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Viruses play a role in causing cardiovascular disease. The viruses and virus classes that most frequent are the common cause of cardiomyopathy are Parvo B19, enteroviruses, adenoviruses, and cytomegalovirus (CMV). Epstein–Barr virus (EBV) and influenza virus cause cardiomyopathy, but less frequently1, 2 than previously mentioned viruses. Recent treatment advances have witnessed the long-term survival of patients with HIV/AIDS, allowing for larger numbers of cardiac manifestations of HIV disease to be observed. Cardiac manifestations of long-term HIV-disease survival include pericardial effusion, myocarditis, dilated cardiomyopathy, endocarditis, pulmonary hypertension, malignant neoplasms, and drug-related cardiotoxicity.3 The relationship between viruses and cardiac tumors, such as cardiac lymphoma and papillary fibroelastoma, has been established.4, 5, 6 Studies have proven that herpes simplex virus (HSV) is also associated with cardiac myxoma.7

Myxoma is the most common benign neoplasm of the heart.8, 9, 10, 11, 12, 13, 14, 15 Cardiac myxoma is associated with many mucopolysaccharidic matrices, including glycosaminoglycans14, 15 and proteoglycans. These glycoproteins may serve as receptors for the entry of HSV-1,16, 17 human papillomavirus (HPV),18 and EBV.19 In addition, the presence of the tumorigenic viruses, HSV and HPV, has been implicated to potentiate neoplastic conditions in mucin-enriched cornea and in conjunctiva.20, 21, 22, 23 HPV may play a major role in the development of rapidly progressive, multifocal transitional cell carcinoma in immunosuppressed patients. E6 and E7 genes of the oncogenic HPV-16 can be transfected into primary human myocardial fibroblasts, immortalizing them. Telomerase activity is detected in these transfected cell lines.24 In addition, EBV has been implicated in the pathogenesis of Burkitt's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, and lymphomas, as well as leiomyosarcomas arising in immunocompromised individuals.25 All of the above characteristics imply that HPV and EBV may play a role in cardiac myxoma.

We have previously reported apoptosis in cardiac myxoma; however, the exact relationship between apoptosis and cardiac myxomas is poorly understood. This investigation attempts to clarify the mechanisms leading to the development of apoptosis and its relationship to HPV and EBV.

Materials and methods

This study, conducted between June 2001 and December 2003, included 15 consecutive patients with cardiac myxoma who were treated by surgical excision at the Chung Gang Memorial Hospital, Taiwan. At that time, we obtained detailed clinical parameters. Information on age, sex, presenting symptoms, echocardiographic characteristics, and surgical procedures was obtained from patient medical records. Follow-up data were gathered from clinical records and from standardized telephone interviews. Tumor size and morphologic features for each patient were retrieved from surgical pathology reports. All sections were stained with hematoxylin–eosin and examined for vascular proliferation, inflammation, cellularity, hyaline, calcification, thrombosis, fibrosis, and Gamna–Gandy bodies. The clinical and pathologic features of the cardiac myxomas were assessed statistically to identify morphological features related to embolism, atrial fibrillation, and patient age on diagnosis. In addition, echocardiographic features were compared using the pathologic features.

Our institutional review board approved the study and all patients signed an informed consent form prior to enrollment into the study.

Genomic DNA Extraction and Electrophoresis

Genomic DNA was extracted using proteinase K and the phenol chloroform extraction method.13, 26, 27 Samples were further purified through the use of a DNAeasy kit (Qiangen Inc., Venlo, The Netherlands). Finally, 1 μg of DNA solution was eluted and 1 μg of the aliquot was used for polymerase chain reaction (PCR) amplification. For electrophoresis, 1 μg of DNA was labeled with Escherichia Coli DNA polymerase and resolved with 1.5% agarose gel. The gel was dried and exposed for autoradiography.13

HPV Genotyping by Genechip

The SPF1/GP6+ consensus primers were used to amplify a fragment of approximately 184 bp in L1 open-reading frame first. Next, each PCR experiment was performed with several positive and negative controls.26 The quality of isolated DNA was checked with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR. The resulting amplimers of 15 μg were then hybridized with an HPV genechip (Easychip HPV Genotyping Array, King Car, Taiwan). In all, 39 types of HPV ligonucleotide probes of 20- and 30-mer, with an approximately 100–200 poly-T tail, were immobilized on a nylon membrane.26

EBV Genotyping by PCR

For PCR amplification, oligonucleotide primers for detecting LMP-1 gene (sense BN1 [5′-AGC GAC TCT GCT GGA AAT GAT-3′] or antisense BN2 [5′-TGA TTA GCT AAG GCA TTC CCA-3′]) were used to examine the presence of extracted DNA.27 The prepared sample was amplified for 35 cycles using the following procedures: denaturation at 94°C for 40 s, annealing at 50°C for 1 min, and extension at 72°C for 90 s in a programmable thermal controller (PTC-100, MJ Research Co.) without the overlay of mineral oil. Amplification of a genomic region in the hemoglobin gene (HEMHC-1 [5′-CGT CTC CTT TCC TCC GGA-3′] or HEMHC-2 [5′-CAC AGT GAC CTT CCC ATC-3′] served as a marker for the presence of intact genomic DNA. Negative control samples containing water were always processed in a manner parallel to the patient samples. DNA from the B95.8 cell line was used as the EBV-positive control sample.27

TUNEL Assay for Apoptosis

Samples from patients were assessed for apoptosis using the terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay.13 In order to detect DNA fragmentation in situ, the TUNEL assay was performed on deparaffinized 5 μm-thick myxoma sections according to the guidelines from the manufacturer (DNA Fragmentation Detection Kit; Oncogene Research). The DNAase I-treated section displayed positive control for TUNEL staining for apoptotic nuclei.

Immunohistochemical Stain for Caspase-3

We used the caspase-3 (H-277; Santa Cruz Biotechnology, Inc.) assay per manufacturer protocol.

The fraction of TUNEL and caspase-3-positive cells determined per 100 cardiac myxoma cells as follows: none (0) 1–20% (+), 21–40% (++), and 41–90% (+++).13 The Student's t-test was utilized to make statistical comparison between the proportion groups.

Results

Clinical Findings

The patient population comprised eight (54%) women and seven (47%) men, with a mean age of 45 years, ranging in age from 30 to 70 years. All patients were cases of sporadic myxomas rather than familial myxoma. The breakdown for initial patient presentations was dyspnea (53%), asymptomatic (27%), stroke (7%), chest pain (7%), and fever (7%). For all patients participating in the study, cardiac myxoma tumors were confined to the left atrium. Tumors ranged in weight from 5 to 54 g (mean 28.6). No patients were noted to be experiencing atrial fibrillation prior to or during the study. All chest radiographs displayed nonspecific changes. All patients were mildly anemic, with average hemoglobin 11.3 mg/dl, most with patients experiencing mild mitral valve and tricuspid valve regurgitation. There were no perioperative mortalities or embolizations during the study period. At the time of this writing, all of the patients were alive. The mean time from patient treatment to the time of the authoring of this article was 10 months, with a range of 1–4.5 years. There were no patients with metastases or a recurrence of disease. Preoperative diagnosis was made by the use of transthoracic echocardiography.

Pathologic Findings

Microscopic examination revealed high levels of vascular proliferation (74%), hyaline formation (74%), congestive areas (67%), inflammation (54%), and multiple giant cells (47%) in the sample patients. On occasion, we noted hemorrhagic foci (14%) and calcification (14%). There were no cases of cardiac lithomyxoma in this study.

Expression of Apoptosis in Cardiac Myxoma

All cardiac myxomas patients had apoptosis-positive tissue samples (Figure 1), according to the following subgroups: 22% in the 11–20% apoptosis group, 22% in the 21–60% apoptosis group, and 66% in the >60% apoptosis group. The occurrence of apoptosis did not correlate with all clinical disease parameters, such as stroke, recurrence, or unusual sites other than the left atrium.

Figure 1
figure 1

The pictures display typical apoptotic changes in cardiac myxoma. ((a) negative control; (b) positive apoptosis stain, arrows) (× 200).

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DNA Ladders Detected by Gel Electrophoresis

Nucleosomal fragmentation was studied using the end-labeling method as per manufacturer protocols. Following an autoradiogram-exposure time of 36 h, myxomas revealed DNA ladders from agarose gel electrophoresis (data not shown). Conversely, no DNA laddering was noted from normal heart tissues of the same time period.

Expression of Caspase-3 in Cardiac Myxomas

All immunochemical studies of caspase-3 were positive (Figure 2) and compatible with the apoptosis according to the following subgroups: 14% in the 11–20% apoptosis group, 20% in the 21–60% apoptosis group, and 67% in the >60% apoptosis group. This suggests that the caspase-3 pathway is activated in cardiac myxoma.

Figure 2
figure 2

Representative pictures showing typical caspase-3 staining in cardiac myxoma ((a) negative control; (b) positive apoptosis stain, arrows) (× 200).

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Negative Detection of HPV or EBV in Cardiac Myxoma

Detection limit of this assay was determined by serial dilution of plasmid DNA containing the specific segment of L1 open-reading frame. In the process, we used 39 HPV types and cell lines containing known HPV genotype, such as HeLa and CaSki cells, including between 10 and 100 copies per sample. None of the 39 (0%) HPV-type DNA sequences were detected on the genechip, and none of the EBV DNA sequences by PCR.

Discussion

Myxoma is a benign neoplasm among cardiac tumors.8, 9, 10, 11, 12, 13, 14, 15 The morphologic studies, such as those on vascular proliferation, hyaline formation, congestive areas, inflammation, hemorrhagic foci, calcification, and multiple giant cells, were not statistically significant for clinical presentations as in previous reports.8, 9, 10, 11, 12 Moreover, the findings of apoptotic changes in cardiac myxoma were compatible with previous studies.13, 28

Apoptosis, commonly referred to as programmed cell death, involves the continuous destruction of nonfunctional cells.29 Several pathways can be involved in programmed cell death. However, the molecular mechanisms that govern these processes remain unclear. Although apoptosis is involved in myxomas,13, 28, 30 exactly how apoptosis and cardiac myxomas are related is poorly understood. Our first step was to provide clear evidence for a caspase-3-dependent pathway of apoptosis in cardiac myxoma by immunochemical studies. Secondly, this study attempts to clarify the relationship between cardiac myxoma, apoptosis, and HPV/EBV.31

One major pathway of apoptosis is in response to viral infection. Viruses have therefore evolved multiple distinct mechanisms for modulating host cell apoptosis. Previous studies have examined the consequences of HSV infection, including the induction of apoptosis.31 Caspase-dependent apoptosis is involved in HSV-associated injury,32 in adrenal cells, neuronal cells, corneal cells, hepatic cells, and dendritic cells as well.33, 34, 35, 36, 37 Recently, the association between cardiac myxoma and HSV-1 has been determined.7 Thus, we assume that HSV-1 might induce apoptosis in myxoma. However, our findings do not support the hypothesis that apoptotic myxoma cells are related to HPV or EBV; there are even some similarities between HSV and HPV.16, 17, 18, 19, 20, 21, 22, 23

This investigation has several limitations. First, this retrospective study had a relatively small sample group. Second, the definition of apoptosis, as opposed to necrosis, remains controversial. We performed DNA fragmentation assays to verify the presence of apoptosis. While necrosis is an unregulated process causing cell demise, apoptosis is ordered and regulated. Third, the quality of DNA extracted from the samples might have been inadequate, even though we checked each sample first by the housekeeping gene, GADPH.

Conclusions

Results of this study demonstrate that caspase-3-dependent apoptosis has a novel pathological pattern in cardiac myxomas. Although the apoptosis in cardiac myxoma may not require HPV or EBV, this finding has potential significance for therapeutic treatment based on the administration of these genes for tumor-suppressive and apoptosis-inducing activity.38, 39