Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable myocardial disease accounting for ventricular tachycardia and sudden death in the young and arising from areas of fibrofatty replacement of predominantly right ventricular myocardium. That some patients manifest life-threatening ventricular tachycardia in the absence of substantial myocardial replacement suggests that gap junction remodeling might be acting synergistically to ventricular remodeling to promote arrhythmogenesis. Hence, we sought to verify gap junction composition and distribution by analyzing the expression and occurrence of specific gap junction proteins (connexins [Cxs]) in patients with ARVC. Right ventricular endomyocardial biopsy specimens were taken from 16 patients with definite ARVC (age 48 ± 16 years) and analyzed for Cx40, Cx43, and Cx45 messenger ribonucleic acid expression (relative to glyceraldehyde-3-phosphate-dehydrogenase messenger ribonucleic acid expression). The results were compared to those obtained from nondiseased donor hearts (n = 6; age 32 ± 11 years). The patients with ARVC showed a significant reduction in the messenger ribonucleic acid expression of Cx40 (p <0.0001) and Cx45 (p <0.0001) compared to that of the controls. The expression of Cx43 was similar in patients with ARVC and controls (p = 0.098). Mutations in plakophilin-2 were identified in 7 of 16 patients (25%). The Cx expression levels were comparable between the mutation carriers and noncarriers (p = NS). In conclusion, ARVC features alterations in the expression of Cxs and their distribution at cardiac intercalated discs. Apart from the deposition of extracellular matrix, the potential loss of gap junctions and shift in the composition of gap junctional Cxs in the ventricular conduction system might further contribute to the development of ventricular arrhythmias in patients with ARVC.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary myocardial disease accounting for recurrent ventricular tachycardia and sudden cardiac death in a young population. Molecular genetic analyses have identified mutations in genes encoding for different desmosomal proteins, supporting the hypothesis that genetically impaired desmosomal function could result in detachment and death of cardiac myocytes, especially in the setting of mechanical stress. At the bipolar ends of cardiac myocytes, desmosomes are co-located with the electrical coupling nexus, the gap junction. Gap junctions comprise clusters of transmembrane channels, which, among others, are thought to play key roles in electrical impulse propagation and conduction velocity. The connexins (Cxs) predominantly expressed by cardiac myocytes are Cx43, Cx40, and Cx45. A reduction of Cx43 expression at the intercellular junction has been reported in small series of selected patients with ARVC with syndromic (Naxos disease in 4 patients ; Carvajal syndrome in 1 patient ) and nonsyndromic disease manifestation and in in vitro studies. This suggests that mutant desmosomes affect the gap junctional integrity, which, in turn, could promote electrical instability and facilitate the occurrence of life-threatening ventricular tachyarrhythmias. However, little is known about the expression pattern of the other main cardiac Cxs, Cx40 and Cx45, in patients with ARVC. Therefore, the present study investigated the spatial distribution of these transmembrane channels and the expression of Cxs in patients with ARVC compared to nonfailing controls (donor heart tissue).
Methods
A total of 16 male patients with ARVC (mean age 48 ± 16 years) were consecutively enrolled in the present study. Detailed clinical characteristics are listed in Table 1 . The diagnosis of ARVC was established in accordance with the proposed diagnostic criteria, with major criteria counting as 2 and minor criteria as 1 score point; ≥4 points from the different diagnostic groups were required for the diagnosis of ARVC. All patients (100%) underwent detailed noninvasive (12-lead electrocardiography at rest, transthoracic echocardiography) and invasive (right/left ventriculography, coronary angiography, endomyocardial biopsy, programmed ventricular stimulation) investigation. Angiographically extensive disease, defined as the combination of right ventricular severe dilation and regional right ventricular dysfunction (>2 right ventricular areas) was present in 6 patients (38%; patients 9, 11 to 13, 15, and 16; Table 1 ).
| Pt. No. | Age (yrs) | Task Force Score ∗ | Major Criteria (n) | Minor Criteria (n) | Initial Clinical Arrhythmia | PVS | ICD | Mutation | Cx40 † | Cx43 † | Cx45 † |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 37 | 4 | 1 A | 2 D,E | sVT | + | + | – | 0.355 | 0.384 | 0.052 |
| 2 | 58 | 7 | 3 A,B,D | 1 E | sVT | + | + | – | 0.000 | 0.326 | 0.040 |
| 3 | 29 | 8 | 3 A,B,F | 2 C,D | VF | + | + | PKP2 (R79X) | 0.494 | 0.400 | 0.161 |
| 4 | 30 | 5 | 2 A,F | 1 C | Syncope | + | − | PKP2 (P544Lfs ∗ ) | RD | 0.479 | 0.000 |
| 5 | 51 | 8 | 3 A,B,F | 2 C,D | sVT | + | + | JUP (S39_K40insS) | 0.203 | 0.400 | 0.170 |
| 6 | 57 | 6 | 2 A,B | 2 D,E | nsVT | + | + | – | 0.276 | 0.486 | 0.110 |
| 7 | 67 | 6 | 2 B,F | 2 A,C | VF | − | + | DSC2 (R146L) | 0.425 | 0.397 | 0.104 |
| 8 | 37 | 9 | 4 A,B,E,F | 1 C | sVT | − | − | PKP2 (C796R) | 0.000 | 0.292 | 0.000 |
| 9 | 28 | 9 | 3 A,B,D | 3 C,E,F | VF | + | + | – | 0.121 | 0.518 | 0.026 |
| 10 | 24 | 6 | 2 A,F | 2 C,E | nsVT | − | + | PKP2 (N696Kfs ∗ ) | 0.441 | 0.316 | 0.074 |
| 11 | 66 | 7 | 3 A,B,D | 1 E | sVT | + | + | – | 0.396 | 0.327 | 0.188 |
| 12 | 65 | 10 | 4 A,B,D,F | 2 C,E | sVT | + | + | PKP2 (R651X) | 0.000 | 0.449 | 0.000 |
| 13 | 61 | 8 | 3 A,B,D | 2 C,E | sVT | + | − | – | 0.382 | 0.452 | 0.097 |
| 14 | 40 | 6 | 3 A,B,D | 0 | Syncope | − | − | – | 0.344 | 0.463 | 0.013 |
| 15 | 69 | 10 | 4 A,B,D,F | 2 C,E | sVT | + | + | PKP2 (S615F) | 0.507 | 0.557 | RD |
| 16 | 50 | 7 | 3 A,B,F | 1 C | VF | + | + | PKP2 (R79X) | 0.217 | 0.436 | 0.217 |
| Mean ± SD | 48 ± 16 | 7 ± 2 | 3 ± 1 | 2 ± 1 | 0.28 ± 0.18 | 0.42 ± 0.08 | 0.08 ± 0.07 |
∗ Task Force criteria according to Marcus et al ; adapted diagnostic categories: A = structural alterations; B = tissue characteristics; C = repolarization abnormalities; D = depolarization abnormalities; E = arrhythmias; F = family history (see text for details).
Molecular genetic analyses of venous blood samples from 16 patients with ARVC and 380 unrelated controls were performed to detect pathogenic mutations in genes encoding key desmosomal proteins and thereby the major ARVC subforms (plakophilin-2, subform ARVD9/ PKP2 ; desmocollin-2, ARVD11/ DSC2 ; desmoglein-2, ARVD10/ DSG2 ; desmoplakin, ARVD8/ DSP ; and plakoglobin, ARVD12/ JUP ). Direct sequencing of the entire coding regions of the plakophilin-2 and other ARVC genes and adjacent intronic sequences was performed as previously reported ; the primer sequences are available on request.
All coding regions and intron boundaries of the 5 genes were directly sequenced and compared with the reference sequences. Identified DNA variants were confirmed by at least another independent polymerase chain reaction (PCR). To address the pathogenicity of altered amino acid exchanges, the presence of mutant alleles was first analyzed in an ethnically matched control population (380 unrelated controls) and, second, in the ExomeVariantServer project of the National Heart, Lung, and Blood Institute (Bethesda, Maryland) that encompasses about 5,000 subjects. In addition, mutation were analyzed for presence in current data and for similar predicted effects in pathogenicity programs (e.g., Sorting Intolerant from Tolerant [SIFT], http://sift.bii.a-star.edu.sg ; Polymorphism Phenotyping v2 [PolyPhen2], http://geneitcs.bwh.harvad.edu/pph2/ ). A variant was considered pathogenic if absent in controls (including the ExomeVariantServer project) and predicted by pathogenicity programs.
The local ethics committee approved the study protocol, all patients provided written informed consent before enrollment in the present study.
Right ventricular septal endomyocardial biopsies were performed in all patients with ARVC during diagnostic right heart catheterization according to current guidelines. Immediately after removal, the specimens were snap frozen in liquid nitrogen or prepared for cryosectioning. Nonfailing human septal myocardium (normal ejection fraction found on echocardiographic examination; absence of cardiovascular medication in patient history; inotropic support only with dopamine but not high-dose isoprenaline or noradrenalin) was obtained from 6 ethnically matched donors who had experienced brain death as a result of traumatic injury but whose hearts could not be transplanted for technical or logistical reasons.
Messenger ribonucleic acid (mRNA) was isolated from the myocardial biopsy specimens by magnetic separation (Mini-MACS System, Miltenyi Biotech, Bergisch-Gladbach, Germany). In brief, the specimens were prepared immediately after biopsy using a standardized protocol; the tissues were homogenized in appropriate amounts of lysis/binding buffer using an ultrasonic homogenizator. A 50-μl aliquot of Oligo (dT) microbeads was added to the tissue lysate, which was then pipetted on top of a separation column. Subsequently, the column was rinsed repeatedly with lysis/binding buffer and wash buffer to remove the proteins and DNA. Finally, the mRNA was eluted with 120 μl of 65°C warm hydrogen peroxide. Until use, the resulting mRNA solution was stored at −70°C. The mRNA of Cx40, Cx43, Cx45, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was assessed in the control and biopsy specimens using reverse transcriptase-PCR. In brief, 20 ng of mRNA was reverse transcribed using Superscript II, according to the manufacturer’s instructions (Life Technologies, Eggenstein, Germany). The RT products (2 μl) were brought to a volume of 100 μl containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 0.1 mM of dNTP, and 2.5 U of Taq Polymerase (Life Technologies GmbH, Darmstadt, Germany). For PCR, 0.5 μM of each primer was used (additional details on primers used available on request). Amplification was performed in a UNO thermocycler (Biometra, Göttingen, Germany) after an initial denaturation at 95°C for 10 minutes for 39 cycles (GAPDH, 30 cycles) using the following cycles: denaturation at 95°C for 60 seconds; primer annealing at 60°C (GAPDH 56°C) for 70 seconds; primer extension at 72°C for 60 seconds; and a final extension of 72°C for 10 minutes. Aliquots of the PCR solution were analyzed using standard agarose gel electrophoresis, a video documentation system, and ImageQuant software (Molecular Dynamics, Krefeld, Germany).
The Cxs were detected as follows: for Cx43, mouse anti-rat Cx43 (1:500, MAB3068, Chemicon, Temecula, California); for Cx40, rabbit anti-mouse Cx40 (1:200, Cx40-A, Alpha Diagnostic, San Antonio, Texas); and for Cx45, rabbit anti-human Cx45 (1:200, AB1745, Chemicon) was used. As an internal control for tissue quality, we performed immunostaining for N-cadherin (1:400, mouse anti-human, GC-4, Sigma-Aldrich, St. Louis, Missouri). The secondary antibody/detection system was donkey anti-rabbit conjugated to fluorescein isothiocyanate (1:250, Molecular Probes, Life Technologies, Norwalk, Connecticut) and donkey anti-mouse conjugated to rhodamin or Cy3 (1:500, Chemicon).
The immunohistochemical staining procedure was performed with modifications, as previously described. In brief, cryosections were incubated with the respective antibody at room temperature for 1 hour or overnight at 4°C. After washing thoroughly in phosphate-buffered saline, the sections were treated with the secondary antibody for 1 hour at room temperature. After washing again, the slides were mounted with fluoromount mounting medium (Dako, Carpinteria, California). Negative controls included substitution of the primary antibody with rabbit immunoglobulins or omission of the primary antibody. The results were documented using a fluorescence microscope (AXIOPHOT2, Carl Zeiss Microscopy, Thornwood, New York) fitted with the appropriate filter blocks for detection of fluorescein isothiocyanate and rhodamin/Cy3 fluorescence.
To evaluate the general morphology and occurrence of collagenous matrix standard hematoxylin-eosin and Picrosirius red staining were performed. The sections were examined using a Zeiss microscope (AXIOPHOT2). The area of collagenous matrix was calculated using the ImageJ software (National Institutes of Health, Bethesda, Maryland) for nonfailing controls and ARVC samples.
For statistical analysis, Origin, version 5.0, and PASW Statistics, version 18, for Windows (SPSS, Chicago, Illinois) were used. Data are presented as mean ± SD. Comparisons between the ratios of Cx/GAPDH mRNA of donor tissues and biopsies were performed using the nonparametric Mann-Whitney U test for independent samples. All statistical analyses were intended to be exploratory rather than confirmatory. p Values <0.05 were considered significant. No adjustment for multiple testing was performed.
Results
The clinical and immunohistochemical characteristics of the study patients are summarized in Tables 1 and 2 . Molecular genetic analyses revealed mutations in 9 of 16 patients with ARVC (56%). The mutations were predominantly identified in the gene coding for plakophilin-2 (7 patients [70%]; Table 1 ). Of the 9 ARVC mutations, 56% (all plakophilin-2) were predicted loss-of-function mutations, and 1 mutation was an amino acid insertion in plakoglobin. All mutations, including 3 missense mutations (2 times plakophilin-2; 1 times desmocollin-2) were not present in the control set or noted in the ExomeVariantServer project; pathogenicity was supported using prediction programs.
| Patients (n) | ARVC | Controls 6 (100%) | p Value ∗ | ||||
|---|---|---|---|---|---|---|---|
| All Patients 16 (100%) | PKP2 7 (44%) | DSC2 1 (6%) | JUP 1 (6%) | No Mutation 7 (44%) | |||
| Connexin 40 | 0.28 ± 0.18 | 0.28 ± 0.24 | 0.43 | 0.20 | 0.27 ± 0.15 | 0.67 ± 0.11 | <0.0001 † |
| Connexin 43 | 0.42 ± 0.08 | 0.42 ± 0.09 | 0.40 | 0.40 | 0.42 ± 0.08 | 0.49 ± 0.10 | 0.098 † |
| Connexin 45 | 0.08 ± 0.07 | 0.08 ± 0.09 | 0.10 | 0.17 | 0.08 ± 0.06 | 0.51 ± 0.12 | <0.000 † |
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