JOURNAL OF PATHOLOGY, VOL.
167: 341-347 (1992)
SUCCESSIVE INFECTION OF COXSACKIEVIRUS B3 AND ENCEPHALOMYOCARDITIS VIRUS: AN ANIMAL MODEL OF CHRONIC MYOCARDITIS IKUTARO OKADA*, AKIRA MATSUMORI*, NOBUYOSHI T O M I O K A ~AND CHUICHI KAWAI*
*Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto, Japan, t Cardiovascular Division, Otsu Red Cross Hospital, Otsu, Japan Received8 October 1991 Accepted 18 December 1991
SUMMARY Successive infection of coxsackievirus B3 and encephalomyocarditis virus was investigated as a disease model of chronic myocarditis. Four-week-old C3H/He mice were inoculated with coxsackievirus B3 and then inoculated with encephalomyocarditis virus at 8 weeks old. The hearts were evaluated on histopathological changes compared with those of non-infected mice and mice infected with either virus alone. At 10 weeks old, the hearts of the mice infected successively with both viruses showed co-existence of fibrosis surrounding calcified lesions and marked cellular infiltration with myocardial necrosis. These findings resembled chronic active myocarditis in humans, unlike the lesions due to either virus alone. At 12 weeks old, the hearts of all the infected mice showed fibrosis with scarce cellular infiltration. The successively infected hearts also showed a significantly higher heart weight to body weight ratio than that of the non-infected control mice, and localized wall thinning in the damaged regions. Thus, we conclude that successive infection additively causes myocardial damage that resembles chronic myocarditis and may produce a heart condition similar to dilated cardiomyopathy. KEY
WORDS-Chronic myocarditis, dilated cardiomyopathy, coxsackievirus B3, encephalomyocarditis virus, successive infection
INTRODUCTION Many viruses, especially enteroviruses, have been implicated as aetiologic in human myocarditis.’ Virus infection has also been suggested as one of the causes of dilated c a r d i o m y ~ p a t h y .However, ~?~ it is often difficult to identify the virus clinically in cases of acute viral my~carditis.~ The detection of neutralizing antibodies cannot be reliably used for the identification of aetiologic enteroviruses because enteroviruses are common human infectious agents’ and the use of enterovirus-specific IgM to identify an aetiologic enterovirus is subject to difficulties in inter~retation.~-’ These serologicalfindings suggested Addressee for correspondence: Chuichi Kawai, MD, Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Kawaracho Shogoin, Sakyo-ku, Kyoto 606, Japan.
0022-341 7/92/07034147 $08.50 0 1992 by John Wiley & Sons, Ltd.
that infections of several kinds of viruses cause human myocarditis and dilated cardiomyopathy. Experimental murine myocarditis can also be induced by inoculation of group B Coxsackie viruses and encephalomyocarditis virus (EMCV). However, all of the animal models of viral myocarditis are models ofacute viral myocarditis. On the other hand, in humans the diagnosis of myocarditis is made as myocarditis with or without fibrosis at the first myocardial biopsy, and on-going (persistent), resolving (healing), or resolved (healed) myocarditis with or without fibrosis at subsequent biopsies.’ New methods of diagnosing viral myocarditis have emerged recently. Several investigators have reported the detection of enteroviral RNA for slotblot, in situ or Northern blot hybridization using nucleic acid probes complementary to viral genomes in experimental models or in human hearts obtained at biopsy, autopsy, or transplantation,’-’’ and
342
I. OKADA E T A L
recently by enzymatic amplification using the polymerase chain r e a ~ t i o n . ' ~ .These '~ studies were conducted mainly to investigate the possibility of persistence of the virus genome in the heart in myocarditis or dilated cardiomyopathy as it relates to the pathogenesis of these diseases. However, since these studies used nucleic acid probes which could detect many kinds of viruses, several kinds of viruses might be involved.16. In this study, we investigated the possibility of successiveinfection as a cause of chronic myocardi tis or dilated cardiomyopathy. The experiment was performed to see whether successiveinfection additively produced myocardial lesions resembling chronic myocarditis of humans. METHODS
Experiment 2 (Table I)-We examined the hearts 4 weeks after EMCV inoculation (12 weeks of age) to study the hearts in the healed stage of experimental myocarditis. At 4 weeks of age, mice (n=25) were inoculated with 4 x lo4pfu CVB3. The surviving mice were randomized into two groups. The mice of one group (C, n = 8) were observed without additional virus inoculation. The other group of mice (CE, n = 14) was inoculated with 4OOpfu EMCV at 8 weeks of age. The mice of another group (E, n = 9) were inoculated with EMCV 400 pfu at 8 weeks of age. Non-infected control mice (N, n = 6 ) were also killed at 12 weeks of age. All of the mice were weighed and then killed by neck dislocation. The hearts were excised aseptically, weighed, fixed in 10 per cent formalin solution and embedded in paraffin. Sections were cut and stained with haematoxylin and eosin. Myocardial cellular infiltration, myocardial cell necrosis, and fibrosis were scored blindly on a scale of 1 + to 4 + in terms of severity as described previously.'8 Briefly, a 1 score indicated a limited focal distribution; a 4 score indicated the presence of multiple lesions over the entire heart; while scores of 2 and 3 + were used to described intermediate severity.
Virus Coxsackievirus B3 (CVB3, Nancy strain) and encephalomyocarditis virus (EMCV, M variant) were obtained from the American Type Culture Collection. Virus stocks were prepared by plating CVB3 or EMCV onto confluent cultures of FL (human amnion) cells and their titres were determined by plaque assay as described previously. l7The virusstockshadtitresof4 x 107forCVB3and4x lo6 Statistical analysis for EMCV plaque-forming units (pfu) per ml. Virus One-way analysis of variance was applied to was stored at - 70°C until use. compare the mean values of multiple groups.
+ +
Virus infection of mice Male inbred C3H/He mice were obtained from Shizuoka Agricultural Cooperative Association (Shizuoka, Japan). Mice were housed in groups of at most five per cage and given water and rodent chow.
+
RESULTS
Experiment 1 We examined the incidence of myocardial lesions in experiment 1; the results are summarized in Table Experiment 1 (Table I)-At 4 weeks of age, mice I. All of the hearts of the mice which were killed 7 ( n = 38) were inoculated intraperitoneally with days after CVB3 inoculation at 4 weeks of age 0.1 ml of CVB3, previously diluted in phosphate- (group C5) showed marked cellular infiltration and buffered saline (PBS) to a concentration of 4 x lo4 myocardial cell necrosis. Six weeks after CVB3 pfu/O.1 ml. The mice of group C5 (n = 6) were killed inoculation (group ClO), the hearts showed dense 7 days after CVB3 inoculation. The other mice were fibrosis and calcification without ap arent cellular observed until 8 weeks of age. The surviving mice infiltration as described previously.l'The hearts of (n = 26) were randomized into three groups. The group CClO which were inoculated with CVB3 mice of group C10 (n=6) were observed until 6 twice also showed fibrosis and calcification without weeks after CVB3 inoculation. The mice of group cellular infiltration. Two weeks after EMCV inocuCClO ( n = 5 ) were inoculated again with CVB3 lation alone (group ElO), four of the ten hearts (4 x lo4 pfu) at 8 weeks of age. The other surviving showed massive cellular infiltration and myocardial mice were inoculated with EMCV, 10 ( n = 10) or 400 cell necrosis without apparent fibrosis. However, (n=5) pfu, at 8 weeks of age (group CEIO). The the rest of the hearts did not show any histological mice of group E l 0 (n = 12) were inoculated with changes (Table I). EMCV (10 pfu) at 8 weeks of age and then killed at On the other hand, the hearts of the mice inoculated successively with both CVB3 and EMCV 10 weeks of age.
343
CHRONIC MYOCARDITIS: AN ANIMAL MODEL
Table I-Experimental infections and histological scores of the hearts of the surviving mice No. of hearts showing each histological scores
Group Experiment I C5 ( n = 6) C 10 ( n = 6) CClO(n=5) CElO (n= 14) E10(n= 10)
1st
2nd
Infiltration
Necrosis
Fibrosis
infection (4 weeks old)
infection (8 weeks old)
0 1+2+3+4+
0 1+2+ 3+4+
0 1+2+3+4+
0 5 4 6 6
2 1 1 6 0
1 0 0 2 0
2 0 0 0 3
1 0 0 0 1
0 6 4 9 6
2 0 1 5 0
0 0 0 0 1
3 0 0 0 3
1 0 0 0 01
6 2 1 4 0
0 4 4 5 0
0 0 0 5 0
0 0 0 0 0
0 0 0 0 0
8 12 4 6
0 I 2 0
0 0 0 0
0 0 0 0
0 8 013 0 6 0 6
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
3 0 0 6
2 5 3 0
3 8 3 0
0 0 0 0
0 0 0 0
CVB3 CVB3 CVB3 CVB3 -
~
CVB3 EMCV EMCV
Experiment 2
C(n=8) CE(n=13) E(n=6) N ( n = 6)
CVB3 CVB3
~
-
EMCV EMCV
-
-
Numbers of the hearts showing each histological score are expressed in each column. A 0 score indicates no myocardial lesion; a score of 1 + indicates a limited focal distribution; a score of 4+ indicates the presence of multiple lesions over the entire heart; while scores of 2 + and 3 + are used to describe intermediate severity. The mice of group C5 were killed 1week after CVB3 infection (5 weeks ofage)/ miceofgroupsC10,CC10,CElO,and Elowere killedat 1Oweeksofage;and thoseofgroupsC,CE, E,andNwere killedat 12 weeks of age.
(group CE10) showed two different types of lesion (Fig. 1). One type of lesion involved cellular infiltration and myocardial cell necrosis, as we observed in group E10. The other involved dense fibrosis as in group C10. These two lesions overlapped each other in part. These changes suggested additional myocardial lesions due to EMCV inoculation after CVB3 inoculation and also resembled chronic active myocarditis. Additional lesions were seen in 8 of 14hearts in group CElO (Table I). Experiment 2
We examined the hearts 4 weeks after EMCV virus inoculation (Table I). At 12 weeks of age, most of the hearts infected with either or both viruses showed apparent fibrotic lesions in the myocardium (Fig. 2). When the fibrotic lesions were relatively large, the ventricular wall of the damaged area showed thinning and ventricular dilation (Fig. 3) compared with non-infected controls (Fig. 4). We also examined indices for congestive heart failure. No apparent pericardial or pleural effusions, ascites, or liver swelling were seen in any of the surviving mice (1 2 weeks of age). However, some
of the mice that died before the end of the experiment showed dilated and thin-walled hearts (Fig. 5), massive pericardial and pleural effusions, ascites, and liver swelling. Most of these mice died between 10 and 17 days after CVB3 or EMCV inoculation. We measured body weights and heart weights and calculated the ratio of heart weight to body weight as another index of congestive heart failure. The mean value of the heart weight of any one group of infected mice (C: 112.4k 12.0; CE: 101.2f 11.1; E: 108.049.4; mean f SD, mg) was not significantly different from that of the non-infected control (N: 101.7 k4.4 mg). However, the heart weight of group C was significantly heavier than that of group CE (P
167: 341-347 (1992)
SUCCESSIVE INFECTION OF COXSACKIEVIRUS B3 AND ENCEPHALOMYOCARDITIS VIRUS: AN ANIMAL MODEL OF CHRONIC MYOCARDITIS IKUTARO OKADA*, AKIRA MATSUMORI*, NOBUYOSHI T O M I O K A ~AND CHUICHI KAWAI*
*Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto, Japan, t Cardiovascular Division, Otsu Red Cross Hospital, Otsu, Japan Received8 October 1991 Accepted 18 December 1991
SUMMARY Successive infection of coxsackievirus B3 and encephalomyocarditis virus was investigated as a disease model of chronic myocarditis. Four-week-old C3H/He mice were inoculated with coxsackievirus B3 and then inoculated with encephalomyocarditis virus at 8 weeks old. The hearts were evaluated on histopathological changes compared with those of non-infected mice and mice infected with either virus alone. At 10 weeks old, the hearts of the mice infected successively with both viruses showed co-existence of fibrosis surrounding calcified lesions and marked cellular infiltration with myocardial necrosis. These findings resembled chronic active myocarditis in humans, unlike the lesions due to either virus alone. At 12 weeks old, the hearts of all the infected mice showed fibrosis with scarce cellular infiltration. The successively infected hearts also showed a significantly higher heart weight to body weight ratio than that of the non-infected control mice, and localized wall thinning in the damaged regions. Thus, we conclude that successive infection additively causes myocardial damage that resembles chronic myocarditis and may produce a heart condition similar to dilated cardiomyopathy. KEY
WORDS-Chronic myocarditis, dilated cardiomyopathy, coxsackievirus B3, encephalomyocarditis virus, successive infection
INTRODUCTION Many viruses, especially enteroviruses, have been implicated as aetiologic in human myocarditis.’ Virus infection has also been suggested as one of the causes of dilated c a r d i o m y ~ p a t h y .However, ~?~ it is often difficult to identify the virus clinically in cases of acute viral my~carditis.~ The detection of neutralizing antibodies cannot be reliably used for the identification of aetiologic enteroviruses because enteroviruses are common human infectious agents’ and the use of enterovirus-specific IgM to identify an aetiologic enterovirus is subject to difficulties in inter~retation.~-’ These serologicalfindings suggested Addressee for correspondence: Chuichi Kawai, MD, Third Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Kawaracho Shogoin, Sakyo-ku, Kyoto 606, Japan.
0022-341 7/92/07034147 $08.50 0 1992 by John Wiley & Sons, Ltd.
that infections of several kinds of viruses cause human myocarditis and dilated cardiomyopathy. Experimental murine myocarditis can also be induced by inoculation of group B Coxsackie viruses and encephalomyocarditis virus (EMCV). However, all of the animal models of viral myocarditis are models ofacute viral myocarditis. On the other hand, in humans the diagnosis of myocarditis is made as myocarditis with or without fibrosis at the first myocardial biopsy, and on-going (persistent), resolving (healing), or resolved (healed) myocarditis with or without fibrosis at subsequent biopsies.’ New methods of diagnosing viral myocarditis have emerged recently. Several investigators have reported the detection of enteroviral RNA for slotblot, in situ or Northern blot hybridization using nucleic acid probes complementary to viral genomes in experimental models or in human hearts obtained at biopsy, autopsy, or transplantation,’-’’ and
342
I. OKADA E T A L
recently by enzymatic amplification using the polymerase chain r e a ~ t i o n . ' ~ .These '~ studies were conducted mainly to investigate the possibility of persistence of the virus genome in the heart in myocarditis or dilated cardiomyopathy as it relates to the pathogenesis of these diseases. However, since these studies used nucleic acid probes which could detect many kinds of viruses, several kinds of viruses might be involved.16. In this study, we investigated the possibility of successiveinfection as a cause of chronic myocardi tis or dilated cardiomyopathy. The experiment was performed to see whether successiveinfection additively produced myocardial lesions resembling chronic myocarditis of humans. METHODS
Experiment 2 (Table I)-We examined the hearts 4 weeks after EMCV inoculation (12 weeks of age) to study the hearts in the healed stage of experimental myocarditis. At 4 weeks of age, mice (n=25) were inoculated with 4 x lo4pfu CVB3. The surviving mice were randomized into two groups. The mice of one group (C, n = 8) were observed without additional virus inoculation. The other group of mice (CE, n = 14) was inoculated with 4OOpfu EMCV at 8 weeks of age. The mice of another group (E, n = 9) were inoculated with EMCV 400 pfu at 8 weeks of age. Non-infected control mice (N, n = 6 ) were also killed at 12 weeks of age. All of the mice were weighed and then killed by neck dislocation. The hearts were excised aseptically, weighed, fixed in 10 per cent formalin solution and embedded in paraffin. Sections were cut and stained with haematoxylin and eosin. Myocardial cellular infiltration, myocardial cell necrosis, and fibrosis were scored blindly on a scale of 1 + to 4 + in terms of severity as described previously.'8 Briefly, a 1 score indicated a limited focal distribution; a 4 score indicated the presence of multiple lesions over the entire heart; while scores of 2 and 3 + were used to described intermediate severity.
Virus Coxsackievirus B3 (CVB3, Nancy strain) and encephalomyocarditis virus (EMCV, M variant) were obtained from the American Type Culture Collection. Virus stocks were prepared by plating CVB3 or EMCV onto confluent cultures of FL (human amnion) cells and their titres were determined by plaque assay as described previously. l7The virusstockshadtitresof4 x 107forCVB3and4x lo6 Statistical analysis for EMCV plaque-forming units (pfu) per ml. Virus One-way analysis of variance was applied to was stored at - 70°C until use. compare the mean values of multiple groups.
+ +
Virus infection of mice Male inbred C3H/He mice were obtained from Shizuoka Agricultural Cooperative Association (Shizuoka, Japan). Mice were housed in groups of at most five per cage and given water and rodent chow.
+
RESULTS
Experiment 1 We examined the incidence of myocardial lesions in experiment 1; the results are summarized in Table Experiment 1 (Table I)-At 4 weeks of age, mice I. All of the hearts of the mice which were killed 7 ( n = 38) were inoculated intraperitoneally with days after CVB3 inoculation at 4 weeks of age 0.1 ml of CVB3, previously diluted in phosphate- (group C5) showed marked cellular infiltration and buffered saline (PBS) to a concentration of 4 x lo4 myocardial cell necrosis. Six weeks after CVB3 pfu/O.1 ml. The mice of group C5 (n = 6) were killed inoculation (group ClO), the hearts showed dense 7 days after CVB3 inoculation. The other mice were fibrosis and calcification without ap arent cellular observed until 8 weeks of age. The surviving mice infiltration as described previously.l'The hearts of (n = 26) were randomized into three groups. The group CClO which were inoculated with CVB3 mice of group C10 (n=6) were observed until 6 twice also showed fibrosis and calcification without weeks after CVB3 inoculation. The mice of group cellular infiltration. Two weeks after EMCV inocuCClO ( n = 5 ) were inoculated again with CVB3 lation alone (group ElO), four of the ten hearts (4 x lo4 pfu) at 8 weeks of age. The other surviving showed massive cellular infiltration and myocardial mice were inoculated with EMCV, 10 ( n = 10) or 400 cell necrosis without apparent fibrosis. However, (n=5) pfu, at 8 weeks of age (group CEIO). The the rest of the hearts did not show any histological mice of group E l 0 (n = 12) were inoculated with changes (Table I). EMCV (10 pfu) at 8 weeks of age and then killed at On the other hand, the hearts of the mice inoculated successively with both CVB3 and EMCV 10 weeks of age.
343
CHRONIC MYOCARDITIS: AN ANIMAL MODEL
Table I-Experimental infections and histological scores of the hearts of the surviving mice No. of hearts showing each histological scores
Group Experiment I C5 ( n = 6) C 10 ( n = 6) CClO(n=5) CElO (n= 14) E10(n= 10)
1st
2nd
Infiltration
Necrosis
Fibrosis
infection (4 weeks old)
infection (8 weeks old)
0 1+2+3+4+
0 1+2+ 3+4+
0 1+2+3+4+
0 5 4 6 6
2 1 1 6 0
1 0 0 2 0
2 0 0 0 3
1 0 0 0 1
0 6 4 9 6
2 0 1 5 0
0 0 0 0 1
3 0 0 0 3
1 0 0 0 01
6 2 1 4 0
0 4 4 5 0
0 0 0 5 0
0 0 0 0 0
0 0 0 0 0
8 12 4 6
0 I 2 0
0 0 0 0
0 0 0 0
0 8 013 0 6 0 6
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
3 0 0 6
2 5 3 0
3 8 3 0
0 0 0 0
0 0 0 0
CVB3 CVB3 CVB3 CVB3 -
~
CVB3 EMCV EMCV
Experiment 2
C(n=8) CE(n=13) E(n=6) N ( n = 6)
CVB3 CVB3
~
-
EMCV EMCV
-
-
Numbers of the hearts showing each histological score are expressed in each column. A 0 score indicates no myocardial lesion; a score of 1 + indicates a limited focal distribution; a score of 4+ indicates the presence of multiple lesions over the entire heart; while scores of 2 + and 3 + are used to describe intermediate severity. The mice of group C5 were killed 1week after CVB3 infection (5 weeks ofage)/ miceofgroupsC10,CC10,CElO,and Elowere killedat 1Oweeksofage;and thoseofgroupsC,CE, E,andNwere killedat 12 weeks of age.
(group CE10) showed two different types of lesion (Fig. 1). One type of lesion involved cellular infiltration and myocardial cell necrosis, as we observed in group E10. The other involved dense fibrosis as in group C10. These two lesions overlapped each other in part. These changes suggested additional myocardial lesions due to EMCV inoculation after CVB3 inoculation and also resembled chronic active myocarditis. Additional lesions were seen in 8 of 14hearts in group CElO (Table I). Experiment 2
We examined the hearts 4 weeks after EMCV virus inoculation (Table I). At 12 weeks of age, most of the hearts infected with either or both viruses showed apparent fibrotic lesions in the myocardium (Fig. 2). When the fibrotic lesions were relatively large, the ventricular wall of the damaged area showed thinning and ventricular dilation (Fig. 3) compared with non-infected controls (Fig. 4). We also examined indices for congestive heart failure. No apparent pericardial or pleural effusions, ascites, or liver swelling were seen in any of the surviving mice (1 2 weeks of age). However, some
of the mice that died before the end of the experiment showed dilated and thin-walled hearts (Fig. 5), massive pericardial and pleural effusions, ascites, and liver swelling. Most of these mice died between 10 and 17 days after CVB3 or EMCV inoculation. We measured body weights and heart weights and calculated the ratio of heart weight to body weight as another index of congestive heart failure. The mean value of the heart weight of any one group of infected mice (C: 112.4k 12.0; CE: 101.2f 11.1; E: 108.049.4; mean f SD, mg) was not significantly different from that of the non-infected control (N: 101.7 k4.4 mg). However, the heart weight of group C was significantly heavier than that of group CE (P
