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Plakoglobin localization was examined in this study in biopsies of 11 ARVC patients, 8 of which had mutations in desmosomal genes.It was compared with 14 biopsies of patients with heart disease other than ARVC and 10 healthy controls.
To resolve the existing discrepancies, we designed a cross-species study in which variables were reduced as much as possible by adjusting immunosignals to within the dynamic detection range, by normalization of recordings using control reactions in the same tissue sections and by recording entire data sets with identical settings in single sessions.Hearts were dissected, cut in half, and fixed in 4% buffered formaldehyde overnight.The tissues were then dehydrated using a graded isopropanol series and were embedded in paraffin according to standard protocols.Diagnosis of ARVC was based on clinical and genetic parameters that have been defined by the task force.This classification distinguishes six categories concerning global or regional dysfunction and structural alterations (I), tissue characterization of wall (II), repolarization abnormalities (III), depolarization abnormalities (IV), arrhythmias (V), and family history (VI). Reduced plakoglobin staining was detectable in ARVC only with one antibody directed against a defined epitope but not with three other antibodies reacting with different epitopes of plakoglobin.
Reduced plakoglobin staining in intercalated discs of heart tissue from human ARVC patients and in a murine ARVC model is caused by alterations in epitope accessibility and not by protein relocalization.
The animals were fed standard rodent lab diet (Sniff) and had free access to food and water.
The animal experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) Nordrhein-Westfalen under the reference number 8.87–.09.114.
Most mutations have been identified in the plakophilin 2 gene, followed by mutations in the genes encoding desmoglein 2 and desmoplakin and the fewest mutations in the genes coding for plakoglobin, desmocollin 2 and the desmosome-associated intermediate filament protein desmin.
In addition, mutations in the non-desmosomal proteins transforming growth factor β3, ryanodine receptor 2, transmembrane protein 43, phospholamban, lamin, and titin have been reported with highly variable prevalence in different populations.
A mixture of primary antibodies diluted in PBS containing 1.5% (w/v) BSA was applied over night at 4°C, and unbound antibodies were removed by two times washing for 10 min in TBST buffer containing 50 m M Tris/HCl (p H 7.5), 0.05% (v/v) Tween 20, and 0.3 M Na Cl.