Arrhythmogenic Cardiomyopathy Research Articles

Historical background of formation of ideas about arrhythmogenic cardiomyopathy (literature review)

Historical background of formation of ideas about arrhythmogenic cardiomyopathy (literature review)

Unveiling the Spectrum of Minor Genes in Cardiomyopathies: A Narrative Review.

Hereditary cardiomyopathies (CMPs), including arrhythmogenic cardiomyopathy (ACM), dilated cardiomyopathy (DCM), and hypertrophic cardiomyopathy (HCM), represent a group of heart disorders that significantly contribute to cardiovascular morbidity and mortality and are often driven by genetic factors. Recent advances in next-generation sequencing (NGS) technology have enabled the identification of rare variants in both well-established and minor genes associated with CMPs. Nowadays, a set of core genes is included in diagnostic panels for ACM, DCM, and HCM. On the other hand, despite their lesser-known status, variants in the minor genes may contribute to disease mechanisms and influence prognosis. This review evaluates the current evidence supporting the involvement of the minor genes in CMPs, considering their potential pathogenicity and clinical significance. A comprehensive analysis of databases, such as ClinGen, ClinVar, and GeneReviews, along with recent literature and diagnostic guidelines provides a thorough overview of the genetic landscape of minor genes in CMPs and offers guidance in clinical practice, evaluating each case individually based on the clinical referral, and insights for future research. Given the increasing knowledge on these less understood genetic factors, future studies are essential to clearly assess their roles, ultimately leading to improved diagnostic precision and therapeutic strategies in hereditary CMPs.

Desmoplakin mutations in cardiac fibroblasts cause TGFβ1-mediated pathological fibrogenesis in desmoplakin cardiomyopathy via beclin-1 regulation.

Pathological fibrosis is a major finding in cardiovascular diseases and can result in arrhythmia and heart failure. Desmosome gene mutations can lead to arrhythmogenic cardiomyopathy (ACM). Among ACM, pathogenic desmoplakin ( DSP ) variants cause a distinctive cardiomyopathy with excessive cardiac fibrosis that could precede ventricular dysfunction. DSP variants are also linked to other fibrotic diseases. Whether DSP plays any role in pathological fibrosis remain unknown. Mesenchymal stromal cells (MSCs) are resident fibroblast-like cells that are responsible for fibrogenesis in most organs, including hearts. We first used unbiased genome-wide analyses to generate cardiac fibroblasts-like, induced pluripotent stem cell-derived MSCs from normal donors and ACM patients with DSP mutations. We then studied the fibrogenic responses of cardiac MSCs to transforming growth factor beta-1 (TGF-β1) using Western/Co-IP, autophagy assay, gene knockdowns/over-expressions, genomic analyses, mouse DSP knockdown models, immunostaining, and qPCR. TGFβ1 induced excessive accumulations of vimentin (VIM)/fibrillar collagens, and over-activated fibrotic genes in DSP- mutant MSCs when compared to normal MSCs. In normal MSCs, VIMs bind to wild-type DSP during normal fibrogenesis after TGFβ1. DSP- mutant MSCs exhibited a haplo-insufficient phenotype with increased DSP-unbound VIMs that sequestered beclin-1 (BECN1) from activating autophagy and caveolin-1 (CAV1)-mediated endocytosis. Decreased autophagy caused collagen accumulations and diminished CAV1 endocytosis resulted in abnormal CAV1 plaque formation that over-activated fibrotic genes [ COL1A1, COL3A1, and fibronectin ( FN )] via heightened p38 activities after TGFβ1. Genome-wide analysis and DSP knockdown in mouse fibroblasts confirmed this novel role of DSP mutations in pathological fibrosis. Overexpression of VIM-binding domains of DSP could suppress pathological fibrosis by increasing collagen autophagic degradation and decreasing fibrotic gene expressions. Our data reveal that DSP deficiency in MSCs/fibroblasts leads to exaggerated fibrogenesis in DSP-cardiomyopathy by decreasing BECN1 availability for autophagy and CAV1-endocytosis. Overexpression of VIM binding domains of DSP could be a new strategy to treat pathological fibrosis.

Hyperactivation of ATF4/TGF-β1 signaling contributes to the progressive cardiac fibrosis in Arrhythmogenic cardiomyopathy caused by DSG2 Variant

BackgroundArrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy characterized with progressive cardiac fibrosis and heart failure. However, the exact mechanism driving the progression of cardiac fibrosis and heart failure in ACM remains elusive. This study aims to investigate the underlying mechanisms of progressive cardiac fibrosis in ACM caused by newly identified Desmoglein-2 (DSG2) variation.MethodsWe identified homozygous DSG2F531C variant in a family with 8 ACM patients using whole-exome sequencing and generated Dsg2F536C knock-in mice. Neonatal and adult mouse ventricular myocytes isolated from Dsg2F536C knock-in mice were used. We performed functional, transcriptomic and mass spectrometry analyses to evaluate the mechanisms of ACM caused by DSG2F531C variant.ResultsAll eight patients with ACM were homozygous for DSG2F531C variant. Dsg2F536C/F536C mice displayed cardiac enlargement, dysfunction, and progressive cardiac fibrosis in both ventricles. Mechanistic investigations revealed that the variant DSG2-F536C protein underwent misfolding, leading to its recognition by BiP within the endoplasmic reticulum, which triggered endoplasmic reticulum stress, activated the PERK-ATF4 signaling pathway and increased ATF4 levels in cardiomyocytes. Increased ATF4 facilitated the expression of TGF-β1 in cardiomyocytes, thereby activating cardiac fibroblasts through paracrine signaling and ultimately promoting cardiac fibrosis in Dsg2F536C/F536C mice. Notably, inhibition of the PERK-ATF4 signaling attenuated progressive cardiac fibrosis and cardiac systolic dysfunction in Dsg2F536C/F536C mice.ConclusionsHyperactivation of the ATF4/TGF-β1 signaling in cardiomyocytes emerges as a novel mechanism underlying progressive cardiac fibrosis in ACM. Targeting the ATF4/TGF-β1 signaling may be a novel therapeutic target for managing ACM.

Association of Multiple Nonhypertrophic Cardiomyopathy-Related Genetic Variants and Outcomes in Patients With Hypertrophic Cardiomyopathy.

Approximately 10% of hypertrophic cardiomyopathy (HCM) patients have left ventricular systolic dysfunction (end-stage HCM) leading to severe heart-failure; however, risk stratification to identify patients at risk of progressing to end-stage HCM remains insufficient. In this study, the authors sought to elucidate whether the coexistence of other cardiovascular disease (CVD)-related variants is associated with progression to end-stage HCM in patients with HCM harboring pathogenic or likely pathogenic (P/LP) sarcomeric variants. The authors performed genetic analysis of 83 CVD-related genes in HCM patients from a Japanese multicenter cohort. P/LP variants in 8 major sarcomeric genes (MYBPC3, MYH7, TNNT2, TNNI3, TPM1, MYL2, MYL3, and ACTC1) definitive for HCM were defined as "sarcomeric variants." In addition, P/LP variants associated with other CVDs, such as dilated cardiomyopathy and arrhythmogenic cardiomyopathy, were referred to as "other CVD-related variants." Among 394 HCM patients, 139 carried P/LP sarcomeric variants: 11 (7.9%) carried other CVD-related variants, 6 (4.3%) multiple sarcomeric variants, and 122 (87.8%) single sarcomeric variants. In a multivariable Cox regression analysis, presence of multiple sarcomeric variants (adjusted HR [aHR]: 3.35 [95%CI: 1.25-8.95]; P = 0.016) and coexistence of other CVD-related variants (aHR: 2.80 [95%CI: 1.16-6.78]; P = 0.022) were independently associated with progression to end-stage HCM. Coexisting other CVD-related variants were also associated with heart failure events (aHR: 2.75 [95%CI: 1.27-5.94]; P = 0.010). Approximately 8% of sarcomeric HCM patients carried other CVD-related variants, which were associated with progression to end-stage HCM and heart failure events. Comprehensive surveillance of CVD-related variants within sarcomeric HCM patients contributes to risk stratification and understanding of mechanisms underlying end-stage HCM.

Sudden Cardiac Death in Athletes: Magnitude, Causes, and Prevention Strategies - A Literature Review

Sudden cardiac death (SCD) in athletes, though rare, is the leading cause of exercise-related mortality and poses significant concern due to the symbolic health and vitality of athletes. This review examines the epidemiology, causes, and prevention strategies of SCD in athletes. SCD, defined as sudden death presumed to be of cardiac origin within one hour of symptom onset, affects athletes variably depending on age, race, and type of sports. Key causes include hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) in younger athletes, and coronary artery disease (CAD) in older athletes. Ion channelopathies and structural heart diseases also contribute significantly. Preparticipation screening (PPE) involving ECGs, lifestyle adjustments, and medical interventions are critical in prevention. Continuous research and adaptive guidelines are essential for safeguarding athletes against these fatal events. Keywords: sudden cardiac death, athletes, sports, exercise, cardiovascular diseases, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, coronary artery disease, ion channelopathies, long QT syndrome, Brugada syndrome, preparticipation screening, electrocardiogram (ECG), risk factors, prevention strategies.

Natural History, Phenotype Spectrum and Clinical Outcomes of Desmin (DES)-Associated Cardiomyopathy.

Pathogenic/likely pathogenic (P/LP) desmin (DES) variants cause heterogeneous cardiomyopathy and/or skeletal myopathy phenotypes. Limited data suggest a high incidence of major adverse cardiac events (MACE), including cardiac conduction disease (CCD), sustained ventricular arrhythmias (VA), and heart failure (HF) events (HF hospitalization, LVAD/cardiac transplant, HF-related death), in patients with P/LP DES variants. However, pleiotropic presentation and small cohort sizes have limited clinical phenotype and outcome characterization. We aimed to describe the natural history, phenotype spectrum, familial penetrance and outcomes in patients with P/LP DES variants through a systematic review and individual patient data meta-analysis using published reports. We searched Medline (PubMed) and Embase for studies that evaluated cardiac phenotypes in patients with P/LP DES variants. Cardiomyopathy diagnosis or occurrence of MACE were considered evidence of cardiac involvement/penetrance. Lifetime event-free survival from CCD, sustained VA, HF events, and composite MACE was assessed. Out of 4,212 screened publications, 71 met the inclusion criteria. A total of 230 patients were included (52.6% male, 52.2% probands, median age: 31 years [22.0; 42.8] at first evaluation, median follow-up: 3 years [0; 11.0]). Overall, 124 (53.9%) patients were diagnosed with cardiomyopathy, predominantly dilated cardiomyopathy (14.8%), followed by restrictive cardiomyopathy (13.5%), whereas other forms were less common: arrhythmogenic cardiomyopathy (7.0%), hypertrophic cardiomyopathy (6.1%), arrhythmogenic right ventricular cardiomyopathy (5.2%), and other forms (7.4%). Overall, 132 (57.4%) patients developed MACE, with 96 [41.7%] having CCD, 36 [15.7%] sustained VA, and 43 [18.7%] HF events. Familial penetrance of cardiac disease was 63.6% among relatives with P/LP DES variants. Male sex was associated with increased risk of sustained VA (HR 2.28, p=0.02) and HF events (HR 2.45, p=0.008). DES cardiomyopathy exhibits heterogeneous phenotypes and distinct natural history, characterized by high familial penetrance and substantial MACE burden. Male patients face higher risk of sustained VA events.

Correlation between Epsilon Wave and Late Potentials in Arrhythmogenic Right Ventricular Cardiomyopathy-Do Late Potentials Define the Epsilon Wave?

Introduction: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder characterised by progressive fibrosis predominantly of the right ventricular (RV) myocardium, resulting in life-threatening arrhythmias and heart failure. The diagnosis is challenging due to a wide spectrum of clinical symptoms. The important role of ECG was covered in the current diagnostic criteria. The role of the epsilon wave (EW) is still under discussion. Aim: The aim of the study was to examine a potential association between the EW and late ventricular potentials (LPs) in ARVC patients (pts). The correlation between RV dilatation or dysfunction and LPs/EW was also analysed. Methods: The ARVC group consisted of 81 pts (53 men, aged 20-78 years) fulfilling 2010 International Task Force Criteria. 12-lead ECG, LPs, Holter, and ECHO were performed in all pts. The presence of EW was analysed in ECG by 3 investigators. LPs were detected by signal-averaged ECG (SAECG). SAECG was considered positive for LPs when at least two of the three following criteria were met: (1) the filtered QRS duration (fQRS) ≥ 114 msec; (2) the duration of the final QRS fragment in which low-amplitude signals lower than 40 μV are recorded (LAS-40 > 38 msec); and (3) the root mean square amplitude of the last 40 milliseconds of the fQRS complex (RMS-40 < 20 μV). The results were compared with a reference group consisting of 53 patients with RV damage in the course of atrial septum defect (ASD) or Ebstein's Anomaly (EA). Results: In the ARVC group, a significant relationship was observed between the occurrence of EW and the presence of LPs. EW was more common in the LP+ than in the LP- patients (48.1% vs. 6.9%, p < 0001; OR 12.5; 95% CI [2.691-58.063]). In ARVC pts, RVOT > 36 mm, RVIT > 41 mm, and RV S' < 9 cm/s were observed significantly more often in the LPs+ than in the LPs- group (OR [95% CI]: 8.3 [2.9-1.5], 6.4 [2.2-19.0] and 3.6 [1.1-12.2], respectively). In the ARVC group, any of fQRS > 114 ms, LAS > 38 ms, and RMS < 20 μV were significantly more frequent in EW+ pts. In multivariate analysis, the independent factors of the EW were LAS-40 and RV S'. In the LPs- subgroup, RVOT > 36 mm was more frequent in ASD/EA than in ARVC (70.4% vs. 25%, p = 0.002). Similarly, in the LPs- subgroup, RVIT > 41 mm was encountered more frequently in ASD/EA than in ARVC (85.2% vs. 48.3%, p = 0.004). Conclusions: In ARVC, there is an association between EW and LPs, with both probably resulting from the same process of fibrofatty substitution of the RV myocardium. Although RV dilatation is common in ASD and EA, it does not correlate with LPs.

Prognostic value of electroanatomic-guided endomyocardial biopsy in patients with myocarditis, arrhythmogenic cardiomyopathy and non dilated left ventricular cardiomyopathy

A wide variety of non-invasive and invasive techniques for SCD risk stratification in non ischemic cardiomyopathy (NICM) have been proposed, including left ventricular (LV) ejection fraction, QRS duration, late gadolinium enhancement (LGE) on cardiac magnetic resonance (CMR) and invasive electrophysiologic study with or without three-dimensional electroanatomic mapping (3D-EAM), to identify and characterize the arrhythmogenic substrate. There is still no clear consensus on the risk stratification in this clinical setting.The aim of our study is to characterize the 3D-EAM substrate in patients with the same clinical presentation of unexplained complex VAs and NICM using CMR, three-dimensional electranatomic mapping (3D-EAM) in association with endomyocardial biopsy (EMB) and genetic screening, as a more precise and early diagnostic assessment may provide important subsequent prognostic impact. The study was designed as a prospective multi-center observational evaluation and the patient follow-up was scheduled at 6 months interval. We enrolled 125 patients distinct into four different group by complete diagnostic work-up: myocarditis, non-dilated left ventricular cardiomyopathy, arrhythmogenic cardiomyopathy and control group. The four groups were compared in terms of clinical, imaging and 3D-EAM data. At multivariate analysis sustained VT/VF on admission [HR: 3.64 (1.79–7.4), p < 0.001], total bipolar scar area of left and right ventricle detected by 3D-EAM [HR: 2.24 (1.13–4.49), p = 0.02], histological diagnosis of myocarditis by 3D-EAM guided endomyocardial biopsy (EBM) [HR: 2.79 (1.04–7.44), p = 0.01] were independent predictors of complex VAs or death at follow-up. 3D-EAM guided EMB represent not only a valid diagnostic tool to identify the arrhythmogenic substrate in patients with NICM and ventricular arrhythmic phenotype but also an important predictor of complex Vas at long term follow-up.

Combination of FLNC and JUP variants causing arrhythmogenic cardiomyopathy in an Iranian family with different clinical features

BackgroundArrhythmogenic cardiomyopathy (ACM) characterized by progressive myocardial loss and replacement with fibro-fatty tissue is a major cause of sudden cardiac death (SCD). In particular, ACM with predominantly left ventricular involvement, known as arrhythmogenic left ventricular cardiomyopathy (ALVC), has a poor prognosis.MethodsThe proband underwent whole-exome sequencing (WES) to determine the etiology of ALVC. Family members were then analyzed using PCR and Sanger sequencing. Clinical evaluations including 12-lead ECG, transthoracic echocardiography, and cardiac MRI were performed for all available first-degree relatives.ResultsWES identified two variants in the FLNC (c.G3694A) and JUP (c.G1372A) genes, the combination of which results in ALVC and SCD.ConclusionThe present study comprehensively investigates the involvement of two discovered variants of FLNC and JUP in the pathogenesis of ALVC. More study is necessary to elucidate the genetic factors involved in the etiology of ALVC.

Generation of a human induced pluripotent stem cell line UGENTi002-A from an arrhythmogenic cardiomyopathy patient carrying the c.817C>T DSP heterozygous variant and isogenic control using CRISPR/Cas9 editing

Arrhythmogenic cardiomyopathy is a severe genetic heart muscle disease characterized by fibro-fatty replacement of the myocardium. Pathogenic variants causal for this disease are mainly located in desmosomal genes, including desmoplakin (DSP). Renal epithelial cells were isolated from a patient carrying the heterozygous c.817C>T (p.Q273*, nonsense) pathogenic variant in DSP, and subsequently reprogrammed using the Cytotune®-iPS 2.0 Sendai Reprogramming Kit. An isogenic control line was generated using CRISPR/Cas9 genome editing. The resulting induced pluripotent stem cell lines were characterized and displayed the required traits for in vitro disease modeling.

Exploration of Clozapine-Induced Cardiomyopathy and Its Mechanism.

In this study, by pooling the clinical data of patients who died with a history of long-term clozapine use and by examining their hearts, it was found that long-term clozapine use can lead to cardiomyopathy and that its presentation resembles arrhythmogenic cardiomyopathy (ACM), i.e., it exhibits a predominantly right ventricular fatty infiltration with mild left ventricular damage. The transcriptomic data of rat cardiomyocytes after clozapine intervention were analyzed by transcriptomic approach to explore the causes of clozapine cardiomyopathy. The cause of clozapine cardiomyopathy was then explored by a transcriptomic approach, which revealed that its clozapine action on cardiomyocytes enriched cardiomyocyte-related differential genes in biological processes such as muscle development and response to hypoxia, as well as pathways such as fatty acid metabolism and cellular autophagy. Transcriptomic analysis showed that Egr1, Egr2, ler2, Jun, Mapk9, Nr1d2, Atf3, Bhlhe40, Crem, Cry1, Cry2, Dbp were hub genes for clozapine injury to the myocardium, and that these genes may play an important role in the myocardial ACM-like changes caused by clozapine. Combined with the results of pathological examination and transcriptomic analysis, it can be concluded that the long-term action of clozapine on cardiomyocytes leads to cellular autophagy and subsequent structural remodeling of the heart, and in the remodeling affects fatty acid metabolism, which eventually leads to ACM-like changes.

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.

Toward Precision Medicine in the Treatment of Arrhythmogenic Cardiomyopathy

Toward Precision Medicine in the Treatment of Arrhythmogenic Cardiomyopathy

Characterization of a novel SCN5A mutation associated with long QT syndrome and arrhythmogenic right ventricular cardiomyopathy in a family.

Sudden cardiac death represents a significant diagnostic challenge for forensic pathologists, particularly in inherited arrhythmia syndromes or cardiomyopathies resulting from genetic defects. Molecular autopsies can reveal the underlying molecular etiology in such cases. In this study, we investigated a family with a history of sudden cardiac death to elucidate the molecular basis responsible for sudden cardiac death. The proband underwent a comprehensive forensic examination. Family members received thorough clinical evaluations, including electrocardiogram, Holter monitoring, echocardiography, and cardiac magnetic imaging. Whole exome sequencing and genetic analysis were performed on the deceased and her parents. In addition, Western blotting and patch-clamp recordings were employed to evaluate the expression and function of the mutant protein in vitro. Forensic examination diagnosed arrhythmogenic right ventricular cardiomyopathy (ARVC) as the cause of sudden death. Genetic analysis identified a novel missense mutation in SCN5A (p.V1323L), which was assessed as likely pathogenic by the ACMG guideline. Another family member carrying the mutation manifested long QT syndrome and mild cardiac fibrosis. The cellular electrophysiological study demonstrated that the mutation resulted in an enhanced late sodium current, suggesting it was a gain-of-function mutation. This study characterizes a novel SCN5A mutation that putatively causes long QT syndrome and may contribute to the development of ARVC. Our work expands the pathogenic spectrum of SCN5A variants and underscores the importance of molecular autopsy in sudden death cases, especially in those with suspected genetic disorders.

Role of miRNA-mRNA Interactome in Pathophysiology of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy is an inherited entity characterized by irregular cell-cell adhesion, cardiomyocyte death and fibro-fatty replacement of ventricular myocytes, leading to malignant ventricular arrythmias, contractile dysfunction and sudden cardiac death. Pathogenic variants in genes that encode desmosome are the predominant cause of arrhythmogenic cardiomyopathy. Moreover, signalling pathways such as Wnt/ß-catenin and transforming growth factor-β have been involved in the disease progression. However, still little is known about the molecular pathophysiological mechanisms that underlie arrhythmogenic cardiomyopathy pathogenesis. We used mRNA and small RNA sequencing to analyse the transcriptome of health and arrhythmogenic cardiomyopathy of autopsied human hearts. Our results showed 697 differentially expressed genes and eight differentially expressed miRNAs. Functional enrichment revealed mitochondrial respiratory-related pathways, impaired response to oxidative stress, apoptotic signalling pathways and inflammatory response-related and extracellular matrix response pathways. Furthermore, analysis of the miRNA-mRNA interactome identified eleven negatively correlated miRNA-target pairs for arrhythmogenic cardiomyopathy. Our finding revealed novel arrhythmogenic cardiomyopathy-related miRNAs with important regulatory function in disease pathogenesis, highlighting their value as potential key targets for therapeutic approaches.

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

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

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.

Abstract We117: Preclinical Evidence of Long-term Efficacy and Safety of LX2020, an AAV based Plakophilin-2 Gene Therapy, for the Treatment of Arrhythmogenic Cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is a fatal inherited cardiac disease caused by mutations in desmosomal anchoring genes, with plakophilin-2 (PKP2) being the most frequently mutated gene (70%). While AAV9-PKP2 gene therapy shows promise in mitigating ACM-related deficits in mice, high dose toxicity and organ failure of AAV9 in human clinical trials raises safety concerns. Prior studies showed AAVrh.10 exhibiting preferential cardiac gene expression at lower doses that could potentially be safer. We hypothesized that AAVrh.10.PKP2 (LX2020) could restore PKP2 to scaffold the desmosomal complex and ameliorate cardiac deficits with long-term efficacy at relatively lower doses and safety in non-human primates (NHP). We employed LX2020 to assess its efficacy in severe (PKP2 homozygous (Hom)) and mild (PKP2 heterozygous (Het)) adult ACM diseased mice, as well as safety in NHP at doses from 2E13 to 1E14 vg/kg and up to 12 weeks post-administration. A dose-dependent increase in hPKP2 expression was seen in all models, with rescue of desmosomal/gap junction protein deficits at 2E13 and 6E13 vg/kg doses in PKP2-Het and PKP2-Hom mouse hearts. Long term LX2020 efficacy was observed at the same doses in PKP2-Hom mice with dose-dependent improvements in cardiac function, arrhythmia reduction and histological improvements, such that LX2020 treated PKP2-Hom hearts resembled wild-type hearts, indicating a potential for reversal of late-stage pathology. The extended lifespan was striking with 100% of PKP2-Hom mice surviving using 2E13 vg/kg, completely averting premature death. Safety assessments in NHP also revealed no adverse events up to the highest dose of 1E14 vg/kg. These data support advancing LX2020 as a potentially viable treatment for PKP2 ACM patients, based on safety and long-term efficacy in rescuing desmosomal and cardiac deficits in ACM models at lower doses reported in the field.