Desmosomal Proteins Research Articles

The transmembrane domain of the desmosomal cadherin desmoglein-1 governs lipid raft association to promote desmosome adhesive strength.

Cholesterol- and sphingolipid-enriched domains called lipid rafts are hypothesized to selectively coordinate protein complex assembly within the plasma membrane to regulate cellular functions. Desmosomes are mechanically resilient adhesive junctions that associate with lipid raft membrane domains, yet the mechanisms directing raft association of the desmosomal proteins, particularly the transmembrane desmosomal cadherins, are poorly understood. We identified the desmoglein-1 (DSG1) transmembrane domain (TMD) as a key determinant of desmoglein lipid raft association and designed a panel of DSG1TMD variants to assess the contribution of TMD physicochemical properties (length, bulkiness, and palmitoylation) to DSG1 lipid raft association. Sucrose gradient fractionations revealed that TMD length and bulkiness, but not palmitoylation, govern DSG1 lipid raft association. Further, DSG1 raft association determines plakoglobin recruitment to raft domains. Super-resolution imaging and functional assays uncovered a strong relationship between the efficiency of DSG1TMD lipid raft association and the formation of morphologically and functionally robust desmosomes. Lipid raft association regulated both desmosome assembly dynamics and DSG1 cell surface stability, indicating that DSG1 lipid raft association is required for both desmosome formation and maintenance. These studies identify the biophysical properties of desmoglein transmembrane domains as key determinants of lipid raft association and desmosome adhesive function.

Prognostic implications of genotype findings in non-ischemic dilated cardiomyopathy: a network meta-analysis

Abstract Background Evidence on the relative impact of diverse genetic backgrounds associated with non-ischemic dilated cardiomyopathy (DCM) remains contradictory. Aim This study sought to synthesize the available data regarding long-term outcomes of different gene groups in DCM. Methods Electronic databases were systematically screened to identify studies reporting prognostic data on pre-specified gene groups. Those included pathogenic/likely pathogenic (P/LP) variants, truncating titin variants (TTNtv), lamin A/C mutations (LMNA), and desmosomal proteins. Outcomes were divided into composite adverse events (CAEs), major ventricular arrhythmic events (MVAEs) and heart failure events (HFEs). Random effects meta-analyses were conducted to quantify the pooled odds ratio (ORs) for each gene group. A frequentist network meta-analysis was executed to allow indirect within-gene group comparisons. Results Twenty-six studies comprising 7,255 genotyped DCM individuals were included. Patients with P/PL variants had a higher risk for CAEs [OR, 2.10 (95% CI, 1.67-2.65)], MVAEs [OR, 1.86 (95% CI, 1.52-2.26)], and HFEs [OR, 2.01 (95% CI, 1.08-3.73)] than genotype negative patients. The presence of TTNtv was linked to a higher risk for CAEs [OR, 1.78 (95% CI, 1.20-2.63)], but not MVAEs or HFEs. LMNA and desmosomal groups suffered a higher risk for CAEs, MVAEs, and HFEs compared to non-LMNA and non-desmosomal groups respectively (Picture 1). When genes were indirectly compared, the presence of LMNA resulted in a more detrimental effect that TTNtv, with respect to all composite outcomes but no significant difference was found between LMNA and desmosomal genes. Desmosomal genes harbored a higher risk for MVAEs compared to TTNtv (Picture 2). Conclusion Different genetic substrates associated with DCM result in divergent natural histories. Routine utilization of genetic testing should be employed to refine risk stratification and inform therapeutic strategies in DCM.Natural history of DCM based on genotypeNetwork meta-analysis of gene variants

Galectin-3 zebrafish model: identification and pharmacological modulation of arrhythmogenic cardiomyopathy-related phenotypes

Abstract Background Arrhythmogenic Cardiomyopathy (ACM) is rare hereditary cardiac condition characterized by the substitution of ventricular myocardium with fibro-fatty tissue leading to high risk of life-threatening arrhythmias and sudden cardiac death, particularly among young individuals and athletes. Based on genotype-phenotype correlation, the spectrum of the ACM phenotype was shown to be broader than previously believed. Approximately 40% of ACM patients carry one or more putative pathogenic variants in genes encoding desmosomal proteins. Recently LGALS3 gene involvement in ACM early pathogenesis has been advocated. Aim To achieve a comprehensive understanding of LGALS3 role in the pathogenesis of AC. Methods This stable mutant line was generated by CRISPR/Cas9 strategy and then deeply characterized. Phenotyping included heart activity characterization using a recently published software specific for zebrafish, the pyHeart4Fish. Gene expression analysis was carried out by using RNAseq, Real Time PCR and signaling pathway reporter zebrafish lines. Immunofluorescence analysis were performed using and antibody against L-plastin, a leukocyte marker for the detection of inflammatory cells and Acridine Orange/Ethidium Bromide staining to check the possible presence of apoptotic and necrotic events. Results Morphological investigation revealed notable structural abnormalities such as pericardial effusion and/or hemopericardium. The heart activity analysis detected a diminished ventricle contractility and ejection fraction, with bradycardia and occurrence of arrhythmia events. Rna analysis highlighted the dysregulation of Wnt/β-catenin signaling pathway. Moreover, the L-plastin inflammatory marker revealed a significant infiltration of inflammatory cells in the heart as well as an elevated number of apoptotic and necrotic cells (figure 1). The pharmacological treatment, partially rescued a subset of AC-related cardiac phenotypes observed, including heart contractility, bradycardia and arrhythmia. Conclusion Our findings strongly validate our lgals3a zebrafish mutant as an effective model to elucidate its role in ACM related phenotype. This model accurately replicates the cardiac abnormalities and dysregulation in cell signaling that are characteristic of ACM. Further, observed cell death and inflammation and the partial recovery of cardiac phenotypes through pharmacological intervention, underscores the potential of targeting the Wnt/β-catenin signaling pathway as future perspective for ACM.Figure 1

Protein redistribution in buccal cells as a prognostic marker for arrhythmogenic cardiomyopathy in paediatric patients

Abstract Background Mutations in genes encoding desmosomal proteins [plakoglobin (JUP), desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2) and desmocollin-2 (DSC2)] cause complex genetic heart muscle disorders collectively described as arrhythmogenic cardiomyopathies (ACM). Diagnosis and risk stratification, particularly in the paediatric population, remain challenging owing to heterogeneous phenotypic manifestations and reduced genetic penetrance. Immunohistochemical analysis of heart muscle samples has shown that several junctional and signalling molecules are redistributed in patients with ACM, offering diagnostic information. However, the use of this approach in living patients was limited by need for heart samples. Recent studies in adults with ACM showed that the same set of proteins re-localised in the ACM heart also do so in the buccal epithelium. Purpose Given the ease of analysing serial cheek smears, we sought to establish how protein redistribution in buccal cells relates to disease onset and progression in children bearing desmosomal gene mutations. Methods Serial cheek smears from children with desmosomal variants were subjected to immunocytochemistry and confocal microscopy to study the distribution of JUP, DSP and PKP-1 as well as the major gap junction protein connexin43 (Cx43; GJA1) and the major subunit of the NFκB inflammatory pathway; RelA. Results 37 patients Conclusion These data indicate that shifts in junctional/signalling proteins precede the onset of clinical disease. Indeed, in the majority of pre-clinical children, Cx43 and RelA seem to be redistributed first, suggesting that onset of abnormalities in electrical conductivity and inflammatory pathways occur first in the natural disease progression, in agreement with previous clinical observations. Moreover, the data suggest that additional proteins are shifted as the disease progresses. Although further verification is required, this study suggests that analysis of buccal smears may be used as a prognostic/risk stratification test in children with desmosomal variants.

Phase I gene therapy clinical trial design of RP-A601 in adult patients with PKP2-arrhythmogenic cardiomyopathy (PKP2-ACM)

Abstract Background Arrhythmogenic cardiomyopathy (ACM) is a rare, progressive, autosomal dominant disorder characterized by increased risk of ventricular arrhythmias and sudden cardiac death. Up to 45% of all ACM cases are caused by pathogenic variants in the PKP2 gene, which encodes for the desmosomal protein Plakophilin-2. This genetic cardiomyopathy is referred to as PKP2-ACM. AAVrh.74-PKP2a is a recombinant adeno-associated viral (AAV) vector containing the coding sequence of human PKP2 isoform A (PKP2a) and delivers the functional PKP2 gene to cardiomyocytes. Preclinical testing conducted in a clinically relevant mouse model of PKP2-ACM (with survival ≤50 days post disease onset) demonstrated that administration of a single AAVrh.74-PKP2a dose after disease onset extended survival through 5 months with improved right ventricular (RV) function, and decreased cardiac dilation, myocardial fibrosis, and arrhythmias. Additional studies in rodents and nonhuman primates demonstrated AAVrh.74-PKP2a safety. These preclinical findings support clinical initiation. Purpose We describe the design of a phase I trial to assess safety and preliminary efficacy of AAVrh.74-PKP2a (RP-A601) in high-risk adult patients with PKP2-ACM. Methods This study (NCT05885412) is a multi-center, open-label, dose escalation trial evaluating the safety and preliminary efficacy of a single intravenous infusion of RP-A601 in adult patients (age ≥18 yrs) with clinical and genetic PKP2-ACM diagnosis and an implanted ICD. Patients with severe right ventricular dysfunction, LV ejection fraction ≤50% (echocardiogram or cardiac MRI) and/or NYHA Class IV heart failure are excluded. Preliminary efficacy will be assessed at 12 months after RP-A601 infusion and includes evaluation of change in PKP2 myocardial tissue expression and clinical markers of arrhythmia burden. Conclusions PKP2-ACM is a devastating disease associated with high morbidity and mortality. This Phase I trial will evaluate the safety and preliminary efficacy of RP-A601 and was initiated based on robust preclinical efficacy and safety data.

A case of rapidly progressive hair loss due to azathioprine, and the prevalence of NUDT15 variants among Japanese patients with autoimmune blistering diseases: A single-center retrospective observational study.

Autoimmune blistering diseases (AIBDs), classified into pemphigus and pemphigoid, consist of relatively rare skin disorders caused by autoantibodies that target desmosomal and hemidesmosomal proteins, respectively. Although systemic corticosteroids are used as a first-line treatment for AIBDs, azathioprine is frequently co-administered as a steroid-sparing agent. Azathioprine is metabolized into thioguanine nucleotides (TGNs) which are its major active metabolites. The enzyme nudix hydrolase 15 (NUDT15) plays a key role in regulating TGNs. Serious side effects of azathioprine, including leukopenia and alopecia, are known to be particularly problematic in individuals with NUDT15 variants. The single-nucleotide polymorphism c.415C >T (p.Arg139Cys) is one of the most frequent NUDT15 variants associated with severe thiopurine toxicity. Recently, we treated a case of pemphigus vulgaris in a patient with NUDT15 variants in which the patient developed rapidly progressive diffuse hair loss and myelosuppression while receiving azathioprine. Previous reports on NUDT15 polymorphisms mainly focused on patients with inflammatory bowel disease or hematological malignancies, and the prevalence of NUDT15 polymorphisms remains unknown in AIBDs. This highlights the urgent need for research on NUDT15 polymorphisms in AIBDs to achieve a better understanding of the genetic factors influencing adverse reactions to azathioprine. To clarify the prevalence of NUDT15 variants in Japanese patients with AIBDs, we retrospectively reviewed the medical records of 78 patients with AIBDs (26 with bullous pemphigoid, 26 with pemphigus vulgaris, 17 with pemphigus foliaceus, and nine with other AIBDs) who had come to Hokkaido University Hospital between 2018 and 2023. The frequencies of NUDT15 variants of Arg/Arg, Arg/Cys, and Cys/Cys in these patients were approximately 72%, 23%, and 5%, respectively. Our findings indicate a prevalence of NUDT15 variants in AIBD patients that is similar to the prevalences of previous studies on patients with other diseases. These results emphasize the importance of screening for NUDT15 variants prior to initiating azathioprine treatment in Japanese patients with AIBDs.

H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

BackgroundABO blood group antigens (ABH antigens) are carbohydrate chains glycosylated on epithelial and red blood cells. Recent findings suggest reduced ABH expression in psoriasis and atopic dermatitis, a chronic inflammatory skin disease with retained scale. H antigen, a precursor for A and B antigens, is synthesized by fucosyltransferase 1 (FUT1). Desmosomes, critical for skin integrity, are known to require N-glycosylation for stability. We investigate the impact of H antigens, a specific type of glycosylation, on desmosomes in keratinocytes.MethodPrimary human keratinocytes were transfected with FUT1 siRNA or recombinant adenovirus for FUT1 overexpression. Cell adhesion and desmosome characteristics and their underlying mechanisms were analyzed.ResultThe knockdown of FUT1, responsible for H2 antigen expression in the skin, increased cell-cell adhesive strength and desmosome size in primary cultured keratinocytes without altering the overall desmosome structure. Desmosomal proteins, including desmogleins or plakophilin, were upregulated, suggesting enhanced desmosome assembly. Reduced H2 antigen expression via FUT1 knockdown led to increased keratinocyte differentiation, evidenced by elevated expression of differentiation markers. Epidermal growth factor receptor (EGFR) has been described to be associated with FUT1 and promotes cell migration and differentiation. The effects of FUT1 knockdown were recapitulated by an EGFR inhibitor concerning desmosomal proteins and cellular differentiation. Further investigation demonstrated that the FUT1 knockdown reduced EGFR signaling by lowering the levels of EGF ligands rather than directly regulating EGFR activity. Moreover, FUT1 overexpression reversed the effects observed in FUT1 knockdown, resulting in the downregulation of desmosomal proteins and differentiation markers while increasing both mRNA and protein levels of EGFR ligands.ConclusionThe expression level of FUT1 in the epidermis appears to influence cell-cell adhesion and keratinocyte differentiation status, at least partly through regulation of H2 antigen and EGFR ligand expression. These observations imply that the fucosylation of the H2 antigen by FUT1 could play a significant role in maintaining the molecular composition and regulation of desmosomes and suggest a possible involvement of the altered H2 antigen expression in skin diseases, such as psoriasis and atopic dermatitis.

Upregulation of keratin 15 is required for varicella-zoster virus replication in keratinocytes and is attenuated in the live attenuated vOka vaccine strain

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterised by epidermal virus replication in skin and mucosa and the formation of blisters. We have previously shown that VZV infection has a profound effect on keratinocyte differentiation, altering the normal pattern of epidermal gene expression. In particular, VZV infection reduces expression of suprabasal keratins 1 and 10 and desmosomal proteins, disrupting epidermal structure to promote expression of a blistering phenotype. Here, we extend these findings to show that VZV infection upregulates the expression of keratin 15 (KRT15), a marker expressed by basal epidermal keratinocytes and hair follicles stem cells. We demonstrate that KRT15 is essential for VZV replication in the skin, since downregulation of KRT15 inhibits VZV replication in keratinocytes, while KRT15 exogenous overexpression supports viral replication. Importantly, our data show that VZV upregulation of KRT15 depends on the expression of the VZV immediate early gene ORF62. ORF62 is the only regulatory gene that is mutated in the live attenuated VZV vaccine and contains four of the five fixed mutations present in the VZV Oka vaccine. Our data indicate that the mutated vaccine ORF62 is not capable of upregulating KRT15, suggesting that this may contribute to the vaccine attenuation in skin. Taken together our data present a novel association between VZV and KRT15, which may open a new therapeutic window for a topical targeting of VZV replication in the skin via modulation of KRT15.

Heterozygous DSP in-frame deletion in a poodle with syndromic ichthyosis involving additional hair and tooth abnormalities.

Ichthyoses comprise a large heterogeneous group of skin disorders, characterized by generalized scaly and hyperkeratotic skin. We investigated a miniature poodle with early onset generalized scaling, dry and irregularly thickened skin, paw pad hyperkeratosis and abnormalities in hair and teeth. The clinical signs of ichthyosis were confirmed by histopathological examination, which revealed mild epidermal hyperplasia and lamellar orthokeratotic hyperkeratosis. A hereditary condition was suspected and a genetic investigation was initiated. We sequenced the whole genome of the affected dog and searched for potentially causative variants in functional candidate genes for the observed phenotype. The analysis revealed a heterozygous in-frame deletion in DSP, NC_049256.1:g.8804542_8804544del resulting from a de novo mutation event as evidenced by genotyping leukocyte DNA from both parents. The 3 bp deletion is predicted to remove one aspartic acid without disrupting the open reading frame (XM_038584124.1:c.1821_1823del, XP_038440052.1:p.(Asp608del)). The DSP gene encodes desmoplakin, a desmosomal plaque protein, responsible for cell-cell adhesion to provide resistance to mechanical stress in epidermal and cardiac tissues. We hypothesize that the deletion of one amino acid in the N-terminal globular head domain acts in a dominant negative manner and thus impairs the proper connection with other proteins. Several variants in DSP in humans and cattle have been described to result in different phenotypes associated with hair and skin abnormalities, sometimes in combination with variable cardiac and/or dental manifestations. In conclusion, we characterized a new syndromic ichthyosis phenotype in a dog and identified a de novo 3 bp deletion in the DSP gene as causal variant.

Characterization of Disparities, Diagnoses and Treatment Outcomes with Pemphigus Vulgaris

Pemphigus vulgaris (PV) and foliaceus (PF) are classified as rare autoimmune blistering disorders in which IgG antibodies destroy desmosome proteins desmoglein 1 and desmoglein 3 within the stratum spinosum of the epithelial layer of the skin. These disorders once used to be a fatal condition, but the advent of steroids and immunosuppressants has made these diseases manageable. Such treatments, however, only alleviate symptoms and have not been viable in allowing patients to achieve prolonged steroid-free remission. In 2018, the US Food and Drug Administration (FDA) approved the anti-CD20 monoclonal antibody rituximab as the first-line treatment for moderate to severe cases of pemphigus. While rituximab has been generally successful in treating this disease and has led to prolonged steroid-free remission in many patients, treatment disparities and differences in patient outcomes due to social determinants of health have not yet been studied in the context of PV and PF. Such studies have already been conducted with more common dermatological disorders such as hidradenitis suppurativa (HS), which show significant evidence of outcome disparities due to social factors such as race, ethnicity and socioeconomic status. However, such studies have yet to be conducted with pemphigus likely because of how rare the disease is and because the current first-line treatment was FDA approved recently in 2018. The aim of this study is to conduct a retrospective multivariable analysis of 100 patients with pemphigus from UT Southwestern Medical Center and Parkland County Hopital to determine which social factors are the most responsible for any discovered pemphigus treatment outcome disparities. The impacts of this study are far-reaching as any discovered disparities can alert dermatologists to place a special emphasis to address any social factors that may play a role in leading to treatment outcome differences for patients with pemphigus.

Abstract Mo092: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as an In Vitro Model to Evaluate the Efficacy of a Gene Replacement Therapy for Plakophilin-2- Associated Arrhythmogenic Right Ventricular Cardiomyopathy

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disorder characterized by severe arrhythmias and heart dysfunction. Mutations in desmosome gene Plakophilin-2 ( PKP2 ) account for 40% of ARVC cases, with current palliative therapies failing to address the genetic cause. Herein, we generated an in vitro ARVC model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) harboring a pathogenic PKP2 variant (c.2146G>C), as a platform to test a PKP2 -gene replacement approach developed at Tenaya Therapeutics for the treatment of PKP2 -associated ARVC patients. Methods: Three isogenic human iPSC lines (wild-type, heterozygous and homozygous PKP2 mutant) were differentiated to iPSC-CM; and monolayers and engineered heart tissues (EHTs) were generated. Gene and protein expression were assessed by RNA-Seq/RT-qPCR, and immunocytochemistry, respectively. Contractility, electrophysiology and calcium handling were measured. A proprietary adeno-associated virus gene therapy (AAV9: PKP2 ) was used to transduce human iPSC-CM, and changes in gene and protein expression, and contractile function were evaluated. Results: A human iPSC-CM beating monolayer was achieved for all three lines. PKP2 expression was depleted in a genotype-dependent manner, and desmosome structure was disrupted in the mutant lines. Functional assessment of human iPSC-CM monolayers showed impaired contractile properties and abnormal electrophysiological and calcium transients in the mutant lines, such as decreased contraction amplitude and prolonged field and action potential duration. Transcriptional analysis revealed changes in desmosome, gap junctions, sarcomere, ion channels, metabolic and apoptosis gene expression. Characterization of EHTs displayed a deficit in contractility, slower action and field potential kinetics and irregular calcium homeostasis in the mutant lines. The administration of AAV9: PKP2 to the mutant lines restored ion channel and desmosome gene and protein expression, and contractile function. Conclusions: Our PKP2 human iPSC-CM disease model recapitulated the main hallmarks of ARVC phenotype. Administration of AAV9: PKP2 restored desmosome protein expression and contractility. This model lays a foundation for the understanding of the underlying molecular pathophysiological mechanisms of PKP2 -ARVC, and the potential for AAV9: PKP2 as a one-time dose to correct the genetic cause of disease in individuals with PKP2 -associated ARVC.

Prognostic implications of genotype findings in non-ischaemic dilated cardiomyopathy: A network meta-analysis.

Evidence on the relative impact of diverse genetic backgrounds associated with non-ischaemic dilated cardiomyopathy (DCM) remains contradictory. This study sought to synthesize the available data regarding long-term outcomes of different gene groups in DCM. Electronic databases were systematically screened to identify studies reporting prognostic data on pre-specified gene groups. Those included pathogenic/likely pathogenic (P/LP) variants, truncating titin variants (TTNtv), lamin A/C variants (LMNA), and desmosomal proteins. Outcomes were divided into composite adverse events (CAEs), malignant ventricular arrhythmic events (MVAEs) and heart failure events (HFEs). A total of 26 studies (n = 7255) were included in the meta-analysis and 6791 patients with genotyped DCM were analysed. Patients with P/LP variants had a higher risk for CAEs (odds ratio [OR] 2.10, 95% confidence interval [CI] 1.67-2.65), MVAEs (OR 1.86, 95% CI 1.52-2.26), and HFEs (OR 2.01, 95% CI 1.08-3.73) than genotype-negative patients. The presence of TTNtv was linked to a higher risk for CAEs (OR 1.78, 95% CI 1.20-2.63), but not MVAEs or HFEs. LMNA and desmosomal groups suffered a higher risk for CAEs, MVAEs, and HFEs compared to non-LMNA and non-desmosomal groups, respectively. When genes were indirectly compared, the presence of LMNA resulted in a more detrimental effect that TTNtv, with respect to all composite outcomes but no significant difference was found between LMNA and desmosomal genes. Desmosomal genes harboured a higher risk for MVAEs compared to TTNtv. Different genetic substrates associated with DCM result in divergent natural histories. Routine utilization of genetic testing should be employed to refine risk stratification and inform therapeutic strategies in DCM.

Distorted frequency and functionality of natural killer cells in pemphigus vulgaris: A potential therapeutic target

Pemphigus vulgaris (PV) is a rare autoimmune disorder where autoantibodies target the desmosomal proteins resulting in blistering of oral mucosa and skin. While the pathogenesis of PV is mainly mediated by the adaptive immune system, key players of innate immunity are also emerging. This study outlines the phenotypic as well as functional attributes of NK cells in PV. Through in-depth analysis using flow cytometry we identified an increase in the frequency of CD56+ CD3- NK cells and their subtypes in periphery. Along with this there is an increased frequency of IFNγ+ CD56bright CD16dim NK cells. mRNA expression of sorted NK cells for differentially expressed genes, particularly key transcription factors such as T-bet and EOMES, as well as surface receptors like NKG2D and KIR2D, and the cytokine IFNγ, displayed significant upregulation. A significant activation of NK cells was seen in the disease state. The levels of perforin and IFNγ were significantly elevated in the culture supernatants of patients. Additionally, a significantly higher cytotoxicity of NK cells in PV was observed. In lesioned tissues of PV, NK related markers were significantly increased. Lastly, we observed NK cells using confocal microscopy in the tissue biopsies of patients which showed significant infiltration of CD56+ CD3- NK cells at the lesional sites. This study aimed to shed light on the pivotal role of NK cells in the immunopathology of PV, offering a thorough understanding of their behaviour and changes in expression which might help in contributing to the development of novel therapeutics.

S100A11 is involved in the progression of colorectal cancer through the desmosome-catenin-TCF signalingpathway.

Compiling evidence has indicated that S100A11 expression at high levels is closely associated with various cancer species. Consistent with the results reported elsewhere, we have also revealed that S100A11 is highly expressed in squamous cell carcinoma, mesothelioma, and pancreatic cancers and plays a crucial role in cancer progression when secreted into extracellular fluid. Those studies are all focused on the extracellular role of S100A11. However, most of S100A11 is still present within cancer cells, although the intracellular role of S100A11 in cancer cells has not been fully elucidated. Thus, we aimed to investigate S100A11 functions within cancer cells, primarily focusing on colorectal cancer cells, whose S100A11 is abundantly present in cells and still poorly studied cancer for the protein. Our efforts revealed that overexpression of S100A11 promotes proliferation and migration, and downregulation inversely dampens those cancer behaviors. To clarify how intracellular S100A11 aids cancer cell activation, we tried to identify S100A11 binding proteins, resulting in novel binding partners in the inner membrane, many of which are desmosome proteins. Our molecular approach defined that S100A11 regulates the expression level of DSG1, a component protein of desmosome, by which S100A11 activates the TCF pathway via promoting nuclear translocation of γ-catenin from the desmosome. The identified new pathway greatly helps to comprehend S100A11's nature in colorectal cancers and others.

Animal Models and Molecular Pathogenesis of Arrhythmogenic Cardiomyopathy Associated with Pathogenic Variants in Intercalated Disc Genes.

Arrhythmogenic cardiomyopathy (ACM) is a rare genetic cardiac disease characterized by the progressive substitution of myocardium with fibro-fatty tissue. Clinically, ACM shows wide variability among patients; symptoms can include syncope and ventricular tachycardia but also sudden death, with the latter often being its sole manifestation. Approximately half of ACM patients have been found with variations in one or more genes encoding cardiac intercalated discs proteins; the most involved genes are plakophilin 2 (PKP2), desmoglein 2 (DSG2), and desmoplakin (DSP). Cardiac intercalated discs provide mechanical and electro-metabolic coupling among cardiomyocytes. Mechanical communication is guaranteed by the interaction of proteins of desmosomes and adheren junctions in the so-called area composita, whereas electro-metabolic coupling between adjacent cardiac cells depends on gap junctions. Although ACM has been first described almost thirty years ago, the pathogenic mechanism(s) leading to its development are still only partially known. Several studies with different animal models point to the involvement of the Wnt/β-catenin signaling in combination with the Hippo pathway. Here, we present an overview about the existing murine models of ACM harboring variants in intercalated disc components with a particular focus on the underlying pathogenic mechanisms. Prospectively, mechanistic insights into the disease pathogenesis will lead to the development of effective targeted therapies for ACM.

A novel DSP zebrafish model reveals training- and drug-induced modulation of arrhythmogenic cardiomyopathy phenotypes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Associazione Italiana per la Ricerca sul Cancro [AIRC grant IG-2017-19928], Italian Ministry of University and Research [grant PRIN 20173ZWACS] Introduction Arrhythmogenic cardiomyopathy (AC) is an inherited disorder characterized by progressive loss of the ventricular myocardium causing life-threatening ventricular arrhythmias, syncope and sudden cardiac death in young and athletes. About 40% of AC cases carry one or more mutations in genes encoding for desmosomal proteins, including Desmoplakin (Dsp). Methods We generated the first stable Dsp knock-out (KO) zebrafish line able to model cardiac alterations and cell signalling dysregulation, characteristic of the AC disease. Results Our stable Dsp knock-out (KO) zebrafish line was characterized by cardiac alterations, edema and bradycardia at larval stages. Histological analysis of mutated adult hearts showed reduced contractile structures and abnormal shape of the ventricle, with thinning of the myocardial layer, vessels dilation and presence of adipocytes within the myocardium. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes. Intensive physical training protocol caused a global worsening of the cardiac phenotype, accelerating the progression of the disease. Of note, we detected a decrease of Wnt/β-catenin signalling, recently associated with AC pathogenesis, as well as Hippo/YAP-TAZ and TGF-β pathway dysregulation. Pharmacological treatment of mutated larvae with SB216763, a Wnt/β-catenin agonist, rescued pathway expression and cardiac abnormalities, stabilizing the heart rhythm. Conclusion Our Dsp KO zebrafish line recapitulates many AC features observed in human patients, pointing at zebrafish as a suitable system for in vivo analysis of environmental modulators, such as the physical exercise, and the screening of pathway-targeted drugs, especially related to the Wnt/β-catenin signalling cascade.

Arrhythmogenic cardiomyopathy related phenotypes are detected and pharmacologically modulated in Galectin-3 zebrafish models

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of University and Research Introduction Arrhythmogenic Cardiomyopathy (AC) is a rare inherited cardiac disorder characterized by fibro-fatty replacement and progressive loss of the ventricular myocardium, causing life-threatening arrhythmias, syncope and sudden cardiac death in young and athletes. About 50% of AC cases carry one or more mutations in genes encoding for desmosomal proteins. However, mutations in non-desmosomal genes have been identified in 1-3% of AC patients, such as Galectin-3 (Gal-3). Purpose For a better understanding of Gal-3 role in AC pathogenesis, we have generated a stable Gal-3 knock-out (KO) zebrafish line, able to reproduce cardiac abnormalities and cell signaling dysregulation characteristic of AC, on which environmental factors and candidate drugs can be tested. Methods The zebrafish Gal-3 mutant line was generated by CRISPR/Cas9 strategy and then phenotypically characterized. Phenotyping included heart activity characterization, by pyHeart4Fish software, and gene expression analysis using Real Time PCR and signaling pathway reporters. Immunofluorescence analysis was performed using an antibody against L-plastin, a leukocyte marker for the detection of inflammatory cells, and Acridine Orange/Ethidium Bromide (AO/EB) staining to check for apoptotic and necrotic events. Transmission Electron Microscopy (TEM) analysis was carried out to detect possible ultrastructural alterations. A pathway-directed pharmacological treatment was applied to try to rescue pathological phenotypes. Results The Gal-3 KO zebrafish line, analysed at larval stage, presented structural abnormalities, pericardial effusion and/or hemopericardium associated with reduced contractility, bradycardia and presence of arrhythmia events. In addition to Wnt/β-catenin signaling dysregulation, as seen in other AC forms, we also observed cell death and inflammation. Specifically, the analysis of the L-plastin inflammatory marker showed remarkable infiltration of inflammatory cells in the cardiac region of Gal-3 mutants, whereas AO/EB staining demonstrated elevated number of apoptotic and necrotic cells. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes in mutant adult hearts. Of note, pharmacological treatment at larval stage, using a Wnt/β-catenin signaling agonist, partially rescued a set of AC cardiac phenotypes (contractility, bradycardia, arrhythmia). Conclusions Overall, the Gal-3 KO zebrafish line confirms in zebrafish the strict connection between Gal-3 mutation and AC-like cardiac alterations. Moreover, cell death and inflammation, known to be involved in the remodelling of the myocardium, were observed. The pharmacological rescue of the Wnt/β-catenin signaling partially recovered a set of cardiac phenotypes, confirming this pathway as a key modulator of AC progression. In conclusion, our results strongly support the usefulness of the Gal-3 zebrafish mutant to clarify Gal-3 involvement in AC, offering future perspectives for AC treatment.

PKP2 induced by YAP/TEAD4 promotes malignant progression of gastric cancer.

Gastric cancer (GC) exhibits significant heterogeneity and its prognosis remains dismal. Therefore, it is essential to investigate new approaches for diagnosing and treating GC. Desmosome proteins are crucial for the advancement and growth of cancer. Plakophilin-2 (PKP2), a member of the desmosome protein family, frequently exhibits aberrant expression and is strongly associated with many tumor types' progression. In this study, we found upregulation of PKP2 in GC. Further correlation analysis showed a notable association between increased PKP2 expression and both tumor stage and poor prognosis in individuals diagnosed with gastric adenocarcinoma. In addition, our research revealed that the Yes-associated protein1 (YAP1)/TEAD4 complex could stimulate the transcriptional expression of PKP2 in GC. Elevated PKP2 levels facilitate activation of the AKT/mammalian target of rapamycin signaling pathway, thereby promoting the malignant progression of GC. By constructing a mouse model, we ultimately validated the molecular mechanism and function of PKP2 in GC. Taken together, these discoveries suggest that PKP2, as a direct gene target of YAP/TEAD4 regulation, has the potential to be used as an indication of GC progression and prognosis. PKP2 is expected to be a promising therapeutic target for GC.

Impact of functional gene groups on outcomes after ablation of ventricular tachycardia in patients with inherited left ventricular cardiomyopathy: an international multicentre study

Abstract Background Advances in genetic testing has enabled identification of pathogenic variants of different functional gene groups associated with non-dilated left ventricular cardiomyopathy (NDLCM)/dilated cardiomyopathy (DCM) and ventricular tachycardia (VT). However, there is limited data on the impact of functional groups on VT ablation outcome. Aim We aimed to assess the impact of likely pathogenic/pathogenic variants (LP/Pv) of different functional gene groups on heart failure (HF) outcomes and VT recurrence in patients with NDLCM/DCM after VT ablation. Methods Patients with NDLCM/DCM who underwent genetic testing (next-generation sequencing) and were referred for VT ablation were enrolled at 6 participating centers. Patients were followed for VT recurrence, HF-related adverse outcomes (death, left ventricular assist device [LVAD], or heart transplantation due to HF), and all-cause mortality. Results A total of 239 patients (age 55±14yrs; male n=193; LVEF 40±12%; amiodarone at admission n=103; ICD n=180) were included. LP/Pv were identified in 128 patients (54%), predominantly found in genes encoding sarcomeric (n=36/128, [28%]; titin n=20, myosin binding protein C n=12), followed by nuclear membrane (n=27/128, [22%]; lamin A/C; n=26), desmosomal (n=23/128, [18%]; desmoplakin n=11, plakophilin n=9), cytoskeleton (n=12/128, [9%]; filamin C n=8), and sarcoplasmic reticulum proteins (n=12/128, [9%]; phospholamban n=10). Variants of unknown significance (n=32) were grouped with patients who tested negative. Acute complete ablation success, defined as non-VT inducibility of any sustained monomorphic VT, was achieved in 113 patients (47%), 63 (56%) with LP/Pv. During a median follow-up of 32 months, 108 patients (45%) had VT recurrence, 41 (17%) had HF-related adverse outcomes (death with/without LVAD in 34, LVAD in 2, heart transplantation in 5) and 47 (20%) died. Patients with LP/Pv were more likely to have VT recurrence, HF-related adverse outcomes and had a higher mortality compared to those without (VT, 68 [53%] vs. 40 [36%]; HF-related adverse outcomes, 30 [23%] vs. 11 [10%]; mortality, 30 [23%] vs. 17 [15%]; log-rank, P<0.01 for all). As compared to patients without LP/Pv, those with LP/Pv in genes encoding nuclear membrane or sarcomeric proteins had significantly worse (P<0.01 for both) and those with LP/Pv in genes encoding desmosomal proteins had similar HF and VT recurrence outcomes (Figure). Of interest, patients with LP/Pv in genes encoding cytoskeleton proteins had favorable HF-related adverse outcomes, but worse VT recurrence (P=0.02) compared to those without LP/Pv (Figure). Conclusion In patients with NDLCM/DCM and VT, the impact of LP/Pv on HF and VT-recurrence outcomes after ablation differs across functional groups. Integrating genetic testing in the comprehensive evaluation of patients with NDLCM/DCM undergoing VT ablation may facilitate personalized ablation strategies and early consideration of alternative HF treatments.

Absence of Epidermal Antibodies in Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Patients but Beware of Single Positive Results.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare and potentially life-threatening mucocutaneous blistering diseases that clinically can resemble autoimmune bullous diseases. Moreover, it has been shown that autoantibodies against epidermal proteins are present in SJS/TEN. To establish the presence of antibodies against desmosomal and hemidesmosomal proteins in confirmed SJS/TEN patients. Serum of SJS/TEN patients diagnosed based on clinical criteria, e.g., epidermal detachment with erosions and severe mucosal lesions, (suspicion of) a culprit drug, and matching histologic results was evaluated by various techniques, e.g., indirect immunofluorescence on monkey esophagus, salt split skin and rat bladder, immunoblotting (IB) and immunoprecipitation (IP), ELISAs against desmogleins and BP180, keratinocyte footprint assay, and keratinocyte binding assay. A total of 28 patients were included in this study, 15 men and 13 women with a mean age of 56 years. In most patients, none of the serological tests were positive. In two patients, an elevated DSG3 titer was found suspicious for pemphigus vulgaris. Three patients had elevated NC16a titers, suggesting bullous pemphigoid. However, in all these patients, no other tests were positive and in these patients, the biopsy for direct immunofluorescence showed no evidence for an autoimmune bullous disease. Three patients showed reactivity against rat bladder rat bladder; these were, however, completely negative for A2ML1, envoplakin, and periplakin in the IB as well as the IP. Serological analysis for desmosomal and hemidesmosomal antibodies is reliable to rule an autoimmune bullous disease in patients with suspected SJS/TEN. However, one should not rely on one single test method since false positive results can occur. Moreover, this study also makes it less plausible that antibodies against desmosomal and/or hemidesmosomal components are involved in the pathogenesis of SJS/TEN.