Mutations In Desmosomal Genes Research Articles

Cardiac sympathetic neurons are additional cells affected in genetically determined arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (AC) is a familial cardiac disease, mainly caused by mutations in desmosomal genes, which accounts for most cases of stress-related arrhythmic sudden death, in young and athletes. AC hearts display fibro-fatty lesions that generate the arrhythmic substrate and cause contractile dysfunction. A correlation between physical/emotional stresses and arrhythmias supports the involvement of sympathetic neurons (SNs) in the disease, but this has not been confirmed previously. Here, we combined molecular, in vitro and ex vivo analyses to determine the role of AC-linked DSG2 downregulation on SN biology and assess cardiac sympathetic innervation in desmoglein-2mutant (Dsg2mut/mut) mice. Molecular assays showed that SNs express DSG2, implying that DSG2-mutation carriers would harbour the mutant protein in SNs. Confocal immunofluorescence of heart sections and 3-D reconstruction of SN network in clarified heart blocks revealed significant changes in the physiologialc SN topology, with massive hyperinnervation of the intact subepicardial layers and heterogeneous distribution of neurons in fibrotic areas. Cardiac SNs isolated from Dsg2mut/mut neonatal mice, prior to the establishment of cardiac innervation, show alterations in axonal sprouting, process development and distribution of varicosities. Consistently, virus-assisted DSG2 downregulation replicated, in PC12-derived SNs, the phenotypic alterations displayed by Dsg2mut/mut primary neurons, corroborating that AC-linked Dsg2 variants may affect SNs. Our results reveal that altered sympathetic innervation is an unrecognized feature of AC hearts, which may result from the combination of cell-autonomous and context-dependent factors implicated in myocardial remodelling. Our results favour the concept that AC is a disease of multiple cell types also hitting cardiac SNs. KEY POINTS: Arrhythmogenic cardiomyopathy is a genetically determined cardiac disease, which accounts for most cases of stress-related arrhythmic sudden death. Arrhythmogenic cardiomyopathy linked to mutations in desmoglein-2 (DSG2) is frequent and leads to a left-dominant form of the disease. Arrhythmogenic cardiomyopathy has been approached thus far as a disease of cardiomyocytes, but we here unveil that DSG2 is expressed, in addition to cardiomyocytes, by cardiac and extracardiac sympathetic neurons, although not organized into desmosomes. AC-linked DSG2 downregulation primarily affect sympathetic neurons, resulting in the significant increase in cardiac innervation density, accompanied by alterations in sympathetic neuron distribution. Our data supports the notion that AC develops with the contribution of several 'desmosomal protein-carrying' cell types and systems.

Abstract Mo046: GJA1-20k Restores Connexin-43 Trafficking and β-Catenin Signaling in Myocytes Lacking Desmoplakin

Background: Arrhythmogenic Cardiomyopathy (ACM) is an insidious hereditary heart disease that results in structural and electrical cardiac abnormalities leading to arrhythmogenesis and heart failure. ACM is characterized by cardiomyocyte loss, decreased cellular coupling, and fibrofatty replacement. The disease arises from mutations in desmosomal genes, such as plakophilin (PKP), desmoglein (DSG), and desmoplakin (DSP). The pathogenesis of this disease is linked to dysfunction at the level of several cellular processes and pathways, including Wnt/β-catenin signaling. Recent evidence has shown that GJA1-20k, a truncated isoform of Connexin-43 (Cx43) generated by internal translation, has a therapeutic role in preserving Cx43 trafficking and cell-cell coupling in ACM of DSG origin. This study aims to investigate the role of GJA1-20k in protecting against ACM of DSP origin by evaluating its effect in cell models on Cx43 trafficking and Wnt/β-catenin signaling. Methods: DSP knockdown was performed on HEK cells and mouse neonatal cardiomyocytes. The role of GJA1-20k was determined using imaging, biochemical, and molecular biology techniques. Results: Protein levels of Cx43 and β-catenin were decreased on Western blot (WB) upon DSP knockdown. These findings were corroborated through imaging wherein the signal intensity for Cx43 at cellular junctions and β-catenin at the membrane was significantly decreased compared to the control group. β-catenin signal intensity was also decreased within the nucleus. Upon administration of GJA1-20k, protein levels of Cx43 were normalized on WB. Moreover, the ratio of phosphorylated β-catenin (inactive) to total β-catenin was decreased. Imaging studies identified a significant increase in Cx43 and β-catenin at the membrane in ACM models treated with GJA1-20k. This was also accompanied by an increase in β-catenin signal intensity within the nucleus. A TOP-flash luciferase assay was performed and revealed a significant increase in β-catenin transcriptional activity in the presence of GJA1-20k despite DSP knockdown. Conclusions: These results suggest that GJA1-20k is able to rescue Cx43 trafficking and recover Wnt/β-catenin signaling in cells and cardiomyocytes lacking DSP. The ability of GJA1-20k to restore dysfunctional genetic pathways involved in arrhythmogenesis and fibro-adipogenesis make it an attractive potential candidate for targeted therapy against ACM.

Abstract Tu060: Unveiling Arrhythmogenic Right Ventricular Dysplasia In Pregnancy

Introduction: Arrhythmogenic right ventricular dysplasia (ARVD) is a rare autosomal dominant disease, caused by desmosomal gene mutations, and characterized by fibrofatty replacement of right ventricular (RV) myocardium with left ventricular (LV) involvement in advanced stages. It is mostly diagnosed between the second and forth decades of life and can present with ventricular tachycardia and arrhythmic sudden death. Here we present a case of ARVD diagnosis in a female in her forth pregnancy. Case description: A 28-year-old female, on her 27 th week of gestation (G4P3) was admitted to the hospital with preterm premature rupture of membranes. She also endorsed intermittent chest pressure, palpitations and shortness of breath. Her vital signs were stable, and electrocardiogram (ECG) revealed normal sinus rhythm with frequent premature ventricular contractions (PVCs) and T wave inversions in leads V1- V3. Laboratory studies showed normal troponin and D-dimer levels, and slightly elevated brain natriuretic peptide 240 pg/mL. Patient notably had frequent runs of monomorphic non-sustained ventricular tachycardia (NSVT) arising from RV apical territory, with left bundle branch morphology on telemetry. An echocardiogram revealed a dilated right atrium and RV with hypokinesis of RV; LV size and function were normal. Overall, patient met the revised task force diagnostic criteria for ARVD based on PVC morphology, abnormal ECG and dilated and hypokinetic RV. She had no sustained or hemodynamically unstable VT, no history of syncope or family history of sudden cardiac death and did not meet class I indication for implantable cardioverter defibrillator. She was started on oral metoprolol succinate 25 mg daily with which her arrhythmias significantly improved. She was recommended a cardiac magnetic resonance imaging and genetic testing for confirmation and to assess familial implications. Conclusion: Recognition of ARVD during pregnancy is challenging given the overlapping symptoms of normal pregnancy-related changes. Hormonal surge and sympathetic drive may have driven the increased frequency of NSVT in our patient and beta blocker was effective in managing these. Early recognition and tailored management are crucial to ensure the well-being of both the mother and the fetus. A close follow-up is necessary to understand the long-term implications for the patient and potential genetic predisposition in the family.

Zebrafish dspb-/- mutant as model for arrhythmogenic cardiomyopathy - impact of physical activity

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III Background Zebrafish has been reported as a model to study the Arrythmogenic cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. Purpose Our aim was the characterization of dspb-/- mutants, studying the impact of physical activity in development of the disease. Methods The characterization of phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) and adult (8 months old) individuals. The impact of physical activity was determined through moderated training periods (1 month) and endurance assays using a swimming tunnel. Results Viability assays showed that dspb mutants suffer a premature death, with significant higher mortality rates than the wildtype group (survival WT 89.7% vs dspb-/- 61.3%, p<0.01). Larvae cardiac function showed dspb mutants had a higher heart rate (WT 106.8 ± 26.8 vs dspb-/- 141.0 ± 7.2, p<0.01) while echocardiography showed differences in heart rate (WT 127.2 ± 49,8 vs dspb-/- 219.2 ± 26.8, p<0.05), systolic (WT 26.9 ± 5.3 vs dspb-/- 18.5 ± 5.2, p<0.05) and diastolic (WT 37.6 ± 8.4 vs dspb-/- 23.7 ± 5.4, p<0.05) areas. Gene expression analysis in adult heart showed dspb expression drastically decreased (WT 0.9 ± 0.1 vs dspb-/- 0.4 ± 0.01, p<0.01), whilst pkp2 expression increased (WT 0.9 ± 0.1 vs dspb-/- 2.59 ±0.22, p<0.001) as well as dspa gene expression (wt 0.9 ± 0.1 vs dspb-/- 4.41 ± 0.18, p< 0.001) as a compensation mechanism. Moderated long term physical activity had a positive impact in heart rate (dspb-/-pre 219.2 ± 26.8 vs dspb-/-post 138 ± 7.5, p<0.01) and diastolic area (dspb-/-pre 24 ± 5 vs dspb-/-post 32 ± 2.1, p<0.05). Adult mutants showed a better endurance showing improvement in immobile cumulative duration (dspb-/-pre 849.3 ± 29.3 vs dspb-/-post 179.8 ± 29.7, p<0.01) and immobile frequency (dspb-/-pre 5399.5 ± 1127.8 vs dspb-/-post 1310 ± 481.9, p<0.01). Conclusion The dspb-/- zebrafish presented pathological cardiac phenotype at larvae and adult stages, and less endurance in intensity exercises. The moderate exercise has turned out to be a beneficial modulator of the phenotype.

Zebrafish dspb -/- tert +/- mutant as model for arrhythmogenic cardiomyopathy. Impact of aging in the development of the disease

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III Background/Objectives Zebrafish has been reported as model to study the Arrythmogenic Cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic Cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. On the other hand, tert gene enables telomerase activity and telomere maintenance and mutations in this gene have been used for studying aging effects. After multiple crossings of our dspb model with tert mutants we have obtained dspb-/-tert+/- model line. Purpose Our aim was the characterization of dspb-/-tert+/- mutants, studying the impact of aging in the development of the disease. Methods The model was obtained by crossing dspb-/- and tert-/- mutants, crossing the heterozygous mutants and genotyping for the selection of the dspb-/-tert+/- mutants. The characterization of the phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) young adult (8 months old) and old adult (3 years old) individuals. Results Viability assays showed that dspb-/-tert+/- mutants suffer a premature death, with much lower survival rates than the dspb-/- group (survival dspb-/- 61.3% vs dspb-/-tert+/- 35.6%, p<0.01). Larvae heart rate analysis showed no significant differences. Echocardiography in young adults showed no significant differences between dspb-/-tert+/- and dspb-/- mutants. In old adults, echocardiography showed differences in heart rate (dspb-/- 142.5 ± 15.5 vs dspb-/-tert+/ -97.5 ± 3.5, p<0.01), respiratory frequency (dspb-/- 288.7 ± 36.1 vs dspb-/-tert+/- 95 ± 49.4, p<0.01) and atrial contraction velocity (dspb-/-155.7 ± 26.09 vs dspb-/-tert+/ -227.2 ± 17.6, p<0.05). Gene expression analysis in larvae showed that dspb expression drastically decreased (dspb-/- 0.3 ± 0.1 vs dspb-/-tert+/ -0.01 ± 0.008, p<0.01). In adults, dspb expression drastically decreased in heart tissue (dspb-/- 0.4 ± 0.01 vs dspb-/-tert+/- 0.06 ± 0.004, p<0.01) and skin (dspb-/ -0.34 ± 0.02 vs dspb-/-tert+/- 0.25 ± 0.01, p<0.01). Conclusion The dspb-/-tert+/- zebrafish presented worse survival rates, lower dspb gene expression levels and worse pathological cardiac phenotype at adult stages than dspb-/- mutants, proving that age is an important factor in the development of the disease and proving that dspb-/-tert+/- zebrafish is an appropriate model to study the impact of aging.

Abstract 17747: Gynecological Manifestations in a Patient With Arrhythmogenic Right Ventricular Cardiomyopathy: A Desmosomal Link?

Introduction: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is inherited cardiomyopathy which is caused by desmosomal protein gene mutations, primarily in the desmoplakin (DSP), plakophilin-2 (PKP2), and desmoglein-2 (DSG2) genes. It is characterized pathologically by myocardial atrophy, fibrofatty replacement, and fibrosis, precipitating ventricular arrhythmias (VA). Desmosomal mutations have been linked to impaired follicular development and leiomyoma formation. However, there is little information about the association of their expression in myocardial with ovarian or uterine tissues. As incidences of ARVC/D in women increase, it is crucial to determine if women with the gynecological manifestations of these mutations are at a higher risk of developing ARCV/D. Case: We report a case of a 32-year-old female who presented with exercise-induced palpitations and syncopal episodes. She has a history of severe dysmenorrhea but has no medical conditions otherwise. On monitoring, she was found to have a high burden of ventricular premature complexes (VPCs). VAs were thought to have precipitated her syncopal episodes. She was started on antiarrhythmics. Echocardiogram demonstrated right ventricular dilatation and wall thinning. The patient later underwent genetic testing, which confirmed ARVC-associated mutations. Her dysmenorrhea was managed with combined oral contraceptives and nonsteroidal anti-inflammatory drugs. Pelvic ultrasound revealed leiomyomas, which were surgically removed. Discussion: This case supports the possible complex interplay between cardiovascular and gynecological conditions in patients with desmosomal mutations. Desmosomes are specialized protein complexes crucial in maintaining tissue's structural and mechanical stability. Mutations in desmosome genes disrupt the normal functioning of these protein complexes, leading to impaired cell-cell adhesion and increased vulnerability to tissue damage. The next step is to conduct studies to better understand the risk of developing cardiovascular disease in patients presenting with these gynecological conditions and vice versa to provide early monitoring and treatment.

Modern view on genetic aspects of arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is a genetically determined disease with a high risk of sudden cardiac death (SCD). The spectrum of genetic causes of this disease is quite wide and includes mutations in both desmosomal and non-desmosomal genes. A positive result of a genetic examination is used as a diagnostic tool in patients with ACM, so its correct interpretation is one of the key factors in a personalized approach to managing a patient with this pathology. The review presents generalized modern ideas about the genetic nature of ACM.

Disease modeling of desmosome-related cardiomyopathy using induced pluripotent stem cell-derived cardiomyocytes

Cardiomyopathy is a pathological condition characterized by cardiac pump failure due to myocardial dysfunction and the major cause of advanced heart failure requiring heart transplantation. Although optimized medical therapies have been developed for heart failure during the last few decades, some patients with cardiomyopathy exhibit advanced heart failure and are refractory to medical therapies. Desmosome, which is a dynamic cell-to-cell junctional component, maintains the structural integrity of heart tissues. Genetic mutations in desmosomal genes cause arrhythmogenic cardiomyopathy (AC), a rare inheritable disease, and predispose patients to sudden cardiac death and heart failure. Recent advances in sequencing technologies have elucidated the genetic basis of cardiomyopathies and revealed that desmosome-related cardiomyopathy is concealed in broad cardiomyopathies. Among desmosomal genes, mutations in PKP2 (which encodes PKP2) are most frequently identified in patients with AC. PKP2 deficiency causes various pathological cardiac phenotypes. Human cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs) in combination with genome editing, which allows the precise arrangement of the targeted genome, are powerful experimental tools for studying disease. This review summarizes the current issues associated with practical medicine for advanced heart failure and the recent advances in disease modeling using iPSC-derived cardiomyocytes targeting desmosome-related cardiomyopathy caused by PKP2 deficiency.

Desmosomal protein degradation as an underlying cause of arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited progressive cardiac disease. Many patients with ACM harbor mutations in desmosomal genes, predominantly in plakophilin-2 (PKP2). Although the genetic basis of ACM is well characterized, the underlying disease-driving mechanisms remain unresolved. Explanted hearts from patients with ACM had less PKP2 compared with healthy hearts, which correlated with reduced expression of desmosomal and adherens junction (AJ) proteins. These proteins were also disorganized in areas of fibrotic remodeling. In vitro data from human-induced pluripotent stem cell-derived cardiomyocytes and microtissues carrying the heterozygous PKP2 c.2013delC pathogenic mutation also displayed impaired contractility. Knockin mice carrying the equivalent heterozygous Pkp2 c.1755delA mutation recapitulated changes in desmosomal and AJ proteins and displayed cardiac dysfunction and fibrosis with age. Global proteomics analysis of 4-month-old heterozygous Pkp2 c.1755delA hearts indicated involvement of the ubiquitin-proteasome system (UPS) in ACM pathogenesis. Inhibition of the UPS in mutant mice increased area composita proteins and improved calcium dynamics in isolated cardiomyocytes. Additional proteomics analyses identified lysine ubiquitination sites on the desmosomal proteins, which were more ubiquitinated in mutant mice. In summary, we show that a plakophilin-2 mutation can lead to decreased desmosomal and AJ protein expression through a UPS-dependent mechanism, which preceded cardiac remodeling. These findings suggest that targeting protein degradation and improving desmosomal protein stability may be a potential therapeutic strategy for the treatment of ACM.

462 RELATIONSHIP BETWEEN PATIENT GENOTYPE, VENTRICULAR INVOLVEMENT AND SUDDEN CARDIAC DEATH IN PATIENTS WITH ARRHYTHMOGENIC CARDIOMYOPATHY

Abstract Introduction Arrhythmogenic cardiomyopathy (AC) is an important cause of sudden cardiac death (SCD). AC is characterized by areas of fibro-fatty replacement of the myocardium of one or both ventricles, which can be characterized through cardiovascular magnetic resonance (CMR). Several mutations have been associated with the development of AC, most commonly in desmosomal genes. The relationship between the genotype and the disease phenotype (i.e., the extent and severity of myocardial disease and patient outcome) is unclear. Methods We evaluated 280 consecutive patients evaluated in a single CMR laboratory over a 10-year timespan and diagnosed with AC according to the Padua criteria, based on a retrospective evaluation of the electronic health records. All these patients had undergone a genetic testing through Sanger sequencing or Next Generation Sequencing (after 2012). Pathogenic or likely pathogenic mutations and variants of unknown significance were considered. CMR scans performed at diagnosis were examined by an expert blinded to the results of genetic testing to retrieve the location and extent of the areas of fibro-fatty replacement and late gadolinium enhancement (LGE), biventricular volumes and function. Follow-up data were also retrieved. The primary endpoint was SCD or aborted SCD (following successful defibrillator discharge). The secondary endpoint included SCD or aborted SCD plus sustained or non-sustained ventricular tachycardia episodes. Results Patients were more often males (67%), with a mean age of 48±16 years. Gene mutations related to cardiomyopathy were found in 123 patients, and 85 patients harbored mutations in desmosomal genes (DSP, n=41; DSG2, n=16; PKP2, n=16; DSC2, n=6; JUP, n=6), while the other mutations were in genes including TMEM43, LMNA, SCN5A, MYH7, and RYR2. Patients with mutations in desmosomal genes had more often LGE in the left ventricle (LV) than patients with other mutations or with no mutations detected (66% vs. 58% vs. 46%, respectively; p=0.025). Patients with mutations in the DSP gene had a greater LGE extent in terms of number of segments affected (p=0.011), or percent of the LV (p<0.001) compared with patients with other desmosomal mutations or no mutations. No differences between patients with desmosomal or non-desmosomal mutations or no mutations were found in terms of fibro-fatty replacement in the LV (51% vs. 32% vs. 48%; p=0.577), nor in terms of LGE presence or fibro-fatty replacement of the right ventricle (RV; data not shown). Median follow-up duration was 4.4 years (interquartile range 1.8-6.4); 22% of patients underwent defibrillator implantation. Primary endpoint events were recorded in 21 patients (7.5%), with 20 aborted SCD episodes. The secondary endpoint occurred in 58 patients (21%). At Cox regression analysis, the mutation status did not reach statistical significance as univariate predictor of the two endpoints, but LGE in the LV, LGE in ≥2 LV segments, or LGE >9% (considered one at the time) independently predicted both endpoints. Conclusions In patients with AC, mutations in desmosomal genes are found in almost half of patients, and are associated with the presence and a larger extent of LGE in the LV. In turn, the presence and extent of LGE are independent predictor of SCD or aborted SCD.

Novel view of arrhythmogenic cardiomyopathy as a “multicellular disease” affecting cardiac sympathetic innervation

Arrhythmogenic Cardiomyopathy (AC) is an incurable familial cardiac disease, mainly due to mutations in desmosomal genes, accounting for most cases of stress-related arrhythmic sudden death, in the young and athletes. AC hearts display cardiomyocyte (CM) death and diffuse fibro-fatty lesions, which compromise cardiac contractile performance and generate the substrate for arrhythmias. Correlation between physical/emotional stresses and arrhythmias incriminates sympathetic neurons (SNs), which may be the sought-after killers in disguise.

Cardiac resynchronization therapy: association between genetically determinated dilated cardiomyopathy and dissinchronopathy

Abstract Background Patients with non-ischemic dilated cardiomyopathy (DCM) and left bundle branch block (LBBB) often undergoes cardiac resynchronization therapy (CRT). Despite CRT only a small group of patients restore normal left ventricular geometry and function. Many predictors of response to CRT have been described, but there are still insufficient data on the role of genetic mutations in sarcomere, cytoskeletal or desmosomal genes. Purpose To asses the role of genetic background as predictor of response after CRT in patient DCM and LBBB. Methods We retrospectively analyzed DCM patients with successful CRT implantation and available genetic testing consecutively included in the CRT-Registry of our Institution. DCM was defined by the presence of left ventricular or biventricular dilatation and systolic dysfunction in the absence of abnormal loading conditions or coronary artery disease sufficient to cause global systolic impairment. Changes in main echocardiographic parameters: left ventricular ejection fraction (LVEF), LV End Systolic and End Diastolic Volume (LVESV and LVEDV), where compared between basal and 24–48 months of follow up. LV remodeling was defined by relative changes (Δ) in LV End Systolic Volume (LVESV) [(follow-up LVESV − PRE LVESV) / PRE LVESV × 100)]. Patients were considered super-responders (SR) if LVEF at 24–48 months was>50%. Genetic testing was done by Next Generation DNA and all variants were validated with bidirectional Sanger sequencing. Variants of uncertain significance (VUS) were excluded from the analysis. Patients being carrier of pathogenic (P) or likely pathogenic (LP) variants were considered affected by genetic DCM (GEN+ DCM). Patients non carrier of P/LP variants and with no evidence of familial disease were considered affected by non-genetically determined DCM (GEN− DCM). Results 73 patients met the inclusion criteria, 23 patients were GEN+ DCM, the remaining 50 patients had negative genetic test and no family history of DCM. Baseline characteristics were similar between groups. GEN− group was characterized by a greater improvement in LVEF (39±12 vs 29±12, p=0,006) and a greater relative reduction in LVESV (ΔLVESV 43% vs 18%, p=0,007). GEN− patients had a 33 ml higher mean LVESV reduction at 24–48 months after CRT, indipendently of their respective baseline values. 20 patients (27% of the total population) suffered of heart failure related death, heart transplantation or left ventricular assistance device implantation during follow up and the prevalence was significantly higher in GEN+ group (43% vs 20%, p=0,037). Coherently, patients in the GEN+ group showed a lower prevalence of super responders (4%, vs 24%, p=0,02) after CRT. Conclusions Non-genetically determined DCM DCM is associated with a better clinical and instrumental response to CRT and a higher probability of being super responders. Furthermore, our data support the hypothesis about dyssyncronopathy as a specific clinical entity inside DCM diseases. Funding Acknowledgement Type of funding sources: None.

C63 PHENOTYPE – GENOTYPE RELATIONSHIP IN ARRHYTHMOGENIC CARDIOMYOPATHY

Abstract Background Arrhythmogenic cardiomyopathy (ACM) is a primary disease of the myocardium with arrhythmic manifestations and fibro–fatty replacement either of the right (RV) or the left ventricle (LV) at the cardiac magnetic resonance (CMR). Over the last decade, different mutations in cardiac genes associated with heterogeneous phenotypes have been identified. Aim To investigate the genotype–phenotype relationships in ACM patients. Firstly, the phenotypic expression was defined in definite mutation carriers. Secondly, the prognostic significance of mutations was assessed across different phenotypes. Methods The study population included 281 patients with suspected ACM, based on family history, clinical and electrocardiographic evaluation, echocardiographic and CMR findings (Fig.1), studied at our Institution since 2012. All patients underwent genetic evaluation using Sanger sequencing and NGS of mutations in desmosomal (desmoplakin [DSP], plakophilin–2[PKP2], plakoglobin[JUP], desmoglein–2 [DSG2], desmocollin–2 [DSC2]) or non desmosomal genes. The composite endpoint included cardiac death, sustained and non–sustained ventricular tachycardia (VT), ventricular fibrillation (VF), appropriate defibrillator shock/antitachycardia pacing (ATP). Results The genetic test was positive in 113 patients (40%), 82 (73%) for desmosomal genes (41 DSP, 15 DSG2, 15 PKP2, 5 JUP, 6 DSC2), and 31 (27%) for non–desmosomal genes (4 titin, 2 TMEM 43, 2 lamin A/C, 23 others). Gene–positive compared to gene–negative (n = 168, 60%) patients showed a higher prevalence of LV ejection fraction (EF)<50% (14%vs.6%, p<0.011), LV and RV LGE (62%vs.46%, p < 0.001, 20%vs.12%, p<0.02), RV dilatation (27%vs.17%, p<0.041) (Table 1). DSG2 mutations were associated mainly to biventricular (7/15,46%) or RV involvement (4/15,26%), while DSP mutations to LV involvement (24/41,58%). During a median 36–month follow–up (IQ.range 12–56), 55 patients (19%) experienced the composite endpoint (35 non–sustained VT; 11 sustained VT – 3 ATP, 1 defibrillator–shock–; 9 VF, all treated by defibrillator–shock). At Kaplan–Meier analysis, DSP mutations were associated with the worst prognosis (Fig. 3). Conclusion Diverse genotypes were differently associated with LV dysfunction/LGE, RV dilatation/LGE. DSP mutations were associated with higher arrhythmic risk. Genetic screening may result in a better risk stratification, useful for clinical decision–making.

A novel desmoplakin mutation causes dilated cardiomyopathy with palmoplantar keratoderma as an early clinical sign.

BackgroundPPKs represent a heterogeneous group of disorders with hyperkeratosis of palmar and/or plantar skin. PPK, hair shaft abnormalities, cardiomyopathy and arrhythmias can be caused by mutations in desmosomal genes, e.g. desmoplakin (DSP). PPK should trigger genetic testing to reveal mutations with possible related cardiac disease.ObjectivesTo report a large multigenerational family with a novel DSP mutation associated with early‐onset PPK and adult‐onset cardiomyopathy and arrhythmias.MethodsA custom‐designed in‐house panel of 35 PPK related genes was used to screen mutations in the index patient with focal PPK. The identified DSP mutation was verified by Sanger sequencing. DNA samples from 20 members of the large multigenerational family were sequenced for the DSP mutation. Medical records were reviewed. Clinical dermatological evaluation was performed, including light microscopy of hair samples. Cardiac evaluation included clinical examination, echocardiography, cardiac magnetic resonance imaging (CMR), electrocardiogram (ECG), Holter monitoring and laboratory tests.ResultsWe identified a novel autosomal dominant truncating DSP c.2493delA p.(Glu831Aspfs*33) mutation associated with dilated cardiomyopathy (DCM) with arrhythmia susceptibility and focal PPK as an early cutaneous sign. The mutation was found in nine affected family members, but not in any unaffected members. Onset of dermatological findings preceded cardiac symptoms which were variable and occurred at adult age.ConclusionsWe report a novel truncating DSP mutation causing focal PPK with varying severity and left ventricular dilatation and ventricular extrasystoles. This finding emphasizes the importance of genetic diagnosis in patients with PPK for clinical counselling and management of cardiomyopathies and arrhythmias.

Clinical and genetic features of arrhythmogenic cardiomyopathy: diagnosis, management and the heart failure perspective

BackgroundArrhythmogenic cardiomyopathy (ACM) is an emerging new concept of a life-threatening heart muscle disorder due not only to desmosome gene mutations but also to non-desmosome genes, such as filamin C, lamin A/C, phospholamban, transmembrane protein 43, titin, SCN5A, and RNA binding motif protein 20.Multi-modality imaging along with genetic testing are important tools for risk stratification to tailor treatment to a single patient. Cardiac magnetic resonance imaging (CMR) with late gadolinium enhancement (LGE) is the gold standard for evaluating left and right ventricular structure and function, edema, and fibrosis. The identification of regional fibrosis with LGE has prognostic value.The management of ACM involves several aspects: treatment of arrhythmias and heart failure, risk stratification, implantable cardioverter-defibrillator (ICD) placement, exercise restrictions, and lifestyle changes. The decision for ICD placement in ACM patients is not well established and should be made weighing risks and benefits. However, the presence of specific genotypes can allow a precision medicine approach. In ACM patients with only mild left ventricular dysfunction but phospholamban, filamin C, or lamin A/C mutations, an ICD is now considered a reasonable approach. Aim of reviewWe sought to provide an overview of clinical and genetic features of arrhythmogenic cardiomyopathy providing epidemiology, imaging, diagnostic, and treatment information, using a systematic genetic approach. Key Scientific Concepts of ReviewACM is due not only to desmosome gene mutations but also to non-desmosome genes, such as filamin C, lamin A/C, phospholamban, transmembrane protein 43, titin, SCN5A, and RNA binding motif protein 20. The phenotypes of ACM spans from isolated right ventricular to biventricular or predominant left ventricular involvement. Due to heterogeneity, ACM remains an important diagnostic challenge requiring a multimodality imaging approach. -Genetic assessment may implement a precision medicine approach and should always be considered in the risk assessment and management.

Combination of arrhythmogenic right ventricular dysplasia with left ventricular non-compaction as a special form of cardiomyopathy: clinic, diagnostics, genetic, natural course

Abstract Background A few cases of combination of arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) with left ventricular noncompaction (LVNC) have been described. Aims To study the genetics, diagnostical features and clinical course of the combination of ARVC with LVNC. Materials and methods 58 patients with ARVC diagnosis (26 men; mean age 39.1±14.2 years; mean follow-up period – 21.5 [6; 60] months) and 125 patients with LVNC (74 men; mean age 46.4±15.1 years; mean follow-up period – 14 [3; 40] months). All patients underwent electrocardiogram (ECG), echocardiography, 24-h ECG monitoring. Heart MRI was performed in 53 (91.4%) patients with ARVC and 60 (48%) with LVNC, heart CT – in 18 (31%) patients with ARVC and 89 (71.2%) with LVNC. For all patients with combination of ARVC and LVNC DNA-diagnostic was performed using both ARVC (PKP2, DSG2, DSP, DSC2, JUP, TMEM43, TGFB3, PLN, LMNA, DES, CTTNA3, EMD, SCN5A, LDB3, CRYAB, FLNC) and LVNC (MYH7, MYBPC3, TAZ, TPM1, LDB3, MYL2, MYL3, ACTC1, TNNT2, TNI3) gene panels. Results Combination of ARVC and LVNC was found in 9 patients (15.5% of patients form ARVC cohort and 7.2% from LVNC cohort). These patients were distinguished from patients with isolated ARVC or LVNC by aggressive ventricular arrhythmias (frequent premature ventricular beats, sustained ventricular tachycardia, significantly worse antiarrhythmic therapy effect, appropriate shocks of implanted cardioverter-defibrillators (ICD) in all patients with ICD). Patients with combination of ARVC+LVNC were also distinguished from patients with isolated LVNC by the dilatation of RV, low QRS voltage on ECG, presence of AV block, absence of signs of LV hypertrophy on ECG. LV dilatation with reduction of its ejection fraction distinguished patients with mixed cardiomyopathy from patients with isolated ARVC. Potentially pathogenic variants (IV-V classes of pathogenicity) and variants of unclear clinical significance (III class of pathogenicity) were found in both desmosomal and non-desmosomal genes in 78% of patients, including 3 (33%) in DSP gene. Conclusions The combination of ARVC and LVNC can be caused by mutations in both desmosomal and non-desmosomal genes and has typical features: aggressive, resistant ventricular rhythm abnormalities leading to appropriate ICD shocks and a high risk of sudden cardiac death. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Genetic testing of patients was supported by Grant No. 16-15-10421 of the Russian Science Foundation

Abstract MP218: Cop9 Signalosome Subunit 6 Restricts Desmosomal Proteome Degradation To Prevent Desmosomal Targeted Cardiac Disease

Dysregulated protein degradative pathways are increasingly recognized as mediators of human cardiac disease. This pathway may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication. Genetic mutations in desmosomal genes resulting in the destabilization/breakdown of the desmosomal proteome are a central hallmark of all genetic-based desmosomal-targeted diseases, including the cardiac disease arrhythmogenic right ventricular (RV) dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here we identified a desmosomal resident regulatory complex, composed of subunit 6 of the COP9 signalosome (CSN6), enzymatically restricted neddylation and targets desmosomal proteome. Pharmacological restoration of CSN enzymatic function (via neddylation inhibitors) could rescue desmosomal protein loss in CSN6 deficient cardiomyocytes. Through the generation of two novel mouse models, we showed that cardiomyocyte-restricted CSN6 loss in mice selectively accelerated desmosomal destruction to trigger classic disease features associated with ARVD/C. We further showed that disruption of CSN6-mediated (neddylation) pathways underlined ARVD/C as CSN6 binding, localization, levels and function were impacted in hearts of classic ARVD/C mouse models and ARVD/C patients impacted by desmosomal loss and mutations, respectively. We anticipate our findings have broad implications towards understanding mechanisms driving desmosome degradation in other desmosomal-based diseases, such as cancers.

Abstract P449: Connexin43 As A Therapeutic For Arrhythmogenic Right Ventricular Cardiomyopathy

Limited efforts have been focused on the interventions which could therapeutically alter arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), a fatal cardiac disease of the desmosomal (mechanical) cell-cell junction. The desmosome is a critical target for intervention as mutations in desmosomal genes underlie 40-50% of ARVD/C populations and its dysregulation is associated with severe cardiac electrical and structural alterations, which facilitate myocardial failure, arrhythmias and premature death in these populations. Cardiomyocyte reduction of the predominant ventricular gap junction protein connexin43 is a molecular alteration that underlies desmosomal deficits and arrhythmias in ARVD/C. However, the role of connexin43 in structural alterations associated with ARVD/C remains unclear. We intervened with connexin43 reduction in human and mouse models of ARVD/C via connexin43 restoration strategies, which revealed beneficial effects in both ARVD/C models. We show ARVD/C human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes that recapitulate desmosomal structural defects and reveal connexin43 diminution alterations that are reflective of disease found in donor ARVD/C hearts. Connexin43 restoration was sufficient to rescue cardiac physiological deficits and increase desmosomal gene expressions in ARVD/C hiPSC derived cardiomyocytes, encompassing structural alterations. In vivo studies exploiting a mouse model of ARVD/C harboring severe desmosomal structural alterations revealed that cardiac connexin43 restoration was sufficient to prolong lifespan and restore cardiac desmosomal proteins. Herein, we provide evidence for non-canonical functions for connexin43, classically associated with electrical function, in the mechanical modulation of junctions. Our findings have broad implications in exploiting connexin43 as a therapeutic in advanced diseases associated with cardiac structural defects.

Neuropeptide Y promotes adipogenesis of human cardiac mesenchymal stromal cells in arrhythmogenic cardiomyopathy

BackgroundArrhythmogenic Cardiomyopathy (AC) is a familial cardiac disease, mainly caused by mutations in desmosomal genes. AC hearts show fibro-fatty myocardial replacement, which favors stress-related life-threatening arrhythmias, predominantly in the young and athletes. AC lacks effective therapies, as its pathogenesis is poorly understood. Recently, we showed that cardiac Mesenchymal Stromal Cells (cMSCs) contribute to adipose tissue in human AC hearts, although the underlying mechanisms are still unclear. PurposeWe hypothesize that the sympathetic neurotransmitter, Neuropeptide Y (NPY), participates to cMSC adipogenesis in human AC. MethodsFor translation of our findings, we combined in vitro cytochemical, molecular and pharmacologic assays on human cMSCs, from myocardial biopsies of healthy controls and AC patients, with the use of existing drugs to interfere with the predicted AC mechanisms. Sympathetic innervation was inspected in human autoptic heart samples, and NPY plasma levels measured in healthy and AC subjects. ResultsAC cMSCs expressed higher levels of pro-adipogenic isotypes of NPY-receptors (i.e. Y1-R, Y5-R). Consistently, NPY enhanced adipogenesis in AC cMSCs, which was blocked by FDA-approved Y1-R and Y5-R antagonists. AC-associated PKP2 reduction directly caused NPY-dependent adipogenesis in cMSCs. In support of the involvement of sympathetic neurons (SNs) and NPY in AC myocardial remodeling, patients had elevated NPY plasma levels and, in human AC hearts, SNs accumulated in fatty areas and were close to cMSCs. ConclusionsIndependently from the disease origin, AC causes in cMSCs a targetable gain of responsiveness to NPY, which leads to increased adipogenesis, thus playing a role in AC myocardial remodeling.

The Novel Desmin Variant p.Leu115Ile Is Associated With a Unique Form of Biventricular Arrhythmogenic Cardiomyopathy

BackgroundArrhythmogenic cardiomyopathy (AC) is a heritable myocardial disorder and a major cause of sudden cardiac death. It is typically caused by mutations in desmosomal genes. Desmin gene (DES) variants have been previously reported in AC but with insufficient evidence to support their pathogenicity. MethodsWe aimed to assess a large AC patient cohort for DES mutations and describe a unique phenotype associated with a recurring variant in three families. A cohort of 138 probands with a diagnosis of AC and no identifiable desmosomal gene mutations were prospectively screened by whole-exome sequencing. ResultsA single DES variant (p.Leu115Ile, c.343C>A) was identified in 3 index patients (2%). We assessed the clinical phenotypes within their families and confirmed cosegregation. One carrier required heart transplantation, 2 died suddenly, and 1 died of noncardiac causes. All cases had right- and left-ventricular (LV) involvement. LV late gadolinium enhancement was present in all, and circumferential subepicardial distribution was confirmed on histology. A significant burden of ventricular arrhythmias was noted. Desmin aggregates were not observed macroscopically, but analysis of the desmin filament formation in transfected cardiomyocytes derived from induced pluripotent stem cells, and SW13 cells revealed cytoplasmic aggregation of mutant desmin. Atomic force microscopy revealed that the mutant form accumulates into short protofilaments and small fibrous aggregates. ConclusionsDES p.Leu115Ile leads to disruption of the desmin filament network and causes a malignant biventricular form of AC, characterized by LV dysfunction and a circumferential subepicardial distribution of myocardial fibrosis.