Stefania Rizzo, Elisabeth M Lodder, Arie O Verkerk, Rianne Wolswinkel, Leander Beekman, Kalliopi Pilichou, Cristina Basso, Carol A Remme, Gaetano Thiene and Connie R Bezzina
Background: We sought to evaluate the pathological substrate of electrical instability in the pre- phenotypic stage of arrhythmogenic cardiomyopathy (AC), before myocardial cell death and fibro- fatty replacement onset.
Design: We studied transgenic mice carrying mutation of desmoglein2 (dsg2) in the adhesive extracellular domain (Tg-NS). Gross, histological and ultrastructural analyses were used in the pre- phenotypic stage of the diseases, i.e., between 2 and 6 weeks. The structure and molecular composition of the ID was assessed by electron microscopy (EM) and by immunofluorescence. Mice with cardiac over expression of wild-type dsg2 (Tg-WT) and wild-type mice served as controls. Surface ECGs, electrical epicardial mapping and patch-clamp experiments were performed to determine ventricular conduction and arrhythmia susceptibility.
Results: At gross and histologic examination, Tg-NS hearts appeared normal, with no evidence of replacement-type fibrosis. Immunofluorescence uncovered no differences in the level and localization of junctional proteins between Tg-NS/L mice and controls. Ultrastructural examination of the myocardium ruled out cardiomyopathic changes, including cell necrosis, focal myofibrillar lysis, dilated sarcoplasmatic reticulum and T-tubules, and mitochondrial clustering at this age. Widening of the intercellular spaces at the level of desmosomes/adherens junctions was seen in otherwise morphologically normal cardiomyocytes in 25% of TgNS at 2 weeks and in 100% at 6 weeks and the percentage of widened cellular junctions increased with age (from about 10% at an age of 2 weeks to 60% at 6 weeks). Morphometric analysis showed that the intercellular space was significantly widened in Tg-NS/L mice (149 +/- 80 nm) compared with controls (32 3.5nm), p<0.05. None of the control mice (Tg-WT, WT) displayed any intercellular space widening. QRS- prolongation and inducible ventricular arrhythmias were observed in mutant mice. A reduced action potential (AP) upstroke velocity due to a lower Na(+) current density was also observed at this stage of the disease
Conclusions: Dsg-2 mutant mice display ultrastructural evidence of desmosomes /adherens junctions widening, before development of cardiomyopathic changes. This coincided with conduction slowing and inducible ventricular arrhythmias thus emphasizing the importance of ID integrity for proper electrical conduction.