JOURNAL OF PATHOLOGY, VOL.
163: 129-1 31 (1991)
ENTEROVIRUSES AND IDIOPATHIC DILATED CARDIOMYOPATHY HUGH R. COCHRANE, FELICITY E. B. MAY, THOMAS ASHCROFT* AND JOHN H. DARK?
Division of Pathology, School ofPathologica1 Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne; Departments of* Pathology and TCardiothoracic Surgery, Freeman Hospital, Newcastle upon Tyne NE7 7DN, U.K. Received 11 September 1990 Accepted2 October 1990
SUMMARY Previous studies have suggested that some cases of idiopathic dilated cardiomyopathy (IDC) are due to persistent viral infection following an episode of viral myocarditis. Viral RNA sequences have recently been detected in material from patients with IDC using molecular biological techniques. We tested 40 samples from recipients’ hearts explanted at cardiac transplantation for the presence of enteroviral RNA sequences, using a Northern blotting technique. Material from 19 cases of IDC and 21 cases of non-cardiomyopathic cardiac failure was examined together with Coxsackie-virus-infected neonatal mouse heart as a positive control and non-infected adult mouse heart as a negative control. A sharp band of viral RNA was detected in the positive control sample. N o hybridization signal attributable to viral RNA was obtained for the negative control or for any of the test samples. We conclude that the role of enteroviruses in the pathogenesis of cardiomyopathy is not fully established and that further study is warranted. KEY WoRDs-Dilated
cardiomyopathy, enteroviruses, Coxsackie viruses.
INTRODUCTION
METHODS
Idiopathic dilated cardiomyopathy (IDC) is a condition of unknown aetiology. Evidence from clinical follow-up studies and serologic surveys of patients with dilated cardiomyopathy suggests that a proportion of cases may be due to persistent viral infection following an episode of viral myocarditis.’ Recently molecular biological techniques have been applied to test this contention. Bowles et al. demonstrated enteroviral RNA sequences in a proportion of extracts from myocardial biopsy samples from patients with myocarditis and/or IDC using a slotblot hybridization technique.’ Enteroviral RNA sequences have also been demonstrated with this technique in recipients’ hearts removed at cardiac transplantation from patients with IDC.3 In situ hybridization has also been used to demonstrate enteroviral RNA in myocardial biopsies and recipients’ hearts from patients with IDC.4
We studied material derived from the Freeman Hospital heart transplant programme to determine whether enteroviral RNA sequences were present in cellular RNA from recipients’ hearts removed because of IDC or other non-cardiomyopathic causes of heart failure. Tissue samples were snap-frozen in liquid nitrogen at the time of transplantation. Cellular RNA was obtained by phenol-chloroform extraction following homogenization of pulverized tissue samples in a lithium chloride-urea solution. In total 40 samples were available for analysis, including 19 cases of IDC and 21 non-cardiomyopathic samples (Table I). Ten pg of extracted RNA was separated by formaldehyde agarose gel electrophoresis. A positive control sample of Coxsackie-virus-B3-infected neonatal mouse heart RNA and a negative control of non-infected adult mouse heart RNA were also run. After electrophoresis, RNA was transferred to nylon membranes by Northern blotting. Hybridiz-
Addressee for correspondence: Dr H. R. Cochrane, Department of Pathology, Freeman Hospital, High Heaton, Newcastle upon Tyne NE7 7DN, U.K.
0022-341 7/91/020129-03 $05.00 0 1991 by John Wiley & Sons, Ltd.
130
H. R. COCHRANE ETAL.
Table I-Details
of transplant recipients
Cardiomyopathic Case No.
Sex
Age at transplantation (years)
F M M M F F M M M F M M F M M F F M M
28 46 52 54 34 27 17 35 50 34 48 7 53 38 43 50 53 21 45
Non-cardiomyopathic Age at transplantation (years)
Case No.
Sex
29 33 41 60 61
M M M M M
44
62 65 70 71 76 82 87 88 91 92 101 103 108 112 113 114
M F F F F M M M F M M M M M M M
51 54 36 55 55 51 55 53 21 53 39 47 51 48 50 42
Diagnosis
~
17 23 27 30 35 40 48 58 59 63 64 68 75 80 81 85 86 90 111
48 41 55 43
IHD IHD IHD IHD Mitral valve replacement; biventricular hypertrophy; left ventricular fibrosis IHD IHD Primary pulmonary hypertension IHD IHD IHD IHD IHD Cystic fibrosis IHD Granulomatous myocarditis IHD IHD IHD IHD IHD
IHD = Ischaemic heart disease
Vira I
-
RNA
C83C I I I N Fig. I-Autoradiograph showing Coxsackie-virus-B3-infected mouse heart RNA (CB3), cardiomyopathic heart RNA (C), ischaemic heart RNA (I), and non-infected adult mouse heart RNA (N)
ation was carried out using a group-specific enteroviral probe. A 53 1-base-pair Pst I fragment ofcDNA from the 5' end of Coxsackie virus B4 inserted into the Pst I site of the multicloning region of pGEM-I was kindly supplied by Dr Owen Jenkins (Department of Microbiology, University of Reading). A 32P-labelled RNA probe was constructed using the Riboprobe system (Promega Biotec, U.S.A.). Hybridization was carried out for 72 h at 55°C with added radioactivity at a specific activity of
5 x lo6cpm per ml of hybridization solution. After hybridization, filters were washed in several changes of 0.1 x SSC, 0.1 per cent SDS at 75°C and exposed to preflashed X-ray film at -70°C for varying periods of time.
RESULTS A sharp band of viral RNA was detected in the positive control sample. No hybridization signal attributable to viral RNA was obtained for the negative control sample or for any of the 40 test samples (Fig. 1). DISCUSSION Previous studies have shown enteroviral sequences in approximately one-half of myocardial biopsy samples from patients with IDC and/or
131
ENTEROVIRUSES A N D DILATED CARDIOMYOPATHY
m y o c a r d i t i ~and ~ , ~ one-third of explanted (recipients’) hearts from patients with IDC.3,4It is surprising, therefore, that we were unable to demonstrate enteroviral RNA in any of our test samples from cardiomyopathic patients. It is possible that the technique used in this study, while detecting the viral RNA in recently infected mouse heart, was insufficiently sensitive to detect the small amounts of viral RNA persisting after the acute phase of viral infection. If persistent viral RNA were fragmented rather than intact, it would be difficult to detect hybridization above the general background signal; however, enteroviral RNA has been reported in IDC samples using the slot-blot t e c h n i q ~ ewhich , ~ is not necessarily more sensitive than the techniques used here. Also, pilot studies in our department using a nick-translated cDNA probe revealed a sharp band of viral RNA in the positive control sample without a comparable or indeed smaller band in the remaining samples tested. Archard et al.’ tested more than one biopsy sample in a number of their cases and found the presence of viral RNA to be focal. This explanation is unlikely to account for the negative findings in our study, as RNA was extracted from relatively large tissue samples of up to 1 g. We conclude that the role of enteroviruses in the pathogenesis of cardiomyopathy is not yet fully
established and that further study is warranted in this field using the sensitive molecular biological techniques now available. ACKNOWLEDGEMENTS
We are grateful to Professor C. R. Madeley and Mr R. F. Laidler of the Department of Virology, Royal Victoria Infirmary, Newcastle upon Tyne for advice and assistance. We thank Mr E. Bulmer for photographic assistance and Miss L. C. Brown for typing the manuscript. REFERENCES 1. Abelmann WH, Love11 BH. The challenge of cardiomyopathy. J Am Coil Cardioi 1989; 13: 1219-1239. 2. Bowles NE, Richardson PJ, Olsen EGJ, Archard LC. Detection of Coxsackie-B-virus-specificRNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1986;i: 112&1123. 3. Bowles NE, Rose ML, Taylor P, et ai. End-stage dilated cardiomyopathy-persistence of enterovirus RNA in myocardium at cardiac transplantation and lack of immune response. Circulation 1989; 80: I 128-1 136. 4. Kandolf R. The impact of recombinant DNA technology on the study of enterovirus heart disease. In: Bendinelli M, Friedman H, eds. Coxsackieviruses-A General Update. New York: Plenum, 1988; 293-318. 5 . Archard LC, Richardson PJ, Olsen EGJ, Dubowitz V, Sewry C, Bowles NE. The role of Coxsackie B virus in the oathoeenesis of mvocarditis. cardiomyopathy and inflammatory muscle disease. Eiochem S OSymp ~ 1987; 5 3 5 1 4 2 .
.
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163: 129-1 31 (1991)
ENTEROVIRUSES AND IDIOPATHIC DILATED CARDIOMYOPATHY HUGH R. COCHRANE, FELICITY E. B. MAY, THOMAS ASHCROFT* AND JOHN H. DARK?
Division of Pathology, School ofPathologica1 Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne; Departments of* Pathology and TCardiothoracic Surgery, Freeman Hospital, Newcastle upon Tyne NE7 7DN, U.K. Received 11 September 1990 Accepted2 October 1990
SUMMARY Previous studies have suggested that some cases of idiopathic dilated cardiomyopathy (IDC) are due to persistent viral infection following an episode of viral myocarditis. Viral RNA sequences have recently been detected in material from patients with IDC using molecular biological techniques. We tested 40 samples from recipients’ hearts explanted at cardiac transplantation for the presence of enteroviral RNA sequences, using a Northern blotting technique. Material from 19 cases of IDC and 21 cases of non-cardiomyopathic cardiac failure was examined together with Coxsackie-virus-infected neonatal mouse heart as a positive control and non-infected adult mouse heart as a negative control. A sharp band of viral RNA was detected in the positive control sample. N o hybridization signal attributable to viral RNA was obtained for the negative control or for any of the test samples. We conclude that the role of enteroviruses in the pathogenesis of cardiomyopathy is not fully established and that further study is warranted. KEY WoRDs-Dilated
cardiomyopathy, enteroviruses, Coxsackie viruses.
INTRODUCTION
METHODS
Idiopathic dilated cardiomyopathy (IDC) is a condition of unknown aetiology. Evidence from clinical follow-up studies and serologic surveys of patients with dilated cardiomyopathy suggests that a proportion of cases may be due to persistent viral infection following an episode of viral myocarditis.’ Recently molecular biological techniques have been applied to test this contention. Bowles et al. demonstrated enteroviral RNA sequences in a proportion of extracts from myocardial biopsy samples from patients with myocarditis and/or IDC using a slotblot hybridization technique.’ Enteroviral RNA sequences have also been demonstrated with this technique in recipients’ hearts removed at cardiac transplantation from patients with IDC.3 In situ hybridization has also been used to demonstrate enteroviral RNA in myocardial biopsies and recipients’ hearts from patients with IDC.4
We studied material derived from the Freeman Hospital heart transplant programme to determine whether enteroviral RNA sequences were present in cellular RNA from recipients’ hearts removed because of IDC or other non-cardiomyopathic causes of heart failure. Tissue samples were snap-frozen in liquid nitrogen at the time of transplantation. Cellular RNA was obtained by phenol-chloroform extraction following homogenization of pulverized tissue samples in a lithium chloride-urea solution. In total 40 samples were available for analysis, including 19 cases of IDC and 21 non-cardiomyopathic samples (Table I). Ten pg of extracted RNA was separated by formaldehyde agarose gel electrophoresis. A positive control sample of Coxsackie-virus-B3-infected neonatal mouse heart RNA and a negative control of non-infected adult mouse heart RNA were also run. After electrophoresis, RNA was transferred to nylon membranes by Northern blotting. Hybridiz-
Addressee for correspondence: Dr H. R. Cochrane, Department of Pathology, Freeman Hospital, High Heaton, Newcastle upon Tyne NE7 7DN, U.K.
0022-341 7/91/020129-03 $05.00 0 1991 by John Wiley & Sons, Ltd.
130
H. R. COCHRANE ETAL.
Table I-Details
of transplant recipients
Cardiomyopathic Case No.
Sex
Age at transplantation (years)
F M M M F F M M M F M M F M M F F M M
28 46 52 54 34 27 17 35 50 34 48 7 53 38 43 50 53 21 45
Non-cardiomyopathic Age at transplantation (years)
Case No.
Sex
29 33 41 60 61
M M M M M
44
62 65 70 71 76 82 87 88 91 92 101 103 108 112 113 114
M F F F F M M M F M M M M M M M
51 54 36 55 55 51 55 53 21 53 39 47 51 48 50 42
Diagnosis
~
17 23 27 30 35 40 48 58 59 63 64 68 75 80 81 85 86 90 111
48 41 55 43
IHD IHD IHD IHD Mitral valve replacement; biventricular hypertrophy; left ventricular fibrosis IHD IHD Primary pulmonary hypertension IHD IHD IHD IHD IHD Cystic fibrosis IHD Granulomatous myocarditis IHD IHD IHD IHD IHD
IHD = Ischaemic heart disease
Vira I
-
RNA
C83C I I I N Fig. I-Autoradiograph showing Coxsackie-virus-B3-infected mouse heart RNA (CB3), cardiomyopathic heart RNA (C), ischaemic heart RNA (I), and non-infected adult mouse heart RNA (N)
ation was carried out using a group-specific enteroviral probe. A 53 1-base-pair Pst I fragment ofcDNA from the 5' end of Coxsackie virus B4 inserted into the Pst I site of the multicloning region of pGEM-I was kindly supplied by Dr Owen Jenkins (Department of Microbiology, University of Reading). A 32P-labelled RNA probe was constructed using the Riboprobe system (Promega Biotec, U.S.A.). Hybridization was carried out for 72 h at 55°C with added radioactivity at a specific activity of
5 x lo6cpm per ml of hybridization solution. After hybridization, filters were washed in several changes of 0.1 x SSC, 0.1 per cent SDS at 75°C and exposed to preflashed X-ray film at -70°C for varying periods of time.
RESULTS A sharp band of viral RNA was detected in the positive control sample. No hybridization signal attributable to viral RNA was obtained for the negative control sample or for any of the 40 test samples (Fig. 1). DISCUSSION Previous studies have shown enteroviral sequences in approximately one-half of myocardial biopsy samples from patients with IDC and/or
131
ENTEROVIRUSES A N D DILATED CARDIOMYOPATHY
m y o c a r d i t i ~and ~ , ~ one-third of explanted (recipients’) hearts from patients with IDC.3,4It is surprising, therefore, that we were unable to demonstrate enteroviral RNA in any of our test samples from cardiomyopathic patients. It is possible that the technique used in this study, while detecting the viral RNA in recently infected mouse heart, was insufficiently sensitive to detect the small amounts of viral RNA persisting after the acute phase of viral infection. If persistent viral RNA were fragmented rather than intact, it would be difficult to detect hybridization above the general background signal; however, enteroviral RNA has been reported in IDC samples using the slot-blot t e c h n i q ~ ewhich , ~ is not necessarily more sensitive than the techniques used here. Also, pilot studies in our department using a nick-translated cDNA probe revealed a sharp band of viral RNA in the positive control sample without a comparable or indeed smaller band in the remaining samples tested. Archard et al.’ tested more than one biopsy sample in a number of their cases and found the presence of viral RNA to be focal. This explanation is unlikely to account for the negative findings in our study, as RNA was extracted from relatively large tissue samples of up to 1 g. We conclude that the role of enteroviruses in the pathogenesis of cardiomyopathy is not yet fully
established and that further study is warranted in this field using the sensitive molecular biological techniques now available. ACKNOWLEDGEMENTS
We are grateful to Professor C. R. Madeley and Mr R. F. Laidler of the Department of Virology, Royal Victoria Infirmary, Newcastle upon Tyne for advice and assistance. We thank Mr E. Bulmer for photographic assistance and Miss L. C. Brown for typing the manuscript. REFERENCES 1. Abelmann WH, Love11 BH. The challenge of cardiomyopathy. J Am Coil Cardioi 1989; 13: 1219-1239. 2. Bowles NE, Richardson PJ, Olsen EGJ, Archard LC. Detection of Coxsackie-B-virus-specificRNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1986;i: 112&1123. 3. Bowles NE, Rose ML, Taylor P, et ai. End-stage dilated cardiomyopathy-persistence of enterovirus RNA in myocardium at cardiac transplantation and lack of immune response. Circulation 1989; 80: I 128-1 136. 4. Kandolf R. The impact of recombinant DNA technology on the study of enterovirus heart disease. In: Bendinelli M, Friedman H, eds. Coxsackieviruses-A General Update. New York: Plenum, 1988; 293-318. 5 . Archard LC, Richardson PJ, Olsen EGJ, Dubowitz V, Sewry C, Bowles NE. The role of Coxsackie B virus in the oathoeenesis of mvocarditis. cardiomyopathy and inflammatory muscle disease. Eiochem S OSymp ~ 1987; 5 3 5 1 4 2 .
.
-
