Expression of Coxsackievirus and Adenovirus Receptor in Hearts of Rats With Experimental Autoimmune Myocarditis
Abstract—The expression of coxsackievirus and adenovirus receptor (CAR) was dominant in the brains and hearts of mice until the newborn phase. There is no detailed information concerning the relation between the expression of CAR and development of hearts. It is also uncertain whether CAR is able to be induced in adult hearts after cardiac injury. We demonstrated that CAR was abundant in the hearts of newborn rats but was barely detectable in the hearts of adult rats. The expression of CAR in rat hearts with experimental autoimmune myocarditis, which was induced by immunization of purified cardiac myosin, was serially investigated. Active myocarditis was observed from day 15 after immunization. By immunohistochemistry, cardiomyocytes were strongly stained for CAR antibody from days 24 to 42. CAR mRNA was also detected from days 18 to 30 by using reverse transcription–polymerase chain reaction. In the next experiment, the induction of CAR on isolated cardiomyocytes was investigated. CAR was barely detectable in cultured cardiomyocytes by Western blot analysis after isolation. This molecule gradually appeared along with the creation of clusters and beating of cardiomyocytes. Furthermore, the induction of CAR in cultured cardiomyocytes increased after supplement with conditioned medium of rat splenocytes activated by concanavalin A. In conclusion, rat CAR is expressed strongly in the hearts of newborn rats and is suppressed in those of adult rats. The expression of CAR is enhanced during the active phase of experimental autoimmune myocarditis and is induced by inflammatory mediators. CAR may play a role in cell-to-cell contact and adhesion of cardiomyocytes.
Coxsackievirus and adenovirus receptor (CAR) was recently identified, and cDNA of this molecule was isolated in humans and mice.1 2 3 CAR exhibits both tissue- and species-specific expression. In mice, CAR was present in the brain, liver, heart, lungs, and kidneys.1 2 In our previous study, CAR was strongly expressed in the brains and hearts of mice until the newborn phase; its expression then decreased and subsequently became undetectable in adult mice.4 These observations imply that the expression of CAR exhibits developmental changes and that this molecule may play roles in differentiation and development of the brain and heart.
CAR structurally belongs to the immunoglobulin superfamily, similar to intercellular adhesion molecule-1 (ICAM-1) and neural cell adhesion molecule (N-CAM). Both ICAM-1 and N-CAM have been demonstrated to be related to either the normal development of the heart or the pathogenesis of cardiovascular diseases.5 6 7 The physiological functions of CAR have not yet been elucidated.3 Whether CAR plays a role in left ventricular remodeling after myocardial injury has not yet been determined. In the present study, we demonstrated that CAR was reexpressed in the hearts of adult rats with rat experimental autoimmune myocarditis, which is an animal model of human giant cell myocarditis leading to dilated cardiomyopathy. Furthermore, we examined the expression of CAR in cultured rat cardiomyocytes supplemented with conditioned medium of rat splenocytes activated by concanavalin A (Con A) to elucidate the mechanisms of the induction of CAR, accompanied by inflammation.
Materials and Methods
Experimental autoimmune myocarditis was elicited in Lewis rats by immunization of purified pig cardiac myosin as previously described.8 On day 0, 15 rats were immunized with antigen-adjuvant emulsion. Another group of 15 rats was immunized with saline as the control.
Newborn rats were killed on day 1 after birth, and their hearts were removed. Myosin-immunized rats were killed on days 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 49, and 56 after immunization. Macroscopic findings were scored in 3 grades as previously described.8 Hearts were then removed and weighed to calculate the heart weight/body weight ratio. The ventricular muscle was used for the following analysis.
Preparation of Antibody
Seventeen amino acid residues (KTQYNQVPSEDFERAPQ) in an intracellular domain of CAR were selected. Antibodies against CAR were prepared as previously described.9
Histopathology and Immunohistochemistry
CAR was immunohistochemically detected by use of purified rabbit anti-murine CAR antibodies. Positive findings for CAR produced a brown color. The severity of inflammation was graded from 0 (no inflammation) to 3 (severe inflammation) as previously described.10 The expression of CAR was graded as follows: −, negative; ±, weakly positive; +, moderately positive; and ++, strongly positive.
Total RNA was extracted from rat hearts, and reverse transcription (RT)–polymerase chain reaction (PCR) was performed with primers (ATGGATCCTACACCCGAACAGAGGATCG [sense] and GCGAATTCGCGTCGCCAGACTTGACAT [antisense]). β-Actin cDNA was amplified as the control. Thirty-three cycles of amplification reactions (94°C denaturation, 53°C annealing, and 72°C extension) were performed.
Preparation of Conditioned Medium of Rat Splenocytes Activated by Con A
Single-splenocyte suspensions of adult rat11 were cultured for 2 days in the presence of 10 μg/mL of Con A, and the supernatant was obtained (Con A medium).
Preparation of Cultured Cardiomyocytes
Cardiomyocytes were prepared from neonatal rat ventricles by the modified method of Libby.12 The cardiomyocytes were divided into 4 groups. Group 1 was not cultured. Group 2 was cultured for 24 hours. Groups 3 and 4 were cultured for 96 hours. Group 4 was cultured in standard medium for an initial 24 hours and then cultured in Con A medium (10% of culture medium) for 72 hours.
Western Blot Analysis
Total protein was prepared from the left ventricle of 1-day-old and adult rats and from cultured cardiomyocytes with cell lysis buffer. Protein (15 μg) from total cell lysates13 was separated by SDS-PAGE and analyzed by immunoblotting with the polyclonal anti-CAR antibodies. The bands of CAR were analyzed with NIH Image and expressed in relation to the control values.
Data are presented as mean±SD. Statistical assessment of the significance among groups was made by 1-way ANOVA followed by the Bonferroni-Dunn method. A value of P<0.05 was considered significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
Expression of CAR in Hearts of Newborn and Adult Rats
To confirm that the PCR product was an amplified fragment of rat CAR cDNA, the PCR product on day 21 was directly sequenced. Because the sequence of the PCR product had a homology of 93% with murine CAR, we interpreted this product as rat CAR (sequence not shown). RT-PCR revealed that CAR mRNA was expressed abundantly in the hearts of newborn rats, whereas only a slight expression of CAR was detected in the hearts of normal adult rats (Figure 1⇓). Immunohistochemical staining showed that the expression of CAR was strong in the hearts of newborn rats but was undetectable in those of adult rats (Figure 2⇓).
Expression of CAR in Rat Hearts With Myocarditis
Clinical Course of Myosin-Immunized Rats
One of the myosin-immunized rats died on day 18, during the active phase of myocarditis. This rat showed macroscopic evidence of severe myocarditis and was thought to have died of heart failure. No control rats died spontaneously throughout the period of this experiment (Table⇓).
The extent of discolored areas of the hearts and the appearance of pericardial effusion are summarized in the Table⇑. From the 15th day after immunization, discolored areas were observed on the cardiac surface of the myosin-immunized rats. Hearts of the myosin-immunized rats were markedly enlarged, as shown by heart weight/body weight ratios >4.0; control rat heart weight/body weight ratios ranged from 2.89 to 3.83, with a mean of 3.16. Pericardial effusion was observed in myosin-immunized rats on days 18, 21, and 24 (Table⇑).
Microscopic findings are summarized in the Table⇑. There were no microscopic abnormalities in the hearts of either the control rats or the myosin-immunized rats on day 9. Small lesions of mononuclear cell infiltration were observed in the hearts of the myosin-immunized rats on day 12. Thereafter, inflammatory lesions spread extensively, and interstitial edema became evident. From day 21, in addition to mononuclear cell infiltration, myocardial necrosis was observed. Fibrosis became prominent on day 36. Most of the infiltrated cells disappeared on day 49, and the inflammatory lesions were replaced by fibrosis.
CAR mRNA was identified in the hearts of the myosin-immunized rats on days 18 to 30 and peaked on days 21 and 24. CAR mRNA was not detectable after day 33 by RT-PCR (Figure 3⇓). CAR mRNA was not detected in the control group throughout the experiment by our RT-PCR (data are not shown).
Immunohistochemical staining for CAR was negative in the hearts of control rats. Hearts of the rats with experimental autoimmune myocarditis were stained with anti-CAR antibodies from day 24. Immunoreactivity of CAR was found in the cardiomyocytes of rats with experimental autoimmune myocarditis, not only in the inflammatory lesions but also in the intact area, where infiltration cells and myocardial degeneration were not found. Scar tissue and the vascular wall were not stained. The staining was most intense in the hearts obtained on day 33, after which immunoreaction for CAR gradually decreased (Figure 4⇓).
Induction of CAR in Cultured Cardiomyocytes
Although CAR was expressed abundantly in the hearts of the newborn rats, only a slight expression of CAR was detected in cultured rat cardiomyocytes after isolation from the newborn rats by Western blot analysis (Figure 5A⇓). The expression of CAR was suppressed after isolation, but then it gradually increased according to the creation of clusters and beating of cardiomyocytes. Furthermore, the induction of CAR was more significantly enhanced in cultured cardiomyocytes treated with Con A medium than those treated with culture medium (Figure 5B⇓).
CAR structurally belongs to the immunoglobulin superfamily, and its physiological function has not yet been clarified. The immunoglobulin superfamily includes many cell-surface proteins that mediate cell-to-cell recognition or antigen recognition in the immune system. N-CAM, which also belongs to the immunoglobulin superfamily, was expressed prominently in the embryonic phase, and its expression was suppressed in the hearts of adult rats.14 N-CAM immunoreactivity was reexpressed in transplanted extrinsically denervated human hearts and also in the hypertrophic myocardium of rats.15 16 From these observations, N-CAM was considered to play roles in both development of the heart and innervation to the heart. The expression of CAR was high in the myocardium of newborn rats and low in the myocardium of adult rats in the present study. This implied that CAR might act on myocardial morphogenesis similar to N-CAM. Although CAR was abundantly expressed in the hearts of newborn rats, the expression of CAR decreased in cultured cardiomyocytes after isolation from these hearts. CAR gradually appeared again as cardiomyocytes began to make clusters and start a beat. On the basis of these results, we suggest that CAR may play roles in cell-to-cell contact and adhesion.
Rat experimental autoimmune myocarditis is inducible by purified cardiac myosin and is an animal model of human giant cell myocarditis, which leads to dilated cardiomyopathy. 17 T cells play an important role in inducing myocarditis in this model.11 We examined the expression of CAR in experimental autoimmune myocarditis of rats to clarify whether CAR was reenhanced in diseased hearts. In the course of experimental autoimmune myocarditis, the expression of CAR in the hearts was low or undetectable before the onset of the disease, and then its expression became apparent during the active phase of myocarditis. Next, the expression of CAR decreased in the chronic phase. ICAM-1, which also belongs to the immunoglobulin superfamily, was also induced in myocardial cells of mice with acute viral myocarditis.7 Its expression coincided with cell infiltration. ICAM-1 on cardiac myocytes, together with myosin heavy chain antigens, was demonstrated to be associated with cell-mediated myocardial injuries by activated lymphocytes.7 On the other hand, the expression of rat CAR was preceded by several days of massive cell infiltration. Accordingly, the expression of CAR in myocardial cells might not be related to myocardial injuries in acute myocarditis. CAR may be associated with the healing phase of myocarditis or regeneration of damaged myocardium.
The mechanism responsible for the induction of CAR had not been elucidated. In the present study, CAR was induced when myocytes were treated with conditioned medium of rat splenocytes activated by Con A. This result implied that the expression of CAR was induced by inflammatory mediators. We previously revealed the time course of the expression of various cytokine mRNA in experimental autoimmune myocarditis.18 19 mRNA of proinflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, and interleukin-1β, and inducible nitric oxide synthase were expressed in the myocardium of experimental autoimmune myocarditis from the beginning of the acute inflammatory phase. CAR mRNA was reexpressed after the expression of these cytokines. The present study suggests that inflammatory mediators play a role in the induction of CAR in experimental autoimmune myocarditis. Further study is necessary to identify the specific mediators inducing CAR.
In conclusion, the expression of CAR was enhanced in experimental autoimmune myocarditis. Our results suggest that CAR is induced by inflammatory mediators. This molecule may contribute to cell-to-cell contact and to the adhesion of cardiomyocytes and act on myocardial regeneration.
- Received August 19, 1999.
- Accepted November 5, 1999.
- © 2000 American Heart Association, Inc.
Bergelson JM, Krithivas A, Celi L, Droguett G, Horwitz MS, Wickham T, Crowell RL, Finberg RW. The murine CAR homolog is a receptor for coxsackie B viruses and adenoviruses. J Virol. 1998;72:415–419.
Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science. 1997;275:1320–1323.
Tomko RP, Xu R, Philipson L. HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci U S A. 1997;94:3352–3356.
Hotta Y, Masuko M, Hanyu T, Abe TK, Honda T, Tominaga K, Odani S, Kuwano R. Immunohistochemical analysis of new adhesion molecule gmp46. Neurosci Res. 1998;22(suppl):S313. Abstract.
Hillis GS, Flapan AD. Cell adhesion molecules in cardiovascular disease: a clinical perspective. Heart. 1998;79:429–431.
Seko Y, Matsuda H, Kato K, Hashimoto Y, Yagita H, Okumura K, Yazaki Y. Expression of intracellular adhesion molecule-1 in murine hearts with acute myocarditis caused by coxsackievirus B3. J Clin Invest. 1993;91:1327–1336.
Kodama M, Zhang S, Hanawa H, Saeki M, Inomata T, Suzuki K, Koyama S, Shibata A. Effects of 15-deoxyspergualin on experimental autoimmune giant cell myocarditis of the rat. Circulation. 1995;91:1116–1122.
Kodama M, Matsumoto Y, Fujiwara M. In vivo lymphocyte-mediated myocardial injuries demonstrated by adoptive transfer of experimental autoimmune myocarditis. Circulation. 1992;85:1918–1926.
Gordon L, Wharton J, Moore SE, Walsh FS, Moscoso JG, Penketh R, Wallwork J, Taylor KM, Yacoub MH, Polak JM. Myocardial localization and isoforms of neural cell adhesion molecule (N-CAM) in the developing and transplanted human heart. J Clin Invest. 1990;86:1293–1300.
Kodama M, Hanawa H, Saeki M, Hosono H, Inomata T, Suzuki K, Sibata A. Rat dilated cardiomyopathy after autoimmune giant cell myocarditis. Circ Res. 1994;75:278–284.
Hirono S, Islam MO, Nakazawa M, Yoshida Y, Kodama M, Shibata A, Izumi T, Imai S. Expression of inducible nitric oxide synthase in rat experimental autoimmune myocarditis with special reference to changes in cardiac hemodynamics. Circ Res. 1997;80:11–20.
Okura Y, Yamamoto T, Goto S, Inomata T, Hirono S, Hanawa H, Feng L, Wilson CB, Kihara I, Izumi T, Shibata A, Aizawa Y, Seki S, Abo T. Characterization of cytokine and iNOS mRNA expression in situ during the course of experimental autoimmune myocarditis in rats. J Mol Cell Cardiol. 1997;29:491–502.