Cardiovascular Research

Cardiovascular Research 1998 38(1):158-168; doi:10.1016/S0008-6363(97)00323-4
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Expression of apoptosis-related proteins in experimental coxsackievirus myocarditis

James T Colston, Bysani Chandrasekar and Gregory L Freeman^*

Division of Cardiology, University of Texas Health Science Center at San Antonio, and South Texas Veterans Health Care System, Audie L. Murphy Division, 7703 Floyd Curl Drive, San Antonio, TX 78284-7872, USA

^* Corresponding author. Tel.: +1 (210) 567 4600; Fax +1 (210) 567 6960; E-mail: freeman@uthscsa.edu

Received 15 August 1997; accepted 26 November 1997

	Abstract

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Objective: The extent to which apoptosis contributes to myocyte cell loss during acute carditic viral infection is unknown. To assess whether apoptosis occurs in acute viral myocarditis, and how it is modulated, we studied mice inoculated with coxsackievirus B3 (CVB3). Methods: Five CD1 and C3H.HeJ (C3H) mice/group were sacrificed as saline vehicle-injected controls, and at 1, 2, and 3 weeks post-inoculation (p.i.) with 5x10⁶ pfu CVB3. Histopathological status and terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assays quantified inflammation, necrosis and apoptosis in myocardium. Apoptosis-related protein immunoreactivity defined presence and location of Bax, Fas, Fas Ligand (FasL), Bcl-2, interleukin-1β converting enzyme (ICE), inducible nitric oxide synthase (iNOS) and the proto-oncogene p53. Results: Both strains exhibited significant histopathology at all time points. Saline-injected control animals showed no signs of inflammation and no significant difference in apoptosis-related protein immunoreactivity was observed between strains. Myocardial TUNEL-positive cells were exceedingly rare though apoptosis was present in thymic medulla and spleen follicles. Pro-apoptotic proteins Bax, Fas, and FasL were present in all groups though no clear correlation with histopathology was apparent. By contrast, the anti-apoptotic protein Bcl-2 showed mild immunoreactivity in controls, which increased following infection and correlated well with histopathological scores in both strains. Myocardial iNOS immunoreactivity displayed a similar though weaker staining pattern to Bcl-2 over the 3 week study period in both strains. Neither ICE nor p53 immunoreactivity could be demonstrated in myocardium. Conclusion: Thus, despite marked inflammatory activity, myocyte apoptosis is rare in acute CVB3 myocarditis in CD1 and C3H.HeJ mice.

KEYWORDS CD1; C3H.HeJ; Mice; Coxsackievirus; Myocarditis; Apoptosis; Immunohistochemistry

	1 Introduction

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Coxsackieviruses of the B type affect multiple organ systems and generally are associated with focal myopathies in susceptible populations. In heart, coxsackievirus infections result in myocardial inflammation characterized by significant extravascular immune cell infiltrate and necrosis [1]. This inflammatory process is typically associated with elevated levels of pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor- (TNF-), and IL-6 [2]. In addition, the inducible form of nitric oxide synthase (iNOS) is upregulated following coxsackievirus infection, which together with induced cytokines, have a negative influence on myocardial function [2–4]. This situation is exacerbated by the significant loss of myocytes to necrotic processes in the heart. Recent attention has focused on a second mechanism of cell death, apoptosis [5, 6]. Many of the inflammatory mediators present in coxsackievirus myocarditis, in addition to affecting heart function adversely, have been shown to stimulate apoptosis in some cell populations. It is not known whether apoptosis contributes significantly to myocardial cell loss in acute coxsackievirus myocarditis.

Programmed cell death, or apoptosis, is regulated by several factors which determine the relative susceptibility of a cell to this process. Infiltrating cytotoxic T lymphocytes can induce either necrosis or apoptosis in a target cell depending on the apoptotic factors present in that cell [7]. Several pro-apoptotic proteins have been identified, which include the Bcl-2 homologue Bax, Fas, and Fas ligand (FasL). Additional factors include the tumor suppressor p53 which is induced in response to DNA damage in some cells, and interleukin-1β converting enzyme (ICE) which is reportedly involved in the intracellular transduction of Fas- and TNF-mediated death signals [8]. Several other factors have been identified that prevent or delay apoptosis including Bcl-2 and its anti-apoptotic homologues.

Given the highly inflammatory substrate present in experimental coxsackievirus myocarditis, we were interested in whether apoptosis was taking place in those hearts. In addition, we wanted to evaluate the expression of factors known to regulate programmed cell death. We chose coxsackievirus B3 inoculation in strains of mice known to have differing susceptibilities to this virus. This allowed us to see whether factors known to modulate programmed cell death correlate temporally with inflammatory state, and whether they respond to both onset and resolution of inflammation. Our results suggest that myocardial immunoreactivity of the anti-apoptotic factor Bcl-2 parallels signs of tissue inflammation, and that despite the presence of pro-apoptotic factors, very little apoptosis occurs in the hearts of CD1 and C3H mice.

	2 Materials and methods

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
2.1 Experimental animals, diet, and inoculation with coxsackie (CVB3_m) virus
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985) [9]. Four week-old male mice of the CD1 (Charles River Laboratories, Boston) and the C3H.HeJ strain (Jackson Laboratories, Bar Harbor, ME) were maintained in the Laboratory Animal Resources facilities of the University of Texas Health Science Center at San Antonio. Water and nutritionally adequate mouse chow were available ad libitum. Mice were maintained in plastic cages and a 12 h light/dark cycle was followed. Mice were inoculated I.P. with 5x10⁶ pfu of virus; the time of inoculation was defined as day zero. Control mice were inoculated with normal saline vehicle.

2.2 Tissue collection and preparation
Mice were sacrificed 1, 2, and 3 weeks post inoculation (p.i.) under anesthesia (0.01–0.02 ml of solution containing 65 mg/ml ketamine, 2.2 mg/ml acepromazine, 13 mg/ml xylazine). Hearts were removed rapidly and fixed for 24 h in 10% buffered formalin solution. Additionally, thymus, spleen, and pancreas samples were collected and treated in the same manner. Following fixation and paraffin embedding, 5 µm thick sections were cut and mounted onto poly-L-lysine-coated glass slides.

2.3 Immunohistochemistry
Five micrometer thick paraffin-embedded sections were used for immunohistochemistry (DAKO PAP kit; DAKO, Carpenteria, CA). Sections were deparaffinized in xylene (5 min, x3), and rehydrated through graded ethanol (100% for 5 min, x2; 95% for 3 min; 70% for 3 min; dH₂O for 5 min). Epitope unmasking was achieved by either of the following 2 methods:

Method 1: For immunostaining of Bax, Fas, FasL, Bcl-2, ICE, and p53, slides were incubated in 0.01 mol/l citrate buffer, pH 6, at 95–100°C for 15 min followed by a wash in dH₂O for 5 min. Endogenous peroxidase activity was quenched by incubating sections in 3% H₂O₂ for 5 min followed by washing in Tris-buffered saline (TBS; 0.05 mol/l Tris, 0.15 mol/l NaCl, pH 7.4; 2 min; x4).

Method 2: For immunostaining of ICE, p53, and iNOS (NOSII), and TUNEL assay, tissue sections were incubated in a humidified chamber with proteinase K (20 µg/ml; Sigma) for 15 min at 23°C, and washed in dH₂O (2 min; x4) followed by incubation in TBS for 5 min at 23°C. Endogenous peroxidase activity was quenched by incubating sections in 3% H₂O₂ for 5 min followed by washing in TBS (2 min; x4).

Tissue sections were then blocked for 1 h at 23°C in a humidified chamber with blocking solution (0.05% Tween-20, 0.5% BSA, in TBS) containing either (1) preimmune normal goat serum (for rabbit polyclonal antibodies; Bax, Fas, FasL, Bcl-2, iNOS), or (2) preimmune normal rabbit serum (for goat polyclonal antibodies; ICE, p53). After blocking, primary antibodies were diluted in the appropriate blocking solution at the following concentrations: Fas, FasL, Bax, or p53, 2 µg/ml; ICE, 1 µg/ml; Bcl-2, 3 µg/ml; iNOS, 5 µg/ml. All primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and are reported to cross-react with mouse epitopes. Optimal antibody concentrations were determined empirically in separate experiments.

Tissue sections were incubated in a humidified chamber with primary antibody for 1 h at 23°C followed by washing in TBST buffer (TBS+0.05% Tween-20; 5 min, x3). Sections were then incubated in a humidified chamber with appropriate secondary antibody (either rabbit anti-goat or swine anti-rabbit Ig; DAKO) for 20 min at 23°C. After washing in TBST buffer (5 min, x3), sections were incubated in a humidified chamber with PAP (soluble horseradish peroxidase-[rabbit or goat] anti-horseradish peroxidase) conjugate for 20 min at 23°C, and washed in TBST buffer (5 min, x3). The sections were then incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAB; DAKO®Liquid DAB) substrate. Tissue sections were counterstained with Mayer's hematoxylin (Fluka, Ronkonkoma, NY). Sections were dehydrated through graded ethanol solutions followed by xylenes, then mounted permanently using Permount (Sigma) and glass coverslips. Specific staining of target antigen was controlled in each experiment by (1) omission of primary antibody, (2) incubation with rabbit or swine preimmune serum in place of primary antibody, and (3) use of primary antibody following neutralization with its control peptide antigen (Santa Cruz Biotechnology, Inc.). In addition, since secondary lymphoid tissues have been shown to exhibit active apoptotic processes, thymus and spleen specimens were collected and mounted on the same slides as the heart specimens and served as positive controls.

2.4 Terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay
TUNEL assays were performed using the ApopTag detection system (ApopTag® In Situ Detection Kit, Oncor, Inc., Gaithersburg, MD). Briefly, tissue sections were treated as previously described under Method 2 (Immunohistochemistry). Sections were then incubated for 1 min at room temperature in equilibration buffer. Equilibration buffer was replaced by terminal deoxynucleotidyl transferase (TdT) solution (32 µl TdT+152 µl reaction buffer) and incubated in a humidified chamber for 1 h at 37°C. This concentration of TdT enzyme was determined to be optimal in a separate set of experiments using tissue sections prepared from samples used in this study. Slides were placed in a Coplin jar containing stop/wash buffer at 37°C and incubated for 10 min. Slides were then washed in phosphate buffered saline (PBS, pH 7.4; 5 min, x3), incubated with anti-digoxigenin-peroxidase antibody for 30 min at room temperature in a humidified chamber, and washed again with PBS (5 min; x3). Development using DAB substrate was performed as previously described. Tissue sections were then counterstained with methyl green (10 min), dehydrated, and mounted with glass coverslips. TUNEL assays performed on thymus and spleen sections served as positive controls.

2.5 DNA laddering assay
Total DNA was isolated from CD1 and C3H.HeJ mouse hearts and splenocytes (cultured for 24 h) using a single reagent method (DNA Stat 60; Tel-Test, Corp., Friendswood, TX). Isolated DNA was quantified by spectrophotometer, then 10 µg total DNA was size fractionated on a 1.8% agarose gel, stained with 2 µg/ml ethidium bromide and photographed.

2.6 Immunohistochemical and histopathological scoring
Tissue sections were evaluated by light microscopy in blinded fashion. The observations were made by a single observer without the knowledge of the experimental group. Immunohistochemical staining intensity was scored using a semiquantitative scale from 0 to 3 where; 0=none, 1=weak, 2=moderate or intermediate, and 3=strong staining. In order to reduce intra-assay variability, tissue sections were stained with each primary antibody in batch fashion thus ensuring identical reagents and conditions within each assay. Histopathological evaluation of tissue sections was performed using a semiquantitative scale from 0 to 4 where; 0=no immune infiltrate and no necrotic lesions, 1=less than 25%, 2=26 to 50%, 3=51 to 75%, and 4=more than 75% of myocardium had necrotic lesions with extravascular immune cell infiltrate. Data for individual animals represent average values from two tissue sections cut from different areas of the heart (i.e., apex and base of the ventricles) and mounted side by side on the same slide.

2.7 Statistical analysis
Non-parametric statistical tests were used to analyze mean data, which follow an ordinal distribution. To compare across time points, an overall test for differences was performed using Kruskal–Wallis Analysis of Variance. When significant (p<0.05), post hoc pairwise comparisons between control and 1, 2, and 3 weeks p.i. were performed using Mann–Whitney U tests. Comparisons between strains within each antibody and time point were also performed using Mann–Whitney U tests.

	3 Results

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Saline-injected CD1 and C3H control animals did not exhibit any signs of myocardial inflammation. Fig. 1A shows typical uninfected mouse myocardium at 200x original magnification. Fig. 1B shows a typical focal lesion containing moderate extravascular immune infiltrate with mild necrosis in a CD1 mouse heart 2 weeks p.i. Both strains exhibited significant signs of extravascular inflammatory infiltrate with necrosis at all time points following inoculation though with different severity. There were no deaths during the study period.

View larger version (120K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Photomicrographs of representative saline vehicle-injected control (Panel A) and coxsackievirus infected (Panel B; 2 weeks p.i.) CD1 mouse hearts. Panel B shows a typical inflammatory lesion with extravascular immune cell infiltrate. Five micrometer thick paraffin-embedded tissue sections were stained with Meyer's hematoxylin (original magnification: 200x).

 
Shown in Fig. 2, Panels A–C, are representative photomicrographs of Bax, Fas, and FasL protein immunoreactivity in mouse heart. Fig. 3 shows group data for Bax, Fas, and FasL immunoreactivity in the hearts of both strains at control, 1, 2, and 3 weeks p.i. Myocardial vessel immunoreactivity is shown in Table 1. Fig. 2A shows typical Bax immunoreactivity in the myocytes and vessels of a saline injected control C3H animal. As indicated in Fig. 3, we observed mild to moderate levels of Bax staining in the hearts of both strains of mice at all time points. We did not observe significant differences in Bax immunoreactivity in the hearts and blood vessels of either strain over the study period.

View larger version (142K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Representative photomicrographs showing pro-apoptotic protein immunoreactivity (IR) in mouse heart. Immunohistochemistry was performed as described in Section 2. (Panel A) Bax IR in vehicle-injected control C3H mouse heart at 200x original magnification. (Panel B) Fas IR in the heart of a C3H mouse 3 weeks p.i. at 200x original magnification. (Panel C) FasL IR in the heart of a CD1 mouse 2 weeks p.i. showing granular immunostaining pattern along membranes (arrows) at 400x original magnification. Control specimens incubated without the corresponding primary antibodies are shown below each panel. Scale bars represent 10 µm.

 

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Bax, Fas, and FasL immunoreactivity (IR) in hearts from CD1 (left panel) and C3H (right panel) mice at control (C) and at 1, 2, and 3 weeks (wk) p.i. with coxsackievirus B3. Immunohistochemistry with controls was performed as described in Section 2. Slides were scored for Bax, Fas, and FasL IR (0–3 scale) in blinded fashion. Bars represent mean score from 5 animals/group. *p<0.05, p<0.01 vs. vehicle-injected controls from same strain. No significant differences in Bax, Fas, and FasL IR in heart were observed between CD1 and C3H controls.

 

View this table: [in this window] [in a new window]   Table 1 Myocardial vessel immunoreactivity

 
Fig. 2B shows typical Fas immunostaining in myocardium from a C3H mouse 3 weeks p.i. As shown in Fig. 3, though Fas immunoreactivity was observed in the hearts of both strains at all time points, no significant differences in staining were detected except at 2 weeks p.i. in CD1 animals. In addition, as shown in Table 1, no significant differences in vessel Fas immunoreactivity were observed in either strain over the study period.

As indicated by the arrows in Fig. 2C, FasL exhibited an unusual granular staining pattern in the hearts of both strains at all time points. This staining pattern suggests FasL is membrane associated or is localized to the membranes of myocytes by a FasL receptor (e.g., Fas). Myocardial FasL immunoreactivity is shown graphically in Fig. 3. As indicated in Table 1, we observed FasL immunoreactivity in the vascular smooth muscle cells of heart vessels in both strains of mice. Significantly, in vessels, FasL immunoreactivity fell to undetectable levels by 1 week p.i. in both strains (both p<0.01), after which FasL returned to control levels in CD1 but not C3H mice. With few exceptions, no clear temporal pattern of Bax, Fas, and FasL immunostaining was observed in the myocardium of either strain.

Fig. 4, panels A and B, show Bcl-2 immunoreactivity in the heart of a C3H mouse at control and 1 week p.i., respectively. This figure demonstrates the typical cytoplasmic immunostaining pattern that was observed in the hearts of both strains of mice. Fig. 5 shows Bcl-2 immunoreactivity scores for each group plotted against time of infection. Histopathology scores are plotted on the same graph to demonstrate similarities in the temporal patterns of Bcl-2 antigen expression and inflammatory status in the hearts of CD1 and C3H mice. We observed an increase in Bcl-2 immunoreactivity in heart in both strains following viral inoculation. As indicated in Fig. 5, in both strains, Bcl-2 immunoreactivity appeared to follow histopathological status in heart. Additionally, as shown in Table 1, we observed mild to moderate Bcl-2 staining in vascular smooth muscle cells although no significant differences in immunoreactivity were observed in either strain over the study period. These data suggest a possible link between pathology and myocardial Bcl-2 expression that is not necessarily present in vascular smooth muscle.

View larger version (170K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Myocardial Bcl-2 immunoreactivity in vehicle-injected control (Panel A) and infected (1 week p.i.; Panel B) C3H mice. Immunohistochemistry with controls were performed as described in Section 2. Panel B shows increased Bcl-2 immunoreactivity is associated with extravascular immune cell infiltrate in myocardium. Scale bars represent 10 µm.

 

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Bcl-2 immunoreactivity (IR) and histopathological scores in hearts of CD1 (left panel) and C3H (right panel) mice before (C) and at 1, 2, and 3 weeks (wk) p.i. with coxsackievirus B3. Slides were scored for Bcl-2 IR (0–3 scale) and inflammation (0–4 scale) in blinded fashion as described in Section 2. Bars represent mean score from 5 animals/group. *p<0.05, p<0.01 vs. control from same strain. No significant difference in Bcl-2 IR in heart was observed between CD1 and C3H controls.

 
Because inducible nitric oxide (NO) production has been reported to stimulate apoptosis in some cells, we further assessed iNOS antigen expression in the hearts of CD1 and C3H mice before and after viral inoculation. Fig. 6 shows group data for iNOS immunoreactivity in CD1 and C3H hearts plotted against time of infection. These data indicate a temporal expression pattern similar to that of Bcl-2. Moderate to strong iNOS immunostaining was observed in myocardial blood vessels of both strains at control and all time points (Table 1).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 iNOS immunoreactivity (IR) in hearts of CD1 and C3H mice before (C) and at 1, 2, and 3 weeks (wk) p.i. with coxsackievirus B3. Immunohistochemistry was performed as described in Section 2. Slides were scored for iNOS IR (0–3 scale) in blinded fashion. Weak iNOS IR was observed in controls from both strains. iNOS IR was undetectable in hearts from CD1 mice 3 weeks p.i. Bars represent mean score from 5 animals/group. *p<0.05, p<0.01 vs. control from same strain. No significant difference in iNOS IR in heart was observed between CD1 and C3H saline vehicle-injected controls.

 
Since pro-apoptotic proteins were present in the hearts of both strains of mice, we searched for the presence of interleukin-1β converting enzyme (ICE) and p53 immunoreactivity in the hearts of these animals. ICE has been reported to be necessary for the intracellular transduction of Fas-mediated death signals. Despite the presence of Fas and FasL, we did not find intracellular ICE immunoreactivity in the myocardium of either strain of mice. Interestingly, we did find some extracellular ICE immunostaining associated with the epicardium (not shown). The precise source of the positive immunoreactivity could not be determined at the light microscopic level. Because the pro-apoptotic proto-oncogene, p53, is induced in some cells in response to DNA strand breaks and since this damage may be present in inflamed myocardium, especially given the induction of iNOS, we conducted a search for p53 antigen expression in the hearts of CD1 and C3H mice. We found no detectable levels of p53 immunoreactivity in the myocardium of either strain of mouse over the course of this study.

3.1 Terminal transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay
Since pro-apoptotic proteins were present in abundance in myocardium, we conducted a careful and systematic search for cells containing nuclei with fragmented DNA, one hallmark of apoptosis, which were labeled using the TUNEL assay. Tissue sections were also studied by light microscopy under high power (oil immersion) in an attempt to identify vesiculated cells exhibiting apoptotic bodies, another morphological feature of apoptosis. Although we observed some variability in the numbers of TUNEL-positive cells in thymus and spleen, we did observe positively stained cells in nearly all animals. Despite this, we found only two occurrences of TUNEL-positive cells in heart sections. Furthermore, we cannot confirm that the two cells apparently undergoing apoptosis in heart were myocytes. Fig. 7 and inset show one of these TUNEL-positive cells in the heart of a C3H mouse 3 weeks p.i. The absence of significant apoptosis in the hearts of control and infected mice was further supported by DNA laddering results (Fig. 8), which did not exhibit the typical approximately 200 nucleotide banding pattern. Our difficulty in demonstrating TUNEL-positive cells in the hearts of CD1 and C3H mice despite its presence in other tissues over the course of this study, taken together with our immunohistochemical results, indicates that apoptosis in myocardium under these conditions is exceedingly rare.

View larger version (160K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Apoptosis in C3H myocardium (A), thymus (B), and spleen (C) 3 weeks p.i. with coxsackievirus. Panel A shows one cell of unknown type exhibiting both TUNEL-positive staining and morphological characteristics (apoptotic bodies) attributable to apoptosis. The secondary lymphoid tissues shown in Panels B and C (arrow) contained many cells undergoing apoptosis. Original magnifications were 400x and 1000x (inset) with scale bars representing 10 µm.

 

View larger version (87K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 DNA laddering was not detected in control and infected CD1 and C3H mouse hearts. Total DNA was isolated from mouse hearts before and after viral inoculation, size fractionated on a 1.8% agarose gel, and stained with 2 µg/ml ethidium bromide as described in Section 2. Lanes 1 and 8 contain 2 µg each of 100 bp DNA size standard. Lanes 2–7 contain 10 µg total DNA from mouse hearts and cultured splenocytes as indicated.

 

	4 Discussion

Top Abstract 1 Introduction 2 Materials and methods 3 Results 4 Discussion References

 
Results from this study indicate that programmed cell death, or apoptosis, contributes minimally to the myocardial degeneration observed during the acute phase of experimental viral myocarditis in CD1 and C3H mice. We [2], and others [10, 11], have previously demonstrated that hearts of coxsackievirus B3-infected mice exhibit increased expression of proinflammatory cytokines, IL-1β and TNF-, as well as IL-6 and iNOS; all of which have been shown to induce or accelerate apoptosis in susceptible cells [12–15]. Given the potential role of apoptosis in viral infections [16], and the myocardial expression of pro-apoptotic proteins [17], we sought to determine the occurrence of apoptosis in heart and whether differences in susceptibility to viral myocarditis could be attributed to differences in the expression of apoptosis-related antigens.

Immunohistochemical assessment of apoptosis-related proteins in the hearts of coxsackievirus B3-infected mice revealed that increased myocardial inflammation was associated with increased cytoplasmic expression of the apoptosis-opposing protein, Bcl-2. Fig. 5 indicates that, in a fashion similar to that of the histopathology scores, Bcl-2 expression increased modestly in CD1 mice, peaking at 2 weeks p.i. This was followed by a return to control levels by week 3. C3H mice exhibited increased and sustained presence of both inflammation and Bcl-2 immunoreactivity following infection. This increase in Bcl-2 immunoreactivity following infection was not observed in myocardial vessels. These data suggest that in myocytes, and not myocardial vessels, the bcl-2 gene is upregulated following infection. This may be mediated indirectly, by a signal in the inflammatory milieu as demonstrated for the Bcl-2 homologue A1 [18], and/or directly by infective virus or persistent viral genetic material in the myocardium [19, 20]. Kajstura and coworkers [21]showed that bcl-2 mRNA levels correlated with programmed cell death during normal myocardial development in the rat, suggesting that bcl-2 gene expression is a physiologically relevant mechanism to suppress apoptosis in heart.

We observed that cytoplasmic immunostaining of the apoptosis-promoting Bcl-2 homologue, Bax, remained moderate in myocardium, with no significant changes in immunoreactivity following viral inoculation in either strain of mice. Despite a lack of statistical significance, Bax immunoreactivity tended to be lowest at 1 week p.i. in both strains. Bax has been shown to potently induce apoptosis by heterodimerizing with Bcl-2 and opposing its protective effects [22]. Thus, one mechanism for suppression of apoptosis is felt to be the relation between the relative abundance of Bcl-2 and Bax, or their homologues [23–26]. We found an increased expression of Bcl-2 relative to Bax in the hearts of coxsackievirus-infected mice, a combination which may be sufficient to actively suppress apoptosis in heart.

In addition to induction of apoptosis by proinflammatory cytokines, IL-6, and nitric oxide (NO), crosslinking of the Fas (APO-1) receptor by FasL or anti-Fas antibodies has been shown to induce apoptosis in a variety of susceptible cells [27]. Apoptosis may also be induced in target cells by cytotoxic T lymphocytes through a Fas-mediated mechanism [28]. Watanabe-Fukunaga et al. [29]detected abundant Fas mRNA in various tissues including heart. We observed moderate Fas immunoreactivity in both strains of mice at all time points in the study (Fig. 3). There was no clear temporal pattern of Fas staining in either strain, nor was there a correlation with histological signs of inflammation. Fas expression was significantly elevated at 2 weeks p.i. in CD1 mice, otherwise no significant differences were observed in either strain as compared to controls. Fig. 2B shows diffuse cytoplasmic Fas immunostaining in heart.

We report FasL immunoreactivity in heart for the first time. FasL is a member of the tumor necrosis factor family which is reportedly produced as a type II membrane protein expressed in activated splenocytes and thymocytes [30]. Interestingly, as shown in Fig. 2C, FasL exhibited a granular staining pattern along myocardial cell membranes which was observed reproducibly in all animals, including saline-injected controls. This unique, membrane-localized staining pattern was observed only in heart and suggests that either (1) FasL is produced by myocytes and targeted to plasmalemma where it may interact with Fas receptor, and/or (2) soluble FasL is produced by T lymphocytes [31], or testes and becomes bound to Fas expressed on myocytes. The presence of FasL immunostaining in control animals argues against local production by myocytes given the reported absence of FasL mRNA in rodent heart [30, 32]. In addition to myocytes, we found cytoplasmic FasL immunoreactivity in blood vessels (Table 1), and in thymus, spleen, and pancreatic islets (data not shown).

The colocalization of Bax, Fas, and FasL in heart demonstrates that the substrate for apoptosis is present in this tissue. Whether or not programmed cell death is initiated under these conditions likely depends on the presence of protective proteins such as Bcl-2 and its analogues, as well as on the ability of the cells in the tissue to transduce the death signal intracellularly. Prior studies have shown that an essential link in the transduction and execution of Fas- and TNF-mediated apoptosis is the presence of interleukin-1β converting enzyme (ICE). ICE is a cysteine protease which processes inactive pro-IL-1β into mature, active IL-1β [33], and which plays an essential role in programmed cell death [34–37]. We did not find ICE immunoreactivity in myocytes or in vessels of either strain of mice before or after infection, suggesting this signal for apoptosis is not present, and is not induced during inflammation within the myocardium. It is possible that an isoform of ICE not recognized by our antibody, or another enzyme with ICE-like activity could have been present in heart. Interestingly, we did find ICE immunoreactivity in an extracellular distribution in the heart (data not shown). Singer and coworkers [38]reported the presence of ICE on the external membranes and cytoplasmic ground substance of human monocytes. Since this extracellular ICE is unlikely to directly participate in apoptosis, its role remains unclear. The absence of intracellular ICE immunoreactivity indicates this apparatus for transduction of the death signal was not active in the hearts we studied.

Another protein involved in the induction of apoptosis following DNA damage is the tumor suppressor, p53. We found no p53 immunoreactivity in mouse heart. The absence of p53 in heart is consistent with the observed expression patterns of Bcl-2 and Bax. The p53 protein is a negative regulator of bcl-2 [39], whereas it increases the expression of bax which contains four p53 binding motifs [40]. We observed increased expression of Bcl-2 in heart without significant induction of Bax, a pattern which would not be expected following involvement of p53.

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is known to participate in host cell defense against microbial pathogens [41]. Messmer and coworkers [15]have shown that induction of iNOS leads to DNA strand breaks, p53 accumulation, and apoptosis in cultured mouse macrophage-like RAW 264.7 cells, and that this is prevented by iNOS inhibition. We observed mild to moderate iNOS immunoreactivity in myocytes which exhibited a temporal expression pattern similar to Bcl-2 (see Figs. 5 and 6). This induction is consistent with the observation that inflammatory mediators such as TNF- and IL-1β, known to be elevated in viral myocarditis, can stimulate the expression of iNOS [42]. In our study, although iNOS was induced following viral infection, myocyte nuclei containing DNA strand breaks were not found by TUNEL assay and there was no accumulation of p53. Furthermore, despite strong iNOS staining in vessels, no TUNEL-positive nuclei were observed in these structures.

Considerable recent attention has focused on apoptotic processes in the heart. Gottlieb and colleagues [43]have described apoptosis in cultured adult rabbit cardiomyocytes following ischemia/reperfusion injury which is consistent with similar observations reported by Tanaka et al. [44]showing hypoxia-induced apoptosis in cultured neonatal rat cardiomyocytes. Furthermore, Kajstura and coworkers [17]reported that apoptosis constituted the major form of cardiomyocyte cell death observed during the first few hours following epicardial coronary occlusion in rats, whereas necrosis was mainly responsible for cell death by 1 and 2 days post-occlusion. The apparent absence of apoptosis in mice with acute viral myocarditis may reflect fundamental differences in myocardial response to these two insults. It is likely that the post-ischemic burst of reactive oxygen intermediates is far greater than that seen in a more chronic inflammatory state. In addition, differences in species may determine the intrinsic susceptibility of myocytes to programmed cell death. The possibility exists that we did not find apoptotic cardiomyocytes in mice due to our sampling frequency, which was designed to elucidate ongoing apoptotic processes in heart during the acute phase of viral infection as opposed to the more immediate effects of hypoxia on myocardium. Our results are supported, in part, by the observations of Kawano et al. [45]which failed to demonstrate evidence of apoptosis in heart biopsies from three human patients with acute myocarditis. However, our conclusions are based on lines of evidence in addition to direct visualization of apoptosis, since direct demonstration of this process is made difficult due to the transient nature of this process resulting from its rapid induction and the rapid clearance of apoptotic debris [46].

In this study we selected two strains of mice which differ in their susceptibility to coxsackievirus B3 infection; CD1 mice develop acute myocarditis and recover rapidly, whereas C3H mice develop a more severe and persistent myocarditis. Differences in prognosis may result, in part, from the expression of apoptosis-related proteins, including iNOS and Bcl-2, which are more strongly induced in C3H than in CD1 animals. Levine and coworkers [47]have demonstrated that over-expression of bcl-2 inhibits cell lysis by Sindbis virus and results in persistent infection. Although it is unlikely that latent coxsackievirus persists in heart [48], viral suppression of apoptosis has been demonstrated in a number of cases [16, 49]. Suppression of apoptosis during viral infection could have several consequences in heart, including increased inflammation resulting from viral lysis of cells, as opposed to apoptosis which typically does not elicit an immune response. Further studies in other mouse strains, particularly those with limited postviral necrosis and tendencies toward cardiac autoimmunity, may be important to determine whether apoptosis plays a role in these subgroups of myocarditic animals.

We have previously shown that C3H mice express higher levels of IL-1β, IL-6, TNF-, and iNOS in heart than CD1 animals following acute coxsackievirus B3 infection [2]. Furthermore, this increase in expression of inflammatory mediators correlated with increased contractile dysfunction and inflammation in C3H as compared to CD1 mice [2]. Results from this study indicate that apoptosis does not contribute significantly to viral susceptibility and myocardial degeneration in acute experimental coxsackievirus myocarditis. This conclusion is supported by our observation that Bcl-2, which is protective against apoptosis, is upregulated in myocardium while Bax expression remains relatively constant. In addition, neither interleukin-1β converting enzyme nor p53, both of which are involved in the induction of apoptosis, could be detected in heart. And finally, the lack of significant numbers of TUNEL-positive myocytes further supports the notion that apoptosis is rare under these conditions. Further studies are needed to determine which factors control the expression of apoptosis-related proteins and to identify the source of those factors in acute experimental coxsackievirus myocarditis.

Time for primary review 30 days.

	Acknowledgements

 
The authors wish to sincerely thank Charles Gauntt, Ph.D. for generously supplying the virus used for these studies, and Jim Wood for assisting with inoculation. This work was supported by the Research Service of the Department of Veterans Affairs, and by the Cardiovascular Training Grant 2 T32 HL07350 (JTC).

	References

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