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Cardiovascular Research 2006 69(1):186-197; doi:10.1016/j.cardiores.2005.07.012
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Copyright © 2005, European Society of Cardiology

The protective roles of nitric oxide and superoxide dismutase in adriamycin-induced cardiotoxicity

Marsha P. Colea, Luksana Chaiswingb,c, Terry D. Oberleyb, Stephanie E. Edelmannd, Michael T. Piascikd, Shu-Mei Lina, Kinsley K. Kininghame and Daret K. St. Claira,*

aGraduate Centers for Toxicology and Nutritional Sciences, University of Kentucky, Lexington, Kentucky, 464 Health Sciences Research Building, Lexington, KY 40536, USA
bDepartment of Pathology, VA Hospital, University of Wisconsin, Madison, Wisconsin 53705, USA
cFaculty of Medical Technology, Mahidol University, Bangkok, Thailand 10700
dDepartment of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, Kentucky 40536, USA
eDepartment of Pharmacology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25704, USA

* Corresponding author. Tel.: +1 859 257 3956; fax: +1 859 323 1059. Email address: dstcl00{at}pop.uky.edu

Received 7 February 2005; revised 28 June 2005; accepted 14 July 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Treatment with adriamycin (ADR) is associated with cardiotoxicity mediated through the generation of superoxide (O2·–). Because nitric oxide (·NO) reacts with O2·–, generating peroxynitrite, we hypothesized that decreased ·NO production would lead to protection in acute cardiac injury.

Methods: We investigated the role of decreased ·NO levels in exacerbation of ADR-induced cardiotoxicity in vivo using iNOS (–/–) mice. Pathology, biochemical injury markers, and cardiac function were used to assess ADR-induced cardiac injury.

Results: Ultrastructural analysis demonstrated that iNOS (–/–) mice exhibited extensive cytoplasmic swelling and degeneration of mitochondria when compared to wildtype mice following treatment with ADR. Mice lacking iNOS exhibited a decrease in resting indices of cardiac function as well as an impairment in the positive inotropic actions of isoproterenol following treatment with ADR compared to nTg mice. Cardiac troponin, creatine phosphokinase, and lactate dehydrogenase levels were significantly increased after treatment in iNOS (–/–) mice as compared to controls and wildtype mice.

Conclusions: These results indicate that a lack of ·NO production by iNOS caused significantly enhanced cardiac injury. However, when iNOS (–/–) mice were crossed with manganese superoxide dismutase (MnSOD)-overexpressing animals, mitochondrial injury was ameliorated to the level of the wild type. These findings suggest that reduction of ·NO levels mediated by ADR treatment leads to increased cardiac mitochondrial injury that can be attenuated by a compensatory increase in MnSOD.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Adriamycin (ADR) is used to treat a wide range of malignant tumors. One limitation of this drug is an associated dose-dependent cardiac toxicity. Olson and Capen first showed cardiac damage in mitochondria and endoplasmic reticulum following ADR treatment using electron microscopy [1,2]. Although it is perhaps one of the mechanisms of ADR's efficacy, a free radical mediated theory has been proposed to explain ADR-induced cardiac injury [3]. The quinone moiety of this compound is reduced through enzymatic and non-enzymatic mechanisms with concurrent generation of O2·– [4].

Previously we demonstrated that transgenic mice over-expressing human MnSOD (hMnSOD) were protected against ADR-induced myocardial injury when compared to nTg mice [5]. This finding suggests that O2·– radicals formed in mitochondria are related to ADR-induced cardiac injury. We have also reported that inactivation of mitochondrial complex I by ADR-induced O2·– production was prevented by overexpression of hMnSOD [7].

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine through a five electron reduction, ending with formation of L-citrulline and subsequent production of ·NO. Under certain circumstances, such as arginine and essential co-factor deficiencies, as well as the binding of ADR to the reductase domain of NOS III, uncoupling of the enzyme occurs, allowing for production of O2·– [8]. Inducible NOS has been shown to be expressed in all nucleated cells; this enzyme has been identified in the cardiovascular system and its level is regulated by different stimuli [9]. Inducible NOS can be up-regulated through inflammatory cytokines such as interferon gamma or tumor necrosis factors, and oxidative stress [10]. It has been suggested that regulation of iNOS occurs predominantly at transcriptional levels, but studies report that both post-transcriptional and post-translational regulation is possible [11].

Physiologic responses to ·NO in the cardiovascular system include increase in [or modulation of] excitation–contraction coupling, myocardial relaxation and diastolic function, the Frank–Starling response, heart rate, β-adrenergic inotropic response, and myocardial energetics and substrate metabolism [12]. Aside from the essential role as a modulator of physiological myocardial function, ·NO is known to exhibit antioxidant-like actions in lipid bilayers [13–16]. The limitation of diffusion of ·NO in a lipid bilayer is dependent upon its reaction with cellular antioxidants, oxidants, and other free radicals [17].

Because iNOS is thought to be involved in the pathophysiological response to acute tissue injury, in the present study, we used an acute model to study the mechanism of ADR-induced heart injury. We hypothesized that iNOS deficiency would reduce ADR-induced cardiac injury. Unexpectedly, we found that the absence of iNOS caused an exacerbation of cardiac injury as demonstrated by both ultrastructural pathology and biochemical criteria. Moreover, this injury was circumvented by increasing the endogenous level of MnSOD.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Generation of mice deficient in iNOS and overexpressing MnSOD (iNOS (–/–) and iNOS (–/–)–TgM (+/+))
All animal experiments in this study were performed with the approval of the University of Kentucky Animal Care and Use Committee and in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals. Inducible NOS knock-out mice purchased from Jackson Laboratories (Bar Harbor, ME) in the C57BL/6 background were bred into the B6C3 background further than 10th generation. The medium expressing line (TgM (+/–)) [5] was bred to obtain mice with homozygous MnSOD overexpression (TgM (+/+)). The protein and activity levels of MnSOD are increased in transgenic mice as previously demonstrated [5,6]. The iNOS (–/–)–TgM (+/+) cross was generated by sequential selection and back-crossing between iNOS (–/–) and TgM (+/+) mice as described by Chaiswing et al. [6].

2.2 Southern blot analysis of iNOS (–/–) and iNOS (–/–)–TgM (+/+) mice
Genomic DNA was isolated from mouse tail, digested (BamHI or PstI), and separated on 0.7% or 0.9% agarose as previously described [18]. Following transfer, blots were hybridized using 32P labeled iNOS or hMnSOD cDNA as previously described [5].

2.3 Electron microscopy
Heart tissue was isolated in ice cold phosphate buffered saline (PBS), cut into small 1 mm3 pieces, and immediately fixed in half strength Karnovskys fixative and analyzed as previously described [5].

2.4 Animal treatment and tissue preparation
Male mice, ages 8–10 weeks old, received one intraperitoneal injection (i.p.) (20 mg/kg) of ADRIAMYCIN PFS® (Pharmacia and Upjohn, Kalamazoo, MI) or saline. After 3 days, mice were anesthetized using Nembutal® sodium solution (65 mg/kg) (Abbott Laboratories, North Chicago, IL). The heart was excised, washed in ice cold PBS, and immediately frozen in liquid nitrogen. Frozen tissue was ground using a mortar and pestle prior to homogenization.

2.5 Measurement of cardiac function in intact heart preparations
Cardiac contractility was determined using an isolated perfused heart preparation with Krebs–Hensleit buffer as previously described, 3 days following treatment with ADR [19].

2.6 Creatine phosphokinase (CPK) and lactate dehydrogenase activity (LDH)
CPK and LDH activity in serum was analyzed as previously described [5].

2.7 Western blot analysis
Heart tissue homogenate (100 µg) was electrophoresed using SDS-PAGE method as previously described [20]. An affinity purified rabbit anti-GAPDH antibody (1:3000) was purchased from Trevigen (Gaithersburg, MD) and monoclonal mouse anti-cardiac troponin I (TnI) antibody was obtained from Advanced ImmunoChemical (Long Beach, CA).

2.8 Griess Assay
Serum nitrate levels were determined as previously described [6].

2.9 Real time PCR
Total tissue RNA, 6 h following ADR treatment, was isolated with Trizol (1 heart/1 mL) and further purified using the RNeasy Mini, RNA isolation kit (Qiagen, Valencia, CA). Complimentary DNA was obtained using oligo(dT) primers (Clontech Laboratories, Palo Alto, CA) and was stored at –80 °C. Primers are listed below:
Annealing number of
Gene Temp., °C Cycles Primer Sequence
iNOS 55 40 5'-CTGATGGTCAAGATCCAGAGGTCT-3'
3'-CTGCATGTGCTTCATGAAGGACTCT-5'
GAPDH 50 20 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3'
3'-CATGTAGGCCATGAGGTCCACCAC-5'

LightCycler-DNA Master SYBR Green I (Roche, Basel, Switzerland) was used with LightCycler 3.5.3 software to analyze results. Gene expression was normalized with mRNA expression of GAPDH. Samples were analyzed in triplicate using N=3 for each murine genotype and treatment.

2.10 Statistical analysis
Multiple comparisons for each variable were performed using analysis of variance (ANOVA) with the post hoc Fisher's or Tukey's test. When applicable, the unpaired t-test was used to test the significance of each pre-planned comparison. Data was considered statistically significant for P ≥ 0.05. All data is presented as mean ± SEM.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Ultrastructural analysis
Previous studies have established that treatment with ADR induced mitochondrial injury [1,2,5]. To investigate the role of iNOS in ADR-induced injury, mice were treated with a single injection (20 mg/kg) of ADR and heart tissues were examined following a 5-day period. Ultrastructural pathology (Fig. 1) revealed that cardiac muscle from C57BL/6 mice (Fig. 1a) and nTg (B6C3) mice (Fig. 1c) treated with saline had normal morphology with normal mitochondria and prominent myofilaments. Heart tissues from C57BL/6 (Fig. 1b) and nTg (B6C3) mice (Fig. 1d) treated with ADR demonstrated mild cytoplasmic swelling, myelin figures, mitochondria with focal loss of cristae, and focal disorganization of myofilaments. Additionally, nTg (B6C3) mice treated with ADR showed some mitochondria with loss of subcellular structure. Cardiac muscle from homozygous iNOS knockout mice (C57BL/6) treated with saline (Fig. 1e) showed normal morphology with typical mitochondria and myofilaments. However, cardiac tissues from iNOS (–/–) mice (C57BL/6) treated with ADR (Fig. 1f) demonstrated extensive cytoplasmic swelling, focal disruption of mitochondria and myofilaments, as well as extensive degeneration of mitochondria; injury changes observed were more extensive than those observed in nTg mice.


Figure 1
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  		Fig. 1 Inducible NOS (–/–) mice demonstrated increased ultrastructural damage following ADR treatment compared to nTg. (a, b) C57BL/6 mice. Cardiac muscle from mice treated with saline (a) showed normal morphology with unremarkable mitochondria (M) and myofilaments (Myo). Cardiac muscle from mice treated with ADR (b) demonstrated mild cytoplasmic swelling (asterisk), myelin figures (Mye), mitochondria (M) with focal loss of cristae, and focal disorganization of myofilaments (Myo). (c, d) nTg (wildtype B6C3) mice. Cardiac muscle from mice treated with saline (c) showed normal morphology with unremarkable mitochondria (M) and myofilaments (Myo). Cardiac muscle from mice treated with ADR (d) exhibited mild cytoplasmic swelling (asterisk), mitochondria (M) with focal loss of cristae, focal disorganization of myofilaments (Myo), and some mitochondria with loss of subcellular structure (arrow). (e, f) Homozygous iNOS knockout (C57BL/6) mice. Cardiac muscle from mice treated with saline (e) showed normal morphology with unremarkable mitochondria (M) and myofilaments (Myo). Cardiac muscle from mice treated with ADR (f) demonstrated extensive cytoplasmic swelling (asterisk), focal disruption of mitochondria (M) and myofilaments (Myo), as well as extensive degradation of mitochondria (arrows).

 
3.2 Cardiac function in response to isoproterenol following in vivo treatment with ADR
To determine if these structural changes resulted in an impairment of cardiac function, contractile activity was assessed in the isolated perfused heart. Neither the resting heart rate nor the positive chronotropic actions of isoproterenol were significantly altered in ADR treated iNOS (–/–) and nTg mice (Fig. 2a). Wildtype mice treated with ADR showed expected increases in measures of contractile function. Increases in LVDP were observed following infusion of increasing amounts of isoproterenol in nTg control hearts. However, in iNOS deficient mice, a decrease in resting LVDP was observed. Additionally, the ability of isoproterenol to promote increases in these recorded parameters was significantly reduced in iNOS (–/–) mice (Fig. 2b).


Figure 2
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  		Fig. 2 The ability of isoproterenol to increase contractile force was significantly decreased in hearts from iNOS (-/-) mice as compared to nTg following ADR treatment, with no difference in heart rate. (a) Beats/min in 8–10 weeks old nTg ({blacksquare}) and iNOS (–/–) ({square}) mice treated with ADR (n ≥ 4 mice in each group). Data are expressed as mean ± SEM. b. Left ventricular developed pressure (LVDP) in 8–10 weeks old nTg ({blacksquare}) and iNOS (–/–) ({square}) mice treated with ADR (n ≥ 4 mice in each group). Data are expressed as mean ± SEM. *P<0.05 for nTg-ADR versus iNOS (–/–)-ADR for dose of isoproterenol.

 
3.3 CPK, LDH, and troponin serum levels
To verify the consequence of iNOS deficiency on ADR-induced cardiac injury, the levels of biochemical markers including CPK, LDH, and cTnI were assessed. Fig. 3 shows that increased levels of CPK, LDH, and cTnI were detected in serum 3 days following ADR treatment in nTg and iNOS (–/–) mice (P ≥ 0.0001). Serum CPK was increased to a larger degree in iNOS (–/–) mice treated with ADR than nTg mice (P ≥ 0.05) (Fig. 3a). Compared to their respective controls, nTg and iNOS (–/–) mice had significant increases in serum LDH after ADR (P ≥ 0.01 and P ≥ .001) (Fig. 3b). Inducible NOS (–/–) mice demonstrated a greater serum LDH release following treatment with ADR as compared to nTg treated mice (P ≥ 0.01). Using an antibody raised against cTnI, the levels were measured in the serum. Western analysis revealed that nTg mice treated with ADR had a slight increase in cTnI, but they were not found to be statistically significant. Serum from iNOS (–/–) mice treated with ADR showed a statistically significant increase in cTnI (P ≥ 0.0001) as compared to saline control (Fig. 3c).


Figure 3
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  		Fig. 3 CPK, LDH, and cTnI were more significantly elevated in iNOS (–/–) mice as compared to nTg following ADR treatment. (a) Serum creatine phosphokinase (CPK) in 8–10 weeks old wt (nTg) and iNOS (–/–) mice treated with ADR (n=4 mice in each group). Data are expressed as mean ± SEM. *P ≥ 0.0001 versus saline for mice of the same genotype. (b) Serum lactate dehydrogenase (LDH) in 8–10 weeks old wt (nTg) and iNOS (–/–) mice treated with ADR (n=4 mice in each group). Data are expressed as mean ± SEM. *P ≥ 0.01 versus saline for wt (nTg) mice. **P ≥ 0.001 versus saline for iNOS (–/–) mice. (c) Serum cardiac troponin in 8–10 weeks old wt (nTg) and iNOS (–/–) mice treated with ADR for 3 d (n=4 mice in each group). Data are expressed as densitometric mean ± SEM. *P ≥ 0.0001 versus saline for iNOS (–/–) mice.

 
3.4 eNOS protein, serum nitrate levels, and iNOS mRNA gene expression
Adriamycin has been reported to induce eNOS transcription; therefore, Western blot analysis was used to determine whether treatment leads to a compensatory increase in eNOS levels in nTg and mice lacking iNOS [21]. After normalization with actin, there was no change in the level of eNOS protein in nTg or iNOS (–/–) mice at 1, 3, 6, 12, 24, or 72 h following ADR treatment. Fig. 4a shows the typical results at 72 h.


Figure 4
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  		Fig. 4 Serum nitrate levels and iNOS mRNA expression were significantly elevated in nTg, but not iNOS (–/–) mice, with no compensatory increase in eNOS protein after treatment with ADR. (a) Protein extracts isolated from both nTg (wildtype) and iNOS (–/–) mice were analyzed by Western blot using an eNOS antibody three days after ADR treatment (n=5 mice in each group). Quantitation did not reveal any significant differences (data not shown). (b) Serum nitrate concentration (µM) following time course treatment with ADR (n=6 mice for each time point) was determined. nTg (wildtype) mice are represented by the black bars and iNOS (–/–) mice are represented by the white bars. Values are expressed as mean ± SEM. *P ≥ 0.05 versus saline for wt (nTg) mice. (c) Transcript levels of iNOS 6 h following treatment with ADR (n=3 mice for each group) was determined. Mice receiving saline or ADR are represented by the black or white bars, respectively. Values are expressed as fold increase from saline control after normalization with GAPDH transcript levels. *P ≥ 0.0001 versus saline for wt (nTg) mice.

 
The Griess reaction was used to assess the levels of total nitrate and nitrite generated after treatment with ADR. Using a time course study, serum nitrate levels were increased by 1 h following ADR treatment in nTg mice. Significant increases (P ≥ 0.05) at 6, 24, and 72 h following treatment in nTg mice were observed (Fig. 4b). There was no increase at any time point in the level of serum nitrates in iNOS (–/–) mice.

The mRNA level of iNOS was assessed 6 h following treatment with ADR. There was a significant increase (P ≥ 0.0001) in iNOS transcript levels in nTg mice following treatment with ADR as compared to saline-nTg mice (Fig. 4c). There were no significant differences in iNOS mRNA levels in iNOS (–/–), TgM (+) (mice transgenic for MnSOD), and iNOS (–/–)–TgM (+) mice after ADR compared to their respective controls. Both iNOS (–/–) and iNOS (–/–)–TgM (+) mice lacked appreciable iNOS mRNA expression compared to nTg and TgM (+) mice.

3.5 Assessment of reactive oxygen species (ROS)/reactive nitrogen species (RNS) modified proteins
The increases in serum nitrate levels suggest that ADR treatment can lead to an increase in ·NO levels, therefore we detected a product of ·NO with O2·–, ONOO–, which reacts with protein tyrosine residues. Time course studies using Western blot analysis revealed that there was an increase in nitrotyrosine adducted proteins (~40 kDa, one band) between 1 and 3 h following ADR treatment in the nTg mice (Fig. 5a). These increases seen in nTg mice were absent in ADR-treated iNOS (–/–) mice. Quantitation of nitrotyrosine adducted proteins revealed that nTg mice had significant increases at 3, 6, and 12 h (Fig. 5b).


Figure 5
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  		Fig. 5 Nitrotyrosine protein adducts increased in nTg, but not iNOS (–/–) mice after ADR treatment. (a) Protein extracts analyzed by Western blot using an antibody detecting nitrotyrosine (n=3 mice for each time point). Protein extracts were co-hybridized with an antibody detecting GAPDH to assess equal loading of the samples. (b) The autoradiographs of nitrotyrosine levels (a) were quantitated and normalized to GAPDH. nTg (wildtype) mice are represented by the black bars and iNOS (–/–) mice are represented by the white bars. Values are expressed as fold increases from respective saline control. *P ≥ 0.05 versus saline for wt (nTg) mice.

 
3.6 Expression of MnSOD ameliorated ADR-induced cardiotoxicity in iNOS (–/–) mice
Following a 5-day period after receiving one 20 mg/kg dose of ADR, heart tissues from all four genotypes were analyzed for ultrastructural changes (Fig. 6). No pathological changes were found in the heart tissues of saline treated nTg mice (Fig. 6a), overexpressing MnSOD mice (TgM (+/+)) (Fig. 6c), iNOS (–/–) (Fig. 6e), or the cross between MnSOD transgenic and iNOS (–/–) mice (TgM+iNOS) (Fig. 6g). Cardiac tissues from nTg, iNOS (–/–) and TgM+iNOS mice treated with ADR showed increased myocardial damage (Fig. 6b, f, h) compared to saline controls, with changes consisting of mitochondria with myelin figures, mitochondria with loss of cristae, intracytoplasmic vacuolization, and edema. Inducible NOS (–/–) mice treated with ADR (Fig. 6f) showed more extensive ultrastructural changes than other groups. In contrast, TgM (+/+) mice (Fig. 6d) had only mild ultrastructural changes compared to the other ADR treated mouse groups.


Figure 6
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  		Fig. 6 Overexpression of MnSOD ameliorated ADR-induced cardiotoxicity in iNOS (–/–) mice. nTg (wildtype B6C3) mice treated with saline or ADR (a, b), overexpression MnSOD mice (TgM B6C3) treated with saline or ADR (c, d), iNOS (–/–) (B6C3) treated with saline or ADR (e, f), cross breed between overexpression MnSOD and iNOS knockout mice (TgM+iNOS) (B6C3) treated with saline or ADR (g, h). M indicates normal mitochondria, N indicates nucleus, Myo indicates myofibril, I indicates intracytoplasmic edema, V indicates intracytoplasmic vacuolization, arrow indicates mitochondria with myelin figures, and arrowhead indicates mitochondria with the loss of cristae. No pathological changes were found in the heart tissues of experimental mice that were treated with saline (a, c, e, and g). nTg, iNOS and TgM+iNOS mice showed increased myocardial damage (b, f, h), with changes consisting of mitochondria with myelin figures, mitochondria with loss of cristae, intracytoplasmic vacuolization, and edema. Inducible NOS mice treated with ADR (f) showed more extensive ultrastructural changes than other groups. In contrast, TgM mice (d) had only mild ultrastructural changes (loss of cristae) compared to the other ADR treated mouse groups.

 
3.7 MnSOD overexpression enhances cardiac contractility in response to isoproterenol
In order to determine if cardiac function correlated with ultrastructural findings, LVDP was determined as a response to isoproterenol. Fig. 7a and b demonstrates that heart rate and LVDP was not altered in control animals (saline). Treatment with ADR did not alter heart rate in response to isoproterenol (Fig. 7c). However, in mice lacking iNOS there was a decrease in LVDP (Fig. 8d). Importantly, overexpression of MnSOD in mice lacking iNOS restored cardiac function as a measure of LVDP (Fig. 7d).


Figure 7
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  		Fig. 7 The ability of isoproterenol to increase contractile force was significantly improved in hearts from mice lacking iNOS which overexpress MnSOD following ADR treatment, with no difference in heart rate. a Beats/min in 8–10 weeks old nTg ({blacksquare}), iNOS (–/–) ({square}), TgM (+/+) ({blacktriangleup}), and iNOS (–/–)–TgM (+/+) ({triangleup}) control mice (saline) (n ≥ 3 mice in each group). Data are expressed as mean ± SEM. b. Left ventricular developed pressure (LVDP) in 8–10 weeks old nTg ({blacksquare}) and iNOS (–/–) ({square}), TgM (+/+) ({blacktriangleup}), and iNOS (–/–)–TgM (+/+) ({triangleup}) control mice (saline) (n ≥ 3 mice in each group). Data are expressed as mean ± SEM. c. Beats/min in 8–10 weeks old nTg ({blacksquare}), iNOS (–/–) ({square}), TgM (+/+) ({blacktriangleup}), and iNOS (–/–)–TgM (+/+) ({triangleup}) mice treated with ADR (n>4 mice in each group). Data are expressed as mean ± SEM. d. Left ventricular developed pressure (LVDP) in 8–10 weeks old nTg ({blacksquare}) and iNOS (–/–) ({square}), TgM (+/+) ({blacktriangleup}), and iNOS (–/–)–TgM (+/+) ({triangleup}) mice treated with ADR (n>4 mice in each group). Data are expressed as mean ± SEM. *P<0.05 for iNOS (–/–)-ADR versus nTg, TgM (+/+), and iNOS (–/–)–TgM (+/+)-ADR for dose of isoproterenol.

 

Figure 8
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  		Fig. 8 MnSOD overexpression attenuated serum LDH levels after ADR treatment. Serum lactate dehydrogenase (LDH) in 8–10 weeks old wt (nTg), iNOS (–/–), TgM (+/+) and iNOS (–/–)' TgM (+/+) mice treated with ADR (n=10 mice in each group). Data are expressed as mean ± SEM. *P ≥ 0.0001 versus saline of mice for the same genotype. **P ≥ 0.0001 versus wt (nTg) and iNOS–TgM (+/+) ADR treated mice.

 
3.8 Overexpression of MnSOD attenuates serum LDH and cardiac troponin levels after ADR treatment
To verify the cardioprotective effect of MnSOD in the iNOS (–/–) mice, we compared LDH and cardiac troponin serum levels. Analysis of LDH release in serum following treatment with ADR demonstrated that iNOS (–/–) mice had elevated levels compared to nTg mice (P ≥ 0.0001) (Fig. 8). Mice transgenic for MnSOD (TgM (+/+)) demonstrated no significant change in LDH levels after treatment with ADR. Levels of LDH release were attenuated in iNOS (–/–)–TgM (+) mice, comparable to LDH levels after treatment with ADR in nTg mice (P>0.05). Fig. 9 shows an attenuation of cardiac troponin levels in serum with overexpression of MnSOD in mice that lack iNOS (P ≥ 0.0001) following ADR treatment when compared to iNOS deficient mice.


Figure 9
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  		Fig. 9 Cardiac troponin was significantly decreased in iNOS (–/–) mice overexpressing MnSOD as compared to iNOS (–/–) mice. Serum cardiac troponin in 8–10 weeks old wt (nTg), iNOS (–/–), TgM (++), and iNOS (–/–)–TgM (++) mice treated with ADR for three days (n=4–5 mice in each group). Data are expressed as densitometric mean ± SEM. *P ≥ 0.0001 versus saline treated mice. **P ≥ 0.0001 versus wt (nTg) and iNOS–TgM (+/+) ADR treated mice.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Adriamycin is one of the most frequently used chemotherapeutic drugs in the treatment of cancer. However, the clinical usefulness of ADR is limited by a dose-related cardiac toxicity. Mitochondria have been identified as one of the targets in ADR-induced subcellular damage in heart tissues [1]. Previously, our laboratory has shown that ventricular tissues isolated from wildtype mice treated with ADR exhibited mitochondrial damage in a dose-dependent manner [5]. The present study confirmed and extended our previous results and showed that iNOS null mice had more extensive mitochondrial damage when treated with ADR as compared to wildtype mice. This is the first report to demonstrate that ·NO produced by iNOS contributes to protection of normal tissue injury by ADR in vivo. These ultrastructural results are consistent with measurements of cardiac function and further supported by biochemical parameters analyzed.

Cardiac dysfunction as a response to ADR treatment has been shown previously by Kang et al. [22]. Using an in vivo method, Pacher et al. observed that iNOS (–/–) mice had slightly better cardiac function compared to wildtype 5 days following ADR treatment [23]. One additional difference, other than the obvious difference in methods of ex vivo versus in vivo, is that the background of the mice are different. Mice used for hemodynamic study by Pacher et al. are in the C57BL/6 background, whereas mice used in our experiments are bred more than 10th generation in B6C3 background. It is possible that differences in strains of mice used may contribute to the differences in some parameters measured. Since our functional data are in agreement with the ultrastructural pathology and biochemical data in the same genetic background, we are confident that the observed effect of iNOS is real in our animals. Limitations exist for both in vivo and ex vivo cardiac function measurements, including but not limited to, systemic and/or neurohumoral modulation such as CNS and blood volume, variables extrinsic of the heart. However, cardiac function determined ex vivo provides an indication of cardiac status independent of other systolic factors. Herein, using the isolated perfused heart to assess cardiac function, we observed no change in the resting heart rate in iNOS (–/–) ADR-treated mice when compared to nTg animals. Similarly we noted no difference in the ability of the β-adrenergic receptor agonist, isoproterenol, to promote increases in heart rate. In contrast, resting contractile force and the ability of isoproterenol to increase indicies of contractile function was significantly decreased in mice lacking iNOS as compared to nTg mouse heart following treatment with ADR. This result demonstrates and confirms an enhancement of cardiac damage in iNOS (–/–) mice.

Serum CPK and LDH activities are used clinically to evaluate and diagnose cardiac dysfunction and disease [24]. Fig. 3 shows serum CPK and LDH were significantly elevated in nTg and iNOS (–/–) mice 3 days following treatment with ADR. However, iNOS (–/–) mice treated with ADR had more significant increases in both CPK and LDH as compared to nTg treated mice. This finding directly correlates with the increased ultrastructural damage and cardiac contractility dysfunction exhibited in the iNOS (–/–) mice following ADR treatment as compared to nTg mice. Because serum CPK and LDH can be elevated in many conditions and may not be specific markers for cardiac injury, we chose to probe for a more specific biomarker of cardiac injury, cTnI [25,26]. The levels of cTnI in serum have been found to be directly correlated to infarct magnitude and more specifically to ADR-induced damage [26,27]. Inducible NOS (–/–) mice had higher elevations of cTnI following treatment with ADR as compared to nTg mice (Fig. 3c). These findings are also consistent with the biochemical analysis of CPK and LDH, as well as the ultrastructural results. Specifically, levels of cTnI correlate more closely with the ultrastructural results, indicating that cardiac injury in mice deficient in iNOS is attenuated after expression of MnSOD. Taken together, these results suggest that ADR-induced ·NO production from iNOS may play a role in preventing cardiac injury. The contribution of iNOS to ·NO production in cardiac tissue is supported by the following data: first, iNOS transcript levels and nitrate levels increased in nTg but not in iNOS (–/–) mice; second, eNOS levels in both nTg and iNOS (–/–) mice were unchanged before and after treatment with ADR, and third, nitrotyrosine in nTg, but not in iNOS (–/–) mice, an adduct of tyrosine modification, was increased.

Consistent with our results, Weinstein et al. demonstrated immunohistochemical staining of iNOS and of 3-nitrotyrosine in myocardial tissue after treatment with ADR [28]. Increases in nitrotyrosine can be directly linked to increases in RNS, rather than exclusively ONOO– production [29]. Our results indicated that iNOS transcript levels are increased in nTg mice 6 h following treatment with ADR. Significant nitrate levels were found as early as 6 h and continued to increase afterward and the levels of nitrated proteins, as a measure of nitrotyrosine formation, also increased in nTg mice after ADR (Fig. 5). However, cardiac tissue from iNOS (–/–), TgM (+/+), and iNOS (–/–)–TgM (+/+) ADR-treated mice did not express nitrated proteins, consistent with the lack of increase in iNOS mRNA and nitrotyrosine. These results suggest that overexpression of MnSOD protects against ADR-induced iNOS expression. Taken together, this data would suggest that the increases in nitrotyrosine formation in nTg mice following ADR were dependent mainly on the iNOS activity. Further, the absence of nitrotyrosine formation in iNOS (–/–) mice would indicate that increases in nitrating species found in nTg mice were not involved in causing direct tissue injury or cytotoxic signaling. The clinical significance of these findings is that inhibition of iNOS as a therapeutic attempt to prevent deleterious effects of ·NO (but not necessarily other ROS) in both physiological and pathological circumstances may actually prove to be deleterious.

It is well documented that ·NO can react with O2·–, therefore the level of ·NO can alter the fate of O2·– in vivo [30]. Nitric oxide may aid SOD in the removal of excess O2·–, to prevent generation of hydroxyl radical. Based upon the results reported herein and our previous findings demonstrating protection against ADR-induced injury with overexpression of hMnSOD, we postulated that increased MnSOD may compensate for the lack of iNOS and hence iNOS-mediated ·NO production. Ultrastructural analysis of cardiac tissues from all four genotypes of mice following treatment with ADR indicated that TgM (+/+) mice exhibited the least cardiac damage (Fig. 6). Protection against ADR-induced cardiac injury in TgM (+/+) mice as demonstrated by ultrastructural analysis did not manifest into enhanced cardiac function. However, overexpression of MnSOD restored cardiac function in mice lacking iNOS (Fig. 7b). Importantly, the exacerbation of injury as seen in the iNOS (–/–) mice was attenuated in iNOS (–/–)–TgM (+/+) mice. The level of cardiac injury in MnSOD transgenic mice lacking iNOS was comparable to wildtype mice following treatment, further demonstrating that ADR-induced ·NO production plays a cytoprotective role.

Taken together, the findings reported here demonstrate for the first time that cardiac injury associated with ADR treatment is exacerbated in iNOS (–/–) as compared to nTg mice; this conclusion was supported by: 1) ultrastructural analysis, assessment of cardiac contractile function, and biochemical markers for cardiac injury, 2) the enhanced cardiac injury induced by ADR in iNOS (–/–) mice appears to be O2·– mediated, a finding consistent with the protection effect observed in mice deficient in iNOS, but overexpressing MnSOD, 3) in contrast to iNOS (–/–) mice, nTg mice exhibited the ability to generate endogenous nitrate and increased iNOS mRNA expression, perhaps providing cytoprotective cell signaling events, and 4) overexpression of MnSOD can compensate for the lack of cytoprotective mechanisms resulting from the absence of iNOS. These results signify the intricate relationship between ·NO and SOD in the prevention of cardiac tissue injury induced by cancer chemotherapy.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We thank Dr. Judy Hines for providing the initial steps in generation of the mice and Dr. Brett Jones, Teresa Noel, and Jenny Smith for technical assistance. This work was supported by NIH grants T32DK07778, R01CA80152, R01CA94853, and fellowship support to Luksana Chaiswing from the Thailand Research fund Royal Golden Jubilee program. This research was supported by resources and facilities from the William S. Middleton Veterans Administration Hospital, Madison, WI.


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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