Abnormal connexin43 in arrhythmogenic right ventricular cardiomyopathy caused by plakophilin-2 mutations.

Clinical characteristics of ARVC patients

Patients presenting to our cardiac arrhythmia program with any of non-sustained monomorphic ventricular tachycardia with LBBB morphology, a family history of ARVC or an epsilon wave underwent non-invasive testing included ECG, signal-averaged ECG, 24-hr ambulatory ECG, exercise testing, echocardiogram and magnetic resonance imaging. Those patients with one or more additional positive non-invasive findings underwent an electrophysiology study, right ventricular angiography and endomyocardial biopsy from the RV septum. Patients were approached for research (PKP-2, DSP, DSG) or clinical (PKP-2) genetic testing. Four patients with both a positive genetic diagnosis of a PKP-2 mutation and available residual tissue from endomyocardial study form the basis of this report.

Genetic analysis

Genetic analysis of PKP-2 in the four described patients was performed at two separate institutes. Two patients were sequenced at the DNA Diagnostics Lab at John Hopkins University, Baltimore, while two were determined by The Centre for Applied Genomics at the Hospital for Sick Children in Toronto. For DNA sequencing, polymerase chain reaction (PCR) amplification was performed, followed by genomic DNA sequencing. Twenty-seven patients with ARVC were screened for mutations in PKP-2 via DNA sequencing. PCR was completed using 20–50 ng of DNA in buffer [10mM Tris-HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl 2 , 0.16 mg BSA, 0.01% gelatin], 0.4 mM dNTP’s, 50 ng of each primer and 1 unit of Taq Polymerase (Applied Biosystems, Foster, CA, USA), resulting in total reaction mixtures of 25 μl. Reaction cycles were performed at 95°C for 2 min., 35 cycles at 94°C for 30 sec., 30 sec. of exon-specific temperatures, 72°C for 1 min., followed by 10 min. at 72°C, which correspond to the initial extension, denaturation, annealing, extension and final extension stages, respectively. Purification of DNA was performed using a CleanSeq (Agencourt Biosciences), followed by sequencing involving BigDye 3.1 chemistry on a 3730XL DNA Analyzer (Applied Biosystems, Foster, CA, USA). This study made use of human material and conformed to the principles outlined in the Declaration of Helsinki. Genetic research was performed with patient consent and approval of the Institutional Research Ethics Board.

Acquisition of tissues

Cardiac tissue was available for study from patients who had previously undergone endomyocardial biopsies as part of a clinical assessment for possible ARVC. Following clinical reporting of these specimens as analysed by light and electron microscopy, residual tissue was stored in liquid nitrogen.

Institutional Research Ethics

Board approval for immunofluorescence analysis of these residual samples was obtained. One to two sections per antibody were assessed by confocal immunofluorescence microscopy to characterize N-cadherin (an adherens junction protein with no known survivable mutations) as a marker of the intercalated disk, PKP-2 (the desmosomal protein of interest) and the ventricular gap junctionprotein connexin43. Control ventricular myocardial tissues for immunofluorescence analysis were obtained as frozen blocks from the native hearts of patients coming to heart transplantation for congenital heart disease without primary arrhythmia or myocardial failure ( e.g. valvular disease) in which the myocardium, frozen immediately upon surgical removal of the native heart, was histologically normal.

Immunofluorescence and confocal microscopy

Seven-micrometer cryostat sections from patients and the controls were fixed with acetone at –20°C for 15 min. followed by air-drying for 30 min. The sections were rinsed in phosphate-buffered saline (PBS) and blocked in PBS with 2% normal donkey serum, 1% bovine serum albumin and 0.2% TritonX-100 for 1 hr at room temperature. Connexin43, PKP-2, DSP and N-cadherin antigens were examined following incubation with rabbit anti-DSP (1:250, Serotec, Oxford, UK), mouse anti-PKP-2 (1:2, Progen Biotechnik, Heidelberg) and rabbit anti-connexin43 (1:200, Zymed Laboratories, San Francisco, CA, USA) antibodies for 1 hr. After rinsing with PBS, the sections were incubated with the corresponding secondary donkey antibodies (conjugated with Cy2 and Cy3, respectively) (Jackson ImmunoResearch, West Grove, PA, USA), diluted 1:200 and 1:500, for another 30 min. at room temperature. For double staining, sections were incubated with mouse anti-N-cadherin (1:500, Zymed Laboratories, San Francisco, CA, USA) or rabbit anti-pan cadherin (1:500, Sigma, St. Louis, MO, USA) and corresponding secondary antibody. Finally, DAPI staining was performed to identify nuclei. After a final rinsing step, sections were mounted with a 10% solution of polyvinyl alcohol containing 2.5% 1,4-diazabicyclo-2,2,2-octane (PVA/DABCO, both from Sigma, St. Louis, MO, USA), coverslipped, and visualized using a confocal laser scanning microscope LSM510 META (Carl Zeiss, Jena, Germany).

Quantitative localization and colocalization analysis

Double-stained images were obtained by sequential scanning for each channel to eliminate the crosstalk of chromophors and to ensure the reliable quantification of colocalization. Images for all four patients and two controls were acquired and processed for localization analysis of connexin43 to the intercalated disk. This analysis was performed using Adobe Photoshop on a Macintosh PC, adapting a previously published technique of luminescence analysis [ 18 ]. The outlines of intercalated disks were traced based on N-cadherin-stained samples, and the corresponding immunofluorescence signals for connexin43 and N-cadherin at the intercalated disk were sampled on a luminescence scale from 1 to 255. Approximately 5–10 intercalated disks were examined in roughly 5–10 different field images per sample. Sample background (non-intercalated disk) signal was subtracted and corrected connexin43 luminescence values representing the intercalated disks were expressed as a percentage of the corrected N-Cadherin luminescence [(Cx43 IC Disk –Cx43 Background )/N-Cad IC Disk –N-Cad Background )*100%]. These percentages were then averaged and expressed as a final percentage luminescence in comparison to N-Cadherin for each patient. Quantitative colocalization analysis of antigens was performed using Volocity Improvision 3.7.0 ( http://www.improvision.com ) for patients 2–4 and a single control. Background was corrected to remove the necessary number of pixels at all channels. Pearson’s correlation coefficient (PCC), one of the standard measures of pattern recognition, was employed for colocalization of connexin43 and N-Cadherin.

Transfection of HL-1 mouse myocyte cells with PKP-2 siRNA

In parallel to examining endomyocardial biopsies from ARVC patients, HL-1 atrial myocytes were chosen for in vitro analysis. HL-1 cells have the ability to be continuously passaged and recurrently used in ARVC research [ 19 – 21 ]. These cells maintain the electrophysiological functioning of healthy cardiomyocytes and express near natural levels of connexin43, making them ideal for this particular research [ 21 ]. Ventricular myocytes, although anatomically appropriate for understanding ARVC pathogenesis, would be difficult to use in siRNA experimentation due to their limited lifespan. The sense strands of the commercial hairpin siRNA (GenePharma, Shanghai, China) were homologous to a 19-nucleotide region in the PKP2 mRNA starting at nucleotide 192. The sequence of the siRNA for targeting and negative control siRNA were 5′-GGAUCCAGGAGCAGGUGCAdtdt-3′ and 5′-UGUCCGAACGUGUCACGUTTdtdt-3′ (sense sequence only). siRNA molecules with initial nucleotide hybridization at nucleotides 2121 and 2343 were also employed during the siRNA efficiency screening process. HL-1 myocyte cells were grown in Claycomb medium (JRH Biosciences, Lenexa, KS, USA) supplemented with 10% foetal bovine serum (JRH Biosciences), 0.1 M norepinephrine (Sigma, Saint Louis, MO, USA), 2 mM L-glutamine (Invitrogen, South San Francisco, CA, USA), and penicillin/streptomycin (10 4 U/ml P and 10 4 μg/ml S; Invitrogen, South San Francisco, CA, USA) in a humidified 5% CO 2 -incubator at 37°C. HL-1 cells were plated at 8 × 10 4 cells/well in a 12-well plate and as monolayers on glass cover-slips at a density of approximately 2.5 × 10 4 cells/well in a 24-well plate. The next day, 0.5 μg siRNA was transfected into HL-1 cells using X-tremeGENE siRNA Transfection Reagent (Roche). The media was changed the following day and cells were experimented 96 hrs after transfection.

Immunoblot analysis

Cells in 12-well plates were rinsed twice with PBS, then added to 50 μl of RIPA lysis buffer (20 mM Tris-HCl, pH 8.0; 1% Nonidet P-40, 0.1%SDS, 0.5% sodium deoxycholate, and complete protease inhibitor; Roche), sonicated for 15 sec., and centrifuged at 12,000 rpm for 15 min. Protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Aliquots of 10 μg of protein extracts were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk in Tris-buffered saline (TBS), then probed with a mouse anti-plakophilin-2 antibody (1:2 dilution, PROGEN Biotechnik, Heidelberg, German), mouse anti-N-Cadherin (1:400, Zymed Laboratories, San Francisco, CA, USA), or rabbit anti-connexin43 (1:200, Zymed Laboratories, San Francisco, CA, USA). The secondary antibody against mouse primary antibodies was goat anti-mouse IgG-HRP (1:10,000 dilution, Santa Cruz Biotechnology Inc.); against rabbit primary antibodies, a goat anti-rabbit IgG-HRP (1:5,000 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibody was used, followed by the detection of chemiluminescence.