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(Circulation. 1997;96:22-24.)
© 1997 American Heart Association, Inc.

Articles

Chronic Variant of Myocarditis Associated With Hepatitis C Virus Infection

Masanori Okabe, MD; Keisuke Fukuda, MD; Kikuo Arakawa, MD; ; Masahiro Kikuchi, MD

From the Departments of Internal Medicine (M.O., K.F., K.A.) and Pathology (M.K.), School of Medicine, Fukuoka University, Japan.

Correspondence to Masanori Okabe, MD, Second Department of Internal Medicine, School of Medicine, Fukuoka University, 7-45-1, Nanakuma, Jonan-ku, Fukuoka 814-80, Japan.

	Abstract

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Background Although molecular biological studies suggest a pathogenic link between enterovirus infection and dilated cardiomyopathy (DCM), the frequency of detection of enteroviral RNA is not consistently high in myocardial tissue from patients with DCM. A recent study has suggested that hepatitis C virus (HCV) may also be involved in the development of DCM.

Methods and Results We performed genomic analysis for HCV in three patients with chronic active myocarditis. In all three patients, serum aminotransferase activities remained within normal ranges until the terminal stage of heart failure. At necropsy, all three livers showed evidence of tissue damage caused by chronic congestion, and one liver had evidence of chronic hepatitis. Routinely processed, paraffin-embedded tissue blocks of myocardium and liver were analyzed. Renal specimens were also analyzed to exclude the possibility of myocardial contamination with HCV material from the circulating blood. RNA extracted from the heart, liver, and kidney was subjected to strand-specific reverse transcription and amplified by semi-nested polymerase chain reaction. The target nucleotide sequence was a 178-bp fragment of the highly conserved 5'-noncoding region. Both positive- (genomic) and negative-strand RNA (replicative intermediates) were present in myocardial and liver tissue samples from all three patients. However, negative-strand RNA was undetectable in renal tissue from one patient.

Conclusions These findings suggest that HCV replicated in myocardial tissue of these patients with myocarditis. Thus, HCV infection may contribute to the development of this unusual form of myocarditis.

Key Words: myocarditis • heart failure • polymerase chain reaction • viruses

	Introduction

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Molecular biological studies support the concept that infection with enterovirus, the most common pathogen causing human myocarditis,¹ is related to the development of DCM. However, the frequency of detection of the enteroviral genome has been variably reported in endomyocardial tissue samples obtained from patients with DCM.^{2 3 4} One of these reports suggested that enteroviral infection is not a major cause of DCM.⁴ Matsumori et al⁵ recently suggested that HCV may contribute to the development of DCM.

Chronic myocarditis, which is not a sequela of acute inflammation but rather a myocarditis persisting for years, has been described,^{6 7 8} but it remains ill-defined.^{9 10 11 12} We previously described three autopsy cases with "chronic active myocarditis" that was histologically characterized by numerous lymphocytic clusters and myocardial cell damage.¹³ We believe that these patients belong to a specific subgroup of patients with chronic myocarditis. However, patients with fatal heart failure with histological findings similar to those seen in these three patients have rarely been described. We performed genomic analysis for HCV in myocardial tissue samples obtained from the three patients with this unusual form of myocarditis using a PCR that specifically detects both positive (genomic) and negative (replicative) strands of the HCV RNA.

	Methods

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Patients
Serum aminotransferase activities did not become elevated until the terminal stage of heart failure in all three patients. In contrast, the activities of lactic dehydrogenase and alkaline phosphatase occasionally increased to <20% above the normal range in patient 1.¹³ Clinical evaluation of HCV infection was not performed, because all three patients had died before the discovery of HCV.¹⁴ At necropsy, all three livers showed marked centrilobular necrosis and associated fibrosis, suggesting tissue damage due to chronic systemic congestion. Cirrhosis was not observed, but careful microscopic examination revealed foci of chronic hepatitis in patient 2.¹³

Strand-Specific PCR
Routinely processed autopsy samples of myocardium, liver, and kidney were analyzed. Thin tissue sections were depleted of paraffin in xylene. Total RNA was extracted by a guanidinium isothiocyanate protocol¹⁵ and suspended in a final volume of 45 µL of diethyl pyrocarbonate–treated water. Strand-specific RT was performed in the presence of only one oligonucleotide primer (either sense or antisense primer), followed by semi-nested PCR in the presence of both primers as described by Sherker et al¹⁶ with some modifications. The primers used were chosen from the highly conserved 5'-noncoding region of HCV.¹⁷ The antisense primer (primer A) sequence was 5'-ACTCGCAAGCACCCTATCAG-3', the external sense primer (primer B) sequence was 5'-GAGGAACTACTGTCTTCACG-3', and the internal sense primer (primer C) sequence was 5'-GAGCCATAGTGGTCTGCGGA-3'.

Before RT incubation, the RNA suspension was denatured at 70°C for 7 minutes and quickly chilled on ice. A 10-µL RNA sample was then incubated at 60°C for 60 minutes with a 15-µL reaction volume containing 1 nmol/L of strand-specific primer, 0.5 mmol/L of each dNTP, 1x RT buffer, and 1 U/µL Moloney murine leukemia virus reverse transcriptase (Cosmo Bio). Primer A and primer B were used for synthesis of cDNA of the positive and negative strands, respectively. The RT reaction was stopped by heating of the sample to 95°C for 10 minutes.

Each 5-µL sample of positive- or negative-strand cDNA was supplemented with 50 µL of 1x PCR buffer containing 90 pmol/L of both primer A and primer B and 1 U of Taq DNA polymerase (Roche Molecular Systems). The mixture was overlaid with mineral oil and subjected to 35 cycles of round 1 of PCR (95°C for 80 seconds, 55°C for 60 seconds, and 72°C for 60 seconds). For round 2 of PCR, a 3-µL aliquot of round 1 PCR products was added to a reaction mixture similar to the round 1 PCR mixture but containing 90 pmol/L of primer C (inner primer) instead of primer B (outer primer). The sample was then subjected to 25 cycles of PCR (95°C for 80 seconds, 55°C for 60 seconds, and 72°C for 60 seconds). Round 2 PCR products were analyzed by 3% agarose gel electrophoresis with ethidium bromide staining. The size of the final PCR product was 178 bp. In vitro transcribed positive- and negative-strand RNA was subjected to PCR as a positive control. Nucleic acids extracted from calf sera or water served as a negative control.

	Results

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Both positive- and negative-strand RNA was detected in the myocardial and liver specimens of all three patients (Fig 1). The positive strand was present in the renal specimens of all three patients; however, the negative strand was not detected in patient 3 (Fig 2).

View larger version (53K): [in this window] [in a new window]   Figure 1. Agarose gel electrophoresis with ethidium bromide staining of final products of PCR amplification demonstrating detection of positive and negative strands of HCV RNA in heart and liver. RNA was extracted from autopsy samples, reverse transcribed to cDNA, and amplified by semi-nested PCR with primers from HCV genome. M indicates DNA size markers; S1 through S3, extracts from hearts of patients 1 through 3; S4 through S6, extracts from livers of patients 1 through 3; +, positive-strand detection; -, negative-strand detection; P+, positive control for positive strand; N+, negative control for positive strand; P-, positive control for negative strand; and N-, negative control for negative strand.

View larger version (83K): [in this window] [in a new window]   Figure 2. Agarose gel electrophoresis with ethidium bromide staining of final products of PCR amplification demonstrating detection of positive and negative strands of HCV RNA in kidney. RNA was extracted from autopsy samples, reverse transcribed to cDNA, and amplified by semi-nested PCR with primers from HCV genome. S7 through S9 indicates extracts from kidneys of patients 1 through 3; other abbreviations as in Fig 1.

	Discussion

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The HCV genome is a positive-polarity, single-stranded RNA. Negative-strand RNA is always a constituent of replicable intermediates of HCV.^{16 18} Therefore, the present results suggest that HCV replicated in myocardial tissue. Because of the unavailability of blood samples from the patients, renal specimens were analyzed to exclude the possibility of myocardial contamination with HCV material from the plasma or circulating infected cells.^{16 19} The most important finding in the present study is that PCR analysis of renal specimens ruled out such a possibility in one patient. Enteroviral RNA was not detected by PCR analysis in the same specimens analyzed in the present study (M.O., unpublished data, 1996). It is also noteworthy that HCV RNA was successfully amplified after years of storage (12, 8, and 18 years in patients 1, 2, and 3, respectively¹³ ), because degradation of RNA occurs frequently when paraffin-embedded tissues are used for genomic analysis.^{20 21}

The histological findings of the livers were not inconsistent with chronic HCV infection.²² Since its first description in 1989,¹⁴ much has been learned about the clinical features of HCV infection. HCV infection represents the major cause of parenterally transmitted non-A, non-B hepatitis, which has a remarkably high propensity to produce chronic infection that often leads to cirrhosis and hepatocellular carcinoma. In addition, HCV infection is known to be associated with several extrahepatic immune disorders, including mixed cryoglobulinemia,²³ membranoproliferative glomerulonephritis,²⁴ and lymphocytic sialadenitis resembling Sjögren's syndrome.²⁵ These extrahepatic immune disorders are occasionally observed in the absence of clinically overt liver disease. In our three patients, chronic active myocarditis was the only overt clinical syndrome. HCV-related cardiac manifestations have been described in only two reports in the English literature.^{5 26}

Matsumori et al recently reported a relatively high incidence of HCV antibodies in patients with DCM (6 of 36 patients)⁵ and in those with hypertrophic cardiomyopathy (6 of 35 patients).²⁶ In the series of patients with DCM, 3 of the 6 patients with HCV antibodies had positive-strand HCV RNA present in their myocardium. As discussed in that report, however, possible uptake of HCV material from the circulating blood could not be ruled out in the 3 patients, because they all had genomic HCV RNA present in their serum. Negative-strand RNA was detected in the heart of 1 of the patients,⁵ but the possibility that positive-strand RNA may have acted as a template for synthesis of a "false"-negative strand cannot be excluded.²⁷ In the present study, heat denaturation before cDNA synthesis and RT incubation at an elevated temperature (60°C) were used because these procedures minimized such technical problems.²⁷

The present study is the first report that demonstrates the relationship between HCV infection and myocarditis, because the histological evidence of myocarditis, as defined by the Dallas criteria,¹⁰ was not obtained in the above series of patients with DCM⁵ or hypertrophic cardiomyopathy.²⁷ Chronic HCV infection may have maintained myocarditic activity, or, as has been speculated for other HCV-related extrahepatic manifestations,²⁸ an autoimmune mechanism triggered by HCV infection may have caused the chronic active myocarditis. However, it is unclear why this hepatotropic virus affected the heart without evidence of hepatic involvement. Further evidence, eg, localization of the HCV genome in the myocardium, remains to be obtained.

	Selected Abbreviations and Acronyms

 

DCM = dilated cardiomyopathy HCV = hepatitis C virus PCR = polymerase chain reaction RT = reverse transcription

	Acknowledgments

 
We thank Hiroshi Shijo, MD, First Department of Internal Medicine, School of Medicine, Fukuoka University, for his comments on the histological findings.

Received March 25, 1997; revision received April 24, 1997; accepted April 28, 1997.

	References

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