. Page 18Ta

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