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Laboratory Investigation (2012) 92, 125–134 & 2012 USCAP, Inc All rights reserved 0023-6837/12 $32.00

The mitochondrial respiratory chain has a critical role in the antiviral process in Coxsackievirus B3-induced myocarditis Linda Ebermann1,2, Sylwia Wika1, Inga Klumpe1,2, Elke Hammer3, Karin Klingel4, Dirk Lassner5, Uwe Vo¨lker3, Ulrike Erben6, Heinz Zeichhardt7, Heinz-Peter Schultheiss1 and Andrea Do¨rner1

Well-established differences in Coxsackievirus B3 (CVB3) elimination in resistant C57BL/6 and permissive A.SW/SnJ mice provide suitable models for studying the significance of the link between mitochondrial respiratory chain (RC), antioxidative stress components and -related in the context of myocardial elimination. Distinct myocardial CVB3 titer in C57BL/6 (2.5±1.4 Â 104 plaque-forming units (p.f.u.)/g ) and A.SW/SnJ mice (1.4±0.8 Â 107 p.f.u./g) were associated with differences in the cardiac mitochondrial function 8 days post infection (p.i.). Infected C57BL/6 mouse disclosed increased complex I (CI) and CIII activity, but restricted CII and normal CIV activity of RC. Reduced expression of the antioxidative was accompanied by elevated peroxidation (LPO), indicating . Intrinsic apoptosis was activated demonstrated by elevated levels of Bax, Bcl-2, caspase 3 and DNA degradation. In contrast, all myocardial RC complex activities were restricted in CVB3-infected A.SW/SnJ mice. The antioxidative system provided sufficient protection against oxidative stress shown by an elevated catalase expression and unaltered LPO. Bax and Bcl-2 levels were unchanged in CVB3-infected A.SW/SnJ mice, while caspase 3 was mod- erately increased but no DNA degradation was detectable. Correlation analyses including data from the two mouse strains revealed that reduced CVB3 titer correlated with increased CI and CIII activity, oxidative stress as well as active apoptosis during acute myocarditis (MC). C57BL/6 mice completely eliminated CVB3 and inflammation and normalized all intracellular parameters, while A.SW/SnJ mice showed permanently restricted CI activity in chronic MC 90 days p.i., at which time the replicating virus was no longer detectable but immunological processes were still active. Consequently, the regulation of appears crucial for an effective virus elimination and may be of prognostic and therapeutic significance for patients with virus-induced MC. Laboratory Investigation (2012) 92, 125–134; doi:10.1038/labinvest.2011.145; published online 3 October 2011

KEYWORDS: apoptosis; Coxsackievirus B3; mitochondria; myocarditis; oxidative ; oxidative stress

Coxsackievirus B3 (CVB3), a member of the enteroviral the immunology of the host is crucial for the course of the Picornaviridae, is one of the best-described infectious agents disease, intracellular processes of the infected that de- associated with myocarditis (MC).1 Interestingly, character- termine the differences in the fate of the virus remain elusive. istics of the virus alone do not explain the vast difference in Mitochondria, the center of cellular energy production, clinical outcomes of CVB3-induced MC. Acute inflammatory have been shown to be prominent targets of viral , heart disease is either characterized by rapid virus elimina- suggesting that they have a central role in the host’s cellular tion and recovery or ongoing chronic inflammation asso- defense system.3–5 The mitochondrial respiratory chain (RC), ciated with virus persistence frequently resulting in dilated the antioxidative system and members of the Bcl-2 cardiomyopathy.2 Although it is widely acknowledged that family strongly influence the mitochondrial integrity and

1Department of Cardiology and Pneumonology, Charite´—Campus Benjamin Franklin, Universita¨tsmedizin Berlin, Berlin, Germany; 2Institute of Biochemistry, Freie Universita¨t Berlin, Berlin, Germany; 3Interfaculty Institute for Genetics and Functional , Ernst-Moritz-Arndt Universita¨t, Greifswald, Germany; 4Department of Molecular Pathology, Universita¨tTu¨bingen, Tu¨bingen, Germany; 5Institute for Cardiac Diagnostic and Therapy (IKDT), Berlin, Germany; 6Department of Gastroenterology/Infectiology/Rheumatology, Charite´—Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany and 7Department of Virology, Charite´—Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany Correspondence: Dr A Do¨rner, PhD, Department of Cardiology and Pneumonology, Charite´—Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: [email protected] Received 1 March 2011; revised 5 August 2011; accepted 16 August 2011 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 92 January 2012 125 Mitochondrial function in CVB3 myocarditis L Ebermann et al

consequently need to be examined to understand the link graded 0 for no, 1 for low, 2 for medium and 3 for extensive between mitochondrial function and CVB3 within the immune infiltrations. Myocardial and cellular CVB3 infected heart. titers were determined by plaque-forming assay as previously The RC, the final step of ATP production, transfers elec- described.12 trons from donor molecules via complexes I to IV (CI–CIV) to the acceptor molecule O2, generating the transmembrane RNA Preparation and Real-Time PCR used by the ATP synthase to syn- Total RNA was isolated from myocardium using TRIzol thesize ATP. Imbalance in this tightly coordinated system reagent (Invitrogen, Karlsruhe, Germany), and cDNA was leads to misrouting of and increased generation of generated using standard protocols. Quantitative real-time reactive species (ROS).6,7 ROS are highly reactive and PCR was performed to quantify mRNA expression of tumor damages proteins, and nucleic acids. Components of necrosis factor a (TNFa), interleukin 1b (IL-1b) and trans- the antioxidative stress system such as mitochondrial super- forming growth factor b-1 (TGFb) using the Mastercycler oxide dismutase (SOD2) and catalase activity neutralize these realplex system (Eppendorf, Hamburg, Germany) and the toxic radicals. Maladjustment in RC and disturbance of respective TaqMan Expression Assays from Applied the antioxidative system result in mitochondrial instability Biosystems (Darmstadt, Germany). Gene expression was and apoptosis.8 In those cases, members of the Bcl-2 family normalized in relation to the endogenous housekeeping gene specifically modulate mitochondrial processes initiating hypoxanthine phosphoribosyltransferase 1 (HPRT1). programmed .9 While Bcl-2 suppresses apoptosis, Bax increases the permeability of the inner mitochondrial Mitochondrion Isolation membrane, induces the release of proapoptotic factors that Heart tissue was disrupted with scissors in 10 volumes activate the caspase cascade, initiating cellular degradation. of isolation buffer containing 300 mM sucrose, 5 mM We hypothesize here that the regulation of mitochondrial 3-(N-morpholino)propanesulfonic acid, 1 mM glycol-bis function has a significant impact on the myocardial virus (2-aminoethylether)-N,N,N’, N’-tetraacetic acid, 1 mg/ml load in CVB3-induced MC. To test this hypothesis, we used bovine serum albumin, protease inhibitor mix diluted 1:100 two mouse strains that have been well described for their (Sigma-Aldrich, Steinheim, Germany) pH 7.4. Tissue was different capacity to eliminate CVB3 from the heart. While homogenized in a glass potter and subsequently centrifuged CVB3-infected C57BL/6 mice rapidly eliminate the virus and at 1000 g for 10 min at 4 1C. The supernatant was centrifuged recover from MC, A.SW/SnJ mice develop chronic MC based at 12 000 g for 30 min for mitochondrion preparation. on virus persistence.10,11 These mouse models closely reflect The mitochondrial pellet was washed in 200 ml isolation the etiopathologies in human CVB3-induced MC. buffer and again centrifuged at 12 000 g for 30 min. The mitochondria were resuspended in 50 ml isolation buffer. The MATERIALS AND METHODS supernatant was centrifuged for the preparation of cytosolic Model of Murine MC proteins at 16 000 g for 1 h. concentration was All studies were performed according to national determined by the bicinchoninic acid test (Pierce, Bonn, guidelines and were approved by the institutional animal care Germany). committee. The investigation conforms to the ‘Guide for the Care and Use of Laboratory ’ stated by the US Determination of RC Complex Activities National Institutes of Health. Transfection-derived CVB3 CI activity was measured spectrophotometrically by the (Nancy strain, ATCC VR-30) was propagated on HeLa cells, oxidation of NADH with decylubiquinone at 340 nm, CII isolated and stored at À80 1C until use. Sixteen 6-week-old activity by the oxidation of succinate with ubiquinone at male C57BL/6 mice and 16 age-matched mice of the A.SW/ 600 nm, CIII by the reduction of c with SnJ background were intraperitoneally inoculated with decylubiquinol at 550 nm and CIV by the oxidation of 5 Â 105 plaque-forming units (p.f.u.) of CVB3 in 200 mlof reduced at 550 nm, as described in Jarreta et al.13 phosphate-buffered saline (PBS). A subgroup of age-matched C57BL/6 and A.SW/SnJ mice sham-infected with 200 ml PBS Mass-Spectrometry Analyses served as controls. Eight infected and uninfected mice of each Mitochondrial proteins of individual mice (n ¼ 4 per group) strain were killed during the acute phase of MC at day 8. An were digested by trypsin and the resulting extracts additional set of animals was analyzed in the chronic phase were desalted on reverse phase material.14 LC-ESI-MS/MS 90 days post infection (p.i.). analysis was performed on a LTQ-Orbitrap mass spectro- meter (Thermo Scientific, Bremen, Germany). Proteins Assessment of Heart Inflammation and Viral Load displaying different levels in the two mouse strains were The grade of myocardial inflammation was histologically obtained by label-free quantification analysis performed determined by evaluating hematoxylin/eosin-stained paraffin with Rosetta Elucidator version 3.3.0.1.39 (http://www. sections as described previously by three independent rosettabio.com/products/elucidator; Rosetta Biosoftware, investigators.12 The severity of heart inflammation was MA, USA).

126 Laboratory Investigation | Volume 92 January 2012 | www.laboratoryinvestigation.org Mitochondrial function in CVB3 myocarditis L Ebermann et al

Western Blot Analysis response differed in the strains as seen by diverse cytokine Mitochondrial (3 mg) and cytosolic (30 mg) proteins were patterns. Although TNFa mRNA was equally separated by electrophoresis on 4–12% polyacrylamide transcribed in the two mouse strains (Figure 1c), IL-1b gels and blotted onto a polyvinylidenefluoride membrane. (Figure 1d) and TGFb (Figure 1e) revealed a lower trans- Western blots were performed using a standard protocol with cription level in A.SW/SnJ than in C57BL/6 mice. TGFb did specific primary antibodies against SOD2 (Abcam, Cam- not change yet in infected A.SW/SnJ mice at the analyzed bridge, UK), catalase (Acris, Herford, Germany), Bax, Bcl-2 time point. Simultaneously, the myocardial titer of replicat- (New England Biolabs, Frankfurt, Germany) and caspase 3 ing CVB3 determined by plaque-forming assay was (BD Pharmingen, Heidelberg, Germany), and with HRP- significantly higher in CVB3-permissive A.SW/SnJ than in conjugated swine anti-rabbit as secondary antibody resistant C57BL/6 mice (Figure 1f). (1:10 000, Dako, Glostrup, Denmark). Specific signals were normalized against total loaded protein visualized by Coo- Activities of -Transporting RC Complexes massie blue . To analyze how cardiac mitochondrial RC is regulated in CVB3-induced MC, activities of the electron-transporting RC Detection of LPO CI–CIV were determined in mitochondria isolated from Myocardial lipid peroxidation (LPO), an indicator for oxi- infected and uninfected C57BL/6 and A.SW/SnJ mice. dative stress, was measured using the commercially available Activities were similar in uninfected C57BL/6 and A.SW/SnJ LPO 586 (Oxis International, Portland, OR, USA), which control hearts. However, they displayed profoundly different spectroscopically determined free malondialdehyde and patterns in the two mouse strains after CVB3 infection 4-hydroxyalkenals at 586 nm absorbance. Tissue homogenate (Figure 2). CI and CIII activity were remarkably elevated in was generated in 10 volume ice-cold PBS according to the infected C57BL/6 mouse hearts by 21.7±6.1% (Po0.05) and manufacturer’s protocol. 44.0±10.3% (Po0.05), respectively, while CII activity was moderately restricted by 12.9±4.8% (Po0.05) and CIV TUNEL Staining showed normal functionality (Figure 2a). In contrast, CVB3- A terminal deoxynucleotidyl--mediated dUTP infected A.SW/SnJ hearts revealed a coordinate decrease in nick end-labeling (TUNEL) assay was used to identify all RC complex activities of 16% on average (Figure 2b) double-stranded DNA fragmentation and the characteristics compared with uninfected controls. of DNA degradation by apoptosis. TUNEL staining was To elucidate whether changes in RC complex activities purchased by Roche Diagnostics Deutschland GmbH were related to an altered expression level of RC complex (Mannheim, Germany). Protease K was used for cell per- subunits, mass-spectrometric analyses were performed on meabilization. Alkaline phosphatase-coupled second anti- mitochondrial protein from infected C57BL/6 and A.SW/SnJ body and Fast Red were used as a detection system according hearts. Subunits whose expression differed are listed in to the manufacturer’s protocol. Positive control of the Table 1. Consistent with the observed increase in CI activity TUNEL assay was confirmed by treating the heart sections in infected C57BL/6 hearts, 11 of 45 CI subunits showed with DNase. Negative control of the TUNEL assay was a higher protein content in C57BL/6 than in A.SW/SnJ confirmed by staining the heart tissue in the same manner mitochondria. The amount of the CII subunit SDHC was without primary antibody. lower in the infected C57BL/6 mice. In all, 3 of 11 CIII subunits, UQCRS1, CYC1 and UQCR10, revealed a lower Statistical Evaluation protein amount in infected C57BL/6 than in A.SW/SnJ Two groups were compared using the Student’s t-test or hearts. In total, 2 of 13 CIV subunits, COX7A2 L and Mann–Whitney U-test. Multiple groups were compared COX7A1, were remarkably lower in infected C57BL/6 than in using the one-way ANOVA with the Newmann–Keuls test as A.SW/SnJ hearts, although CIV activity showed normal post test. Differences were considered to be significant function in infected C57BL/6 hearts. when Po0.05. Values are shown as the mean±s.e.m. The Hence, the patterns in expression and function of relationship between two parameters was determined by the the mitochondrial electron-transporting RC complexes are ‘Pearson correlation’. strain-specifically regulated in virus-induced MC.

RESULTS Myocardial Expression of Antioxidative Stress Myocardial Inflammation and CVB3 Load and LPO To validate the induction of MC by CVB3 application in the An imbalance in RC complex activities is frequently linked to two mouse models, we histologically assessed the myocardial increased ROS generation that needs to be neutralized by the pathology. CVB3 infection led to a 100% incidence of antioxidative stress system to avoid oxidative damage. As inflammation in both C57BL/6 and A.SW/SnJ mouse hearts markers for an active antioxidative stress system, we analyzed with an equal degree of myocardial infiltration 8 days p.i. the mitochondrial SOD2 and the cytosolic catalase protein (Figures 1a and b). However, the quality of the immune amount as well as LPO as an indicator for oxidative damage.

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Figure 1 Hematoxylin/eosin-stained heart sections from uninfected (control) and infected ( þ CVB3) C57BL/6 and A.SW/SnJ mice were analyzed for MC at day 8 post infection (p.i.) (a). The severity of MC was graded 0 for no, 1 for low, 2 for medium and 3 for high infiltration with immunocompetent cells. The averages are shown in (b). Open bars show uninfected age-matched controls, filled bars stand for infected animals. The myocardial transcription pattern of energy metabolism-relevant cytokines TNFa (c), IL-1b (d) and TGFb (e) was determined by quantitative PCR in the two mouse strains. Gene expression was normalized in relation to the endogenous housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1). Myocardial CVB3 titer was analyzed by plaque assay from infected C57BL/6 and A.SW/SnJ mice 8 days p.i. (f). *Po0.05; **Po0.01; ***Po0.001.

During infection, the amount of mitochondrial SOD2 hearts (Figure 3f). Hence, CVB3-infected C57BL/6 hearts increased by 29.0±7.9% (Po0.01), while cytosolic catalase were subjected to oxidative stress, whereas infected A.SW/SnJ expression decreased to 41.2±6.5% (Po0.01) in infected hearts were protected from LPO by sufficiently expressing C57BL/6 hearts (Figures 3a and b) compared with controls. antioxidative enzymes. This was accompanied by increased oxidative damage demonstrated by an elevation of LPO of 38.0±14.2% Expression of Mitochondrion-Related Apoptosis (Po0.05) (Figure 3c). Markers in the Acute Phase of MC In contrast, in comparison with uninfected controls CVB3- To further clarify whether deregulation in RC activities and infected A.SW/SnJ hearts doubled the catalase content oxidative stress were associated with mitochondrion-related (206.7±18.9%; Po0.01) but reduced their SOD2 expression apoptosis, pro- and antiapoptotic markers were examined by 26.3±2.9% (Po0.01) (Figures 3d and e), which was still during the acute phase of MC. In comparison with unin- as high as that seen in infected C57BL/6 mouse hearts. The fected controls, infected C57BL/6 mice displayed significantly LPO level remained unaffected in the infected A.SW/SnJ elevated myocardial Bax (150.6±13.2%; Po0.01) and Bcl-2

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Low myocardial CVB3 titers were closely related to elevated CI (r ¼À0.89; Po0.001) (Figure 5a) and CIII activity (r ¼À0.75; Po0.001) (Figure 5b). In addition, reduced catalase expression (r ¼ 0.87; Po0.001) (Figure 5c) was correlated with a depressed virus load in the infected hearts. Increased caspase 3 expression was further linked to virus reduction (r ¼À0.71; Po0.01) (Figure 5d). Thus, an imbalance in RC activities in CVB3-infected hearts that are associated with oxidative stress and apoptosis were linked to a reduced myocardial CVB3 load.

Mitochondrial Parameters in Chronic MC We investigated mitochondrial function in the chronic phase of MC 90 days p.i. that was only evident in A.SW/SnJ mice, while the C57BL/6 mice recovered completely. Although immunological infiltration was no longer histologically detectable in infected A.SW/SnJ mouse hearts, the myocardial immune response was still active as seen by an increased level of TGFb, while TNFa and IL-1b had normalized (Figure 6a). Replicating CVB3 was no longer observed by plaque-forming assay (data not shown). A.SW/SnJ hearts showed permanent restriction of CI activity, whereas CII, CIII and CIV activity reached normal levels (Figure 6b). SOD2 and catalase expression and LPO levels returned to normal (data not shown). Although pro- caspase 3 expression (Figure 6c) was significantly decreased (40.2±7.4%; Po0.01) and no active caspase 3 or apoptotic Figure 2 Respiratory chain complex activity of CI, CII, CIII and CIV were nuclei were detectable (data not shown), both the anti- and measured in isolated mitochondria from uninfected (control, open bars) proapoptotic markers Bcl-2 (145.1±14.7%; Po0.05) and and CVB3-infected (filled bars) C57BL/6 (a) and A.SW/SnJ hearts (b). Data Bax (133.1±3.9; Po0.001) were elevated. are shown as percentages of the uninfected controls. *Po0.05; **Po0.01 vs controls. Thus, resistant C57BL/6 mice recovered completely with normalization of all intracellular parameters described above. A.SW/SnJ mice disclosed a lasting activation of immune protein amounts (181.8±8.9%; Po0.001) (Figures 4a and b). response and disturbance in mitochondrial parameters. This was accompanied by a remarkable enrichment in cas- pase 3 protein. The increase equally affected pro-caspase 3 DISCUSSION and its active form (Figure 4a) and was therefore combined CVB3-resistant C57BL/6 and A.SW/SnJ mice that exhibited a as total caspase 3 (346±52.2%; Po0.001) (Figure 4b). In different quality of immune response and ability in virus addition, TUNEL staining confirmed the presence of apop- elimination revealed distinct mitochondrial function within totic cells in infected C57BL/6 heart tissue (Figure 4e). the infected myocardium. C57BL/6 mice, which quickly Compared with infected C57BL/6 hearts, Bax and Bcl-2 eliminate CVB3 from the heart and recover from MC, protein remained unaffected in the infected A.SW/SnJ mouse showed a strong imbalance in the myocardial mitochondrial hearts despite elevated expression of inactive and active RC, exhaustion of the antioxidative system, pronounced caspase 3 (179.6±5.4%; Po0.01) (Figures 4c and d) which, oxidative stress and strong mitochondrion-related apoptosis, however, appeared to be lower than in the infected C57BL/6 which generates an antiviral intracellular environment in hearts (Po0.001). No apoptotic nuclei were found in the the acute phase of MC. C57BL/6 mice showed high levels of infected A.SW/SnJ mouse hearts (Figure 4e). Thus, the two TNFa, IL-1b and TGFb, cytokines that are known to affect strains revealed significant differences in the regulation of the energy metabolism.15 Interestingly, myocardial IL-1b and mitochondrion-related apoptosis in the acute phase of MC. TGFb transcription were much lower in infected A.SW/SnJ mice developing chronic MC. They displayed decreased but Correlation Between Myocardial CVB3 Titer and balanced RC activities, increased catalase expression as well as Mitochondrial Parameters suppressed oxidative damage and mitochondrial apoptosis in We checked for a direct relationship between mitochondrial acute MC. function and myocardial CVB3 load by correlation analyses Cardiac electron transfer over the mitochondrial RC including the data from C57BL/6 and A.SW/SnJ mice. significantly impacts the modulation of apoptosis,8 a central

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Table 1 Comparison of RC complex subunit expression of CVB3-infected C57BL/6 vs A.SW/SnJ hearts

Name Accession no. Ratio* P-value

Complex I NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 1 (NDUFA1) O35683 1.858 o0.001 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2 (NDUFA2) Q9CQ75 1.142 o0.05 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7 (NDUFA7) Q9Z1P6 1.357 o0.001 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 4 (NDUFB4) Q9CQC7 1.153 o0.001 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9 (NDUFB9) Q9CQJ8 1.295 o0.05 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 (NDUFB10) Q9DCS9 1.130 o0.01 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 11 (NDUFB11) O09111 1.228 o0.01 NADH dehydrogenase [ubiquinone] iron-sulfur protein 4 (NDUFS4) Q9CXZ1 1.324 o0.05 NADH dehydrogenase [ubiquinone] iron-sulfur protein 5 (NDUFS5) Q99LY9 1.233 o0.001 NADH dehydrogenase [ubiquinone] 3 (NDUFV3) Q8BK30 1.221 o0.01 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex assembly factor 2 (NDUFAF1), mimitin Q59J78 3.238 o0.001

Complex II cytochrome b560 subunit (SDHC) Q9CZB0 À1.547 o0.001

Complex III -c1 complex subunit 5 (UQCRFS1) Q9CR68 À1.135 o0.05 Cytochrome b-c1 complex subunit 9 (UQCR10) Q8R1I1 À1.385 o0.001 , protein (CYC1) Q9D0M3 À1.227 o0.05

Complex IV subunit 7A-related protein (COX7A2L) Q61387 À6.357 o0.001 Cytochrome c oxidase polypeptide 7A1 (COX7A1) P56392 À1.153 o0.05

*The ratio was determined by dividing the observed intensities in mitochondrial protein extracts of CVB3-infected C57BL/6 hearts by those of A.SW/SnJ hearts. Minus symbol: amount of subunit is lower in C57BL/6 than in A.SW/SNJ mice.

Figure 3 Protein levels of mitochondrial SOD2 and cytosolic catalase were determined in uninfected (control; open bars) and infected ( þ CVB3; filled bars) C57BL/6 (a, b) and A.SW/SnJ mice (d, e) by western blotting. (a, d) show representative western blots of SOD2 and catalase (upper panels). Specific signals were normalized against total loaded protein visualized by Coomassie blue staining (lower panels). Myocardial lipid peroxidation was spectroscopically measured in C57BL/6 (c) and A.SW/SnJ (f) hearts. *Po0.05; **Po0.01 vs controls.

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effective host response to minimize damage because of oxidative stress in infected C57BL/6 hearts. CI and CIII viral infection, while quickly clearing the invading pathogen. activities are the main producers of ROS within the Imbalance of RC additionally supports apoptosis by mitochondria.7 Huang et al.28 reported that increased CI promoting the production of radicals damaging structural and CIII subunit expression leads to ROS generation that and functional macromolecules.6,7 Underscoring this con- could be blocked by CI-specific knockdown. ROS modify cept, our correlation analysis reveals a strong link between mitochondrial proteins and disturb mitochondrial integrity, unbalanced mitochondrial energy metabolism, a repressed which finally leads to apoptosis29 unless it is sufficiently antioxidative system, enhanced mitochondrion-related neutralized by the antioxidative system. Although the - apoptosis and low myocardial CVB3 titer in the acute phase mitochondrial antioxidative enzyme SOD2 that converts O2 of MC. Although the two infected mouse strains showed an to H2O2 was moderately enhanced in infected C57BL/6 mice, equal grade of inflammation in acute MC, long-lasting dif- the expression of catalase that neutralizes H2O2 was remark- ferences in the cytokine patterns clearly pointed to a distinct ably reduced. Such conditions support ROS accumulation quality of local immune response. TGFb, which is well known that is associated with oxidative stress and apoptosis as for having a significant impact on the mitochondrial function16 observed in infected C57BL/6 hearts. Similar findings were and for exerting a suppressing effect on CVB3 infection,17,18 observed in human MC by Xie et al.30 They demonstrated was differently regulated in C57BL/6 and A.SW/SnJ mice. A that oxidative stress evidenced by elevated plasma LPO and close association between TGFb level and increase in CIII nitric oxide was associated with a decrease in antioxidative activity, Bax and Bcl-2 as well as a decrease in myocardial enzymes like SOD. Moreover, catalase was severely aggravated CVB3 titer confirms the significance of TGFb in CVB3-in- in patients suffering from acute enterovirus-associated MC. duced MC. Thus, not only the quantity of infiltrating cells Oxidative stress is often associated with mitochondrion- but above all the quality of the responses is associated with related apoptosis and has also been observed in infected the diversity of intracellular processes and virus elimination. C57BL/6 mice, characterized by increased Bax, Bcl-2 as Conversely, intracellular processes directly related to the well as caspase 3 activation and DNA degradation. Thus, mitochondrial energy metabolism have been known to imbalanced RC in resistant mice is linked to a prooxidative determine the release of cytokines, thus affecting the immune and proapoptotic environment, both conditions that support system and virus infection.19 virus elimination. C57BL/6 mice disclosed a highly imbalanced RC in the Interestingly, we observed completely different conditions acute phase of MC characterized by enhanced CI and CIII in infected A.SW/SnJ hearts that were unable to efficiently activity but decreased CII and normal CIV activity. Changes remove CVB3 from the heart. Permissive A.SW/SnJ mice in RC activity are in part related to altered subunit expression coordinately downregulated all RC complex activities, which of the RC complexes. The protein amount of several CI is thought to decrease the flow of electrons over the RC31 and subunits was higher in infected C57BL/6 than in A.SW/SnJ lower the risk of electron misrouting and ROS production. hearts. This may not only be due to a downregulation in CI In addition, an activated antioxidative system shown by subunits in A.SW/SnJ hearts as seen in infected permissive increased catalase expression blocked the generation of A/J mice20 but also to subunit upregulation in C57BL/6 mice. oxidative stress and thus LPO production. High catalase In this regard, IL-1b has been shown to enhance CI subunit expression has been shown to have cardioprotective effects in expression .21,22 In agreement with this, our data other heart diseases such as .32,33 In addition, en- revealed a significant association between IL-1b mRNA hanced glutathione peroxidase and thioredoxin transcription, expression and CI activity in the infected hearts. Most of additional antioxidative stress components, were found in the affected CI subunits are accessory proteins that do not CVB3-infected permissive A/J mice with acute MC.20 SOD2 directly belong to CI’s catalytic core.23 However, in expression was reduced in infected A.SW/SnJ hearts but was accessory subunits like NDUFA1, NDUFA2, NDUFS4 have still as high as in infected C57BL/6 mice. Thus, A.SW/SnJ been shown to affect CI activity,24 clearly indicating their mice suppressed oxidative stress by reducing electron flow relevance for CI functionality. In contrast to CI, the subunit over the RC and sustain the antioxidative system. Suppres- expression of CIII and CIV acted contrarily to their activity, sion of oxidative stress preserved mitochondrial integrity and suggesting a compensatory effect against activity-inhibiting aborted severe mitochondrion-related apoptosis indicated agents or RC complex modifications like oxidation and by unaffected Bax and Bcl-2 expression in these animals. The phosphorylation as shown in inflamed hearts and cytokine- increase in myocardial caspase 3 expression and activation in treated isolated cells.25–27 Thus, the regulation of the RC infected A.SW/SnJ mice was much lower than in infected complex activities occurred at least in part on the transla- C57BL/6 mice and considered to be induced by the extrinsic tional level and appears to be related to different myocardial mitochondrion-independent pathway.34 However, its activa- cytokine expression patterns within the infected mouse tion was not sufficient enough to cause DNA degradation. strains. Hence, the suppression of intracellular oxidative stress and The profound imbalance in RC function, including intrinsic apoptosis in the early phase of MC is accompanied enhanced CI and CIII activity, involves a high risk of elevated by high virus replication in the heart.

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Disturbed energy metabolism has also been observed in Our data may help to explain the findings of Alter and chronic experimental and human viral MC.35–37 So far it was Maisch.38 The investigators analyzed myocardial biopsies ambiguous at what point energetic restrictions occur in the from patients with MC and DCM. They did not find apop- progression of chronic MC and whether they are the cause or tosis in patients with enteroviral persistence. In result of cardiac impairment. Our findings demonstrate contrast, they observed elevated concentrations of soluble that alterations in energy production arise already in the Fas-receptors repressing the extrinsic pathway of apoptosis acute phase of MC and persist up to the chronic phase in only in patients with enterovirus persistence.39 Thus, inhibi- permissive A.SW/SnJ mice, whereas resistant C57BL/6 mice tion of Fas-ligand-induced extrinsic apoptosis blocked by recovered completely. soluble Fas-receptors can impair virus clearance and prolong In the chronic phase of MC, A.SW/SnJ mice evidenced elevated Bax and Bcl-2 expression, while caspase 3 expression was significantly reduced and no apoptotic nuclei were visible, showing that extensive did not occur at that time point. No replicating virus was detectable within the heart at that time, but the immune response was still active. Schulze et al.36 have previously demonstrated a tight correlation between disturbed energy metabolism and cardiac performance in CVB3-infected A.SW/SnJ mice, in- dicating the significance of mitochondrial function and the of the disease. Thus, an imbalanced energy metabolism significantly affects not only the antiviral process but also the myocardial function contributing to the severity of the disease. Disturbance in energy metabolism has also been observed in human inflammatory and dilated cardiomyo- pathy,13,35 assuming that similar processes occur in humans.

Figure 6 Biochemical abnormalities in the myocardial cytokine pattern (a), the respiratory chain complex activity (b) and expression of Bcl-2, Bax, Figure 5 The correlation between myocardial CVB3 load shown as and caspase-3 (c) were found in infected A.SW/SnJ mice (filled bars) in log(p.f.u./g tissue) and (a) CI activity, (b) CIII activity, (c) catalase and comparison with uninfected controls (open bars) 90 days p.i. All parameters (d) caspase 3 expression was determined based on the data received returned to baseline in infected C57BL/6 mice and were not graphically from infected C57BL/6 (black dots) and A.SW/SnJ (black rectangles) mice presented. Data are shown as percentages of the uninfected controls. by linear regression (r ¼ regression factor, n ¼ 15–16). *Po0.05; **Po0.01; ***Po0.001 vs controls.

Figure 4 Expression of pro- and antiapoptotic markers was determined in uninfected (control; open bars) and infected ( þ CVB3; filled bars) C57BL/6 (a, b) and A.SW/SnJ mice (c, d) by western blotting. (a, c) show representative western blot analyses of Bax, Bcl-2 as well as pro-caspase 3 and active caspase 3 (upper panels). Specific signals were normalized against total loaded protein visualized by Coomassie blue staining (lower panels). Graph (b, d) display the quantification of proteins in % of uninfected controls. (e) Detection of TUNEL-positive cardiomyocytes in uninfected (control) and CVB3-infected ( þ CVB3) C57BL/6 and A.SW/SnJ hearts. Negative control of the TUNEL assay was confirmed by staining the heart tissue section without primary antibody. Positive control of the TUNEL assay was performed by treating the heart sections with DNase before staining. **Po0.01; ***Po0.001 vs controls. www.laboratoryinvestigation.org | Laboratory Investigation | Volume 92 January 2012 133 Mitochondrial function in CVB3 myocarditis L Ebermann et al

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