Arrhythmogenic Cardiomyopathy Phenotype Research Articles

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

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

Arrhythmogenic Cardiomyopathy: Current Updates and Future Challenges.

Arrhythmogenic cardiomyopathy (ACM) epitomises a genetic anomaly hallmarked by a relentless fibro-fatty transmogrification of cardiac myocytes. Initially typified as a right ventricular-centric disease, contemporary observations elucidate a frequent occurrence of biventricular and left-dominant presentations. The diagnostic labyrinth of ACM emerges from its clinical and imaging properties, often indistinguishable from other cardiomyopathies. Precision in diagnosis, however, is paramount and unlocks the potential for early therapeutic interventions and vital cascade screening for at-risk individuals. Adherence to the criteria established by the 2010 task force remains the cornerstone of ACM diagnosis, demanding a multifaceted assessment incorporating electrophysiological, imaging, genetic, and histological data. Reflecting the evolution of our understanding, these criteria have undergone several revisions to encapsulate the expanding spectrum of ACM phenotypes. This review seeks to crystallise the genetic foundation of ACM, delineate its clinical and radiographic manifestations, and offer an analytical perspective on the current diagnostic criteria. By synthesising these elements, we aim to furnish practitioners with a strategic, evidence-based algorithm to accurately diagnose ACM, thereby optimising patient management and mitigating the intricate challenges of this multifaceted disorder.

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).

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.

The phenotypic and genetic features of arrhythmogenic cardiomyopathy in the pediatric population.

The present study aimed to describe the phenotypic features and genetic spectrum of arrhythmogenic cardiomyopathy (ACM) presented in childhood and test the validity of different diagnostic approaches using Task Force Criteria 2010 (TFC) and recently proposed Padua criteria. Thirteen patients (mean age at diagnosis 13.6 ± 3.7 years) were enrolled using "definite" or "borderline" diagnostic criteria of ACM according to the TFC 2010 and the Padua criteria in patients <18 years old. Clinical data, including family history, 12-lead electrocardiogram (ECG), signal-averaged ECG, 24-h Holter monitoring, imaging techniques, genetic testing, and other relevant information, were collected. All patients were classified into three variants: ACM of right ventricle (ACM-RV; n = 6, 46.1%), biventricular ACM (ACM-BV; n = 3, 23.1%), and ACM of left ventricle (ACM-LV; n = 4, 30.8%). The most common symptoms at presentations were syncope (n = 6; 46.1%) and palpitations (n = 5; 38.5%). All patients had more than 500 premature ventricular contractions per day. Ventricular tachycardia was reported in 10 patients (76.9%), and right ventricular dilatation was registered in 8 patients (61.5%). An implantable cardiac defibrillator was implanted in 61.5% of cases, and three patients with biventricular involvement underwent heart transplantation. Desmosomal mutations were identified in 8 children (53.8%), including four patients with PKP2 variants, two with DSP variants, one with DSG2 variant, and one with JUP. Four patients carried compound heterozygous variants in desmosomal genes associated with left ventricular involvement. Arrhythmias and structural heart disease, such as chamber dilatation, should raise suspicion of different ACM phenotypes. Diagnosis of ACM might be difficult in pediatric patients, especially for ACM-LV and ACM-BV forms. Our study confirmed that using "Padua criteria" in combination with genetic testing improves the diagnostic accuracy of ACM in children.

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.

The arrhythmogenic cardiomyopathy phenotype associated with PKP2 c.1211dup variant.

The arrhythmogenic cardiomyopathy (ACM) phenotype, with life-threatening ventricular arrhythmias and heart failure, varies according to genetic aetiology. We aimed to characterise the phenotype associated with the variant c.1211dup (p.Val406Serfs*4) in the plakophilin‑2 gene (PKP2) and compare it with previously reported Dutch PKP2 founder variants. Clinical data were collected retrospectively from medical records of 106 PKP2 c.1211dup heterozygous carriers. Using data from the Netherlands ACM Registry, c.1211dup was compared with 3 other truncating PKP2 variants (c.235C > T (p.Arg79*), c.397C > T (p.Gln133*) and c.2489+1G > A (p.?)). Of the 106 carriers, 47 (44%) were diagnosed with ACM, at amean age of 41years. By the end of follow-up, 29 (27%) had experienced sustained ventricular arrhythmias and 12 (11%) had developed heart failure, with male carriers showing significantly higher risks than females on these endpoints (p < 0.05). Based on available cardiac magnetic resonance imaging and echocardiographic data, 46% of the carriers showed either right ventricular dilatation and/or dysfunction, whereas asubstantial minority (37%) had some form of left ventricular involvement. Both geographical distribution of carriers and haplotype analysis suggested PKP2 c.1211dup to be afounder variant originating from the South-Western coast of the Netherlands. Finally, aCox proportional hazards model suggested significant differences in ventricular arrhythmia-free survival between 4 PKP2 founder variants, including c.1211dup. The PKP2 c.1211dup variant is aDutch founder variant associated with atypical right-dominant ACM phenotype, but also left ventricular involvement, and apossibly more severe phenotype than other Dutch PKP2 founder variants.

Evolution of surface and endocavitary signals in patients with arrhythmogenic cardiomyopathy at 1 and 5 years

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Small fee from Boston Scientific. Background The use of subcutaneous defibrillator (S-ICD) is a viable alternative to transvenous ICD in patients with Arrhythmogenic Cardiomyopathy (ACM). In transvenous ICD recipients low sensing values may exist at the time of implantation and a progressive decline of signal amplitude may occur later on due to the progressive myocardial fibrofatty replacement. Similarly to what happens at the intracavitary level, in patients with ACM there is a progressive modification of the surface ECG over time. Data on the impact of ECG evolution on S-ICD sensing are still lacking, particularly for different ACM phenotypes. Purpose This study aims to analyze the evolution of surface electrocardiogram (ECG), focusing on R-wave amplitude variation, and intracavitary electrograms (EGMs) in ACM different phenotypes during a 1-year and a 5 five-year follow-up. Methods This study is a retrospective analysis of the data of consecutive patients with ACM who were referred to our centre from January 1992 to October 2021. Using a dedicated software, the ECG analysis was performed in 7 leads: DI, DII, aVF (in view of the similar orientation with the three S-ICD vectors) and V1, V2, V3 and V5. Results We included 69 ACM patients: 28 patients (41%) had a right ventricular involvement (ARVC), 19 (27%) had a dominant LV involvement (ALVC) and 22 (32%) had biventricular involvement. 58 patients (84%) were ICD recipients; 45/58 patients (78%) had a transvenous ICD, and 13/58 (22%) had an S-ICD. During 5-year follow-up, in the whole ACM population, there were no significant changes in the amplitude of the R-wave, S-wave and T-wave. Phenotypic subgroups showed a different R-wave amplitude at baseline: biventricular ACM and ALVC patients had a lower R-wave amplitude in DII (p=0.04; p=0.05) and aVF (p=0.01; p=0.01) compared to ARVC patients. However, within each group, no difference was observed in R-wave amplitude at 1 and 5 years compared to baseline ECG (Figure 1). In the transvenous ICD recipients (45/69 patients) mean sensing decreased progressively over time (9,38 ± 4,24 mV at baseline, 7,70 ± 2,91 mV at 1 year, 1,85 ± 6,86 mV at 5 years). The reduction was not statistically significant at the first year of follow-up (p=0.239) but it reached significance at 5 years (p&amp;lt;0.001). Patients with biventricular ACM had lower sensing values at baseline than ARVC and ALVC (p&amp;lt;0,001). Sensing values in the biventricular patients did not decrease significantly during the 5-year follow-up (p=0.21), whereas in the ARVC and ALVC groups a reduction in sensing values was observed albeit not statistically significant in ALVC (p=0.01 in ARVC and p =0.06 in ALVC) (Figure2). Conclusions These data suggest that surface ECG may be more stable over time compared to EGM, therefore if a patient is suitable for S-ICD implantation S-ICD sensing may remain stable over time. Despite these findings, further studies are needed to support these hypotheses.

Tools to differentiate between Filamin C and Titin truncating variant carriers: value of MRI.

Whereas truncating variants of the giant protein Titin (TTNtv) are the main cause of familial dilated cardiomyopathy (DCM), recently Filamin C truncating variants (FLNCtv) were identified as a cause of arrhythmogenic cardiomyopathy (ACM). Our aim was to characterize and compare clinical and MRI features of TTNtv and FLNCtv in the Belgian population. In index patients referred for genetic testing of ACM/DCM, FLNCtv and TTNtv were found in 17 (3.6%) and 33 (12.3%) subjects, respectively. Further family cascade screening yielded 24 and 19 additional truncating variant carriers in FLNC and TTN, respectively. The main phenotype was ACM in FLNCtv carriers whereas TTNtv carriers showed either an ACM or DCM phenotype. Non-sustained Ventricular Tachycardia was frequent in both populations. MRI data, available in 28/40 FLNCtv and 32/52 TTNtv patients, showed lower Left Ventricular (LV) ejection fraction and lower LV strain in TTNtv patients (p < 0.01). Conversely, both the frequency (68% vs 22%) and extent of non-ischemic myocardial late gadolinium enhancement (LGE) was significantly higher in FLNCtv patients (p < 0.01). Hereby, ring-like LGE was found in 16/19 (84%) FLNCtv versus 1/7 (14%) of TTNtv patients (p < 0.01).In conclusion, a large number of FLNCtv and TTNtv patients present with an ACM phenotype but can be separated by cardiac MRI. Whereas FLNCtv patients often have extensive myocardial fibrosis, typically following a ring-like pattern, LV dysfunction without or limited replacement fibrosis is the common TTNtv phenotype.

Diagnostic delay in arrhythmogenic cardiomyopathy.

Diagnosis of arrhythmogenic cardiomyopathy (ACM) may be challenging, as it comprises diverse phenotypes (right dominant, biventricular, and left dominant), and each may overlap with other clinical entities. The issue of differential diagnosis with conditions mimicking ACM has been previously highlighted; however, a systematic analysis of ACM diagnostic delay, and of its clinical implications, is lacking. Data of all ACM patients from three Italian Cardiomyopathy Referral Centres were reviewed to assess the time from first medical contact to definitive ACM diagnosis; a significant diagnostic delay was defined as a time to ACM diagnosis ≥2 years. Baseline characteristics and clinical course of patients with and without diagnostic delay were compared. Of 174 ACM patients, 31% experienced diagnostic delay, with a median time to diagnosis of 8 years (20% in right-dominant ACM, 33% in left-dominant ACM, and 39% in biventricular). Patients with diagnostic delay, when compared with those without, more frequently exhibited an ACM phenotype with left ventricular (LV) involvement (74 vs. 57%, P = 0.04) and a specific genetic background (none had plakophilin-2 variants). The most common initial (mis)diagnoses were dilated cardiomyopathy (51%), myocarditis (21%), and idiopathic ventricular arrhythmia (9%). At follow-up, all-cause mortality was greater in those with diagnostic delay (P = 0.03). Diagnostic delay is common in patients with ACM, particularly in the presence of LV involvement, and is associated with greater mortality at follow-up. Clinical suspicion and increasing use of tissue characterization by cardiac magnetic resonance in specific clinical settings are of key importance for the timely identification of ACM.

Ca2+ dysregulation in cardiac stromal cells sustains fibro-adipose remodeling in Arrhythmogenic Cardiomyopathy and can be modulated by flecainide

BackgroundCardiac mesenchymal stromal cells (C-MSC) were recently shown to differentiate into adipocytes and myofibroblasts to promote the aberrant remodeling of cardiac tissue that characterizes arrhythmogenic cardiomyopathy (ACM). A calcium (Ca2+) signaling dysfunction, mainly demonstrated in mouse models, is recognized as a mechanism impacting arrhythmic risk in ACM cardiomyocytes. Whether similar mechanisms influence ACM C-MSC fate is still unknown.Thus, we aim to ascertain whether intracellular Ca2+ oscillations and the Ca2+ toolkit are altered in human C-MSC obtained from ACM patients, and to assess their link with C-MSC-specific ACM phenotypes.Methods and resultsACM C-MSC show enhanced spontaneous Ca2+ oscillations and concomitant increased Ca2+/Calmodulin dependent kinase II (CaMKII) activation compared to control cells. This is manly linked to a constitutive activation of Store-Operated Ca2+ Entry (SOCE), which leads to enhanced Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors. By targeting the Ca2+ handling machinery or CaMKII activity, we demonstrated a causative link between Ca2+ oscillations and fibro-adipogenic differentiation of ACM C-MSC. Genetic silencing of the desmosomal gene PKP2 mimics the remodelling of the Ca2+ signalling machinery occurring in ACM C-MSC. The anti-arrhythmic drug flecainide inhibits intracellular Ca2+ oscillations and fibro-adipogenic differentiation by selectively targeting SOCE.ConclusionsAltogether, our results extend the knowledge of Ca2+ dysregulation in ACM to the stromal compartment, as an etiologic mechanism of C-MSC-related ACM phenotypes. A new mode of action of flecainide on a novel mechanistic target is unveiled against the fibro-adipose accumulation in ACM.

Recurrent acute myocarditis: An under-recognized clinical entity associated with the later diagnosis of a genetic arrhythmogenic cardiomyopathy.

Myocardial inflammation has been consistently associated with genetic arrhythmogenic cardiomyopathy (ACM) and it has been hypothesized that episodes mimicking acute myocarditis (AM) could represent early inflammatory phases of the disease. We evaluated the temporal association between recurrent acute myocarditis (RAM) episodes and the later diagnosis of a genetic ACM. Between January 2012 and December 2021, patients with RAM and no previous cardiomyopathy were included (Recurrent Acute Myocarditis Registry, NCT04589156). A follow-up visit including clinical evaluation, resting and stress electrocardiogram, cardiac magnetic resonance imaging, and genetic testing was carried out. Endpoints of the study was the incidence of both ACM diagnosis criteria and ACM genetic mutation at the end of follow-up. Twenty-one patients with RAM were included and follow-up was completed in 19/21 patients (90%). At the end of follow-up, 3.3 ± 2.9 years after the last AM episode, 14/21 (67%) patients with an ACM phenotype (biventricular: 10/14, 71%; left ventricular: 4/14, 29%) underwent genetic testing. A pathogenic or likely pathogenic mutation was found in 8/14 patients (57%), 5/8 in the Desmoplakin gene, 2/8 in the Plakophillin-2 gene, and 1/8 in the Titin gene. Family history of cardiomyopathy or early sudden cardiac death had a positive predictive value of 88% for the presence of an underlying genetic mutation in patients with RAM. RAM is a rare entity associated with the latter diagnosis of an ACM genetic mutation in more than a third of the cases. In those patients, RAM episodes represent early inflammatory phases of the disease. Including RAM episodes in ACM diagnosis criteria might allow early diagnosis and potential therapeutic interventions.

Clinical characteristics of various arrhythmogenic cardiomyopathy phenotypes in the pediatric population: a systematic review and meta-analysis

Arrhythmogenic cardiomyopathy (ACM) is a progressive disease with a high risk of sudden cardiac death (SCD), which is very rare in the pediatric population. Aim. To study the clinical course, outcomes and prognosis in various ACM phenotypes in pediatric patients.Material and methods. During the initial selection, 144 publications were found in the PubMed database. From the initially identified results, an analysis of 7 works was carried out.Results. Positive family history for ACM was revealed in 90% of cases. Every second patient had symptoms of the disease (49,2% (95% confidence interval (CI): 19,3-79,1)). Frequent clinical manifestations of ACM were palpitations, heart failure symptoms, and syncope. The incidence of SCD and sudden cardiac arrest was 7,1% (95% CI: 1,7-12,4) and 5,1% (95% CI: 1,5-8,7), respectively. Implantation of an implantable-cardioverter defibrillator was performed in 40% of cases. Conclusion. We did not reveal any specificity of clinical signs depending on ACM phenotype. However, an earlier onset and an unfavorable course are characteristic of non-classical ACM types. ACM is characterized by a high risk of SCD, so it is extremely important to make a timely diagnosis.

Non Coding RNAs as Regulators of Wnt/β-Catenin and Hippo Pathways in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy histologically characterized by the replacement of myocardium by fibrofatty infiltration, cardiomyocyte loss, and inflammation. ACM has been defined as a desmosomal disease because most of the mutations causing the disease are located in genes encoding desmosomal proteins. Interestingly, the instable structures of these intercellular junctions in this disease are closely related to a perturbed Wnt/β-catenin pathway. Imbalance in the Wnt/β-catenin signaling and also in the crosslinked Hippo pathway leads to the transcription of proadipogenic and profibrotic genes. Aiming to shed light on the mechanisms by which Wnt/β-catenin and Hippo pathways modulate the progression of the pathological ACM phenotype, the study of non-coding RNAs (ncRNAs) has emerged as a potential source of actionable targets. ncRNAs comprise a wide range of RNA species (short, large, linear, circular) which are able to finely tune gene expression and determine the final phenotype. Some share recognition sites, thus referred to as competing endogenous RNAs (ceRNAs), and ensure a coordinating action. Recent cancer research studies regarding the key role of ceRNAs in Wnt/β-catenin and Hippo pathways modulation pave the way to better understanding the molecular mechanisms underlying ACM.

Loss-of-Function FLNC Variants Are Associated With Arrhythmogenic Cardiomyopathy Phenotypes When Identified Through Exome Sequencing of a General Clinical Population.

The FLNC gene has recently garnered attention as a likely cause of arrhythmogenic cardiomyopathy, which is considered an actionable genetic condition. However, the association with disease in an unselected clinical population is unknown. We hypothesized that individuals with loss-of-function variants in FLNC (FLNCLOF) would have increased odds for arrhythmogenic cardiomyopathy-associated phenotypes versus variant-negative controls in the Geisinger MyCode cohort. We identified rare, putative FLNCLOF among 171 948 individuals with exome sequencing linked to health records. Associations with arrhythmogenic cardiomyopathy phenotypes from available diagnoses and cardiac evaluations were investigated. Sixty individuals (0.03%; median age 58 years [47-70 interquartile range], 43% male) harbored 27 unique FLNCLOF. These individuals had significantly increased odds ratios for dilated cardiomyopathy (odds ratio, 4.9 [95% CI, 2.6-7.6]; P<0.001), supraventricular tachycardia (odds ratio, 3.2 [95% CI, 1.1-5.6]; P=0.048), and left-dominant arrhythmogenic cardiomyopathy (odds ratio, 4.2 [95% CI, 1.4-7.9]; P=0.03). Echocardiography revealed reduced left ventricular ejection fraction (52±13% versus 57±9%; P=0.001) associated with FLNCLOF. Overall, at least 9% of FLNCLOF patients demonstrated evidence of penetrant disease. FLNCLOF variants are associated with increased odds of ventricular arrhythmia and dysfunction in an unselected clinical population. These findings support genomic screening of FLNC for actionable secondary findings.

Clinical and Molecular Characteristics of Patients with PLN R14del Cardiomyopathy: State-of-the-Art Review

The deletion of the arginine 14 codon (R14del) in the phospholamban (PLN) gene is a rare cause of arrhythmogenic cardiomyopathy (ACM) and is associated with prevalent ventricular arrhythmias, heart failure, and sudden cardiac death. The pathophysiological mechanism which culminates in the ACM phenotype is multifactorial and mainly based on the alteration of the endoplasmic reticulum proteostasis, mitochondrial dysfunction and compromised Ca2+ cytosolic homeostasis. The symptoms of this condition are usually non-specific and consist of arrhythmia-related or heart failure-related manifestation; however, some peculiar diagnostic clues were detected, such as the T-wave inversion in the lateral leads, low QRS complexes voltages, mid-wall or epicardial fibrosis of the inferolateral wall of the left ventricle, and their presence should raise the suspicion of this condition. The risk stratification for sudden cardiac death is mandatory and several predictors were identified in recent years. However, the management of affected patients is often challenging due to the absence of specific prediction tools and therapies. This review aims to provide the current state of the art of PLN R14del cardiomyopathy, focusing on its pathophysiology, clinical manifestation, risk stratification for sudden cardiac death, and management.

Buccal Mucosa Cells as a Potential Diagnostic Tool to Study Onset and Progression of Arrhythmogenic Cardiomyopathy

In arrhythmogenic cardiomyopathy (ACM) pathogenic variants are found in genes encoding desmosomal proteins and in non-desmosomal genes, such as phospholamban (PLN, p.Arg14del variant). Previous research showed that plakoglobin protein levels and localization in the cardiac tissue of ACM patients, and PLN p.Arg14del patients diagnosed with an ACM phenotype, are disturbed. Moreover, the effects of pathogenic variants in desmosomal genes are reflected in non-cardiac tissues like buccal mucosa cells (BMC) which could serve as a promising new and non-invasive tool to support diagnosis. We collected the BMC of 33 ACM patients, 17 PLN p.Arg14del patients and 34 controls, labelled the BMC with anti-plakoglobin antibodies at different concentrations, and scored their membrane labelling. We found that plakoglobin protein levels were significantly reduced in BMC obtained from diagnosed ACM patients and preclinical variant carriers when compared to controls. This effect was independent from age and sex. Moderate to strong correlations were found with the revised 2010 Task Force Criteria score which is commonly used for ACM diagnosis (rs = −0.67, n = 64, p < 0.0001 and rs = −0.71, n = 64, p < 0.0001). In contrast, plakoglobin scores in PLN p.Arg14del patients were comparable to controls (p > 0.209), which suggests differences in underlying etiology. However, for the individual diagnosis of the ‘classical’ ACM patient, this method might not be discriminative enough to distinguish true patients from variant carriers and controls, because of the high interindividual variability.

Abstract 13147: The Internally Translated Isoform of Connexin 43, GJA1-20k Rescues Cx43 Trafficking and Reduces Arrhythmias in the DSG2 Mutant Model of Arrhythmogenic Cardiomyopathy

Introduction: Arrhythmogenic cardiomyopathy (ACM) is an inherited condition caused by mutations of junctional proteins resulting in heart rhythm abnormalities, heart failure and sudden cardiac death. ACM subjects exhibit a loss of normal trafficking of the gap junction protein Connexin 43 (Cx43) to the intercalated disk. Our group has previously demonstrated that the Cx43 gene, GJA1, undergoes internal translation to form multiple truncations of the full-length protein. The 20 kilodalton isoform, (GJA1-20k) is required for trafficking of Cx43 gap junction channels, and exogenous expression of GJA1-20k increases trafficking of full length Cx43 to the membranes and the intercalated disk. Here, we hypothesize that GJA1-20K will reduce dysrhythmias by improving trafficking of Cx43 to the intercalated disc in a mouse model of ACM. Methods: Desmoglein 2 (DSG2) homozygous knock-out mice, producing a typical ACM phenotype, were injected with a viral vector containing either GJA1-20K-GFP or GFP alone at four weeks of age along with wild-type littermate controls. Echocardiography was performed every four weeks and Telemetry was recorded at ~18-19 weeks of age to record cardiac electrical activity over a continuous 12 hour period. At 20 weeks, the mice were sacrificed, and hearts were harvested for further analysis. Results: Compared to WT littermates DSG2-KO mice that received a GFP viral vector demonstrated over a 100 fold increase in mean number of PVCs observed over a 12 hour period (2 vs 371 respectively p=0.0005). Mice that received the GJA1-20k viral vector in contrast demonstrated significantly fewer PVCs than their GFP treated littermates 43 vs 371. (analysis of variance of Log(#PVC) p =0.015 N = 6-7 mice/group). Quantitative immunofluorescence analysis of hearts confirmed an increase in Cx43 localization at the intercalated disk (as measured as a percent of N-Cadherin-Cx43 colabeling) in mice treated with GJA1-20k relative to GFP (25% vs 6% respectively N= 7 images/mouse and 3 mice/ group) t-test p=0.0009). Conclusions: Our data indicate that GJA1-20K significantly reduces the number of PVCs and recovers Cx43 trafficking to the intercalated disk in a mouse model of ACM. Our results point to a therapeutic option for the treatment of ACM related arrhythmias.

Aberrant accumulation of TMEM43 accompanied by perturbed transmural gene expression in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) caused by TMEM43 p.S358L is a fully penetrant heart disease that results in impaired cardiac function or fatal arrhythmia. However, the molecular mechanism of ACM caused by the TMEM43 variant has not yet been fully elucidated. In this study, we generated knock-in (KI) rats harboring a Tmem43 p.S358L mutation and established induced pluripotent stem cells (iPSCs) from patients based on the identification of TMEM43 p.S358L variant from a family with ACM. The Tmem43-S358L KI rats exhibited ventricular arrhythmia and fibrotic myocardial replacement in the subepicardium, which recapitulated the human ACM phenotype. The four-transmembrane protein TMEM43 with the p.S358L variant (TMEM43S358L ) was found to be modified by N-linked glycosylation in both KI rat cardiomyocytes and patient-specific iPSC-derived cardiomyocytes. TMEM43S358L glycosylation increased under the conditions of enhanced endoplasmic reticulum (ER) stress caused by pharmacological stimulation or age-dependent decline of the ER function. Intriguingly, the specific glycosylation of TMEM43S358L resulted from the altered membrane topology of TMEM43. Moreover, unlike TMEM43WT , which is mainly localized to the ER, TMEM43S358L accumulated at the nuclear envelope of cardiomyocytes with the increase in glycosylation. Finally, our comprehensive transcriptomic analysis demonstrated that the regional differences in gene expression patterns between the inner and outer layers observed in the wild type myocardium were partially diminished in the KI myocardium prior to exhibiting histological changes indicative of ACM. Altogether, these findings suggest that the aberrant accumulation of TMEM43S358L underlies the pathogenesis of ACM caused by TMEM43 p.S358L variant by affecting the transmural gene expression within the myocardium.

Evolving Diagnostic Criteria for Arrhythmogenic Cardiomyopathy

Criteria for diagnosis of arrhythmogenic cardiomyopathy (ACM) were first proposed in 1994 and revised in 2010 by a Task Force. Although the Task Force criteria demonstrated a good accuracy for diagnosis of the original right ventricular phenotype (arrhythmogenic right ventricular cardiomyopathy), they lacked sensitivity for identification of the expanding phenotypic spectrum of ACM, which includes left‐sided variants and did not incorporate late‐gadolinium enhancement findings by cardiac magnetic resonance. The 2020 International criteria (“Padua criteria”) have been developed by International experts with the aim to improve the diagnosis of ACM by providing new criteria for the diagnosis of left ventricular phenotypic features. The key upgrade was the incorporation of tissue characterization findings by cardiac magnetic resonance for noninvasive detection of late‐gadolinium enhancement/myocardial fibrosis that are determinants for characterization of arrhythmogenic biventricular and left ventricular cardiomyopathy. The 2020 International criteria are heavily dependent on cardiac magnetic resonance, which has become mandatory to characterize the ACM phenotype and to exclude other diagnoses. New criteria regarding left ventricular depolarization and repolarization ECG abnormalities and ventricular arrhythmias of left ventricular origin were also provided. This article reviews the evolving approach to diagnosis of ACM, going back to the 1994 and 2010 International Task Force criteria and then grapple with the modern 2020 International criteria.