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Glutathione peroxidase protects mice from viral-induced myocarditis

^a Frank Porter Graham Child Development Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-8180, USA
^b Department of Medical Oncology, City of Hope Medical Center, Duarte, California 91010, USA
^c Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48201, USA

	ABSTRACT

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Glutathione peroxidase 1 (GPX-1) is a selenium-dependent enzyme with antioxidant properties. Previous investigations determined that mice deficient in selenium developed myocarditis when infected with a benign strain of coxsackievirus B3 (CVB3/0). To determine whether this effect was mediated by GPX-1, mice with a disrupted Gpx1 gene (Gpx1^-/-) were infected with CVB3/0. Gpx1^-/- mice developed myocarditis after CVB3/0 infection, whereas infected wild-type mice (Gpx1^+/+) were resistant. Sequencing of viruses recovered from Gpx1^-/--infected mice demonstrated seven nucleotide changes in the viral genome, of which three occurred at the G residue, the most easily oxidized base. No changes were found in virus isolated from Gpx1^+/+ mice. These results demonstrate that GPX-1 provides protection against viral-induced damage in vivo due to mutations in the viral genome of a benign virus.—Beck, M. A., Esworthy, R. S., Ho, Y.-S., Chu, F.-F. Glutathione peroxidase protects mice from viral-induced myocarditis. FASEB J. 12, 1143–1149 (1998)

Key Words: selenium • GPX-1 • malondialdehyde • virus titer • cardiac pathology

	INTRODUCTION

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SELENIUM (Se)² has been recognized as an essential trace element for four decades (1). There are eight well-characterized mammalian selenoproteins, including thioredoxin reductase and four isozymes of glutathione peroxidase (2–5). Keshan disease, an endemic cardiomyopathy affecting women and children residing in specific regions of the People's Republic of China, was found to be associated with Se deficiency (6). However, because of the seasonal and annual incidence of the disease, an infectious cofactor, in addition to a deficiency in Se, was postulated to contribute to the development of Keshan disease (7). A likely candidate for an infectious cofactor of Keshan disease is coxsackievirus. Coxsackieviruses, RNA picornaviruses of approximately 7500 nucleotides, have been isolated from Keshan disease tissues and are known etiologic agents of viral-induced myocarditis (8, 9).

Infection of mice with virulent strains of coxsackievirus B3 (CVB3) results in heart disease similar to human pathology (10). Se- or vitamin E-deficient mice are more severely affected by CVB3 infection than Se- and vitamin E-adequate mice (10, 11). CVB3/0, a benign strain of CVB3 that does not cause myocarditis in Se-adequate mice, induced myocarditis in Se-deficient mice (12). Six point mutations in the genome of virus isolated from the hearts of Se-deficient mice resulted in nucleotide substitutions that were identical to those found in virulent strains of CVB3 (13). These mutations transform a normally benign strain of virus into a strain that induces myocarditis even in mice with normal Se nutriture.

How does a deficiency in Se promote the development of a virulent CVB3 strain from an avirulent strain? We propose that a decrease in GPX-1 activity, a consequence of a deficiency in Se, is the critical step leading to the change in virulence. To test this hypothesis, we infected mice with a disrupted Gpx1 gene (Gpx1^-/- or Gpx1-KO) (14) with the avirulent CVB3/0. We found that, similar to infected Se-deficient mice, an avirulent CVB3 rapidly mutates to a virulent genotype in Gpx1-KO mice.

	METHODS

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Infection of mice
Gpx1-KO mice and wild-type mice have been described elsewhere (14). Mice were maintained under protocols approved by the Institutional Animal Review Board and inoculated at 3–4 wk of age with the noncardiovirulent coxsackievirus B3/0 (CVB3/0) obtained from Steven Tracy, University of Nebraska Medical Center. Virus was obtained by transfection of HeLa cells with a plasmid containing an infectious cDNA copy of the CVB3 genome inserted into a plasmid vector (15). Mice were inoculated with 10⁵ times the median tissue culture infectious dose (TCID₅₀) of CVB3/0 intraperitoneally. At 10 days postinoculation, mice were bled, killed, and their hearts, liver, and spleen were removed.

Histopathology of hearts
One-half of the heart, cut longitudinally, was placed in formalin, embedded in paraffin, sectioned (6 µm), and stained with hematoxylin and eosin. The extent of inflammatory lesions within the myocardium was graded without knowledge of the other experimental variables. Grading was done semiquantitatively according to the relative degree (from heart to heart) of mononuclear cell infiltration and the extent of necrosis.

Virus titers
One-half of the heart was snap frozen on dry ice, weighed, ground in a small volume of RPMI-1640, using a Tenbroek homogenizer, and freeze-thawed three times. The ground tissue was centrifuged (2000xg); the supernate was recovered, titrated on HeLa cell monolayers to TCID₅₀, and reported as the geometric mean titer per gram of tissue.

Neutralizing antibody titers
Neutralizing antibody titers were measured by inhibition of viral cytopathic effects as described previously (10).

Spleen cell proliferation assay
The assay for spleen cell proliferative activity in response to mitogen (concanavalin A) and antigen (CVB3 antigen) has been described in detail elsewhere (10).

Fluorescence-activated cell sorter analysis
Mediastinal lymph nodes were isolated from CVB3/0-infected Gpx1-KO and wild-type mice at 10 days postinoculation. Lymph nodes were teased into single cell suspensions, stained with anti-mouse CD4, and CD8 conjugated with fluorescein. Stained cells were analyzed by fluorescent activated cell sorter (Becton-Dickinson, Rutherford, N.J.).

Malondialdehyde and catalase levels
The entire heart and a fraction of each liver (of equal weight to heart) from both Gpx1-KO and wild-type mice infected 10 days earlier with CVB3/0 were homogenized by Polytron in a buffer composed of 0.15 M KCl, 10 mM MOPS (morpholinopropane sulfonic acid) (pH 7.2), 0.02% BHT (butylated hydroxy toluene) (16–18). A portion of each homogenate was made 0.2% with Triton-X-100. The Triton-X-100 samples were sonicated at ice temperatures, then centrifuged at 15,000 x g. The supernatant was recovered for catalase assays using the method of Beutler (19). The protein content of the samples was determined with the BCA assay (Pierce, Rockford, Ill.) using bovine serum albumin as the standard. The remainder of each homogenate was processed for malondialdehyde (MDA) determination as follows: the 500 x g supernatant (5 min) was centrifuged at 15,000 x g (20 min). The pellet was recovered, rinsed once in the homogenization buffer, and resuspended in 10 vol of the homogenization buffer by sonication at 4°C. The second supernatant was centrifuged at 100,000 x g for 30 min. The pellet was recovered, rinsed once, and resuspended in homogenization buffer by sonication. The 15,000 x g and 100,000 x g pellet fractions were analyzed for MDA content by a TBARS assay (thiobarbituric acid reactive substances). Protein samples (200 µg) were prepared for the TBARS assays according to the method of Schuh et al. (20), with slight modifications suggested by Jentzsch et al. (17). The standard was MDA generated during the acid step from malonaldehyde bis (dimethyl acetyl) (Sigma, St. Louis, Mo.). The TBARS chromogen was extracted into n-butanol (1.25 ml, equal volume to sample). The chromogen sample was read at 535 and 572 nm. The difference in absorption at the two wave lengths was used to quantitate the chromogen using the standard generated from malonaldehyde bis (dimethyl acetyl) (17).

Sequencing of virus
The details of virus sequencing (by direct sequencing of polymerase chain reaction [PCR] -generated DNA) have been reported earlier (13). DNA was sequenced at the UNC-Chapel Hill automated DNA Sequencing Facility on a Model 373A DNA Sequencer (Applied Biosystems, Foster City, Calif.) using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems). At least two separate PCR primer sets and two separate reverse transcriptase and PCR reactions were used to confirm sequence changes.

	RESULTS

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Cardiac pathology of Gpx1-KO mice postinfection
To test the effect of expression of Gpx1 encoding GPX-1 on the development of myocarditis after viral challenge, we inoculated both wild-type (Gpx1^+/+) mice and mice with a disrupted Gpx1 gene (14) with CVB3/0. Other GPX isozymes, GPX3 and GPX4, are not affected in Gpx1-KO mice. Previous work in our laboratory demonstrated that Se-deficient mice develop myocarditis 7 days after CVB3/0 infection, with peak cardiac damage occurring on day 10 postinfection (12). Se-adequate mice do not develop any signs of myocarditis postinfection with the avirulent CVB3/0 strain. Therefore, we killed both wild-type and Gpx1-KO mice at 10 days postinfection, the peak time for cardiac damage. Histological sections of heart were stained and examined by light microscopy. More than half of the Gpx1-KO mice developed mild to moderate myocarditis after CVB3/0 infection, characterized by a mononuclear cell infiltrate and cytolysis ( Fig. 1). In contrast, neither inflammation nor cytolysis were found in the hearts of wild-type mice infected with this virus. Thus, similar to Se-deficient mice, Gpx1-KO mice develop myocarditis when infected with an avirulent strain of CVB3.

View larger version (256K): [in this window] [in a new window]   Figure 1. CVB3/0-induced inflammation. Mice were injected intraperitoneally with 105 tissue culture infectious dose-50 (TCID50) of CVB3/0. Histologic sections of hearts stained with hematoxylin and eosin from CVB3/0-infected wild-type (A) and Gpx1-KO mice (B) at 10 days p.i. Arrow indicates typical inflammatory lesion found scattered throughout the myocardium. C) Anatomically similar portions of the left ventricle were prepared as described in Methods and graded for inflammation. Inflammation was graded on a scale of 0 to 4+. Pathologic scores: 0, no lesions; 1+, foci of mononuclear cell inflammation associated with myocardial cell reactive changes without myocardial cell necrosis; 2+, inflammatory foci clearly associated with myocardial cell reactive changes; 3+, inflammatory foci clearly associated with myocardial cell necrosis and dystrophic calcification; 4+, extensive inflammatory infiltration, necrosis, and dystrophic calcification. Each bar represents the percentage of animals assigned to each grade for each genotype. The graph represents 25 +/+ mice and 29 -/- mice.

Virus titer
To investigate whether the difference in pathology between Gpx1-KO and wild-type mice was due to a difference in viral titer, we determined the geometric mean titers of virus recovered from the hearts of infected animals. The mean viral titer from the hearts of Gpx1-KO mice (3.12±0.7 TCID₅₀) was indistinguishable from the heart virus titer of wild-type mice (3.33±0.9 TCID₅₀). Thus, at 10 days postinfection, the development of myocarditis in the Gpx-1 KO mice was not associated with elevated virus titers in the heart tissue. In contrast, hearts from Se-deficient mice infected with CVB3/0 had 10-fold higher virus titers when compared with Se-adequate mice (12).

Immune functions of infected Gpx1-KO mice
The immune system is involved in viral clearance and contributes to its pathology (21). Se-deficient mice were shown to have impaired immune responses after CVB3 infection. To determine whether Gpx1-KO mice also had impaired immune responses, we examined several immune functions in the infected Gpx1-KO mice and compared them with responses from wild-type mice. Serum titers of virus neutralizing antibody for both Gpx1-KO and wild-type mice were determined at 10 days postinfection. Neutralizing antibody titers in Gpx1-KO mice were less than 20% of those found in wild-type mice ( Fig. 2), indicating that an impairment of the B cell response occurred in the infected Gpx1-KO mice. This is in contrast to Se-deficient mice, in which neutralizing antibody titers were the same as the titers found in Se-adequate mice (12). Functional B cell activity may possibly depend on normal GPX-1 activity during development, as Se-deficient mice were 3 wk old before being fed the Se-deficient diet, an age when much of the immune system has already matured.

View larger version (11K): [in this window] [in a new window]   Figure 2. Serum CVB3/0 neutralizing antibody titers 10 days postinfection. Geometric mean titers (error bars represent standard deviation) were determined by inhibition of viral cytopathic effect (10). Bar for +/+ data represents 14 animals and bar for -/- data represents 36 animals.

T cell responses were determined by measuring [³H]thymidine incorporation in either mitogen (concanavalin A) or CVB3 viral antigen-stimulated splenocytes (12) obtained from CVB3/0-infected Gpx1-KO and wild-type mice. No difference in the splenocyte proliferative responses were found between Gpx1-KO mice for mitogen (stimulation index: [S.I]: 41.8 (4.2) or antigen (S.I.: 22±2.3) and wild-type mice (mitogen S.I.: 39.4±4.6; antigen S.I.: 21±2.2). Again, this is in contrast to Se-deficient mice, in which proliferative responses to mitogen and antigen were significantly decreased (12). Possibly a TH2 response, which drives B cell production of antibody by T cell secretion of interleukin 4 (IL-4) and IL-5, is favored in the Se-deficient animals. However, a TH1 type response, which stimulates T cell activity by secretion of IL-2 and -interferon, might predominate in the Gpx1-KO mice, blunting the TH2 response (22). The observation that viral antibody titers were equivalent in wild-type and knockout mice suggests that the antibody response plays little role in controlling viral replication in the infected mice.

A deficiency in Se has been shown to result in decreased levels of CD4+ T cells (23). To determine whether CD4+ T cells were affected in infected Gpx1-KO mice, we analyzed lymphocytes in mediastinal lymph nodes from Gpx1-KO and wild-type mice for levels of CD4+ and CD8+ T cells by fluorescence-activated cell sorter. No differences in percentages of CD4+ and CD8+ T cells were found between Gpx1-KO and wild-type mice (data not shown).

Analysis of viral sequence from Gpx1-KO and wild-type mice
Our previous work demonstrated that CVB3/0 virus recovered from Se-deficient mice had six point mutations that were identical to those found in virulent CVB3 strains (13). Thus, the change in viral phenotype from avirulent to virulent in the Se-deficient mice was due to genetic change of the virus. To determine whether replication of the benign CVB3 in Gpx1-KO mice would also alter the viral genome, we isolated and sequenced viral RNA from the hearts of a number of Gpx1-KO mice with and without heart pathology and from the hearts of wild-type mice. Seven nucleotides in the CVB3/0 genome that replicated in Gpx1-KO mice had changed ( Table 1). These changes in nucleotide sequence were found only in Gpx1-KO mice with pathology. No sequence changes were found in virus recovered from hearts of Gpx1-KO mice without gross pathology nor from the hearts of wild-type mice. The strict association between changes in the viral nucleotide sequence and induction of cardiomyopathy in Gpx1-KO mice suggests that some, if not all, of these changes are required for virulence.

Six of the seven changes in the viral genome obtained from the hearts of Gpx1-KO mice are identical to those found in virus recovered from the hearts of Se-deficient mice ( Table 1). Thus, it appears that the genetic changes in the virus that occurred in the Se-deficient mice can be attributed to a deficiency in Gpx-1 activity.

Malondialdehyde and catalase levels in infected mice
GPX-1 plays a role in the antioxidant defense strategy of the host. To determine whether increased oxidative stress occurred in the hearts or livers of infected mice, we quantified lipid peroxidation (by measuring MDA levels) and catalase activity, which may be increased in Gpx1-KO mice during viral infection (24, 25). Similar levels of malondialdehyde and catalase activity were found in the hearts and livers of Gpx1-KO and wild-type mice 10 days after viral infection (malondialdehyde in heart microsomes of KO: 225±18 pmol/mg protein in wild-type: 225±36 pmol/mg; heart catalase activity in KO: 21.1 ± 10.8 U/mg protein in wild-type: 23.0 ± 6.0 U/mg protein; liver data not shown). Baseline levels of MDA and catalase in the hearts of uninfected KO and wild-type mice do not differ (14). Thus, measurements of MDA and catalase at 10 days postinfection may not be adequate to detect oxidative stress in this model.

	DISCUSSION

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These data demonstrate that, in a host deficient in GPX-1 activity, an avirulent virus undergoes a phenotypic change to virulence due to point mutations in the genome. Single mutations in various sites in the CVB3 genome have been shown to be associated with cardiovirulence (26, 27). Our previous work demonstrating similar point mutations in Se-deficient mice suggests that the mutations may be driven by oxidative damage.

Glutathione peroxidase 1 (GPX-1), the first selenoprotein identified in mammals (28, 29), is present in both cytosol and mitochondria. The activity of GPX-1 is widespread, with highest levels in liver, kidney, and heart (30, 31). Heart tissue has a very low level of catalase activity, which is the other major enzyme involved in the detoxification of H₂O₂ (32). Although the importance of GPX-1 as an antioxidant has been debated (33, 34), we hypothesized that reduction of GPX-1 activity in Se-deficient mice contributed to the development of myocarditis postinfection with a benign strain of CVB3. The work reported here with the Gpx1-KO mice confirms that GPX-1 plays a critical role in the defense against viral-induced myocarditis. Although many studies demonstrate an antioxidant activity of GPX-1 in cell cultures, there is less evidence for its function as an antioxidant in whole organisms (14, 35). Indeed, because of its rapid response to changes in Se nutriture, GPX-1 has been described as an Se storage protein in rodents (34). This study in Gpx1-KO mice is the first report demonstrating a critical protective role for this enzyme against viral-induced pathogenesis and that other antioxidant protective mechanisms cannot compensate for a lack of GPX-1.

What is the mechanism that allows for the changes in the viral genome to occur in the Se-deficient or Gpx1-KO animal? There are several possibilities. One explanation is direct oxidative damage of viral RNA. Single-stranded RNA has been suggested to be more susceptible than double-stranded DNA to damage by free radicals. Although oxidative damage to RNA has been studied far less than oxidative damage to DNA, a recent report (36) suggested that the Escherichia coli muT protein (which has mammalian homologs) is instrumental in protecting transcriptional fidelity by hydrolyzing oxidized guanine, which mispairs with adenine. Guanine is the site of mutation at 3/7 sites in the virus obtained from Gpx1-KO mice, which likely reflects the high oxidative potential of this nucleotide (37, 38). Unrepaired oxidized bases can cause DNA mispairing and mutation during replication (38, 39), and similar changes may occur at oxidized bases in viral RNA genomes (40). In addition, RNA viruses lack efficient proofreading and postreplicative repair activities (41). Thus, mutation rates of RNA viruses are in the range of 10^-3 to 10^-5 substitutions per copied nucleotide, which is at least 10³-fold greater than the mutation rate for cellular DNA (42, 43). It has been suggested that RNA viruses replicate near the minimal fidelity compatible with maintaining their genetic information, although not all mutations will be viable. Therefore, in an individual virus population, individual genomes that differ in one or more nucleotides will form the average or consensus sequence of the population, or quasispecies. Thus, a high mutation rate coupled with a lack of GPX-1 antioxidant protection increases the likelihood of an accelerated mutation rate of coxsackievirus observed in Gpx1-KO mice.

We hypothesize that the transformation of CVB3/O from avirulence to virulence in Gpx1-KO mice involves several steps: 1) oxidative RNA damage, 2) increased mutagenesis of oxidized viral RNA, 3) lack of proofreading enzymes, and 4) impairment of B and/or T cell function. Our work clearly defines an oxidative protection role for GPX-1 activity and points to the importance of adequate oxidative defense mechanisms of the host to protect from viral challenge. This work demonstrates that oxidative damage can affect not only the host, but can influence the pathogen itself, altering a normally avirulent pathogen into a virulent one that can then cause damage even in mice not oxidatively challenged. Further investigations are needed to identify the critical roles of GPX-1 in resistance to viral pathogenesis.

	ACKNOWLEDGMENTS

 
This work was supported by National Institutes of Health grants DK46921 (F.-F.C.), P30 ESO6693 (Y.-S.H.), and HL54123 (M.A.B.). We thank Q. Shi for excellent technical assistance.

	FOOTNOTES

 
¹ Correspondence: Departments of Pediatrics and Nutrition, FPG Child Development Center, 105 Smith Level Rd., CB #8180, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-8180, USA. E-mail: melinda_beck{at}unc.edu

² Abbreviations: Se, selenium; PCR, polymerase chain reaction; GPX-1, glutathione peroxidase 1; S.I., stimulation index; IL, interleukin; MDA, malondialdehyde; CVB3, coxsackievirus B3; CVB3/0, a benign strain of CVB3; TBARS, thiobarbituric acid reactive substances; TCID₅₀, tissue culture infectious dose-50.

Received for publication January 23, 1998. Accepted for publication March 24, 1998.

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