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Increasing usage of next-generation sequencing techniques pushed during the last decade cardiogenetic diagnostics leading to the identification of a huge number of genetic variants in about 170 genes associated with cardiomyopathies, channelopathies, or syndromes with cardiac involvement. Because of the biochemical and cellular complexity, it is challenging to understand the clinical meaning or even the relevant pathomechanisms of the majority of genetic sequence variants. However, detailed knowledge about the associated molecular pathomechanism is essential for the development of efficient therapeutic strategies in future and genetic counseling. Mutations in DES, encoding the muscle-specific intermediate filament protein desmin, have been identified in different kinds of cardiac and skeletal myopathies. Here, we review the functions of desmin in health and disease with a focus on cardiomyopathies. In addition, we will summarize the genetic and clinical literature about DES mutations and will explain relevant cell and animal models. Moreover, we discuss upcoming perspectives and consequences of novel experimental approaches like genome editing technology, which might open a novel research field contributing to the development of efficient and mutation-specific treatment options.

Background DES mutations cause different cardiac and skeletal myopathies. Most of them are missense mutations. Methods Using a next-generation sequencing cardiac 174 gene panel, we identified a novel heterozygous in-frame indel mutation (DES-c.493_520del28insGCGT, p.Q165_A174delinsAS) in a Caucasian patient with cardiomyopathy in combination with atrioventricular block and skeletal myopathy. This indel mutation is located in the coding region of the first exon. Family anamnesis revealed a history of sudden cardiac death. We performed cell transfection experiments and in vitro assembly experiments to prove the pathogenicity of this novel DES indel mutation. Results These experiments revealed a severe filament formation defect of mutant desmin supporting the pathogenicity. In addition, we labeled a skeletal muscle biopsy from the mutation carrier revealing cytoplasmic desmin positive protein aggregates. In summary, we identified and functionally characterized a pathogenic DES indel mutation causing cardiac and skeletal myopathy. Conclusion Our study has relevance for the clinical and genetic interpretation of further DES indel mutations causing cardiac or skeletal myopathies and might be helpful for risk stratification.

Arrhythmogenic right ventricular cardiomyopathy is a heritable cardiac disease causing severe ventricular arrhythmias, heart failure and sudden cardiac death. It is mainly caused by mutations in genes encoding several structural proteins of the cardiac desmosomes including the DSG2 gene encoding the desmosomal cadherin desmoglein-2. Although the molecular structure of the extracellular domain of desmoglein-2 is known, it remains an open question, how mutations in DSG2 contribute to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy. In the present study, we analyzed the impact of different DSG2 mutations on the glycosylation pattern using de-glycosylation assays, lectin blot analysis and genetic inhibition studies. Remarkably, wildtype and mutant desmoglein-2 displayed different glycosylation patterns, although the investigated DSG2 mutations do not directly affect the consensus sequences of the N-glycosylation sites. Our study reveals complex molecular interactions between DSG2 mutations and N-glycosylations of desmoglein-2, which may contribute to the molecular understanding of the patho-mechanisms associated with arrhythmogenic right ventricular cardiomyopathy. Copyright © 2019. Published by Elsevier Ltd.

In 1990, the Seidman group identified the first pathogenic cardiomyopathy mutation in a large 4-generation family, where several members were affected by hypertrophic cardiomyopathy.1 Since this first report, the number of genes and mutations associated with different cardiomyopathies is increasing from year to year. Currently, mutations in >170 genes associated with different cardiomyopathies, channelopathies, or syndromes with cardiac involvement are described. The huge number of different genes and mutations involved in cardiomyopathies limited routine genetic diagnostics for a long time. For example, Sanger sequencing of TTN, encoding the giant sarcomere protein titin, was difficult, expensive, and time consuming and limited the routine genetic diagnosis.2,3 Therefore, it was not surprising that the development of efficient next-generation sequencing technology pushed also the genetic diagnostics of cardiovascular diseases. Today, cardiovascular next-generation sequencing techniques are implemented in many diagnostic laboratories.4 The availability of next-generation sequencing technology has in the meantime provided the important insight that cardiomyopathies are remarkable heterogeneous disorders with different expressivity and penetrance. The challenges for the future remain the identification of phenotype–genotype relationships and consequences of genotyping for the development of personalized therapies. See Article Tucker and McLellan et al In this context, the contribution of a genetic pathogenesis to restrictive cardiomyopathy (RCM) is incompletely understood. Besides genetic factors, RCM might be a secondary cardiomyopathy and part of a systemic disease like the mineralization disorder pseudoxanthoma elasticum or cardiac amyloidosis. First mutations associated with familial RCM were identified in TNNI3 by the research group of William McKenna in 2003.5 During the …

Cadherins are calcium dependent adhesion proteins that establish the intercellular mechanical contact by bridging the gap to adjacent cells. Desmoglein-2 (Dsg2) is a specific cadherin of the cell-cell contact in cardiac desmosomes. Mutations in the DSG2-gene are regarded to cause arrhythmogenic (right ventricular) cardiomyopathy (ARVC) which is a rare but severe heart muscle disease. The molecular pathomechanisms of the vast majority of DSG2 mutations, however, are unknown. Here, we investigated the homophilic binding of wildtype Dsg2 and two mutations which are associated with ARVC. Using single molecule force spectroscopy and applying Jarzynski’s equality we determined the kinetics and thermodynamics of Dsg2 homophilic binding. Notably, the free energy landscape of Dsg2 dimerization exposes a high activation barrier which is in line with the proposed strand-swapping binding motif. Although the binding motif is not directly affected by the mutations the binding kinetics differ significantly from the wildtype. Furthermore, we applied a dispase based cell dissociation assay using HT1080 cell lines over expressing Dsg2 wildtype and mutants, respectively. Our molecular and cellular results consistently demonstrate that Dsg2 mutations can heavily affect homophilic Dsg2 interactions. Furthermore, the full thermodynamic and kinetic description of Dsg2 dimerization provides a consistent model of the so far discussed homophilic cadherin binding.

Restrictive cardiomyopathy (RCM) is a rare heart disease characterized by diastolic dysfunction and atrial enlargement. The genetic etiology of RCM is not completely known. We identified by a next-generation sequencing panel the novel CRYAB missense mutation c.326A>G, p.D109G in a small family with RCM in combination with skeletal myopathy with an early onset of the disease. CRYAB encodes αB-crystallin, a member of the small heat shock protein family, which is highly expressed in cardiac and skeletal muscle. In addition to in silico prediction analysis, our structural analysis of explanted myocardial tissue of a mutation carrier as well as in vitro cell transfection experiments revealed abnormal protein aggregation of mutant αB-crystallin and desmin, supporting the deleterious effect of this novel mutation. In conclusion, CRYAB appears to be a novel RCM gene, which might have relevance for the molecular diagnosis and the genetic counseling of further affected families in the future.

Background Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is mainly an autosomal dominant disease characterized by fibrofatty infiltration of the right ventricle, leading to ventricular arrhythmias. Mutations in desmosomal proteins can be identified in about half of the patients. The pathogenic mechanisms leading to disease expression remain unclear. Objective The purpose of this study was to investigate myocardial expression profiles of candidate molecules involved in the pathogenesis of ARVC/D. Methods Myocardial messenger RNA (mRNA) expression of 62 junctional molecules, 5 cardiac ion channel molecules, 8 structural molecules, 4 apoptotic molecules, and 6 adipogenic molecules was studied. The averaged expression of candidate mRNAs was compared between ARVC/D samples (n = 10), nonfamilial dilated cardiomyopathy (DCM) samples (n = 10), and healthy control samples (n = 8). Immunohistochemistry and quantitative protein expression analysis were performed. Genetic analysis using next generation sequencing was performed in all patients with ARVC/D. Results Following mRNA levels were significantly increased in patients with ARVC/D compared to those with DCM and healthy controls: phospholamban (P ≤ .001 vs DCM; P ≤ .001 vs controls), healthy tumor protein 53 apoptosis effector (P = .001 vs DCM; P ≤ .001 vs controls), and carnitine palmitoyltransferase 1β (P ≤ .001 vs DCM; P = 0.008 vs controls). Plakophillin-2 (PKP-2) mRNA was downregulated in patients with ARVC/D with PKP-2 mutations compared with patients with ARVC/D without PKP-2 mutations (P = .04). Immunohistochemistry revealed significantly increased protein expression of phospholamban, tumor protein 53 apoptosis effector, and carnitine palmitoyltransferase 1β in patients with ARVC/D and decreased PKP-2 expression in patients with ARVC/D carrying a PKP-2 mutation. Conclusion Changes in the expression profiles of sarcolemmal calcium channel regulation, apoptosis, and adipogenesis suggest that these molecular pathways may play a critical role in the pathogenesis of ARVC/D, independent of the underlying genetic mutations.

Aims Coronary artery disease accounts for the majority of sudden cardiac deaths (SCD) in the older population whereas cardiomyopathies and arrhythmogenic abnormalities predominate in younger SCD victims (

470 Extracardiac endothelium patterns embryonic coronary arterio-venous connections {#article-title-2} Recent reports suggest that mammalian embryonic coronary endothelium (CoE) develops from the sinus venosus and ventricular endocardium. Although several studies in non-mammalian vertebrates have proven that extracardiac endothelial cells also participate in CoE development, the contribution of such cells to mammalian CoE is regarded to be minor and non-significant. Using classic (Wt1Cre) and novel (G2-Gata4Cre) mouse transgenic models for the study of coronary vascular development, we show that extracardiac septum transversum/proepicardiun (ST/PE)-derived endothelial cells are required for proper coronary development. Our results indicate that, at least, 20% of embryonic coronary arterial and capillary endothelial cells derive from the ST/PE. Conditional deletion of the epicardial lineage gene Wt1 in the ST/PE (G2-Gata4Cre) or the endothelium (Tie2Cre) reveals a critical role for these ST/PE-derived endothelial cells in the establishment of transmural ventricular coronary arterio-venous connections. Taken together, our results confirm that embryonic CoE is a developmental mosaic forming from different endothelial sources, and that extracardiac cells are necessary for the completion of coronary morphogenesis. These data contribute to our understanding of some coronary congenital anomalies and suggest embryonic CoE heterogeneity as an ontogenetic factor for adult coronary disease. # 471 DCM-associated RBM20-mutations lead to aberrant splicing of titin and ryanodin receptor 2 in the human myocardium {#article-title-3} Background: Mutations in human RBM20 have previously been shown to cause dilated cardiomyopathy (DCM). RBM20 is predominantly expressed in the heart. Within the nucleus it partially colocalises with other splice factors and binds RNA. Deep sequencing of the cardiac transcriptome of a human RBM20 mutant and of a RBM20 deficient rat model revealed RBM20 dependent regulation of myocardial alternative splicing. We have identified the previously published RBM20 mutation, p.P638L, which is localised in the highly conserved RS-region, in a large German family. The mutation is highly penetrant leading to terminal heart failure and/or to sudden cardiac death. Additionally we have discovered the new RBM20 mutation p.V914A localised outside the RS-region. Purpose: Though it is investigated that RBM20 is involved in alternative splicing in the rat heart little is known on aberrant myocardial splicing in human RBM20-mutation carriers. Goal of this study was to gain insight into the mechanisms leading to cardiomyopathy by analysing explanted human ventricular myocardium of RBM20-mutation carriers. Material and Methods: Cardiac tissue from two RBM20-p.P638L- and one RBM20-p.V914A-DCM-patients and a patient with the RBM20-variant p.D888N was obtained during heart transplantation or the implantation of a ventricular assist device, respectively. Ventricular myocardium was used for RNA extraction, cDNA synthesis and splicing analysis of TTN, RYR2, LDB3, CAMK2D, NEXN and TRDN by Real-Time-PCR. The alternative splicing of Titin was additionally examined by Western Blot using cardiac tissue lysates. Results: We identified aberrant RYR2 splicing in the RBM20 mutant myocardium characterised by an increased inclusion of an additional 24bp-exon. Furthermore we found a decrease of the major cardiac titin splice variant N2B by real-time PCR and Western Blot. Titin Western Blots revealed an increase of the splice variant N2BA in the RBM20 mutants. Of note, an influence on splicing could be detected not only for the RBM20-p.P638L-mutant but also for the mutation p.PV914A localised outside of the conserved RS-domain. The variant p.D888N showed no influence on the splicing of the genes analysed so far. In contrast to the rat hearts, in the human myocardium of RBM20-mutation carriers we did not find splicing defects in TRDN and NEXN. Conclusion: We present here first data on a molecular defect of a novel RBM20-mutation localised outside the conserved RS-region. Furthermore we show the influence of mutant RBM20 on splicing of RYR2 and TTN in the human explanted myocardium of three mutation carriers. The identification of the molecular pathomechanisms of the RBM20-mutations in the affected patients allows a differentiated insight into the disease mechanisms and might offer potential targets for curative treatment in the future. Furthermore the analysis of the splicing pattern helps to distinguish pathogenic variants like RBM20-p.PV914A from rare polymorphisms. # 472 The impact of missense versus nonsense mutations in arrhythmogenic cardiomyopathy phenotype {#article-title-4} Background: Arrhythmogenic Cardiomyopathy (AC) is an inherited heart muscle disease associated with mutations in five genes mainly encoding desmosomal components. Missense mutations leading to a single nucleotide substitution have disputed pathogenicity in inherited cardiomyopathies whereas truncating and splice site mutations are considered disease-causing due to their impact on form and function of the resulting protein. Purpose: Herein we describe the clinical features of two probands and their family members in whom a missense and a nonsense mutation in two desmosome-encoding genes co-segregate. Methods: A Next Generation Sequencing-based strategy comprising 150 inherited cardiomyopathy related genes was applied on the two index female cases (63y, 70y). Libraries were prepared according to the manufacturer’s instructions and sequenced on a MiSeq platform (Illumina). The Illumina Variant Studio software was used to filter and prioritize variants on the basis of genotype quality, allele frequencies, predicted pathogenicity, literature information. Subsequent conventional sequencing was used for cascade mutation screening. HL-1 cells have been transfected to ascertain localization of the mutated proteins. Results: Two unrelated female probands (63 y-old, 70 y-old) carried a missense mutation in DSG2 (p.Asn266Ser). The 63 y-old proband underwent cardiac transplantation (family A). The 70 y-old proband carried an additional nonsense mutation in DSP (c.88G>A, p.Gln113X), as well as her 64 y-old sister. Eight of the fifteen relatives from both families carried the DSG2 mutation: seven of them exhibited typical clinical AC features, whereas two young family members were unaffected at the ages of 6 and 12, respectively. The two DSP mutation carriers of family B were unaffected at the age of 44 and 66. No sudden cardiac death events occurred in both families. Haplotype analysis indicated that all the DSG2 mutation carriers shared the same haplotype suggesting a common founder allele. In vitro experiments on the cardiomyocyte cell line HL-1 showed correct localization of the mutated DSG2, whereas the mutated DSP was absent at the membrane level. Conclusions: This study is calling into question whether nonsense mutations are of high relevance with respect to the missense ones in the pathogenesis of AC. Our results highlight the dominant negative effect of a known missense DSG2 mutation with a presumed common founder effect, whereas the pathogenic role of the DSP nonsense mutation needs further studies to elucidate its contribution to the disease phenotype.