Aufklärung von Pathomechanismen Desmoglein 2 Mutation verursachter Kardiomyopathie

  • Identification of pathomechanisms in desmoglein 2 related cardiomyopathy

Kant, Sebastian; Leube, Rudolf E. (Thesis advisor); Bohrmann, Johannes (Thesis advisor)

Aachen (2016)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen, 2016


Desmosomes are prominent cell-cell contacts of all epithelia and heart muscle. They connect intermediate filaments of neighboring cells. Mutations of genes coding for desmosomal proteins have been identified in hereditary skin and heart diseases. In this thesis the pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC) was examined with the goal to define sequential stages of ARVC and to assign them to characteristic morphological and molecular alterations.To this end transgenic mouse lines were established and analysed with targeted mutations of the desmosomal adhesion protein desmoglein 2 (Dsg2). The knock-in mouse strain DSG2 MT/MT encoding a Dsg2 mutant lacking part of its extracellular adhesive domain was analyzed. In addition, the cardiomyocyte-specific knock-out strain DSG2 cKO/cKO was generated and characterized. Some of the DSG2 MT/MT mice die already during embryogenesis while the survivors are born with morphologically inconspicuous hearts. These animals develop, however, extensive calcifying lesions on the basis of necrotic cardiomyocytes starting at 2-4 weeks. Immune cells infiltrate these areas and collagen fibres are deposited. The immune reaction subsides for the most part until the age of 12 weeks when mature scars are formed. The ensuing chronic phase is characterized by interstitial fibrosis. DSG2 cKO/cKO animals present with an almost identical course of disease. The similarity of the developing phenotype in both strains shows that the primary loss of adhesion is most important for disease initiation and not Dsg2-dependent signaling pathways.Many publications have implicated the junctional plaque protein plakoglobin in ARVC pathogenesis, since plakoglobin is not only responsible for anchorage of actin and intermediate filaments but also influences wnt-pathway in the nuclues. The proposed pathomechanism stringently relies on reduced plakoglobin in the intercalated disc (ICD). To test this quantitative immunofluorescence analyses were performed to determine the expression level of plakoglobin in the ICD in relation to other ICD proteins in our murine ARVC models. Except for a decrease of Dsg2 in the DSG2 MT/MT mice and a loss of Dsg2 in the DSG2 cKO/cKO mice we could not detect alterations. In the case of plakoglobin two carboxyterminal antibodies did not show any alteration but an aminoterminal antibody revealed significant reduction. This observation could be confirmed in human ARVC tissue samples. An explanation is that the aminoterminal epitope of plakoglobin is lost in the pathologically altered ICDs. An influence on the wnt-signaling pathway, however, can not be derived from this observation.An increased mRNA expression was noted for skeletal muscle actin (ACTA1) prior to lesion formation which increased further during disease progression. The mRNA expression was highest in the vicinity of newly-formed lesions that produced TGF-β. In addition, accumulation of actin at ICDs was noted in these areas. Activation of SRF signaling through Mrtf-a could be identified as a cause of the early increase in ACTA1. Furthermore, cell culture experiments suggest that this ACTA1 expression is due to Srf/Mrtf-a activation in a cardiomyocyte-specific manner. The lack of typical desmosomes in both mutant strains suggests a primary adhesive defect through the presence of either the adhesion-deficient Dsg2 or the absence of Dsg2 altogether which likely induces the Mrtf-a/SRF pathway. Scar-induced change of biomechanical properties and the increased perilesional TGF-β levels likely lead to an additional amplifying effect.Taken together, my observation support the hypothesis that alterations of cardiac biomechanics are crucial factors of ARVC pathogenesis whereas a redistribution of plakoglogin is not a major factor. Initial compensatory mechanisms become insufficient during adolescence and in situations of extreme physical activity thereby leading to acute alterations that may be lethal. The underlying pathomechanism amplify in a detrimental cycle whereby structural and functional alterations influence each other. If this critical phase is survived or is therapeutically mitigated, complex mechanisms are in place to support a rather slow further development of the disease.