DSC2 Research Articles

Regulating DSC2 Expression Affects the Proliferation and Apoptosis of Prostate Cancer Cells.

BackgroundProstate cancer threatens the life and health of men in China. Desmocollin-2 (DSC2) is a member of DSC family, abnormal expression of which can affect the invasion and metastasis of tumor cells. The aim of this study was to investigate the role of DSC2 in prostate cancer.Materials and MethodsRegulating DSC2 expression in prostate cancer cells was conducted with transfection. The expression of DSC2, apoptosis-related proteins, cell cycle-related proteins and E-cadherin (E-cad)/β-catenin pathway was detected by Western blot analysis. The proliferation, clone formation ability, migration, invasion and apoptosis of transfected cells were in turn detected by cell counting kit-8 (CCK-8) assay, clone formation assay, wound healing assay, transwell assay and flow cytometry analysis.ResultsDSC2 expression was increased in prostate cancer cells compared with RWPE-1 cells. Inhibition of DSC2 promoted the proliferation, clone formation ability, migration and invasion while suppressed apoptosis of LNCaP cells and PC-3 cells. Inhibition of DSC2 affected the expression of apoptosis-related proteins and cell cycle-related proteins according to the changes of apoptosis and proliferation. Furthermore, inhibition of DSC2 up-regulated the expression of p-β-catenin and EGFR while down-regulated the expression of E-cad. DSC2 overexpression exerted the opposite effect of inhibition of DSC2 on LNCaP cells and PC-3 cells.ConclusionDSC2 expression was increased in prostate cancer cells. In addition, inhibition of DSC2 promoted the proliferation, clone formation ability, migration and invasion while suppressed apoptosis of LNCaP cells and PC-3 cells, which provided the fundamental basis for treatment of prostate cancer.

The PPAR-γ inhibitor T0070907 normalizes impaired electro-mechanical properties of human-induced pluripotent stem cell-derived cardiomyocytes in DSC2 arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) primarily involves right ventricle impairment and lethal arrhythmias. Hereditary forms of ACM are mostly caused by mutations in genes coding for desmosomal proteins. Histopathological hallmark includes fibrofatty replacement of myocardial tissue, which may involve transdifferentiation of cardiomyocytes into adipocyte cells through the PPAR-γ pathway. Comprehensive investigation of cardiomyocytes electro-mechanical properties during this process has never been undertaken. We aimed to assess the implication of the PPAR-γ pathway inhibition using T0070907 inhibitor. We used cardiomyocytes (hiPSC-CM) differentiated from an ACM patient-derived pluripotent stem cells (hiPSC) harbouring a mutation (R132 C) in the DSC2 gene. The control and DSC2 hiPSC were reprogrammed and differentiated into hiPSC-CM to examine gene expression, electro-mechanical properties (patch-clamp and video analysis) and calcium handling (fluorescence imaging) profiles. The DSC2 hiPSC-CM were cultured for 40 days in presence or absence of 1 μM T0070907. The RT-qPCR of both DSC2 patient's heart samples and hiPSC-CM showed an increase of PPAR-γ expression when compared to control cells. When compared to control hiPSC-CM, the DSC2 hiPSC-CM exhibited a shorter action potential duration, due to an increase of K+ current density. Culture of DSC2 hiPSC-CM with the PPAR-γ inhibitor normalized both the action potential duration and K+ current density. Differences were also found in both the calcium transient and mechanical properties between control, DSC2 and DSC2 with PPAR-γ hiPSC-CM. PPAR-γ pathway inhibition by T0070907 normalized the disrupted electro-mechanical properties in DSC2 hiPSC-CM. This result opens interesting perspectives for further studies of the adipogenesis pathway to prevent appearance of fibro-fatty adipose tissue in the ACM patient's heart.

Relevance and diagnostic performance of genes involved in arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disease, affecting preferentially people under 40 years old with ventricular arrhythmia to sudden death. To date, the rate of successful genotyping is about 50%. Desmosomal genes (PKP2, DSC2, DSG2, DSP, JUP) are the most frequently mutated, the implication of the other genes associated with other cardiomyopathies is still debated. The pathogenicity of variants among these genes should be assessed not only based on the rarity because of the presence of rare variants in individuals in the general population. Assess the pathogenicity of variants and the relevance of genes involved in ACM. DNA from 179 patients with ACM were sequenced on a panel of 71 heart disease genes including 11 genes most often described in ACM. The pathogenicity of variants is assessed according to international guidelines (ACMG score) with 2 bioinformatic tools. We performed burden (CAST) and dispersion tests (SKAT, SKAT-O) on the 71 genes between patients and a control population. Among the 179 patients, 34.6% have a pathogenic variant with PKP2 in 42 patients. Furthermore, 17.3% of patients have uncertain significance variants in genes involved in ACM. Burden tests showed a significant enrichment in pathogenic variants in PKP2, DSP, DSC2 and DSG2 ( P < 0.001) and in uncertain significance variants in the PKP2, DSG2 and DSC2 genes ( P < 0.01). No enrichment was observed among other genes. Patients with a variant in genes involved in ACM are diagnosed earlier than patients without variant ( P < 0.05). Only desmosomal genes count a significate proportion of pathogenic variants in patients with ACM compared to control population. Uncertain significance variants in PKP2, DSC2, DSG2 genes seem to contribute to ACM and should be investigated. The age of symptom onset is correlated with the presence of pathogenic variant, thus justifying early family screening for better clinical management.

CAR-T Cell Therapy-An Overview of Targets in Gastric Cancer.

Gastric cancer (GC) is one of the most commonly diagnosed malignancies and, unfortunately, still has a high mortality rate. Recent research points to CAR-T immunotherapy as a promising treatment for this disease. Using genetically engineered T cells designed to target a previously selected antigen, researchers are able to harness the natural anti-tumor activity of T cells. For therapy to be successful, however, it is essential to choose antigens that are present on tumor cells but not on healthy cells. In this review, we present an overview of the most important targets for CAR-T therapy in the context of GC, including their biologic function and therapeutic application. A number of clinical studies point to the following as important markers in GC: human epidermal growth factor receptor 2, carcinoembryonic antigen, mucin 1, epithelial cell adhesion molecule, claudin 18.2, mesothelin, natural-killer receptor group 2 member D, and folate receptor 1. Although these markers have been met with some success, the search for new and improved targets continues. Key among these novel biomarkers are the B7H6 ligand, actin-related protein 2/3 (ARP 2/3), neuropilin-1 (NRP-1), desmocollin 2 (DSC2), anion exchanger 1 (AF1), and cancer-related antigens CA-72-4 and CA-19-9.

O emocionar e o saber ser professor de um coletivo de fisioterapeutas

Objetivo: Conhecer como os profissionais de Fisioterapia, transformam o conteúdo específico de sua área de conhecimento em conhecimento de ensino. Métodos: Estudo qualitativo realizado com dez professores graduados em Fisioterapia que atuam na sala de aula de uma Instituição de Ensino Superior (IES). A opção pelo conversar individual foi o método que nos pareceu adequado para o Estudo e envolveu perguntas com temas como o tempo de trabalho, a formação acadêmica e a decisão pela docência, práticas e técnicas utilizadas em sala de aula, recursos e avaliações do desempenho dos alunos que poderiam nos auxiliar a compreender o problema do estudo. A análise do conversar foi realizada pela técnica do Discurso do Sujeito Coletivo. Resultados: Formou os discursos coletivos que denominamos: DSC1 – O emocionar da docência; DSC2 – A prática da docência. O sujeito fisioterapeuta, pelas interações recorrentes foi sendo constituído na rede de conversação docente e, na convivência, foi se transformando no fazer e no emocionar da docência. Conclusão: Pelos atravessamentos com o fazer docente em sua trajetória de vida e pelas próprias experiências da ação, esses profissionais se produzem na docência na condição de sabedores de um conhecimento específico e de uma emoção que lhes move a fazer o que fazem.

Differential Regulation of Intestinal Epithelial Intercellular Adhesion and Barrier Function by Desmosomal Cadherins

Desmosomal cadherins mediate intercellular adhesion and provide mechanical strength to tissues. Intestinal epithelial cells express desmosomal cadherins Desmoglein‐2 (DSG‐2) and Desmocollin‐2 (DSC‐2). To explore the relative contribution of these cadherins in mediating intercellular adhesion of intestinal epithelial cells, we generated inducible intestinal‐epithelia specific (Villin‐CreERT2) Dsg‐2 and Dsc‐2 knockdown mice (Dsg‐2ERΔIEC; Dsc‐2ERΔIEC). While intestinal mucosal architecture was maintained in Dsg‐2ERΔIEC and Dsc‐2ERΔIEC mice, ultrastructural changes in desmosomal plaques and widened intercellular spaces were observed in these mice compared to littermate floxed control mice. Functional analysis revealed increased paracellular permeability to FITC dextran supporting compromised epithelial barrier function. Interestingly, Dsc‐2ERΔIEC mice showed reduction in desmosome length while Dsg‐2ERΔIEC animals exhibited a higher FITC dextran permeability, suggesting differential dominant roles of DSG‐2 in controlling intestinal epithelial barrier function and of DSC‐2 in regulating intercellular adhesion. Additionally, knockdown of these cadherins in‐vitro in epithelial cells resulted in decreased cell‐cell adhesion and compromised barrier function. Analysis of desmosome mechanical strain using DSG‐2 and DSC‐2 tension sensors revealed that loss of DSC‐2 increased intermediate filaments tension while the loss of DSG‐2 did not show any change in cytokeratin tension. These findings provide new insight on the relative contribution of DSG‐2 and DSC‐2 in controlling intestinal epithelial intercellular adhesion and barrier function.Support or Funding InformationExperiments that lead to this poster were supported by NIH RO1 DK059888, DK055679 to AN. The authors declare no conflict of interest.

Analysis of different proteins in primary open angle glaucoma based on aqueous proteome

Objective To analyze the changes of protein expression in aqueous humor of patients with primary open angle glaucoma (POAG), and to explore the biological markers and potential therapeutic targets associated with disease occurrence. Methods A retrospective case-control study was adopted.Ten patients with age-related cataract and 10 patients with POAG combined with cataract.were collected at the Tianjin Medical University Eye Hospital from October 2016 to December 2017.In the process of surgery, 100 μl of aqueous humor were collected with 1 ml syringe and No.1 needle through the surgical access.Those proteins extracted from aqueous humor were analyzed by quantitative proteomic mass spectrometry.The differential significance test was performed by Maxquant significances A. The differential proteins of the two groups were screened and determined with the conditions of P 2.The biological big data was annotated by the function of GO and KEGG.The study was approved by the Ethics Committee of the Tianjin Medical University Eye Hospital (No.2013KY[L]-10). Patients and their guardians all signed the informed consent. Results Ninty-seven differential proteins were detected in this proteomic analysis, including 48 up-regulated proteins and 49 down-regulated proteins.GO analysis significantly differs in protein function involved in many aspects, some different proteins including lipopolysaccharide-binding protein (LBP), cluster of Differentiation 163(CD163), C-reactive protein (CRP), annexin A1 (ANXA1) were involved in inflammation; redox-related proteins were glutathione S-transferase P (GSTP1), thioredoxin (TXN), some different proteins related with cell adhesion movement were cartilage oligomeric matrix protein (COMP), desmocollin-2 (DSC2) and laminin subunit beta-2 (LAMB2); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1), solid protein (tenascin, TNC) are associated with fibrosis; some different proteins related to nerve growth were reelin (RELN), semaphorin-3F(SEMA3F), semaphorin-4B(SEMA4B). Metabolism-related proteins included pyruvate kinase (PKM), carb oxypeptidase N subunit 2 (CPN2) and so on.KEGG analysis indicated that the expression of NrF2/ERK signaling pathway and TGF-β/NF-κB signaling pathway were different between the two groups. Conclusions The expression of GSTP1 and TXN in the aqueous humor of POAG is significantly decreased, which may regulate cell adhesion and activity and expression of extracellular matrix by regulating NrF2/ERK1/2 and TGF-β/NF-κB signaling pathways.GSTP1 and TXN may serve as a potential biomarker and therapeutic target of POAG. Key words: Primary open angle glaucoma; Aqueous humor; Label free quantitative proteome; Glutathione S-transferase P; Thioredoxin; Transforming growth factor-β; nuclear factor-kappa B; Nuclear factor erythroid 2-related factor 2; extracellular regulated protein kinases

Influence of Vitamin D on Corneal Epithelial Cell Desmosomes and Hemidesmosomes

PurposeWe have observed noticably weak epithelial attachment in vitamin D receptor knockout mice (VDR KO) undergoing epithelial debridement. We hypothesized that VDR KO negatively affects corneal epithelial cell desmosomes and/or hemidesmosomes.MethodsTranscript levels of desmosome and hemidesmosome proteins in VDR KO corneas were assessed by qPCR. Western blotting and immunochemistry were used to detect proteins in cultured cells exposed to 1,25(OH)2D3 and 24R,25(OH)2D3.ResultsVDR KO resulted in decreased corneal desmosomal desmoglein 1 (DSG1) and desmocollin 2 (DSC2) mRNA, and hemidesmosomal plectin mRNA. DSG1 and plectin protein expression were reduced in VDR KO corneas. DSG1 protein expression increased in VDR wild types (VDR WT) and VDR KO mouse primary epithelial cells (MPCEC) treated with 1,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 treatment resulted in increased plectin and integrin β4 levels in VDR WT MPCEC, and decreased levels in VDR KO MPCEC. Treatment of human corneal epithelial cells (HCEC) with 1,25(OH)2D3 and 24R,25(OH)2D3 resulted in increased DSC2 and DSG1 protein expression. Plectin and integrin β4 were only increased in 24R,25(OH)2D3 treated HCEC.ConclusionsVDR KO results in reduced desmosomal and hemidesmosomal mRNA and protein levels. 1,25(OH)2D3 and 24R,25(OH)2D3 increased DSG1 protein in all cells tested. For hemidesmosome proteins, 24R,25(OH)2D3 increased plectin and integrin β4 protein expression in VDR WT and HCEC, with decreased expression in VDR KO MPCEC. Thus, vitamin D3 is involved in desmosome and hemidesmosome junction formation/regulation, and their decreased expression likely contributes to the loosely adherent corneal epithelium in VDR KO mice. Our data indicate the presence of a VDR-independent pathway.

Incorporation of desmocollin-2 into the plasma membrane requires N-glycosylation at multiple sites.

Desmocollin‐2 (DSC2) is a desmosomal protein of the cadherin family. Desmosomes are multiprotein complexes, which are involved in cell adhesion of cardiomyocytes and of keratinocytes. The molecular structure of the complete extracellular domain (ECD) of DSC2 was recently described, revealing three disulfide bridges, four N‐glycosylation sites, and four O‐mannosylation sites. However, the functional relevance of these post‐translational modifications for the protein trafficking of DSC2 to the plasma membrane is still unknown. Here, we generated a set of DSC2 mutants, in which we systematically exchanged all N‐glycosylation sites, O‐mannosylation sites, and disulfide bridges within the ECD and investigated the resulting subcellular localization by confocal laser scanning microscopy. Of note, all single and double N‐glycosylation‐ deficient mutants were efficiently incorporated into the plasma membrane, indicating that the absence of these glycosylation sites has a minor effect on the protein trafficking of DSC2. However, the exchange of multiple N‐glycosylation sites resulted in intracellular accumulation. Colocalization analysis using cell compartment trackers revealed that N‐glycosylation‐ deficient DSC2 mutants were retained within the Golgi apparatus. In contrast, elimination of the four O‐mannosylation sites or the disulfide bridges in the ECD has no obvious effect on the intracellular protein processing of DSC2. These experiments underscore the importance of N‐glycosylation at multiple sites of DSC2 for efficient intracellular transport to the plasma membrane.

Desmocollin 3 has a tumor suppressive activity through inhibition of AKT pathway in colorectal cancer

Desmocollin 3 (DSC3) is a transmembrane adhesion protein of desmosomes and involved in carcinogenesis in various cancer types. Downregulation of DSC3 has been reported in colorectal cancer (CRC). However, the function of DSC3 in CRC has not yet been elucidated. In this study, we performed cell-based functional analysis after DSC3 overexpression by stable transfection and knockdown by siRNA in CRC cells. It turned out that overexpression of DSC3 reduced cell proliferation, colony forming ability, induced G0/G1 cell cycle arrest and promoted apoptosis. Further pathway analysis showed that overexpression of DSC3 significantly inhibited the activity of AKT pathway and increased the expression of E-cadherin as well as p53 and p21. In contrast, siRNA-mediated knockdown of DSC3 increased cell proliferation and colony formation, activated the AKT pathway and decreased the expression of E-cadherin as well as p53 and p21. Additionally, in primary CRC patient samples, the expression of DSC3 protein was significantly related to the expression of desmocollin 1 (DSC1) and desmocollin 2 (DSC2) as well as E-cadherin (p < 0.001 respectively). Taken together, our data reveal that DSC3 suppresses CRC cell growth through inhibition of AKT pathway and regulation of E-cadherin. DSC3 may serve as a novel therapeutic target for CRC.

The aberrant expression or disruption of desmocollin2 in human diseases.

The desmosome is a member of intercellular junctions that named 'anchoring junction', which contributes to the integrity and homeostasis of tissue structure and function of multicellular living organisms. As an important component of the desmosome and the most widely distributed isoform of desmocollins (DSCs), desmocollin2 (DSC2) has been demonstrated to be essential for the unity of epithelial cells, and served as a vital regulator in signaling processes such as epithelial morphogenesis, differentiation, wound healing, cell apoptosis, migration, and proliferation. Recent studies suggested that the aberrant expression or disruption of DSC2 might lead to some disorders, including heart disorders, certain cancers, and some other human diseases. The distinctions in expression and regulation mechanisms of DSC2 in different human diseases provided a potential target for diagnosis and individualized treatment. Further research is required to certify the signaling capacity of DSC2 and to shed light on the down-stream consequences of the signaling for us to understand the new area of DSC2 biology and the development of certain diseases. This review summarizes the molecular structure and dynamics of desmosome and DSC2, the relationship between the disruption or aberrant expression of DSC2 and human diseases and related molecular mechanisms, as well as the possible prospects.

Sudden death in mild hypertrophic cardiomyopathy with compound DSG2/DSC2/MYH6 mutations: Revisiting phenotype after genetic assessment in a master runner athlete

Cardiomyopathies represent a well-known cause of heart failure and sudden death. Although cardiomyopathies are generally categorized in distinct nosographic entities, characterized by single gene-to-disease causal relationships, recently, oligogenic mutations have also been associated to relevant cardiac clinical features. We report the case of a master athlete carrying trigenic mutations in desmoglein-2 (DSG2), desmocollin-2 (DSC2) and heavy chain myosin 6 (MYH6), which determine a mild hypertrophic phenotype associated both to ventricular tachyarrhythmias and atrio-ventricular block. We discuss the differential diagnosis and prognostic approach in patient affected by complex cardiomyopathy phenotype, along with the importance of sport restriction and sudden death prevention.

Comparative proteomic analyses using iTRAQ-labeling provides insights into fiber diversity in sheep and goats

The structural component of wool and hair fibers, such as keratin-associated proteins (KAPs), has been well described, but the genetic determinants of fiber diameter are largely unknown. Here, we have used an iTRAQ-based proteomic approach to investigate differences in protein abundance among 18 samples from sheep and goats across a diverse range of fibers. We identified proteins with different abundance and are associated with variation in fiber features. Proteins with different abundance are mainly keratin or keratin-associated proteins (KRTAP11-1, KRT6A, KRT38), or are related to hair growth (DSC2, DSG3, EEF2, CALML5, TCHH, SELENBP1) and fatty acid synthesis (FABP4, FABP5). RNA-seq further confirmed the functional importance of the DSC2 gene in the determination of woolly phenotype in goat fibers. This comprehensive analysis of fibers from major fiber-producing animals is the first to provide a list of candidate proteins that are involved in fiber formation. This list will be valuable asset for future studies into the molecular mechanisms that underlie fiber diversity. Biological significanceProteins are the basis for animal-derived hair fibers, yet proteins conferring fiber structure and characteristics in sheep and goats are largely elusive. By examining 27 fibers samples representing 9 fiber types from sheep and goats through the iTRAQ approach, we show a list of differentially abundant proteins that are important to hair structural component, or genes related to hair growth and fatty acid synthesis. RNA-seq further validated the DSC2 gene is key to the woolly/straight hair phenotype in goats.

Large Genomic Rearrangements of Desmosomal Genes in Italian Arrhythmogenic Cardiomyopathy Patients.

Arrhythmogenic cardiomyopathy (AC) is an inherited heart muscle disease associated with point mutations in genes encoding for cardiac desmosome proteins. Conventional mutation screening is positive in ≈50% of probands. Copy number variations (CNVs) have recently been linked to AC pointing to the need to determine the prevalence of CNVs in desmosomal genes and to evaluate disease penetrance by cosegregation analysis in family members. A total of 160 AC genotype-negative probands for 5 AC desmosomal genes by conventional mutation screening underwent multiplex ligation-dependent probe amplification. Nine heterozygous CNVs were identified in 11 (6.9%) of the 160 probands. Five carried a deletion of the entire plakophilin-2 (PKP2) gene, 2 a deletion of only PKP2 exon 4, 1 a deletion of the PKP2 exons 6 to 11, 1 a PKP2 duplication of 5' untranslated region till exon 1, 1 the desmocollin-2 (DSC2) duplication of exons 7 to 9, and 1 a large deletion of chromosome 18 comprising both DSC2 and desmoglein-2 genes. All probands were affected by moderate-severe forms of the disease, whereas 10 (32%) of the 31 family members carrying one of these deletions fulfilled the diagnostic criteria. Genomic rearrangements were detected in ≈7% of AC probands negative for pathogenic point mutations in desmosomal genes, highlighting the potential of CNVs analysis to substantially increase the diagnostic yield of genetic testing. Genotype-phenotype correlation demonstrated the presence of the disease in about one third of family members carrying the CNV, underlying the role of other factors in the development and progression of the disease.

MicroRNA-130a Regulation of Desmocollin 2 in a Novel Model of Arrhythmogenic Cardiomyopathy.

MicroRNAs are small noncoding RNA molecules that play a critical role in regulating physiological and disease processes. Recent studies have now recognized microRNAs as an important player in cardiac arrhythmogenesis. Molecular insight into arrhythmogenic cardiomyopathy (AC) has primarily focused on mutations in desmosome proteins. To our knowledge, models of AC due to microRNA dysregulation have not been reported. Previously, we reported on miR-130a mediated down-regulation of Connexin43. Here, we investigate miR-130a-mediated translational repression of Desmocollin2 (DSC2), as it has a predicted target site for miR-130a. DSC2 is an important protein for cell adhesion, which has been shown to be dysregulated in human AC. After induction of miR-130a, transgenic mice demonstrated right ventricular dilation. Surface ECG revealed spontaneous premature ventricular complexes confirming an arrhythmogenic phenotype in αMHC-miR130a mice. Using total protein from whole ventricular lysate, western blot analysis demonstrated an 80% reduction in DSC2 levels in transgenic myocardium. Furthermore, immunofluorescent staining confirmed downregulation of DSC2 in transgenic compared with littermate control myocardium. In transgenic hearts, histologic findings revealed fibrosis and lipid accumulation within both ventricles. To validate DSC2 as a direct target of miR-130a, we performed in vitro target assays in 3T3 fibroblasts, known to express miR-130a. Using a luciferase reporter fused to the 3UTR of DSC2 compared with a control, we found a 42% reduction in luciferase activity with the DSC2 3UTR. This reduction was reversed upon selective inhibition of miR-130a. Overexpression of miR-130a results in a disease phenotype characteristic of AC and therefore, may serve as potential model for microRNA-induced AC.

Unique genetic background and outcome of non-Caucasian Japanese probands with arrhythmogenic right ventricular dysplasia/cardiomyopathy.

BackgroundArrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited cardiomyopathy mainly caused by desmosomal gene mutation. More than half of Caucasian probands have desmosomal mutations, which lead to earlier onset of ventricular arrhythmias. Among non‐Caucasians, the genetic background of ARVD/C probands and its prognostic impact remain unclear.Methods and ResultsWe genotyped 99 unrelated Japanese ARVD/C probands for plakophilin 2 (PKP2), desmoglein 2 (DSG2), desmoplakin (DSP), and desmocollin 2 (DSC2) between 2005 and 2014. Seventy‐five probands who fulfilled “definite” category according to the 2010 Task Force Criteria (TFC) were enrolled and followed up for 6.4 years. Sixty‐four percent of probands had desmosomal mutations; DSG2 was predominant (48% of mutations) followed by PKP2 (38%). DSG2 mutations were almost missense, whereas over 90% of PKP2 mutations were truncating mutations. Lethal ventricular arrhythmias (VAs, sustained ventricular tachycardia/fibrillation) occurred in 57% of probands as the first manifestation and 71% at the end of follow‐up. Five died during follow‐up. Truncating mutation carriers exhibited earlier lethal VAs onset compared to missense mutation carriers or mutation negatives (age at onset 35 ± 12, 49 ± 16, and 50 ± 19 years, respectively, P < 0.05 in each). Cox proportional hazard analysis revealed for the first time that, compared to mutation negatives, truncating mutation carriers had higher risk for lethal VAs, and especially for onset by their 40s, in an age‐dependent manner (RR = 4.6, P < 0.01 by their 40s; RR = 2.9, P = 0.01 by their 50s).ConclusionThe genetic background of Japanese ARVD/C probands is distinct from that of Caucasian probands, leading to distinct prognosis. The most affected gene mutations in Japanese probands were missense mutations in DSG2 leading to modest outcome, whereas PKP2 truncating mutations were the second most and might be a strong marker for lethal VAs in non‐Caucasian Japanese ARVD/C probands.

Transgenic mice overexpressing desmocollin-2 (DSC2) develop cardiomyopathy associated with myocardial inflammation and fibrotic remodeling.

BackgroundArrhythmogenic cardiomyopathy is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure, mainly as a result of mutations in cardiac desmosomal genes. Desmosomes are cell-cell junctions mediating adhesion of cardiomyocytes; however, the molecular and cellular mechanisms underlying the disease remain widely unknown. Desmocollin-2 is a desmosomal cadherin serving as an anchor molecule required to reconstitute homeostatic intercellular adhesion with desmoglein-2. Cardiac specific lack of desmoglein-2 leads to severe cardiomyopathy, whereas overexpression does not. In contrast, the corresponding data for desmocollin-2 are incomplete, in particular from the view of protein overexpression. Therefore, we developed a mouse model overexpressing desmocollin-2 to determine its potential contribution to cardiomyopathy and intercellular adhesion pathology.Methods and resultsWe generated transgenic mice overexpressing DSC2 in cardiac myocytes. Transgenic mice developed a severe cardiac dysfunction over 5 to 13 weeks as indicated by 2D-echocardiography measurements. Corresponding histology and immunohistochemistry demonstrated fibrosis, necrosis and calcification which were mainly localized in patches near the epi- and endocardium of both ventricles. Expressions of endogenous desmosomal proteins were markedly reduced in fibrotic areas but appear to be unchanged in non-fibrotic areas. Furthermore, gene expression data indicate an early up-regulation of inflammatory and fibrotic remodeling pathways between 2 to 3.5 weeks of age.ConclusionCardiac specific overexpression of desmocollin-2 induces necrosis, acute inflammation and patchy cardiac fibrotic remodeling leading to fulminant biventricular cardiomyopathy.

Expression of Desmoglein 2, Desmocollin 3 and Plakophilin 2 in Placenta and Bone Marrow-Derived Mesenchymal Stromal Cells.

Many controversial results exist when comparing mesenchymal stromal cells (MSCs) derived from different sources. Reasons include not only variables in tissue origin, but also methods of cell preparation or choice of expansion media which can strongly influence the expression and hence, function of the cells. In this short report we aimed to investigate the expression of the cell anchoring proteins desmoglein 2, desmocollin 3 and plakophilin 2 in early passage placenta-derived MSCs of fetal (fetal pMSCs) and maternal (maternal pMSCs) origins versus adult bone marrow-derived MSCs (bmMSCs) that were expanded and cultured under the same good manufacturing practice (GMP) conditions. Comprehensive gene expression microarray analysis profiling indicated differential expression of these genes in the different MSC-derived types with fetal pMSCs expressing the highest levels of PKP2, DSC3 and DSG2, followed by maternal pMSCs, while bmMSCs expressed the lowest levels. A higher expression of PKP2 and DSC3 genes in fetal pMSCs was confirmed by qRT-PCR suggesting neonatal increases in the expression of these desmosomal genes vs. adult MSCs. Intracellular desmocollin 3 and desmoglein 2 expression was observed by flow cytometry and cytoplasmic plakophilin 2 by immunofluorescence in all three MSC sources. These data suggest that fetal pMSCs, maternal pMSCs and bmMSCs may anchor intermediate filaments to the plasma membrane via desmocollin 3, desmoglein 2 and plakophilin 2.

0193: Inflammation and desmosome instability: a deadly combination in arrhythmogenic right ventricular dysplasia

Mutations in the DSC2 gene cause Arrhythmogenic Right Ventricular Dysplasia (ARVD). The mechanisms by which these mutations confer histologic substrate remain unknown. It has been suggested that endogenous caspase-1 activity promotes the magnitude of the inflammatory response. Fragments of cardiac tissue from 2 patients were collected after macroscopic and histopathological examination the patients’ hearts. A 63-year-old patient with ARVD caused by a DSC2 mutation (p.Arg132Cys) underwent heart transplantation. The diseased heart genome-wide expression profiling was compared with that of a non-diseased donor heart. The retained genes of interest were listed and classified according to their biological functions using Ingenuity Pathways Analysis (Ingenuity Systems Inc., Mountain View, CA). RT-qPCR and Western Blot (WB) were used to confirmed genes of interest. Transcriptomic and RT-PCR data first revealed a down-expression of DSC2 mRNA and an unexpected overexpression of DSC3 mRNA in the posterior right ventricle of the ARVC patient (Figure) In the ARVD heart, DSC3 was found to be cleaved. Prediction software revealed that this cleavage could be due to Caspase-1. We observed an overexpression of Casp1 and other elements of inflammasome (CARD16, NLRP3) in the posterior RV of the ARVC patient. A down-expression of DSG2, DSP and RyR2 was also documented. Biological pathways analysis identified a relationship of Casp1 with apoptosis (BCL2, FOS), fibrogenesis (Col3A1, Col4A1, Col4A2, mmP9) and adipogenesis (IGF1, ADIPOQ) in the ARVC heart. These findings suggest that in ARVD patients with a DSC2 variant: 1) there is a switch DSC2/DSC3 2) endogenous caspase-1 activity, by cleaving DSC3, contributes to desmosome dysfunction. Abstract 0193 – Figure: RT-PCR of DSC3 in heart samples

Oxidized low-density lipoprotein attenuated desmoglein 1 and desmocollin 2 expression via LOX-1/Ca2+/PKC-β signal in human umbilical vein endothelial cells

Numerous studies have reported the presence of oxidized LDL (ox-LDL) and expression of its lectin-like receptor, LOX-1, have been shown in atherosclerotic regions. The present study aims to investigate the effects of ox-LDL on expression of desmoglein 1 (DSG1) and desmocollin 2 (DSC2) in endothelial cells, and to explore the role of LOX-1 mediated signal in the permeability injury associated with DSG1 and DSC2 disruption induced by oxidized lipoprotein. RT-PCR and Western blotting were applied to determine the mRNA and protein expression levels of DSG1 and DSC2 in human umbilical vein endothelial cells (HUVECs) respectively. Immunoreactivities of DSG1 and DSC2 were detected by laser scanning confocal microscope (LSCM). HUVEC monolayers permeability was evaluated by FITC-labeled LDL in transwell assay system. The possible signal was assessed using in vitro blocking LOX-1 or Ca2+ channel or PKC. The DSG1 and DSC2 expression were decreased by ox-LDL in concentration- and time-dependent manner. The effects of ox-LDL were mediated by its endothelial receptor, LOX-1. In parallel experiments, ox-LDL increased the influx of extracellular calcium, activation of protein kinase C (PKC) and permeability to LDL, which was inhibited by the LOX-1blocking antibody (10 μg/ml), Ca2+ channel blocker (Diltiazem, 50 μmol/L) and PKC-β inhibitor (hispidin, 4 μmol/L). These results suggested that ox-LDL-induced decrease in DSG1 and DSC2 expression and monolayer barrier injury via calcium uptake and PKC-β activation following up-regulation of LOX-1 is one of the mechanisms of inducing greater permeability in HUVECs.