Material and methods

The clinical diagnosis of ARVC was performed in 20 consecutive patients according to the criteria of McKenna et al,²⁶ and confirmed by endomyocardial biopsy according to the histological parameters of Angelini et al.²⁷ There were 11 male and nine female patients, mean age 40 (SD, 16) years. Baseline electrocardiography (ECG) showed inverted T waves on right precordial leads in eight patients, monomorphic or polymorphic left bundle branch block ventricular arrhythmias in 16, and atrial flutter or fibrillation in four. A previous syncopal episode had occurred in 10 patients. A family history for ARVC was ascertained in nine patients. Five patients had a recent clinical onset of the disease (≤ 6 months) and in four patients there was a history of a flu like syndrome. Serological studies, including neutralising enteroviral antibodies, were not diagnostic in any of the patients.

The control group included 20 patients with other cardiac diseases: ischaemic heart disease in five, valvular disease in five, restrictive cardiomyopathy in five, amyloid cardiomyopathy in two, sarcoidosis in two, and cardiomyopathy as a result of radiation in one. Table 1 shows the clinicopathological features of the patients and control subjects enrolled in our study. Four cases of myocarditis, previously diagnosed by PCR as enteroviral myocarditis, and coxsackie virus B3 infected KB cells (cell line derived from oral, human, epidermoid carcinoma) were used as positive controls.

At least six samples (∼ 1 mg ) were obtained from all patients; four were fixed in formalin, whereas two samples were immediately frozen in liquid nitrogen and stored at −70°C. Molecular studies were performed using both buffered formalin fixed and frozen tissue samples. It has been shown that PCR can be used successfully for the detection of some infectious agents, even when myocardial tissues have been formalin fixed.^28,29 Blood samples collected at the same time or shortly after were also analysed.

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL ANALYSES

Sections (6 μm thick) of paraffin wax embedded tissues were stained according to the haematoxylin and eosin and Azan Mallory techniques.

To characterise the cellular infiltrates, paraffin wax embedded sections were stained with a panel of monoclonal antibodies: CD45 for leucocyte common antigen, CD45RO for T cells, CD43 for T cells and myeloid lineage, CD20 for B cells, and CD68 for macrophages (all from Dako, Glostrup, Denmark), according to the avidin–biotin peroxidase complex (ABC) method (Vector, Burlingame, California, USA).

RNA EXTRACTION

Frozen and formalin fixed biopsies were homogenised in an RNAzol solution (0.8 ml)³⁰ by means of a motorised pestle. The pestle was changed between each sample. Chloroform/isoamyl alcohol solution (80 μl of 49/1 dilution) was added and samples were kept on ice for 15 minutes before centrifuging at 12 000 ×g. The top aqueous phase was placed into a new tube, 400 μl isopropyl alcohol was added to precipitate the nucleic acid, and the solution was left at −20°C for at least three hours before centrifuging at 12 000 ×g for 15 minutes. The RNA pellet was washed with 75% ethanol, dried, and resuspended in 10 μl DEPC distilled H₂O.

OLIGONUCLEOTIDES

The oligonucleotides used to ascertain the quality of the extracted RNA were complementary to glyceraldehyde-3-phosphate dehydrogenase (3GPDH) mRNA (table 2). These primers amplify all human RNA samples.³¹ Two set of primers were used for enterovirus nested PCR analysis: OL 68 and OL 252 for the first stage PCR and OL24 and 253 for the nested PCR. These were specific to a region common to several enteroviruses (their sequences and predicted PCR amplicon sizes are given in table 2). The primers were the same of those reported by Khan et al.²⁵

REVERSE TRANSCRIPTION

For viral genome RNA detection the total RNA extracted (at least 1 μg) from the clinical biopsy was reverse transcribed in a total volume of 50 μl containing the following components: 50 mM Tris/HCl (pH 8.3), 70 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM dNTPs (Pharmacia, Cambridge, UK), 25 units of human placental ribonucleoside inhibitor (Amersham International, Little Chalfont, Buckinghamshire, UK), primer OL68 (0.1 nmol), and 200 units of Superscript II RNase H free reverse transcriptase (Gibco BRL, Paisley, UK). The reaction was carried out at 37°C for one hour.

POLYMERASE CHAIN REACTION

For the first PCR assay, 15 μl of each reverse transcribed sample was amplified in a total volume of 100 μl. The following conditions were used: 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM dNTPs, primers OL252 and OL68 (0.1 nmol each), and 2.5 units of Taq polymerase (Boehringer Mannheim, Mannheim, Germany). Thirty five cycles were carried out as follows: denaturation for 1.5 minutes at 94°C, primer annealing for 1.5 minutes at 55°C, and template extension for three minutes at 72°C. An aliquot of 5 μl of the first amplification reaction was amplified further with the nested pair (OL24 and OL253, 0.1 nmol each) in 100 μl of reaction mixture. The reaction was subjected to the above described amplification times except for a reduction of the template extension time to two minutes. Two other annealing temperatures—58°C (high stringency) and 50°C (low stringency)—were used in each PCR reaction. The amplicons were then electrophoresed on a 3% NuSIEVE/1% Seakem TM agarose gel (FMC Bio Products, Rockland, Maine, USA) and stained with ethidium bromide.

Before performing nested PCR for enterovirus, an independent reverse transcription PCR (RT–PCR) amplification for 3GPDH was performed using the previously described protocol.³¹

Precautions were taken to avoid false positives as a result of contamination by PCR product carry over, including physical separation procedures, the use of aliquoted reagents and positive displacement pipettes, and ultraviolet irradiation of non-disposable equipment.

SENSITIVITY

To assess the sensitivity of the nested PCR, serial 10-fold dilutions of the supernatant of coxsackie virus B3 infected KB cells were used to infect monolayers of KB cells to determine virus titre by the plaque assay technique.³² The amount of virus corresponding to a single plaque forming units (PFU) was serially diluted and aliquots with 10⁻¹ to 10⁻⁶ were mixed with approximately 1 mg of normal mouse myocardial tissue. From each sample, total RNA was extracted, reverse transcribed, and amplified as described above. A positive result on amplification was clearly evident with dilutions greater than 10⁻² PFU. Most of the samples with 10⁻³ (nine of 10) showed a positive result and conversely most of the dilutions with 10⁻⁴ (one of 10) gave a negative result. Applying the minimum χ² methods described by Taswell³³ to our data, it was found that our amplification technique can detect about 1800 single RNA molecules/PFU. Other authors have obtained similar results using nested PCR for enterovirus detection.³⁴

Results

HISTOLOGICAL AND IMMUNOHISTOCHEMICAL FINDINGS

At endomyocardial biopsy, a diagnostic amount of fibro-fatty tissue causing myocardial atrophy with residual myocytes less than 45%²⁷ was found in all samples. Residual myocytes were scattered throughout the fatty or fibro-fatty infiltration (fig 1A and B) and often showed pycnotic nuclei and cytoplasmic shrinkage. Foci of inflammatory infiltration were seen in four biopsies. Immunohistochemical staining revealed mixed inflammatory infiltrates (CD45, CD 45RO, CD43, and CD68 positive cells) with a high prevalence of T cells (fig 1C).

ADEQUACY OF NUCLEIC ACID EXTRACTION

All samples were analysed using 3GPDH primers to ensure adequate nucleic acid extraction. The mRNA of this housekeeping gene was amplified successfully from all clinical samples.

DETECTION OF ENTEROVIRUS IN PATIENTS WITH ARVC AND CONTROLS

Table 1 summarised the results of the nested PCR analyses performed in each case. The endomyocardial biopsies from patients with ARVC and control subjects were all negative by nested PCR (fig 2). Negative results obtained in each sample were analysed twice in different experiments, and nested PCR was performed under different stringency conditions.

Coxsackie virus type B neutralising antibodies were not increased in the serum of any of the patients. Blood samples did not reveal any amplification product.

Amplification was always obtained in samples from patients with coxsackie B3 virus myocarditis as well as in coxsackie virus B3 infected KB cells.

Discussion

Despite the evidence of familial occurrence,^9,10 chromosomal gene mapping,^11–16 and apoptosis as a mechanism of progressive myocyte death,^17,18 the aetiopathogenesis of ARVC remains speculative.³ The frequent finding of inflammatory infiltrates in up to two thirds of cases at histology supports the hypothesis that ARVC might be inflammatory in origin.⁴ In the clinical series of Blomström-Lundqvist et al,³⁵ 40% of patients with ARVC developed ventricular tachycardia, in the setting of an infectious disorder with a clinical picture and laboratory tests consistent with acute myocardial damage.

Interestingly, some authors reported the association of ARVC and myocarditis even in the same family.^22,36,37 Such findings suggest a link between inherited and acquired (“inflammatory”) forms of the disease. Genetic factors might play a role not only in influencing susceptibility to infections or to autoimmunity, but also in determining the site of cardiac involvement, namely the epimyocardium of the right ventricle.

Various infectious agents have been claimed to be involved in ARVC, such as Trypanosoma cruzi,³⁸ rubella virus, adenovirus, herpes simplex, cytomegalovirus, Epstein-Barr virus, influenza, para-influenza, and parvovirus,²² but none has been documented in the right ventricular myocardium itself. Why inflammatory lesions are mainly confined to the right ventricle remains to be clarified. It is noteworthy that some experimental forms of myocarditis are limited exclusively to the right ventricle. Matsumori and Kawai reported selective right ventricular perimyocarditis in coxsackie B virus infected BALB/c mice³⁹; in a later stage this led to wall thinning and right ventricular aneurysms with gross and histological pictures similar to those seen in human ARVC. ARVC presents with a broad spectrum of clinical features and different substrates: whether a single pathogenetic mechanism or several genetic and/or environmental factors account for the same elective right ventricular involvement remains to be elucidated.

We used nested PCR to examine the presence of enteroviral genomes in the myocardial tissue of 20 patients with clinically and histologically confirmed ARVC. The method used enables the detection of low copy numbers of targets, and is useful when small amounts of tissue are available. At least two frozen and/or four formalin fixed endomyocardial biopsies were analysed by nested PCR to avoid false negatives as a result of insufficient infected material. Recently, some authors have reported that studies using single biopsies produce consistently lower estimates for enterovirus than studies that examine multiple biopsies.⁴⁰

However, the failure to reveal enteroviral sequences in our samples, which were both frozen and formalin fixed, might be explained by a lower sensitivity of PCR as a result of extraction of RNA combined with RT. RNA molecules are highly labile because of the presence of ubiquitous endogenous and exogenous ribonucleases. This is particularly true for viral RNA. The yield of viral RNA available for RT–PCR is influenced by several factors such as tissue preservation, fixation, the thickness of paraffin wax embedded sections, and the protocols used for RNA extraction and RT. However, the protocols for RNA extraction and RT that we used have improved the sensitivity of this technique, and it is now possible to detect virus genomes using paraffin wax embedded material.^28,29

Our study is the first prospective study investigating the role of enterovirus in a consecutive series of both familial and non-familial patients with ARVC. Despite the very high sensitivity of the technique and the number of processed sample biopsies, positive signals were not obtained in any of the 20 patients with ARVC. To the best of our knowledge there are only four published reports that have investigated the presence of enteroviruses in the myocardium of patients with ARVC.^21–24 One was an adult patient and was considered a false positive because the sequenced PCR product was identical to that of the positive control, a finding consistent with cross contamination.²¹ Two were children and both studies were negative.²² More recently, Heim and colleagues²³ and Grumbach and colleagues²⁴ studied patients with a clinical diagnosis of ARVC; enterovirus RNA was detected by nested PCR in 50% and 37.5%, respectively. However, the accuracy of the clinical diagnoses is questionable because there was no family history, and the onset of first symptoms occurred at mean age of 59 years, with a mean history duration of only 45 and 15 months, respectively. The possibility that myocardial infection might have been merely incidental in the setting of a systemic viral infection cannot be excluded.

With regard to our negative findings, we can rule out the possibility of the virus having been eliminated because we included patients with both recent onset and chronic disease. We cannot exclude the possibility that an infective aetiopathogenesis with viruses other than those investigated might be involved and these should be searched for.

Recently, apoptosis has been shown to be a mechanism accounting for progressive myocyte death in ARVC in both postmortem¹⁷ and biopsy¹⁸ material. Whether this mode of death is related to the presence of viral infection or is simply caused by other triggers, such as an internal genetic clock, remains to be clarified.

Infiltrating cytotoxic T cells, usually present after viral infection, can induce either necrosis or apoptosis in a target cell depending on the apoptotic factors present in that cell.⁴¹ Some pro-apoptotic proteins, such as the Bcl2 homologue Bax, Fas, and Fas ligand, and several anti-apoptotic factors, including Bcl2, have now been identified. Some authors have demonstrated that, despite pronounced inflammatory activity, myocyte apoptosis is rare in experimental coxsackie virus myocarditis, and the myocardial inflammation is associated with increased cytoplasmic expression of Bcl2 protein.⁴²

Therefore, future molecular studies should look for the presence of other infective agents already known to trigger apoptosis.^43–45