Pathogenesis Of Arrhythmogenic Right Ventricular Cardiomyopathy Research Articles

Cardiac Inflammation As an Underlying Trigger for the Pathological Deficits Associated with Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disorder that causes cardiomyocyte loss, resulting in myocardial fibro-fatty deposition leading to cardiac dysfunction and sudden cardiac death, particularly in young patients and athletes, with no available treatments. The genetics of ARVC follow an autosomal dominant pattern, although severe cases (especially in pediatric populations) have been reported as recessive. Mutations in genes encoding components of desmosomes (specialized cell-cell junction that act as mechanical anchor between cardiac muscle cells) resulting in loss of desmosomal proteins underlie approximately 40-60% genetic cases of ARVC. Studies highlight that cardiac inflammation may be a critical and early component of ARVC as patients harboring mutations in the desmosomal gene desmoplakin (DSP) present with recurrent myocardial episodes (observed as myocarditis); however, the mechanisms and whether inflammation is a driver of ARVC remain unclear. We hypothesize that desmosomal gene mutations/loss/byproducts alter the acute inflammatory response on the cardiomyocyte cell surface leading to chronic immune intolerance and triggering of ARVC disease. To test our hypothesis, we injected DSP heterozygous deficient mice (harboring 50% loss of DSP without overt signs of ARVC) with a fragment of α-myosin and pertussis toxin to stimulate the immune system to assess its impact on ARVC pathogenesis. We show that DSP heterozygous mice exposed to acute immunological challenge triggered a destructive cardiomyocyte immune response in DSP heterozygous deficient hearts, reminiscent of fulminant myocarditis. Furthermore, we show that this acute immunological challenge was sufficient to trigger classic ARVC disease features in DSP heterozygous deficient mice as exemplified by: (i) enlarged ventricular (mostly right ventricular) chambers and heart size, (ii) fibrotic remodeling, with more clearly striking effects in the right ventricle (RV), (iii) epicardial fat tissue accumulation, (iv) electrophysiological deficits, such as premature ventricular contractions and QRS prolongation as well as (v) right and left ventricular physiological deficits associated with the bi-ventricular form of ARVC, although the RV appeared to be more significantly impacted. Our studies highlight the development of a novel DSP-related model of myocarditis. Using this model, we show that immunological stress is sufficient to lead to loss of cardiomyocyte immune tolerance in DSP heterozygous deficient mice with compromised desmosomal integrity to drive ARVC pathogenesis. More broadly these studies suggest that the immune system maybe an important driver of ARVC pathogenesis in patients with underlying desmosomal gene mutations/heterozygous loss, especially in the setting of autosomal dominant genetics. Work in the laboratory of FS is supported by grants from the NIH (HL142251, HL162369) and industry (LEXEO Therapeutics Inc.). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Drosophila melanogaster model for TMEM43-related arrhythmogenic right ventricular cardiomyopathy type 5

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a severe cardiac disease that leads to heart failure or sudden cardiac death (SCD). For the pathogenesis of ARVC, various mutations in at least eight different genes have been identified. A rare form of ARVC is associated with the mutation TMEM43 p.S358L, which is a fully penetrant variant in male carriers. TMEM43 p.S358 is homologous to CG8111 p.S333 in Drosophila melanogaster. We established CRISPR/Cas9-mediated CG8111 knock-out mutants in Drosophila, as well as transgenic fly lines carrying an overexpression construct of the CG8111 p.S333L substitution. Knock-out flies developed normally, whereas the overexpression of CG8111 p.S333L caused growth defects, loss of body weight, cardiac arrhythmias, and premature death. An evaluation of a series of model mutants that replaced S333 by selected amino acids proved that the conserved serine is critical for the physiological function of CG8111. Metabolomic and proteomic analyses revealed that the S333 in CG8111 is essential to proper energy homeostasis and lipid metabolism in the fly. Of note, metabolic impairments were also found in the murine Tmem43 disease model, and fibrofatty replacement is a hallmark of human ARVC5. These findings contribute to a more comprehensive understanding of the molecular functions of CG8111 in Drosophila, and can represent a valuable basis to assess the aetiology of the human TMEM43 p.S358L variant in more detail.

Arrhythmogenic right ventricular cardiomyopathy in dogs

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease seen in dogs, cats, and humans. A common entity in Boxers and the related English bulldog, the disease is characterized by fatty or fibrofatty replacement of the myocardium, ventricular arrhythmias, and the potential for syncope or sudden death. In some individuals, concomitant left ventricular involvement results in systolic dysfunction and a progression to congestive heart failure. The clinical and pathological characteristics of ARVC share many similarities in dogs and humans, and Boxers serve as an important spontaneous model of the disease.Although multiple mechanisms have been implicated in the pathogenesis of ARVC, the disease is ultimately considered to be a disorder of the desmosome. Multiple causal genetic mutations have been identified in people, and over 50% of affected humans have an identifiable mutation in desmosomal proteins. To date, only a single genetic mutation has been associated with ARVC in Boxer dogs. Other as-yet-undiscovered genetic mutations and epigenetic modifiers of the disease are likely. Treatment of ARVC in dogs is focused on controlling ventricular arrhythmias and associated clinical signs. This article will review the pathophysiology, clinical diagnosis, treatment, and prognosis of ARVC in the dog.

Different Expressions of Pericardial Fluid MicroRNAs in Patients With Arrhythmogenic Right Ventricular Cardiomyopathy and Ischemic Heart Disease Undergoing Ventricular Tachycardia Ablation.

Introduction: Pericardial fluid is enriched with biologically active molecules of cardiovascular origin including microRNAs. Investigation of the disease-specific extracellular microRNAs could shed light on the molecular processes underlying disease development. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease characterized by life-threatening arrhythmias and progressive heart failure development. The current data about the association between microRNAs and ARVC development are limited.Methods and Results: We performed small RNA sequence analysis of microRNAs of pericardial fluid samples obtained during transcutaneous epicardial access for ventricular tachycardia (VT) ablation of six patients with definite ARVC and three post-infarction VT patients. Disease-associated microRNAs of pericardial fluid were identified. Five microRNAs (hsa-miR-1-3p, hsa-miR-21-5p, hsa-miR-122-5p, hsa-miR-206, and hsa-miR-3679-5p) were found to be differentially expressed between patients with ARVC and patients with post-infarction VT. Enrichment analysis of differentially expressed microRNAs revealed their close linkage to cardiac diseases.Conclusion: Our data extend the knowledge of pericardial fluid microRNA composition and highlight five pericardial fluid microRNAs potentially linked to ARVC pathogenesis. Further studies are required to confirm the use of pericardial fluid RNA sequencing in differential diagnosis of ARVC.

Deciphering the Role of Wnt and Rho Signaling Pathway in iPSC-Derived ARVC Cardiomyocytes by In Silico Mathematical Modeling.

Arrhythmogenic Right Ventricular cardiomyopathy (ARVC) is an inherited cardiac muscle disease linked to genetic deficiency in components of the desmosomes. The disease is characterized by progressive fibro-fatty replacement of the right ventricle, which acts as a substrate for arrhythmias and sudden cardiac death. The molecular mechanisms underpinning ARVC are largely unknown. Here we propose a mathematical model for investigating the molecular dynamics underlying heart remodeling and the loss of cardiac myocytes identity during ARVC. Our methodology is based on three computational models: firstly, in the context of the Wnt pathway, we examined two different competition mechanisms between β-catenin and Plakoglobin (PG) and their role in the expression of adipogenic program. Secondly, we investigated the role of RhoA-ROCK pathway in ARVC pathogenesis, and thirdly we analyzed the interplay between Wnt and RhoA-ROCK pathways in the context of the ARVC phenotype. We conclude with the following remark: both Wnt/β-catenin and RhoA-ROCK pathways must be inactive for a significant increase of PPARγ expression, suggesting that a crosstalk mechanism might be responsible for mediating ARVC pathogenesis.

Myocarditis: An Important Presentation of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC)

Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) traditionally presents with ventricular arrhythmias or, less commonly, heart failure. Myocardial inflammation has been implicated in the pathogenesis of ARVC, but clinical myocarditis in ARVC is not well described. We describe myocarditis as a presenting phenotype of ARVC, and hypothesized that patients with this presentation have distinct clinical characteristics. Methods Using the Johns Hopkins ARVC Registry, we examined patients diagnosed with myocarditis at index presentation based on European Society of Cardiology criteria, who were subsequently diagnosed with ARVC by Task Force Criteria (TFC). Results Of the 12 patients (all female, median age 20) initially diagnosed with myocarditis, the majority presented with chest pain (n=7, 58%) or ventricular arrhythmia (n=3, 25%). All patients had troponin elevations and left ventricular function was reduced in 5 (42%). Cardiac magnetic resonance imaging demonstrated LV delayed gadolinium enhancement and/or pericardial enhancement in 10 (83%), however only 3 (25%) patients had RV abnormalities. Treatment of suspected myocarditis during index presentation consisted of steroids (n=3/12, 25%) and/or non-steroidal anti-inflammatory drugs (NSAIDs) (n=4/12, 33%). Pathogenic genetic variants were identified in 11 (92%) patients: 10 desmoplakin (DSP) and 1 desmoglein-2 (DSG2). The clinical course timeline for each patient is shown in Figure 1. Patients were diagnosed with ARVC 1.8 years (IQR 2.7 years) after myocarditis presentation. After diagnosis, an additional 5 (42%) patients were recommended for implantable cardiac defibrillator and screening led to 17 family member diagnoses of ARVC. Conclusions Myocarditis may be the initial presentation of ARVC in some patients. These patients may have distinct characteristics such as female predominance, LV involvement and DSP gene variants. Genetic testing is key to ARVC diagnosis, especially in left ventricle dominant forms, and should be considered in appropriately selected patients presenting with myocarditis.

Intracellular calcium current disorder and disease phenotype in OBSCN mutant iPSC-based cardiomyocytes in arrhythmogenic right ventricular cardiomyopathy.

Obscurin participates in the development of striated muscles and maintenance of the functional sarcoplasmic reticulum. However, the role of obscurin in arrhythmogenic right ventricular cardiomyopathy (ARVC) is not well understood. We aimed to study the novel obscurin mutations in the pathogenesis of ARVC and the underlying mechanisms.Methods: We generated induced pluripotent stem cells (iPSC) through retroviral reprogramming of peripheral blood mononuclear cells isolated from a 46-year-old female diagnosed with ARVC, carrying a mutation in OBSCN. The cells differentiated into functional iPSC-based cardiomyocytes (iPSC-CMs), whose phenotype was determined by transmission electron microscopy, electrophysiological description, immunofluorescence staining, and Oil Red O staining. Molecular characterization was performed by bioinformatic analyses, and identification by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.Results: ARVC-iPSC-CMs mutation in OBSCN showed significant accumulation of lipids, increased pleomorphism, irregular Z-bands, and increased L type calcium currents. Functional enrichment analysis identified pathways involved in focal adhesion and structure formation; the adipocytokines and PPAR signaling pathways were also activated in the ARVC group. Moreover, our results from ultra-high-resolution microscopy, qRT-PCR and Western blotting confirmed that the mutant OBSCN protein and its anchor protein, Ank1.5, showed structural disorder and decreased expression, but there was increased expression of junctional protein N-Cadherin. Further analysis revealed the gene expression of other desmosomal proteins in ARVC-iPSC-CMs was also decreased but some adipogenesis pathway-related proteins (PPARγ, C/EBPα, and FABP4) were increased.Conclusion: A novel frameshift mutation in OBSCN caused phenotypic alteration accompanied by disrupted localization and decreased expression of its anchoring protein Ank1.5. Furthermore, there was an accumulation of lipids with an increase in fatty fibrosis area and myocardial structural disorder, possibly leading to dysrhythmia in calcium channel-related myocardial contraction. These observations suggested the possibility of attenuating ARVC progression by therapeutic modulation of OBSCN expression.

P5024Pathogenic mechanism of DSG2-F531C mutation caused arrhythmogenic right ventricular cardiomyopathy

Abstract Objective Mutations in the Desmoglein-2 (DSG2) are associated with arrhythmogenic right ventricular cardiomyopathy (ARVC). We previously identified a highly conserved F531C (c.1592T>G) variant of the cardiac desmosome gene DSG2 in the proband with a positive family history. The aim of this study is to investigate the mechanism(s) of DSG2-F531C variant in the pathogenesis of ARVC. Methods A large Han Chinese family with five generations was enrolled in this study, physical and clinical examinations were performed. Seven members were diagnosed with ARVC. Subsequently five desmosomal genes (PKP2, DSG2, DSP, DSC2 and JUP), TMEM43 and PLN were sequenced directly from genomic DNA. Seven patients diagnosed with ARVC were homozygous for DSG2-F531C, while five members of the family with normal phenotype were heterozygous for DSG2-F531C. To further explore the role of DSG2-F531C in ARVC pathogenesis, we generated a knock-in (KI) mouse model, which was carrying the mouse equivalent form (DSG2-F536C) of this variant. Results The homozygous KI mice (DSG2-F536C+/+) revealed a robust cardiomyopathy phenotype with left ventricular systolic dysfunction, histopathology showed accumulation of collagen, spotty calcification and fat droplets with extensive fibrosis in ventricular tissue in all DSG2-F536C+/+ mice. Further detections showed widening of the intercalated disc and regional loss of DSG2 in the hearts of these mice. The marked reduction in immunoreactive signal for sodium voltage-gated channel alpha subunit 5 (NaV1.5) was observed with no apparent changes in plakoglobin and connexin-43. In addition, dramatically elevated expression of transforming growth factor β1 (TGF-β1) was also found in the DSG2 mutant mice. Intriguingly, the heterozygous KI mice (DSG2-F536C+/−) expressed a similar level of TGF-β1 versus homozygous ones (DSG2-F536C+/+), but exhibited slight changes in morphology. Conclusions The DSG2-F536C KI mice produce a similar pathological phenotype of human ARVC, which supports DSG2-F531C as a causative mutation for ARVC. Additionally, the DSG2-F536C+/+ mutation induced cardiac fibrosis through the activation of TGF-β1 signaling will open new opportunities for further mechanistic and therapeutic studies in DSG2-related ARVC. Acknowledgement/Funding National Key R&D Program of China [2017YFC0909400); National Key Basic Research Program of China [973 program No. 2013CB531105]

Rare non-coding Desmoglein-2 variant contributes to Arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to variants in the coding sequence of desmosomal genes. The potential contribution of non-coding desmoglein-2 (DSG2) variants for development of ARVC is undescribed. We sequenced 1450 base pairs upstream of ATG in the DSG2 gene in 65 unrelated patients diagnosed with ARVC (10 borderline cases). Identified variants was evaluated by cosegregation and allele population frequency analysis, in silico tools, immunohistological investigations of myocardial biopsies, gene reporter assays, electrophoretic mobility shift assays (EMSA), and chromatin immunoprecipitation. The genetic analysis identified one novel, rare heterozygous DSG2 upstream variant (−317G > A) in a genetically unexplained ARVC patient. The variant segregated with signs of disease, was absent in publicly available databases, and affected a predicted binding site for activating protein-1 (AP-1). Immunohistochemical analysis of a myocardial biopsy from the −317G > A patient showed a marked reduction in DSG2 protein levels compared to healthy controls. Luciferase reporter gene assays showed promoter activity of the identified DSG2 upstream region and a general reduction in transcriptional activity in the presence of the minor DSG2_A allele (p < .01). Moreover, the DSG2_A allele reduced DSG2 activation by TGF-beta1 and a protein kinase C pathway activator (PMA; all p < .001 vs. DSG2_G). EMSAs showed altered transcription factor binding in presence of the DSG2_A allele. Chromatin immunoprecipitation assays in wild type epithelial cells identified AP-1 components c-FOS and c-JUN at the -317 locus. In conclusion, the non-coding DSG2 promoter variant -317G > A reduces DSG2 transcription in vitro and reduced myocardial DSG2 protein levels were observed in vivo. Our data support a contribution of non-coding DSG2 variants to the pathogenesis of ARVC.

Identification of Cadherin 2 ( CDH2 ) Mutations in Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically heterogeneous condition caused by mutations in genes encoding desmosomal proteins in up to 60% of cases. The 40% of genotype-negative cases point to the need of identifying novel genetic substrates by studying genotype-negative ARVC families. Whole exome sequencing was performed on 2 cousins with ARVC. Validation of 13 heterozygous variants that survived internal quality and frequency filters was performed by Sanger sequencing. These variants were also genotyped in all family members to establish genotype-phenotype cosegregation. High-resolution melting analysis followed by Sanger sequencing was used to screen for mutations in cadherin 2 (CDH2) gene in unrelated genotype-negative patients with ARVC. In a 3-generation family, we identified by whole exome sequencing a novel mutation in CDH2 (c.686A>C, p.Gln229Pro) that cosegregated with ARVC in affected family members. The CDH2 c.686A>C variant was not present in >200 000 chromosomes available through public databases, which changes a conserved amino acid of cadherin 2 protein and is supported as the causal mutation by parametric linkage analysis. We subsequently screened 73 genotype-negative ARVC probands tested previously for mutations in known ARVC genes and found an additional likely pathogenic variant in CDH2 (c.1219G>A, p.Asp407Asn). CDH2 encodes cadherin 2 (also known as N-cadherin), a protein that plays a vital role in cell adhesion, making it a biologically plausible candidate gene in ARVC pathogenesis. These data implicate CDH2 mutations as novel genetic causes of ARVC and contribute to a more complete identification of disease genes involved in cardiomyopathy.

Rho-Kinase Inhibition During Early Cardiac Development Causes Arrhythmogenic Right Ventricular Cardiomyopathy in Mice.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by fibrofatty changes of the right ventricle, ventricular arrhythmias, and sudden death. Though ARVC is currently regarded as a disease of the desmosome, desmosomal gene mutations have been identified only in half of ARVC patients, suggesting the involvement of other associated mechanisms. Rho-kinase signaling is involved in the regulation of intracellular transport and organizes cytoskeletal filaments, which supports desmosomal protein complex at the myocardial cell-cell junctions. Here, we explored whether inhibition of Rho-kinase signaling is involved in the pathogenesis of ARVC. Using 2 novel mouse models with SM22α- or αMHC-restricted overexpression of dominant-negative Rho-kinase, we show that mice with Rho-kinase inhibition in the developing heart (SM22α-restricted) spontaneously develop cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, resulting in premature sudden death, phenotypes fulfilling the criteria of ARVC in humans. Rho-kinase inhibition in the developing heart results in the development of ARVC phenotypes in dominant-negative Rho-kinase mice through 3 mechanisms: (1) reduction of cardiac cell proliferation and ventricular wall thickness, (2) stimulation of the expression of the proadipogenic noncanonical Wnt ligand, Wnt5b, and the major adipogenic transcription factor, PPARγ (peroxisome proliferator activated receptor-γ), and inhibition of Wnt/β-catenin signaling, and (3) development of desmosomal abnormalities. These mechanisms lead to the development of cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, ultimately resulting in sudden premature death in this ARVC mouse model. This study demonstrates a novel crucial role of Rho-kinase inhibition during cardiac development in the pathogenesis of ARVC in mice.

Traditional vs. genetic pathogenesis of arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC), a predominantly familial and autosomal dominant inherited heart muscle disorder, is pathologically characterized by progressive right ventricular myocardial atrophy and fibrofatty replacement and clinically by ventricular arrhythmias with left bundle branch block morphology. Symptoms poorly reflect disease severity, with disease commonly first manifesting as sudden death among the young. The inflammatory and apoptotic theories first put forth to explain ARVC pathogenesis do not explain all cases, and advances in genetic technology have allowed to elucidate genetic mechanisms, with desmosomal mutations attracting much attention. As reviewed here, various non-mutually exclusive pathogenetic mechanisms therefore appear to underlie ARVC.

IASPP, a previously unidentified regulator of desmosomes, prevents arrhythmogenic right ventricular cardiomyopathy (ARVC)-induced sudden death

Desmosomes are anchoring junctions that exist in cells that endure physical stress such as cardiac myocytes. The importance of desmosomes in maintaining the homeostasis of the myocardium is underscored by frequent mutations of desmosome components found in human patients and animal models. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a phenotype caused by mutations in desmosomal components in ∼ 50% of patients, however, the causes in the remaining 50% of patients still remain unknown. A deficiency of inhibitor of apoptosis-stimulating protein of p53 (iASPP), an evolutionarily conserved inhibitor of p53, caused by spontaneous mutation recently has been associated with a lethal autosomal recessive cardiomyopathy in Poll Hereford calves and Wa3 mice. However, the molecular mechanisms that mediate this putative function of iASPP are completely unknown. Here, we show that iASPP is expressed at intercalated discs in human and mouse postmitotic cardiomyocytes. iASPP interacts with desmoplakin and desmin in cardiomyocytes to maintain the integrity of desmosomes and intermediate filament networks in vitro and in vivo. iASPP deficiency specifically induces right ventricular dilatation in mouse embryos at embryonic day 16.5. iASPP-deficient mice with exon 8 deletion (Ppp1r13l(Δ8/Δ8)) die of sudden cardiac death, displaying features of ARVC. Intercalated discs in cardiomyocytes from four of six human ARVC cases show reduced or loss of iASPP. ARVC-derived desmoplakin mutants DSP-1-V30M and DSP-1-S299R exhibit weaker binding to iASPP. These data demonstrate that by interacting with desmoplakin and desmin, iASPP is an important regulator of desmosomal function both in vitro and in vivo. This newly identified property of iASPP may provide new molecular insight into the pathogenesis of ARVC.

Intercalated discs and arrhythmogenic cardiomyopathy.

Heart tissue is subjected to high mechanical stress. Different junctional complexes exist within the intercalated disc (ID) at the site of end-to-end contacts between cardiomyocytes. These junctions are essential for adhesive integrity, morphogenesis, differentiation, and maintenance of cardiac tissue. Recent findings of molecular interactions among intercellular adhesion molecules, gap junctions, and the voltage-gated sodium channel complex suggest that IDs should be considered an organelle in which macromolecular complexes interact specifically to maintain cardiac structure and cardiomyocyte synchrony. It is within this organelle that most of the mutated proteins involved in arrhythmogenic right ventricular cardiomyopathy (ARVC) reside. This inherited cardiomyopathy is characterized by both structural and electric abnormalities of the heart, particularly in young people and athletes. This review highlights recent advances in understanding the link between ID alterations and the molecular genetics and pathogenesis of ARVC. Cardiomyocytes are extensively interconnected at their ends through their IDs, a complex region composed of different kinds of intercellular junctions essential for electric, mechanical, and signaling communication between adjacent cells and, hence, for maintaining correct heart function and growth. Although traditionally depicted as a composition of different separate units, recent data indicate that the ID of cardiomyocytes should be considered a single functional unit in which macromolecular complexes interact mechanically and electrically to maintain cardiomyocyte rigidity and synchrony. The ID in vertebrates was originally described as consisting of 3 main junctional complexes: desmosomes, adherens junctions (AJ, also called fascia adherens in cardiac muscle), and gap junctions. It has been proposed that while gap junctions, being essential for chemical and electric coupling of neighboring cells, represent the electric component of ID, desmosomes together with AJ form the mechanical intercellular junctions in cardiomyocytes.1 Desmosomes and AJ are highly specialized anchoring junctions. They are particularly important for maintenance of adhesion and integrity of tissues exposed to …

TMEM43 mutation p.S358L alters intercalated disc protein expression and reduces conduction velocity in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.

Functional characterization of a newly identified LMNA mutant in HL‐1 cardiomyocytes (1073.7)

A novel mutation in the Lamin A/C gene (LMNA c.418_438dup) was detected in the index patient and in additional family members with diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) and history of sudden cardiac death. The functional characterization of this LMNA mutant was performed in cultured HL‐1 cardiomyocytes expressing EGFP‐tagged wild‐type and mutated LMNA constructs and subjected to confocal microscopy analysis and hypoxic stress conditions in 100% N2 for 8h. Mutated LMNA was clearly expressed in aggregates of different sizes and not uniformly distributed along the nuclear envelope as WT LMNA. Moreover, the mutated LMNA variant causes perturbation in nuclear shape and Nuclear Pore Complexes organization.Of note, we observed that under hypoxic conditions nuclear envelopes expressing mutated LMNA become leaky, leading a nuclear fluorescent marker to escape into the cytoplasm. This indicates that, under cell stressing conditions, the nucleo‐cytoplasmic compartmentalization is affected in cardiomyocytes expressing this LMNA mutation, inducing, as final fatal consequence, cell apoptosis.In conclusion, we not only open new avenues to gain more insights in the pathogenesis of ARVC and to conceive novel therapeutic strategies but we also shed lights on the role of nuclear Lamin A in the physio‐pathology of cardiomyocytes.Grant Funding Source: Supported by Fondo per gli Investimenti della Ricerca di Base‐Rete Nazionale di Proteomica (RBRN07BM

Abstract 18363: Activation of the Hippo Pathway, Associated With Suppression the Canonical Wnt Signaling, Contributes to the Pathogenesis of Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an enigmatic disease characterized by excess fibro-adipocytes in the heart, cardiac arrhythmias and heart failure. Molecular genetic studies have identified mutations in several desmosome proteins as causes of ARVC. Our objective is to delineate the molecular links between the desmosomes and fibro-adiposis in ARVC. Expression levels and localization of plakophilin 2 (PKP2), plakoglobin (JUP), desmocollin 2 (DSC2), desmosome proteins and connexin 43 (GJA1), a member of Gap junction, in the human hearts with ARVC, were reduced. To test whether molecular remodeling of the junctions affected signaling pathways, we analyzed the Hippo kinase pathway, which is regulated by cell-cell contact and known to suppress the canonical Wnt signaling, the latter implicated in the pathogenesis of ARVC. Accordingly, Merlin, an upstream regulator of the Hippo kinase cascade, was activated and YAP, the downstream effector was inactivated by phosphorylation. To gain mechanistic insights, expression of Pkp2, the most common causal gene for ARVC, was stably knocked down using shRNA in the HL-1 cells. Transcriptome analysis showed reduced mRNA levels of the canonical Wnt and Hippo targets including Ctgf, an inhibitor of adipogenesis. KD of PKP2 was associated with a dramatic reduction in pPKC-α level, in accord with the known function of PKP2 as a platform for phosphorylation of PKC-α. Reduced pPKCα, an inhibitor of Merlin through phosphorylation, was associated with activation of Merlin and cascade phosphorylation of MST1, LATS1/2 and YAP, constituents of the Hippo pathway. Consequently, signaling through TEAD, the transcription factor of the Hippo pathway, was reduced and sequestration of pβ-catenin in the cytoplasm was increased, leading to suppression of the canonical Wnt signaling and enhanced adipogenesis. KD of pLAT1/2 using shRNAs partially rescued the molecular phenotype. Thus, human ARVC is characterized by molecular remodeling of the intercalated discs, which results in inactivation of PKCα, phosphorylation of the Hippo kinases, suppression of the canonical Wnt signaling and enhanced adipogenesis. The findings implicate the Hippo pathway in the pathogenesis of ARVC.

Silencing of desmoplakin decreases connexin43/Nav1.5 expression and sodium current in HL-1 cardiomyocytes

Desmosomes and gap junctions are situated in the intercalated disks of cardiac muscle and maintain the integrity of mechanical coupling and electrical impulse conduction between cells. The desmosomal plakin protein, desmoplakin (DSP), also plays a crucial role in the stability of these interconnected components as well as gap junction connexin proteins. In addition to cell‑to‑cell junctions, other molecules, including voltage‑gated sodium channels (Nav1.5) are present in the intercalated disk and support the contraction of cardiac muscle. Mutations in genes encoding desmosome proteins may result in fatal arrhythmias, including arrhythmogenic right ventricular cardiomyopathy (ARVC). Therefore, the aim of the present study was to determine whether the presence of DSP is necessary for the normal function and localization of gap junction protein connexin43 (Cx43) and Nav1.5. To examine this hypothesis, RNA interference was utilized to knock down the expression of DSP in HL‑1 cells and the content, distribution and function of Cx43 and Nav1.5 was assessed. Western blotting and flow cytometry experiments revealed that Cx43 and Nav1.5 expression decreased following DSP silencing. In addition, immunofluorescence studies demonstrated that a loss of DSP expression led to an abnormal distribution of Cx43 and Nav1.5, while scrape‑loading dye/transfer revealed a decrease in dye transfer in DSP siRNA‑treated cells. The sodium current was also recorded by the whole‑cell patch clamp technique. The results indicated that DSP suppression decreased sodium current and slowed conduction velocity in cultured cells. The present study indicates that impaired mechanical coupling largely affects electrical synchrony, further uncovering the pathogenesis of ARVC.

Mutated Desmoglein-2 Proteins are Incorporated into Desmosomes and Exhibit Dominant-Negative Effects in Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary cardiac condition associated with ventricular arrhythmias, heart failure, and sudden death. The most frequent ARVC genes encode desmosomal proteins of which mutations in desmoglein-2 (DSG2), account for 10%-20% of cases. This study aimed to investigate how DSG2 mutations contribute to the pathogenesis of ARVC. Initial mutation analysis of DSG2 in 71 probands identified the first family reported with recessively inherited ARVC due to a missense mutation. In addition, three recognized DSG2 mutations were identified in 12 families. These results and further mutation analyses of four additional desmosomal genes indicated that ARVC caused by DSG2 mutations is often transmitted by recessive or digenic inheritance. Because desmosomal proteins are also expressed in skin tissue, keratinocytes served as a cell model to investigate DSG2 protein expression by Western blotting, 2D-PAGE, and liquid chromatography-mass spectrometry. The results showed that heterozygous mutation carriers expressed both mutated and wild-type DSG2 proteins. These findings were consistent with the results obtained by immunohistochemistry of endomyocardial biopsies and epidermal tissue of mutation carriers, which indicated a normal cellular distribution of DSG2. The results suggested a dominant-negative effect of the mutated DSG2 proteins because they were incorporated into the desmosomes.

Abstract 19168: Effect of Endurance Exercise on Transgenic Arrhythomeginc Right Ventricular Cardiomyopathy Mice

Introduction: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder that causes sudden death and right ventricular heart failure in the young. Clinically, it has been suggested that competitive sports may accelerate ARVC pathogenesis in susceptible persons. The goal of this study was to investigate the cause and effect of exercise stress in the development of ARVC using a transgenic (Tg) mouse model with cardiac-restricted overexpression of human mutant R2834H desmoplakin (DSP). Methods: DSP R2834H Tg (Tg-mut), non-transgenic human wild type DSP WT (Tg-wt) and control non-transgenic (Ntg) mice were divided into groups, sedentary and exercise. Mice in the exercise group were subjected to graded increase in intensity of exercise on a treadmill from the age of 4 weeks. Every 4 weeks, echocardiograms (echo) and ECGs (single lead) were recorded. Mice were dissected for tissue sampling and gross morphology. Results: Initial echo data results are as shown in Table 1. When adjusted for age, number of days exercised, gender, interaction of genotype and exercise status, Tg-mut RV was bigger than Ntg mice (P=0.0005). Within the Tg-mut group, the RV diameter was not statistically significantly different between exercise and sedentary mice when adjusted for same variables. Heart weight/body weight ratio was higher in Tg-mut as compared to Ntg &amp; lcub;sedentary (= 0.001) and exercise group (P = 0.0060&amp;rcub; and Tg-wt. Tissue staining showed early development of fibrosis and fat in Tg-mut mice. The only significant ECG finding occurred in the exercise group: Tg-muthadincreased P wave amplitude and decreased R wave amplitude (P = 0.02 and P =0.001, respectively) compared to Ntg mice. Conclusions: We report significant early histopathologic, and ECG changes with exercise in mutant DSP R2834H Tg mice compared to Ntg and Tg-wt mice.