Arrhythmogenic Cardiomyopathy Patients Research Articles

Mechanical and electrical uncoupling: the role of cardiac magnetic resonance imaging in arrhythmogenic cardiomyopathy. Proof of concept.

Arrhythmogenic cardiomyopathy (ACM) is a complex cardiac disorder associated with ventricular arrhythmias. Understanding the relationship between mechanical uncoupling and cardiac structural changes in ACM patients is crucial for improved risk stratification and management. In this study, we enrolled 25 ACM patients (median age 34 years, 72% men) based on the 2019 Modified Task Force and Padua criteria. Patients were categorized by the presence or absence of clinically relevant ventricular tachycardia (crVT), necessitating emergency interventions. Right ventricular-arterial coupling (VAC) was assessed using echocardiography. Low-rank regression splines were employed to model left ventricular ejection fraction (LVEF) and right ventricular ejection fraction (RVEF) in relation to VAC. Positive associations were observed between VAC and LVEF (ρ = 0.472, p = 0.023), RVEF (ρ = 0.522, p = 0.038), and right ventricular (RV) indexed stroke volume (ρ = 0.79, p < 0.001). Patients with crVT exhibited correlations with RV shortening, reduced RVEF (39.6 vs. 32.2%, p = 0.025), increased left ventricular (LV) mass (38.99 vs. 45.55, p = 0.045), and LV end-diastolic volume (LVEDV) (56.99 vs. 68.15 mL/m2, p = 0.045). Positive associations for VAC were noted with LVEDV (p = 0.039) and LV mass (p = 0.039), while negative correlations were observed with RVEF by CMR (p = 0.023) and RV shortening by echocardiography (p = 0.026). Our findings underscore the significance of right VAC in ACM, demonstrating correlations with RV and LVEF, RV stroke volume, and clinically relevant arrhythmias. Insights into RVEF, LV mass, and end-diastolic volume provide valuable contributions to the understanding of ACM pathophysiology and may inform risk assessment strategies.

A Comprehensive Analysis of Non-Desmosomal Rare Genetic Variants in Arrhythmogenic Cardiomyopathy: Integrating in Padua Cohort Literature-Derived Data.

Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disease at risk of sudden death. Genetic testing impacts greatly in ACM diagnosis, but gene-disease associations have yet to be determined for the increasing number of genes included in clinical panels. Genetic variants evaluation was undertaken for the most relevant non-desmosomal disease genes. We retrospectively studied 320 unrelated Italian ACM patients, including 243 cases with predominant right-ventricular (ARVC) and 77 cases with predominant left-ventricular (ALVC) involvement, who did not carry pathogenic/likely pathogenic (P/LP) variants in desmosome-coding genes. The aim was to assess rare genetic variants in transmembrane protein 43 (TMEM43), desmin (DES), phospholamban (PLN), filamin c (FLNC), cadherin 2 (CDH2), and tight junction protein 1 (TJP1), based on current adjudication guidelines and reappraisal on reported literature data. Thirty-five rare genetic variants, including 23 (64%) P/LP, were identified in 39 patients (16/243 ARVC; 23/77 ALVC): 22 FLNC, 9 DES, 2 TMEM43, and 2 CDH2. No P/LP variants were found in PLN and TJP1 genes. Gene-based burden analysis, including P/LP variants reported in literature, showed significant enrichment for TMEM43 (3.79-fold), DES (10.31-fold), PLN (117.8-fold) and FLNC (107-fold). A non-desmosomal rare genetic variant is found in a minority of ARVC patients but in about one third of ALVC patients; as such, clinical decision-making should be driven by genes with robust evidence. More than two thirds of non-desmosomal P/LP variants occur in FLNC.

Cardiomyocyte-stromal cell interplay modulates the pathogenesis of arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Transnational Research Projects on Cardiovascular Diseases Background Arrhythmogenic Cardiomyopathy (ACM) is an inherited heart disorder characterized by a high incidence of sudden death at young age. Mutations linked to ACM occur primarily in desmosomal genes (e.g. PKP2) [1]. Different cell types individually contribute to set the ACM phenotype with either functional abnormalities (cardiomyocytes; CM) [2]; or fibro-fatty substitution (cardiac mesenchymal stromal cells; cMSC) [3]. The relative contribution of the different cell type derangements to determine disease evolution and electrical instability is still unclear. Purpose We hypothesize that the use of a multicellular cardiac system, modelling the functional interaction between CM and cMSC, will allow the understanding of the relative contribution of the different cell types to the ACM phenotypes. Methods Primary cMSC (carrier of the PKP2 mutation c.2013delC) have been collected from a right ventricle biopsy sample; control cMSC obtained from a biobank. iPSC reprogrammed from the same ACM patient and the isogenic line in which the PKP2 mutation was corrected [4] were used. Cardiac cocultures (cc) were assembled by combining 85% iPSC-CM and 15% primary cMSC (as in Figure 1A), either mutated or control, in different combination (as in Figure 1B). Fibro-fatty accumulation have been evaluated by immunofluorescence analysis. Movies of contracting clusters have been collected and analysed by the MUSCLEMOTION tool [5]. Cell-cell communication was studied in silico by InterCellar analysis [6] on the single culture transcriptomes. The assessment of the paracrine interactions was performed on the cc secretome by Olink technology. Results Immunofluorescence analysis highlighted that ACM cc (cc 4) accumulate more lipids and collagen than healthy control (HC) ones (cc 1). Since intermediate fibro-fatty levels were observed in ‘mixed cc’ (cc 2 and 3), we conclude that ACM iPSC-CM are able to influence fibro-adipo-commitment of cMSC. Contraction studies showed a high propensity for cc monolayers which include ACM iPSC-CM (cc3 and cc4) to display arrhythmic events. An increase in the percentage of arrhythmic events was triggered when the ACM cMSC were replacing HC cMSC, indicating that ACM cMSC can affect iPSC-CM rhythm. The most abundant ligand-receptor couplets regarded cell-cell adhesion, cell-matrix coupling, cell differentiation and inflammation. Differential abundance between the four cc was observed mainly for secreted factors involved in cardiac fibrosis, inflammation, adipogenesis / lipid metabolism and heart failure. In particular, DLK-1, secreted only by HC CM, is validated as novel inhibitor of ACM fibro-adipose differentiation. Conclusion Overall, our novel simple multicellular ACM cell model can recapitulate different phenotypes of ACM and contributed to understand the relative contribution of the different cell types to ACM features (e.g. fibro-adipose replacement, contractile defects and proarrhythmic events).

An antisense lncRNA mediated mechanism controls desmoplakin gene expression

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy). Background Desmosomal genes are established as definitive genes for Arrhythmogenic Cardiomyopathy (ACM). The desmoplakin encoding gene, DSP, is amongst the most frequently mutated genes in ACM patients. While rare genetic variants within desmosomal genes can cause ACM, common variants might also affect cardiac conduction traits in the general population. Purpose 1) To explore the association between common genetic variants in ACM desmosomal genes (DSC2, DSG2, PKP2, JUP, DSP) and cardiac conduction traits. 2) To investigate the effect of a lncRNA, identified from downstream analyses, on DSP gene expression. Methods P-wave, PR, QRS and QT intervals were measured using standard 12-lead electrocardiograms (ECG) in 4295 participants to the Cooperative Health Research in South Tyrol (CHRIS) study (data release 2). Samples were genotyped using the Illumina HumanOmniExpressExome array and imputed on the 1000 Genome Phase 1 panel. We conducted a genetic association study on 3458 variants within the linkage disequilibrium blocks that encompass desmosomal genes, assuming an additive genetic model, at 1.9x10-4 statistical significance. Mendelian Randomization (MR) analyses integrating ECG traits and Genotype-Tissue Expression (GTEx) database transcription levels were performed. A specific GapmeR was used to downregulate the antisense RP3-512B11.3 lncRNA expression in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs lysates were analyzed by digital droplet PCR and Wes™ immunoassay system. Results The rs2744389 variant in DSP gene is associated with a shorter QRS interval (-1.10 ms per effect allele copy; p=3.5x10-6). In the GTEx database, rs2744389 is identified as an expression quantitative trait locus for the antisense lncRNA. MR results support a causal effect of the lncRNA on QRS. To assess the hypothesis that the lncRNA regulates DSP gene expression, we identified a Gapmer able to downregulate the lncRNA expression by approximately 60% in hiPSC-CMs. The lncRNA downregulation led to a significant increase of DSP expression, both at mRNA and protein level, compared to hiPSC-CMs treated with a negative control Gapmer (DSP/RPP30: 16.39±3.32 vs 11.84±1.79; p=0.0313), (Desmoplakin/total protein: 2.44±0.29 vs 1.66±0.11; p=0.0156). Conclusions Starting with the identification of an association between a common genetic variant in DSP gene and a significantly shorter QRS interval in a general population sample, we further unrevealed a new mechanism regulating DSP expression through an antisense lncRNA. While requiring further evidence, these findings might pave the way to a potentially novel therapeutic approach for DSP mutations carriers characterized by desmoplakin protein deficit, as for example patients affected by ACM, but also by dilated cardiomyopathy, cardiocutaneous diseases such as the Carvajal syndrome, and some cancer conditions.

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.

Epicardial catheter ablation of ventricular tachycardia in patients of acm - from a university hospital report in switzerland

Abstract Background Arrhythmogenic left ventricular cardiomyopathy (ALVC) as a subtype of arrhythmogenic cardiomyopathy (ACM) is increasingly being recognized. Cardiac magnetic resonance (CMR) has emerged as the primary imaging modality for the diagnosis of ACM. In patients with right-dominant ACM (ARVC) and recurrent sustained ventricular tachycardia (VT), endocardial +/-epicardial ablation yields good clinical outcomes in the long-term. However, studies on VT ablation in ACM with LV involvement are scarce. Purpose We sought to investigate clinical outcomes of VT ablation in ACM patients with LV involvement. Methods The study included patients from the Zurich ACM Registry who met 2020 "Padua Criteria" for ACM and underwent VT ablation between January 2018 and July 2023. Epicardial ablation was performed in patients with recurrent sustained VT despite endocardial ablation, those lacking and endocardial substrate or if the ECG of the VT / substrate on CMR was suggestive of an epicardial origin. Catheter ablation was guided by activation/entrainment mapping for mappable VT, and pace mapping/voltage mapping during sinus rhythm for unmappable VT. Consecutive follow-up of all patients was performed according to our protocol. Results Twenty-one ACM patients underwent n=24 VT ablation procedures. Of those 20 patients, 6 patients (30%) had LV involvement on CMR. Eight (33%) patients underwent epicardial +/- endocardial ablation. Among patients receiving epicardial ablation, 3 patients (38%) had LV involvement. These three underwent a single ablation procedure, which was epicardial only. Total follow-up was 30 months (range 4-43). Three-month sustained VT/VF recurrence rates were 12% in the ACM group with LV involvement vs 25% in ACM without LV involvement. All patients with sustained VT/VF recurrence underwent repeat ablation via an epicardial approach within 3 months and were free of sustained VT/VF thereafter during a median follow-up of 12 months. One-year recurrence rate did not differ between ACM patients with LV involvement vs. without LV involvement (7.7% vs 8.3%, P＝0.117). Seven out of eight epicardial ablations achieved freedom from VT/VF after one-year follow-up. One ARVC patient with previous endocardial ablation had recurrent VT one month after epicardial ablation, which was free of VT after Flecainide combined with beta-blocker. No significant differences were found in the use of beta-blockers and antiarrhythmic drugs between both groups (ACM with LV involvement vs. without LV involvement) at baseline and during one-year follow-up. Conclusions Single or adjuvant epicardial substrate ablation of VT in arrhythmogenic cardiomyopathy is promising in terms of postprocedural VT free survival, especially in patients with left ventricular involvement demonstrated by CMR.Figure 1Figure 2

Chronic inflammatory cardiomyopathy and its overlap with arrhythmogenic cardiomyopathy: long-term follow-up of a prospective single-centre cohort study

Abstract Background Chronic Inflammatory Cardiomyopathy (infl-CMP) may overlap with Arrhythmogenic Cardiomyopathy (ACM), a group of conditions with similar ventricular arrhythmic phenotypes and key imaging, electrocardiographic, histopathological, and genetic features. Purpose This single-centre cohort study aimed to assess the role of pathogenic genetic variants of ACM in long-term follow-up of patients with infl-CMP and ventricular arrhythmic phenotype. Methods In this prospective, single-centre study, 24 consecutive patients with suspected infl-CMP and ventricular arrhythmic phenotype were enrolled at our tertiary referral hospital between 2017 and 2022. Baseline demographic and clinical characteristics of the patients were collected. All patients underwent extensive workup, including electrocardiography, echocardiography, cardiac magnetic resonance, and genetic counselling and testing. Endomyocardial biopsy was performed if a definite diagnosis had not been reached after non-invasive diagnostic workup. Patients were classified according to phenotype as ACM (either definite or borderline), as indicated by the 2010 Padua Criteria, and genetically classified as genotype-positive or genotype-negative according to the presence of pathogenic variants for ACM. Long-term follow-up was performed to assess cardiovascular morbidity and mortality. The primary outcome was the occurrence of in-hospital admission for sustained ventricular tachyarrhythmias. Results At admission, 5 (21%) patients presented with syncope, 19 (79%) with high ventricular burden (&amp;gt;25% premature ventricular contractions) or with sustained ventricular tachyarrhythmias. After complete diagnostic work-up, 17 patients (71%) received a diagnosis of either definite or borderline ACM, while the remaining 7 patients (29%) received a diagnosis of infl-CMP. In 18 patients (75.0%), an endomyocardial biopsy was required as part of the diagnostic workup. In our population, the frequency of pathogenic ACM variants was 41% (8 variants in ACM patients and 2 variants in infl-CMP); in particular, pathogenic genetic variants coexisted in 2 patients (1 in the ACM group: PKP2 and KCNE1 mutations; 1 in the infl-CMP group: DSG2 and TMEM43 mutations). At a mean follow-up of 4.37 (SD ±1.77) years, the primary outcome occurred in 8 patients (33%), all identified as borderline or definite ACM (X²=4.94, p=0.026). Notably, sustained ventricular tachyarrhythmias occurred in 25% of genotype-negative and 50% of genotype-positive patients (X²=1.50, p=0.220). Conclusions Criteria-driven diagnosis of ACM was significantly associated with the occurrence of sustained ventricular tachyarrhythmias at long-term follow up. The presence of pathogenic genetic variants of ACM did not clearly stratify the ventricular arrhythmic risk as a single factor in our cohort of patients with ventricular arrhythmic phenotype.

Mechanical and electrical uncoupling: the role of cardiac magnetic resonance imaging in arrhythmogenic cardiomyopathy proof of concept

Abstract Funding Acknowledgements None. Introduction Arrhythmogenic Cardiomyopathy (ACM) has a high risk of sudden cardiac death, especially under 60 years old (1:5,000 prevalence). ACM involves the replacement of normal heart tissue with fibro-adipose tissue in specific areas of the right ventricle (RV). Cardiac Magnetic Resonance Imaging (CMR) is vital for ACM stratification and prognosis, focusing on RV volume and ejection fraction. Simulations suggest that ACM's abnormal deformation patterns are primarily due to mechanical changes like reduced contractility and increased stiffness, rather than electrical issues. Ventriculo-arterial coupling (VAC) is a valuable metric for assessing RV efficiency, with the TAPSE/sPAP index reflecting the RV's length-force relationship. CMR assists in ACM stratification and prognosis, with VAC potentially serving as a key predictor of outcomes based on mechanical and structural abnormalities identified through CMR. Purpose Examine and establish a relationship between mechanical uncoupling, ventricular arrhythmias, and findings from CMR in patients with ACM. Methods We examined 25 ACM patients (median age 34, 72% men) using the 2019 Modified Task Force and Padua criteria. We investigated two key factors in ACM: Right ventricular-arterial coupling (TAPSE/SPAP ratio) and clinically relevant ventricular tachycardia (requiring cardioversion). We compared clinical, ECG, and imaging data (transthoracic echocardiogram and CMR as the gold standard). We used Spearman rank tests for correlations, Fisher exact tests for nominal characteristics, and Mann-Whitney-U tests for group comparisons. In predefined regression models involving left ventricular ejection fraction (LVEF) and right ventricular ejection fraction (RVEF) function with VAC, we used flexible nonparametric regressions. A p-value &amp;lt;0.05 was considered statistically significant. The analysis was conducted using SPSSv22 and SAS-University-Edition. Results A positive association of VAC with LVEF (ρ=0.472, p=0.023), RVEF (ρ=0.522, p=0.038) and RV indexed stroke volume (ρ=0.79, p&amp;lt;0.001) was found. Low-rank regression splines showed a mainly positive association of LVEF (df=3 F=20.75 p&amp;lt;0.001), RVEF (df=4 F=24.34 p&amp;lt;0.001) and the RV indexed stroke volume, a positive linear association (df=1 F=31.67 p&amp;lt;0.001). In comparison with patients without crVT, those with crVT had a positive correlation with RV shortening, reduced RVEF (39.6 vs. 32.2%. p=0.025), higher LV mass (38.99 vs. 45.55, p=0.045), and LV end-diastolic volume (LVEDV) (56.99 vs. 68.15 ml/m2, p=0.045) by echocardiogram and CMR respectively; notwithstanding positive associations for VAC were seen with LVEDV (p=0.039), LV mass (p=0.039); and negative with RVEF by CMR (p=0.023), and RV shortening by echo (p=0.026). Conclusions We found that right VAC by echo correlates with RV and LV EF by CMR and RV stroke volume in patients with ACM. RVEF, LV mass, and end-diastolic volume correlate with clinically relevant arrhythmias.

3D Fractal Dimension Analysis: Prognostic Value of Right Ventricular Trabecular Complexity in Participants with Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive myocardial fibro-fatty infiltration accompanied by trabecular disarray. Traditionally, two-dimensional (2D) instead of 3D fractal dimension (FD) analysis has been used to evaluate trabecular disarray. However, the prognostic value of trabecular disorder assessed by 3D FD measurement remains unclear. To investigate the prognostic value of right ventricular trabecular complexity in ACM patients using 3D FD analysis based on cardiac MR cine images. Retrospective. 85 ACM patients (mean age: 45 ± 17 years, 52 male). 3.0T/cine imaging, T2-short tau inversion recovery (T2-STIR), and late gadolinium enhancement (LGE). Using cine images, RV (right ventricular) volumetric and functional parameters were obtained. RV trabecular complexity was measured with 3D fractal analysis by box-counting method to calculate 3D-FD. Cox and logistic regression models were established to evaluate the prognostic value of 3D-FD for major adverse cardiac events (MACE). Cox regression and logistic regression to explore the prognostic value of 3D-FD. C-index, time-dependent receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) to evaluate the incremental value of 3D-FD. Intraclass correlation coefficient for interobserver variability. P < 0.05 indicated statistical significance. 26 MACE were recorded during the 60 month follow-up (interquartile range: 48-67 months). RV 3D-FD significantly differed between ACM patients with MACE (2.67, interquartile range: 2.51 ~ 2.81) and without (2.52, interquartile range: 2.40 ~ 2.67) and was a significant independent risk factor for MACE (hazard ratio, 1.02; 95% confidence interval: 1.01, 1.04). In addition, prognostic model fitness was significantly improved after adding 3D-FD to RV global longitudinal strain, LV involvement, and 5-year risk score separately. The myocardial trabecular complexity assessed through 3D FD analysis was found associated with MACE and provided incremental prognostic value beyond conventional ACM risk factors. 4 TECHNICAL EFFICACY: Stage 1.

Intermuscular placement and defibrillation with the extravascular ICD: a single-centre experience

Abstract Background Extravascular (EV) implantable cardiac defibrillators (ICD) with substernal lead have recently proved to be a safe and effective alternative to conventional transvenous (TV) and subcutaneous defibrillators (S-ICD), owing to the possibility to deliver antitachycardia and asystole pacing while saving intravascular hardware. The proposed implantation technique for EV-ICDs is a subcutaneous pocket along the mid axillary line, although an intermuscular ICD placement can potentially reduce the risk of pocket complications, device migration and improve patient comfort and aesthetic outcomes. Purpose exploring the safety and feasibility of extravascular ICDs implantation with a substernal coil and an intermuscular generator placement. Methods Seventeen patients with a class I or IIa ICD indication either for primary (14/17) or secondary (3/17) prevention underwent EV-ICD implantation in our centre as part of the EV ICD Pivotal Study from May 2021 to November 2022. Access and tunnelling along the retrosternal space were performed based on current standard recommendations for EV-ICDs implantation. An intermuscular pocket for generator placement was obtained between the latissimus dorsi and the serratus anterior muscles beyond the left mid-axillary line. Results Mean age, BMI, NYHA class and left ventricular ejection fraction (LVEF) were respectively 53±9yrs, 25.3 ±6 kg/m2, 2 ±0.7, and 39±14%. The main aetiology was non ischemic cardiomyopathy (12/17), including 3 arrhythmogenic cardiomyopathy patients and one with hypertrophic cardiomyopathy. The implantation was successful in 16 (6 females) out of 17 patients, with one implant failure due to inadequate sensing (0,5 mV); the median sensing amplitude was 2,3 mV (min = 1,3 mV; max = 5,8 mV), with R1R2 being the final sensing vector chosen for 14/16 and R2-Can for 2/16. Defibrillation Testing (DFT) was performed in all patients: a 15 J shock terminated induced ventricular fibrillation (VF) in 12/16 patients, 20 J terminated VF in 3, a single patient had VF terminated at 30 J. No patient had infection or skin erosion or complained pain/discomfort at the pocket site along an 18±4 months follow-up. Only 1/16 patients had ventricular tachycardia at follow-up, successfully treated by the device. Conclusions Intermuscular placement of the EV ICD may be a feasible and reliable alternative to a subcutaneous one in terms of defibrillation efficacy, enabling patient comfort and aesthetic acceptance in thin-habit or young patients. Additional long-term data and larger patient cohorts are required to confirm its advantage compared to the subcutaneous placement.

Lead placement in extravascular ICD recipients: the first step to prevent P-wave oversensing

Abstract Background Extravascular (EV) implantable cardiac defibrillators (ICD) with substernal lead have recently proved to be a safe and effective alternative to conventional transvenous (TV) and subcutaneous defibrillators (S-ICDs) 1, owing to the possibility to deliver antitachycardia and asystole pacing while saving intravascular hardware. However, inappropriate shock delivery occurred in 9.7% of patients, and P-wave oversensing accounted for 42% of inappropriate shocks in the EV ICD Pivotal Study. Purpose to investigate the difference of lead placement in patients with and without P-wave oversensing. Method 17 patients undergoing implantation as part of the EV ICD Pivotal Study received the substernal lead placement based on computer tomography imaging (CT) and following the protocol recommendations. Lead placement beyond the left lateral sternum border was attempted whenever possible, based on CT guidance. P-wave detectability (P detected visually but non sensed by the device) on the sensing vector and P-wave oversensing by the device were investigated at each follow-up. The distance of sensing ring electrodes (R1 and R2) from the mid-spine line (line connecting the spinous processes from cervical to lumbar vertebral processes) was measured in each patient on the posterior-anterior chest radiogram. Results Mean age, BMI, NYHA class and left ventricular ejection fraction (LVEF) were respectively 53±9yrs, 25.3 ±6 kg/m2, 2 ±0.7, and 39±14%. The main aetiology was non-ischemic cardiomyopathy (12/17), including 3 arrhythmogenic cardiomyopathy patients and one with hypertrophic cardiomyopathy. The implantation was successful in 16 (6 females) out of 17 patients without any complication, with one implant failure due to inadequate sensing (0,5 mV) despite several attempts at tunnelling in different locations (rightward and leftward of the sternum). One patient with P-wave detectability had lead dislodgement after 2 weeks, and underwent repositioning to a more leftward location, where P-wave was not detectable. Only one patient had P-wave oversensing at follow-up, and received inappropriate shocks (Figure A); the remaining 15 patients neither had detectable P-wave on the sensing vector nor inappropriate shocks (Figure B). The median sensing amplitude at implantation was 2,7±1.4 mV and 2.6±1.2 mV along a 18±4 months follow-up, with R1-R2 being the final sensing vector chosen for 14/16 and R2-Can for 2/16 patients. The distance of R1 and R2 from the mid-spine line was significantly longer in patients with detectable P-wave compared to those without (37±13mm vs 18 mm and 30±15 vs 16 mm, respectively). Conclusions Placement of the EV ICD lead leftward to the left sternum border is safe, and prevents P-wave detection. The mid-spine line represents an easy landmark to guide lead placement during implantation. R1 distance ≥ 25 mm and R2 distance ≥ 20 mm from the mid-spine line provide a reliable marker of freedom from P-wave oversensing.Figure AFigure B

Abstract 15358: Deletion of Csf2 Accelerates Disease Progression in a Mouse Model of Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is a familial heart muscle disease driven by persistent innate immune signaling. We have shown that ventricular fibrosis and contractile dysfunction in Dsg2 mut/ mut mice, a model of ACM with homozygous knock-in of a variant in the gene for the desmosomal protein desmoglein-2, is mediated by activation of NFκB and actions of pro-inflammatory macrophages that express CCR2. To gain further insights into the roles of specific immune signaling pathways in the pathogenesis of ACM, we crossed Dsg2 mut/ mut mice with mice with germline deletion of Csf2 which encodes GM-CSF. This factor acts to mobilize inflammatory cells to sites of injury. Csf2 -/- mice have a normal leukocyte repertoire and no apparent phenotype under basal conditions, but show reduced immune cell infiltration after MI associated with improved healing and cardiac function. Thus, we anticipated delayed onset and less severe disease in Dsg2 mut/ mut Csf2 -/- mice compared with Dsg2 mut/ mut mice. In fact, the opposite occurred. By 8-weeks of age, Dsg2 mut/ mut Csf2 -/- mice showed a marked increase in ventricular fibrosis (18.2±6.2% of section area; n=11; p&lt;0.05 vs all) compared to Dsg2 mut/ mut (2.6±0.4%; n=27) or wildtype (WT) control (0.9±0.1%; n=18) mice. Dsg2 mut/ mut Csf2 -/- mice also had reduced LV ejection fractions (49.6±8.3%; n=11; p&lt;0.05 vs all) compared to Dsg2 mut/ mut (64.5±3.2%; n=27) or WT (81.8±1.0%; n=18) mice. Hearts of 8-week Dsg2 mut/ mut Csf2 -/- mice contained increased levels of pro-inflammatory chemokines (CCL21, CXCL1), matrix metalloproteinases, osteopontin (Spp1) and periostin (Postn). Single nucleus RNA sequencing studies revealed increased numbers of Spp1 + macrophages and Postn + fibroblasts, and a marked decrease in cDC2 dendritic cells in hearts of Dsg2 mut/ mut Csf2 -/- mice compared to Dsg2 mut/ mut or WT mice. cDC2 dendritic cells are known to have anti-inflammatory actions. These results suggest that cDC2 cells, mobilized by GM-CSF, may act to mitigate disease onset and severity in ACM. If so, cDC2 cells could serve as a determinant of disease expression in ACM patients.

Undiagnosed cardiomyopathy: An incidental finding in a recreational soccer player—A case report and literature review

AbstractIntroductionArrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disease characterised by progressive fibrofatty tissue replacement of the myocardium. Several arrhythmogenic cardiomyopathy (ACM) phenotypes are now recognised, including right‐dominant, biventricular, and left‐dominant variants, which led to the development of the 2020 International criteria (Padua criteria).Raising awareness of these variants is crucial as their clinical entity can be concealed. This is particularly important in the athletic population. Exercise can promote the development of the phenotype and accelerate disease progression, resulting in left ventricular (LV) involvement and systolic dysfunction, which can cause ventricular arrhythmias and sudden death.Case DescriptionA late adolescent male was escorted by paramedics to an emergency department following a medium‐speed motor vehicle accident. A multimodality approach was implemented involving a 12‐lead electrocardiogram (ECG), transthoracic echocardiography (ECHO) and cardiac magnetic resonance (CMR) imaging. An unknown cardiomyopathy was revealed with diagnostic clues suggesting biventricular ACM in a recreational soccer player, when using the Padua criteria.ConclusionACM should be considered in the evaluation of an unexplained cardiomyopathy presenting with possible syncopal events. This case describes the clinical features of an ACM patient, emphasising the utility of ECG, ECHO and CMR in determining biventricular involvement.CMR is a powerful tool for the diagnosis of ACM and the identification of myocardial fibrosis with late gadolinium enhancement (LGE). The use of 12‐lead ECG and 2‐dimensional (2D) strain imaging may also raise suspicion of biventricular phenotypes and predict LV involvement with significant myocardial fibrosis.

Sudden Cardiac Death in Biventricular Arrhythmogenic Cardiomyopathy: A New Undescribed Variant of the MYH6 Gene

Arrhythmogenic cardiomyopathy (ACM) may present with sudden cardiac arrest (SCA), and demonstration of a pathogenic variant in ACM-related genes is crucial for its definitive diagnosis. A 42-year-old female patient with family history of sudden cardiac death (SCD) was referred to the cardiomyopathy clinic after two episodes of aborted SCA. In the second episode, the patient was transported under cardiopulmonary resuscitation (downtime of 57 min) until extracorporeal membrane oxygenation was implanted. A thorough diagnostic work-up led to a diagnosis of biventricular ACM. Genetic testing revealed a previously undescribed variant in ACM patients in the MYH6 gene, c.3673G&gt;T p.(Glu 1225*), which inserts a premature stop codon. This was considered a possible pathogenic variant originating a truncated protein, previously undescribed in ACM. The patient’s 23-year-old daughter was positive for the MYH6 variant and had ECG abnormalities suggestive of ACM. This case details the complex differential diagnosis of SCA and explores the current recommendations for the diagnosis of biventricular ACM. The identification of a MYH6 variant in a patient with ACM, recurrent SCA, and family history of SCD appears to support the hypothesis of the pathogenicity of MYH6 variants in ACM, in which the association of phenotype with sarcomere variants is still unclear.

Proposed diagnostic criteria for arrhythmogenic cardiomyopathy: European Task Force consensus report

Arrhythmogenic cardiomyopathy (ACM) is a heart muscle disease characterized by prominent “non-ischemic” myocardial scarring predisposing to ventricular electrical instability. Diagnostic criteria for the original phenotype, arrhythmogenic right ventricular cardiomyopathy (ARVC), were first proposed in 1994 and revised in 2010 by an international Task Force (TF). A 2019 International Expert report appraised these previous criteria, finding good accuracy for diagnosis of ARVC but a lack of sensitivity for identification of the expanding phenotypic disease spectrum, which includes left-sided variants, i.e., biventricular (ABVC) and arrhythmogenic left ventricular cardiomyopathy (ALVC). The ARVC phenotype together with these left-sided variants are now more appropriately named ACM. The lack of diagnostic criteria for the left ventricular (LV) phenotype has resulted in clinical under-recognition of ACM patients over the 4 decades since the disease discovery. In 2020, the “Padua criteria” were proposed for both right- and left-sided ACM phenotypes. The presently proposed criteria represent a refinement of the 2020 Padua criteria and have been developed by an expert European TF to improve the diagnosis of ACM with upgraded and internationally recognized criteria. The growing recognition of the diagnostic role of CMR has led to the incorporation of myocardial tissue characterization findings for detection of myocardial scar using the late‑gadolinium enhancement (LGE) technique to more fully characterize right, biventricular and left disease variants, whether genetic or acquired (phenocopies), and to exclude other “non-scarring” myocardial disease. The “ring-like’ pattern of myocardial LGE/scar is now a recognized diagnostic hallmark of ALVC. Additional diagnostic criteria regarding LV depolarization and repolarization ECG abnormalities and ventricular arrhythmias of LV origin are also provided. These proposed upgrading of diagnostic criteria represents a working framework to improve management of ACM patients.

Patient-specific primary and pluripotent stem cell-derived stromal cells recapitulate key aspects of arrhythmogenic cardiomyopathy

Primary cardiac mesenchymal stromal cells (C-MSCs) can promote the aberrant remodeling of cardiac tissue that characterizes arrhythmogenic cardiomyopathy (ACM) by differentiating into adipocytes and myofibroblasts. These cells’ limitations, including restricted access to primary material and its manipulation have been overcome by the advancement of human induced pluripotent stem cells (hiPSCs), and their ability to differentiate towards the cardiac stromal population. C-MSCs derived from hiPSCs make it possible to work with virtually unlimited numbers of cells that are genetically identical to the cells of origin. We performed in vitro experiments on primary stromal cells (Primary) and hiPSC-derived stromal cells (hiPSC-D) to compare them as tools to model ACM. Both Primary and hiPSC-D cells expressed mesenchymal surface markers and possessed typical MSC differentiation potentials. hiPSC-D expressed desmosomal genes and proteins and shared a similar transcriptomic profile with Primary cells. Furthermore, ACM hiPSC-D exhibited higher propensity to accumulate lipid droplets and collagen compared to healthy control cells, similar to their primary counterparts. Therefore, both Primary and hiPSC-D cardiac stromal cells obtained from ACM patients can be used to model aspects of the disease. The choice of the most suitable model will depend on experimental needs and on the availability of human source samples.

Store-Operated Ca2+ Entry as a Putative Target of Flecainide for the Treatment of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder that may lead patients to sudden cell death through the occurrence of ventricular arrhythmias. ACM is characterised by the progressive substitution of cardiomyocytes with fibrofatty scar tissue that predisposes the heart to life-threatening arrhythmic events. Cardiac mesenchymal stromal cells (C-MSCs) contribute to the ACM by differentiating into fibroblasts and adipocytes, thereby supporting aberrant remodelling of the cardiac structure. Flecainide is an Ic antiarrhythmic drug that can be administered in combination with β-adrenergic blockers to treat ACM due to its ability to target both Nav1.5 and type 2 ryanodine receptors (RyR2). However, a recent study showed that flecainide may also prevent fibro-adipogenic differentiation by inhibiting store-operated Ca2+ entry (SOCE) and thereby suppressing spontaneous Ca2+ oscillations in C-MSCs isolated from human ACM patients (ACM C-hMSCs). Herein, we briefly survey ACM pathogenesis and therapies and then recapitulate the main molecular mechanisms targeted by flecainide to mitigate arrhythmic events, including Nav1.5 and RyR2. Subsequently, we describe the role of spontaneous Ca2+ oscillations in determining MSC fate. Next, we discuss recent work showing that spontaneous Ca2+ oscillations in ACM C-hMSCs are accelerated to stimulate their fibro-adipogenic differentiation. Finally, we describe the evidence that flecainide suppresses spontaneous Ca2+ oscillations and fibro-adipogenic differentiation in ACM C-hMSCs by inhibiting constitutive SOCE.

Abstract P2060: Gja1-20k Expression Increases With Left Ventricular Mechanical Unloading In Patients With Arrhythmogenic Cardiomyopathy And Advanced Heart Failure

GJA1-20k, the internally translated isoform of the gap junction protein Connexin-43 (Cx43) is required for trafficking of the full length Cx43 to the membrane. We recently found that GJA1-20k expression is reduced in patients and mice with arrhythmogenic cardiomyopathy (ACM), and that gene therapy-mediated introduction of GJA1-20k in a mouse model of ACM rescues Cx43 trafficking to the intercalated disc and reduces arrhythmia burden, indicating that loss of GJA1-20k contributes to arrhythmogenesis in ACM. Here, we examined cardiac muscle from patients with a confirmed diagnosis of ACM for GJA1-20k expression before and after left ventricular assist device (LVAD) support. The goal of this study was to determine if left ventricular (LV) unloading benefits endogenous expression of this critical trafficking protein. Cardiac tissue was collected from consecutive, prospectively enrolled patients undergoing LVAD implantation or heart transplantation in hospitals comprising the Utah Cardiac Recovery program and donor patients from hospitals throughout the Intermountain West. Samples were prepared for analysis using a standard lysis buffer and analyzed via Western Blotting using antibodies to GAPDH and Cx43-CT. Consistent with prior findings, cardiac muscle samples from patients with ACM had decreased GJA1-20k expression relative to muscle from healthy donor hearts not allocated for transplantation. However, tissue obtained from the same ACM patients post LVAD support (at the time of heart transplantation) revealed a large and significant increase in GJA1-20k expression compared to the pre LVAD timepoint (1 +/- 0.5 vs 10.2+/- 1, p=0.0013, n=3 ACM pre/post). In contrast to patients with ACM, patients with non-ischemic cardiomyopathy (NICM) that underwent LVAD implantation had no significant change in GJA1-20k expression after LV unloading (1+/- 0.5 vs 1.4+/- 0.7, p= 0.424, n=4 NICM pre/post). These data indicate that LV mechanical unloading increases GJA1-20k expression in hearts of patients with known ACM. Further work will focus on how mechanical unloading regulates GJA1-20k expression and whether mechanical unloading is sufficient to recover Cx43 trafficking and limit arrhythmias in ACM.

Abstract GS.12: A New Mouse Model To Investigate The Early Steps Of Arrhythmogenic Cardiomyopathy

Arrhythmogenic Cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrosis, systolic dysfunction and life-threatening arrhythmias. Mutations in genes of the desmosomal adhesion complex such as desmoglein-2 (Dsg2) are the major cause, but the underlying mechanisms leading to the disease are not well understood. Accordingly, only symptomatic treatment is available. Here, we establish an inducible Dsg2-W2A knock-in mouse model with temporal control of disease onset as new approach to analyse the early processes underlying the ACM phenotype. We have previously characterized a constitutive Dsg2-W2A knock-in model which develops a cardiac phenotype bearing all major criteria of ACM directly after birth. To control the onset of disease, we now generated a mouse line bearing the Dsg2-W2A mutation and a loxP -flanked Dsg2 on separate alleles, leading to functional hemizygosity of the Dsg2-W2A mutation upon tamoxifen-induced Cre expression. Detailed characterization of this iDsg2-W2A model revealed early repolarization changes in ECG recordings starting directly after induction and progression to extensive impairment of electrical conduction over 10 weeks. These changes were paralleled with the appearance of ventricular and supraventricular arrhythmias. Starting two weeks after induction, reduced cardiac output function of the right and later of the left ventricle was detectable via echocardiography. Finally, these mice developed structural changes with loss of cardiomyocytes and ventricular fibrosis similar to ACM patients. This study gives detailed insight on the early steps of ACM showing a sequence from changes in conduction, occurrence of arrhythmia to functional impairment, starting in the right and progressing to the left ventricle, together with structural changes of the heart. We expect this model to be valuable to discern patho-mechanisms in detail and to identify and test novel treatment options for ACM.

Abstract P2169: Identification Of Pro-adhesive Drugs By A Novel High-throughput Approach

Defective cell-cell adhesion contributes to the patho-mechanism of various diseases. In line with this, a mouse model recently developed by our group demonstrate that loss of cell-cell adhesion is an important initial step leading to Arrhythmogenic Cardiomyopathy (ACM), a disease which presents with severe arrhythmia, ventricular fibrosis and impaired cardiac function. Derived from the central role of defective cell-cell adhesion for the patho-mechanism, we here aim to identify new compounds, which restore intercellular adhesion and could serve as potential therapeutics. To screen a high number of agents, we developed an adhesion-based high-throughput approach by adapting a standard cell-cell dissociation assay to 96-well plate format including establishment of an automated acquisition and analysis pipeline. To model loss of intercellular adhesion, cells deficient for the desmosomal adhesion molecule desmoglein-2 were used. After establishing settings to sensitively detect changes in cell-cell adhesion, this method was applied to screen a library of 1946 FDA-approved drugs in three concentrations. Several new compounds strengthening intercellular adhesion were identified in addition to known pro-adhesive drugs, confirming the robustness of our assay. The increased adhesiveness of selected top hits was confirmed in cells expressing ACM patient mutations. In conclusion, we here developed an adhesion-based high-throughput approach capable of identifying adhesion modulators which have the potential to be used as therapeutics for diseases with defective desmosomal adhesion such as ACM.