Arrhythmogenic Phenotype Research Articles

Genotype-phenotype correlations in patients with titin variants and cardiomyopathy from a Swiss cohort

Abstract Background Alterations in titin (TTN) are mostly described with dilated cardiomyopathy (DCM). Few studies identified TTN variants in arrhythmogenic cardiomyopathy (ACM), but the overlap of dilated and arrhythmogenic phenotypes in TTN variants is scarcely studied. Most pathogenic TTN variants are located on the A-band, but there is few knowledge about the phenotypic presentation of I-band and Z-disc (non-A-band) variants. Purpose The aim of this study is to characterize and investigate the outcome of patients with cardiomyopathy and pathogenic (P) or likely pathogenic (LP) TTN variants with a particular focus on the cardiomyopathy phenotype (DCM vs. ACM) at initial presentation and follow-up. Further, we studied the association of the TTN variant (A-band vs. non-A-band regions) and a unique phenotype. Methods This is a retrospective multicenter study from a Swiss cohort including 34 patients with P/LP TTN variants and cardiomyopathy. The variables of the secured anonymized database include patient characteristics, genetic, ECG, echocardiographic, and cardiac magnetic resonance (CMR) data. Ventricular arrhythmia (VA) events were divided into non-sustained ventricular tachycardia (nsVT), sustained ventricular tachycardia (sVT) and sudden cardiac arrest (SCA +/- ventricular fibrillation). Results Of 34 included patients, 68% were male. Median age at presentation was 43.5 years. 29 (85%) patients presented with DCM, 7 (21%) left dominant ACM (2020 Padua criteria) and none ARVC (2010 ARVC Task Force criteria). At median follow-up of 48.5 months, 17 (50%) patients had VA events (15 nsVT, 5 sVT and 3 SCA events). VA events significantly increased over time (p=0.008) and were not associated with gender or left ventricular ejection fraction (LVEF) <35%. Under optimal guideline-directed medical therapy (GDMT) mean LVEF and right ventricular function increased significantly (LVEF: 28.3±14.4% vs. 41.9±9.8% and right ventricular fractional area change (RV FAC): 24.7±7.5% vs. 35.8±4.8%, p<0.05). Late gadolinium enhancement (LGE) on CMR was commonest in the basal septum, but no patient had higher degree atrioventricular block (AVB) at baseline and follow-up. At last follow-up, 8 (24%) patients fulfilled ACM and 13 (38%) DCM criteria (Figure 1). Most TTN variants were located on the A-band (21, 62%), 10 (29%) on the non-A-band regions. Latter had higher rates of sVT (log-rank p=0.041) during follow-up (Figure 2). 20 (59%) had LP and 11 (32%) P variants. P variant carriers were younger at disease presentation compared to LP variant carriers. Conclusion Patients with TTN variants frequently present as DCM, whereas presentation as left dominant ACM is less common. TTN does not seem to be a classical ARVC causing gene. Despite favourable biventricular remodelling under GDMT, ventricular arrhythmic events increase during follow-up. I-band and Z-disk variants are associated with a higher risk of sVT, which may help in the risk stratification process.

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

Ringlike late gadolinium enhancement: prevalence, association with cardiac phenotype, and patient outcome in an unselected cohort referred for cardiovascular magnetic resonance imaging

Abstract Background The identification of a non-ischemic ringlike enhancement in the left ventricular (LV) myocardium by cardiac magnetic resonance imaging (CMR) has emerged as a significant biomarker associated with arrhythmogenic cardiomyopathy (CMP) and adverse patient outcomes. Recently, the term "scarring CMP" has been also proposed to encompass various conditions characterized by a high burden of non-ischemic, often ring-like, myocardial scarring, including both genetic and acquired etiologies. Purpose To systematically evaluate CMR phenotypic traits and etiologic backgrounds in patients with ringlike enhancement and to assess the burden of adverse outcomes during follow-up (FU). Methods This is a single-center, retrospective analysis of prospectively enrolled patients undergoing CMR (from 2002 to 2024). Ringlike late gadolinium enhancement (LGE) was defined as continuous enhancement in three or more adjacent segments based on the AHA 17 segment model. Patients records were reviewed for etiologic classification and FU assessment. Results Among 23.472 patients, 19.696 (84%) underwent LGE assessment. In 152 patients (0.77%; 73% male; mean age 48.5 ± 17 years) a ringlike LGE was identified. The mean number of LV segments with LGE was 10 ± 3. Right ventricular (RV) LGE was present in 23% of subjects. LV fatty metaplasia was detected in 22%. Infero-lateral segments were most frequently affected. CMR showed the following morpho-functional features: LV dilatation (53%), LV hypokinesia (69%), LV hypertrophy (10.5%), LV non-compaction (11%), RV dilation (26%), and RV ejection fraction < 40% (21%). Phenotypic classification according to 2023 ESC guidelines identified dilated CMP phenotype (43%), non-dilated LV CMP phenotype (39%), predominant arrhythmogenic right ventricular phenotype (6%), hypertrophic phenotype (7%). 5% of cases remained unclassified, all of them showing LV dilated non-hypokinetic phenotype. Genetic analysis was performed in 101 patients (66%). Etiologic classification revealed non-desmosomal gene related CMP (24%), desmosomal gene related CMP (8%), inflammatory CMP (16%), genetic neuromuscular diseases (5%), congenital metabolic errors (3%), familiar gene elusive CMP (2%) and unknown etiology (45%). Notably, only 17% of patients classified as having inflammatory CMP and 66% of those with unknown etiology underwent genetic testing, with negative results. The median follow-up duration was 3 (IQR: 1-7) years. All-cause mortality occurred in 10.5% of patients, while 17% experienced sustained ventricular tachycardia, appropriate implantable cardioverter-defibrillator therapies, or sudden cardiac death. Heart transplantation or left ventricular assist device implantation was required in 8% of cases. Conclusion Ringlike LGE is a rare, non-disease-specific feature that can be found in different morpho-functional CMP phenotypes. It is associated with frequent genetic-determined etiology and high burden of arrhythmic and non-arrhythmic outcomes.

Clinical features and outcomes in carriers of pathogenic desmoplakin variants.

Pathogenic variants in the desmoplakin (DSP) gene are associated with the development of a distinct arrhythmogenic cardiomyopathy phenotype not fully captured by either dilated cardiomyopathy (DCM), non-dilated left ventricular cardiomyopathy (NDLVC), or arrhythmogenic right ventricular cardiomyopathy (ARVC). Prior studies have described baseline DSP cardiomyopathy genetic, inflammatory, and structural characteristics. However, cohort sizes have limited full clinical characterization and identification of clinical and demographic predictors of sustained ventricular arrhythmias (VAs), heart failure (HF) hospitalizations, and transplant/death. In particular, the relevance of acute myocarditis-like episodes for subsequent disease course is largely unknown. All patients with pathogenic/likely pathogenic (P/LP) DSP variants in the worldwide DSP-ERADOS Network (26 academic institutions across nine countries) were included. The primary outcomes were the development of sustained VA and HF hospitalizations during follow-up. Fine-Gray regressions were used to test association between clinical and instrumental parameters and the development of outcomes. Eight hundred patients [40.3 ± 17.5 years, 47.5% probands, left ventricular ejection fraction (LVEF) 49.5 ± 13.9%] were included. Over 3.7 [1.4-7.1] years, 139 (17.4%, 3.9%/year) and 72 (9.0%, 1.8%/year) patients experienced sustained VA and HF episodes, respectively. A total of 32.5% of individuals did not fulfil diagnostic criteria for ARVC, DCM, or NDLVC; their VA incidence was 0.5%/year. In multivariable regression, risk features associated with the development of VA were female sex [adjusted hazard ratio (aHR) 1.547; P = .025], prior non-sustained ventricular tachycardia (aHR 1.721; P = .009), prior sustained VA (aHR 1.923; P = .006), and LVEF ≤ 50% (aHR: 1.645; P = .032), while for HF, they were the presence of T-wave inversion in 3+ electrocardiogram leads (aHR 2.036, P = .007) and LVEF ≤ 50% (aHR 3.879; P < .001). Additionally, 70 (8.8%) patients experienced a myocardial injury episode at presentation or during follow-up. These episodes were associated with an increased risk of VA and HF thereafter (HR 2.394; P < .001, and HR 5.064, P < .001, respectively). Patients with P/LP DSP variants experience high rates of sustained VA and HF hospitalizations. These patients demonstrate a distinct clinical phenotype (DSP cardiomyopathy), whose most prominent risk features associated with adverse clinical outcomes are the presence of prior non-sustained ventricular tachycardia or sustained VA, T-wave inversion in 3+ leads on electrocardiogram, LVEF ≤ 50%, and myocardial injury events.

Abstract Mo079: Analyzing the Predicted Impact of Published SCN5A Variants in Juvenile-Onset Sick Sinus Syndrome

Background: Sick sinus syndrome (SSS) is a cardiac condition caused by sinoatrial node dysfunction, leading to arrhythmias due to the inability to transmit signals properly to the upper chambers of the heart. While age-related degeneration of the sinoatrial node is the most common contributing factor to SSS, congenital disorders involving genetic variants can also lead to its development, even in pediatric patients. To explore the genetic basis of SSS in the pediatric population (age &lt; 19 years), we conducted a comprehensive review of case reports to identify associated genetic variants. Our focus centered on variants reported in the sodium channel protein type 5 subunit alpha ( SCN5A ) gene, which has been reported to contribute to various ventricular arrhythmias, including SSS. Methods: A comprehensive literature review of case reports of juvenile-onset SSS was conducted using PubTator/PubMed and the Online Mendelian Inheritance in Man (OMIM) database to identify SCN5A variants. Case reports were selected if they met the following criteria: at least one patient diagnosed with SSS at or under the age of 19 with a detected variant in the SCN5A gene. The structural effects of identified variants were assessed using the HOPE web service. Results: Data compiled from the literature review provided a total of 53 cases related to juvenile-onset SSS and the SCN5A gene. Among the reported patients, many exhibited multiple arrhythmogenic phenotypes alongside SSS, including progressive cardiac conduction disease, idiopathic ventricular tachycardia, and Brugada syndrome. The majority of identified variants were missense, although truncating and insertion/deletion variants were also documented. All three inheritance variations - heterozygous, homozygous, and compound heterozygous - were observed in this set of patients. Although many of the variants had some functional studies performed to determine their pathogenicity, some of the variants did not have a predicted impact. For these variants, we characterized the location and biochemical properties of the variants, comparing them to the variants with known functional impact. Conclusion: Future studies will explore the functional impact associated with the predicted variants through modeling specific mutations in SCN5A , using cardiomyocytes in vitro . Additionally, using further published studies and surveying the protein structure analysis of SCN5A variants will aid in characterizing those that are specific to SSS.

Abstract Or126: Targeting ER stress can attenuate arrhythmia in catecholaminergic polymorphic ventricular tachycardia

Gain-of-function mutations of the sarcoplasmic reticulum (SR) Ca 2+ release channel, the ryanodine receptor (RyR2), are linked to the inherited arrhythmia syndrome catecholaminergic polymorphic ventricular tachycardia (CPVT). Increasing evidence suggests that RyR2 gain-of-function not only disturbs intracellular Ca 2+ homeostasis but drives remodeling of cell signaling and ultrastructure that markedly contributes to the arrhythmogenic phenotype. It is well established that disturbed SR Ca 2+ homeostasis can activate the endoplasmic reticulum (ER) stress response, yet whether RyR2 gain-of-function in CPVT drives ER stress is yet to be explored. The goal of our study was to determine the contribution of ER stress evoked by RyR2 gain-of-function to cardiac arrhythmogenesis. To test this, we created a new rat model of CPVT induced by RyR2-S2222L (+/-) mutation. Simultaneous whole cell patch clamp and Ca 2+ imaging demonstrated that under β-adrenergic stimulation, CPVT ventricular myocytes (VMs) exhibit a high propensity to spontaneous Ca 2+ waves (SCWs) and delayed afterdepolarizations. Importantly, CPVT VMs showed increased XBP1 splicing as a marker of ER stress, as well as increased intra-SR redox stress measured using biosensor ER_roGFPiE. Assessment of ER stress proteins that contribute to SR redox status revealed upregulation of H 2 0 2 -producer enzyme ERO1α, with no compensatory change in expression of H 2 O 2 -degrader Peroxiredoxin-4 (PRDX4). Adenoviral overexpression of PRDX4 in CPVT VMs not only normalized SR redox status but attenuated Ca 2+ mishandling, reducing RyR2 activity and the incidence of proarrhythmic spontaneous Ca 2+ waves. Of note, immunofluorescence and biochemical studies suggest a direct interaction between RyR2 and PRDX4. To test whether targeting ER stress was protective at the whole heart level, we delivered AAV9-αMHC-PRDX4 to CPVT rats and performed ex vivo optical mapping. While CPVT hearts exhibited triggered activity and increased incidence of VT, sustained VT was prevented in PRDX4-injected hearts. Collectively, these data strongly suggest that ER stress contributes to the arrhythmogenic phenotype of CPVT. Targeting ER stress protein PRDX4 has promising therapeutic potential to normalize SR homeostasis, stabilize RyR2 activity and attenuate Ca 2+ -dependent arrhythmogenesis.

Divergent Biochemical Properties and Disparate Impact of Arrhythmogenic Calmodulin Mutations on Zebrafish Cardiac Function.

Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca2+)-binding sensor that plays a vital role in many cellular processes by binding and regulating the activity of over 300 protein targets. In cardiac muscle, CaM modulates directly or indirectly the activity of several proteins that play a key role in excitation-contraction coupling (ECC), such as ryanodine receptor type 2 (RyR2),l-type Ca2+ (Cav1.2), sodium (NaV1.5) and potassium (KV7.1) channels. Many recent clinical and genetic studies have reported a series of CaM mutations in patients with life-threatening arrhythmogenic syndromes, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently showed that four arrhythmogenic CaM mutations (N98I, D132E, D134H, and Q136P) significantly reduce the binding of CaM to RyR2. Herein, we investigate in vivo functional effects of these CaM mutations on the normal zebrafish embryonic heart function by microinjecting complementary RNA corresponding to CaMN98I, CaMD132E, CaMD134H, and CaMQ136P mutants. Expression of CaMD132E and CaMD134H mutants results in significant reduction of the zebrafish heart rate, mimicking a severe form of human bradycardia, whereas expression of CaMQ136P results in an increased heart rate mimicking human ventricular tachycardia. Moreover, analysis of cardiac ventricular rhythm revealed that the CaMD132E and CaMN98I zebrafish groups display an irregular pattern of heart beating and increased amplitude in comparison to the control groups. Furthermore, circular dichroism spectroscopy experiments using recombinant CaM proteins reveals a decreased structural stability of the four mutants compared to the wild-type CaM protein in the presence of Ca2+. Finally, Ca2+-binding studies indicates that all CaM mutations display reduced CaM Ca2+-binding affinities, with CaMD132E exhibiting the most prominent change. Our data suggest that CaM mutations can trigger different arrhythmogenic phenotypes through multiple and complex molecular mechanisms.

HEART RHYTHM DISTURBANCES IN CONNECTIVE TISSUE DYSPLASIA

The article discusses the association between connective tissue dysplasia (CTD) and arrhythmias, particularly focusing on mitral valve prolapse as a potential risk factor for life-threatening ventricular arrhythmias and sudden cardiac death. Various studies emphasize the structural and mechanical abnormalities in the heart that contribute to arrhythmogenic phenotypes in CTD patients, such as fibrosis, mitral annular disjunction, and accessory chordae. Additionally, the text explores the role of autoantibodies, cardiac remodeling, and autoimmune processes in mediating rhythm disturbances and cardiac complications in CTD. The importance of ECG analysis, neural networks, and stress tests in detecting and monitoring arrhythmias in CTD patients is highlighted. The research underscores the significance of understanding the morphological basisand pathophysiological mechanisms of arrhythmias in CTD patients to improve therapeutic strategies and enhance patient outcomes.

A novel DSP zebrafish model reveals training- and drug-induced modulation of arrhythmogenic cardiomyopathy phenotypes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Associazione Italiana per la Ricerca sul Cancro [AIRC grant IG-2017-19928], Italian Ministry of University and Research [grant PRIN 20173ZWACS] Introduction Arrhythmogenic cardiomyopathy (AC) is an inherited disorder characterized by progressive loss of the ventricular myocardium causing life-threatening ventricular arrhythmias, syncope and sudden cardiac death in young and athletes. About 40% of AC cases carry one or more mutations in genes encoding for desmosomal proteins, including Desmoplakin (Dsp). Methods We generated the first stable Dsp knock-out (KO) zebrafish line able to model cardiac alterations and cell signalling dysregulation, characteristic of the AC disease. Results Our stable Dsp knock-out (KO) zebrafish line was characterized by cardiac alterations, edema and bradycardia at larval stages. Histological analysis of mutated adult hearts showed reduced contractile structures and abnormal shape of the ventricle, with thinning of the myocardial layer, vessels dilation and presence of adipocytes within the myocardium. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes. Intensive physical training protocol caused a global worsening of the cardiac phenotype, accelerating the progression of the disease. Of note, we detected a decrease of Wnt/β-catenin signalling, recently associated with AC pathogenesis, as well as Hippo/YAP-TAZ and TGF-β pathway dysregulation. Pharmacological treatment of mutated larvae with SB216763, a Wnt/β-catenin agonist, rescued pathway expression and cardiac abnormalities, stabilizing the heart rhythm. Conclusion Our Dsp KO zebrafish line recapitulates many AC features observed in human patients, pointing at zebrafish as a suitable system for in vivo analysis of environmental modulators, such as the physical exercise, and the screening of pathway-targeted drugs, especially related to the Wnt/β-catenin signalling cascade.

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.

Altered firing activity of intracardiac neurons isolated from mice carrying the Scn5a-1798insD mutation

Abstract Funding Acknowledgements None. Background Dysfunction of the cardiac sodium channel Nav1.5 (encoded by SCN5A) is associated with arrhythmias and sudden cardiac death, the mechanisms of which have been extensively studied in cardiomyocytes. However, patients carrying an SCN5A mutation often display clinical symptoms which are not readily explained solely by alterations in cardiomyocyte electrophysiology. For instance, carriers of the SCN5A-1795insD mutation, which is associated with features of Brugada syndrome and Long QT syndrome type 3 (LQT3), display severe bradycardia leading to excessive QT-prolongation and sudden death especially occurring at rest or during the night, suggesting autonomic triggers. Interestingly, SCN5A is not only present in cardiomyocytes, but also in intracardiac neurons (ICN). ICN modulate cardiac electrophysiological properties and their dysregulation may contribute to arrhythmogenesis. Purpose To explore whether ICN from mice carrying the Scn5a-1798insD mutation (the murine equivalent of SCN5A-1795insD) display electrophysiological alterations that may (in part) contribute to the arrhythmogenic phenotype. Methods and Results ICN were isolated by enzymatic dissociation from fat pads located in the pulmonary veins and sinoatrial node regions of the hearts of wild-type (WT) and Scn5a-1798insD mice. Immunodetection of the cholinergic marker ChAT confirmed the prominent cholinergic phenotype of ICN. In agreement with previous reports in other species, we also detected the presence of Nav1.5 in murine ICN. Nav1.5 expression was mainly observed in ChAT positive cells. To evoke repetitive action potential (AP) firing in single ICN, 500 ms-sustained depolarizing current pulses were applied of increasing amplitude (from 0 pA to 150 pA). Two types of neurons were identified on the basis of firing pattern characteristics: accommodating (neurons that discharged multiple APs decrementing in frequency during the pulse) and tonic (neurons that discharged multiple APs at a high frequency). APs firing frequency did not differ between WT and Scn5a-1798insD accommodating ICN, while it was significantly increased in Scn5a-1798insD tonic ICN as compared to WT (at 100 pA, AP count/500ms in WT CMs: 29.5±1.7, n=24; at 100 pA, AP count/500ms in Scn5a-1798insD: 36.2±2.0, n=18, p=0.02). Conclusions The impact of SCN5A mutations on ICN function are unexplored, but may play an essential role in arrhythmogenesis. The increased AP firing frequency observed in Scn5a-1798insD ICN may lead to increased release of neurotransmitters, such as acetylcholine, that in turn may contribute to the bradycardia observed in patients. Overall, further elucidation of the electrophysiological consequences of inherited Nav1.5 dysfunction in ICN might provide novel therapeutic targets to prevent lethal arrhythmias.

Modeling mutation-specific arrhythmogenic phenotypes in isogenic human iPSC-derived cardiac tissues

Disease modeling using human induced pluripotent stem cells (hiPSCs) from patients with genetic disease is a powerful approach for dissecting pathophysiology and drug discovery. Nevertheless, isogenic controls are required to precisely compare phenotypic outcomes from presumed causative mutations rather than differences in genetic backgrounds. Moreover, 2D cellular models often fail to exhibit authentic disease phenotypes resulting in poor validation in vitro. Here we show that a combination of precision gene editing and bioengineered 3D tissue models can establish advanced isogenic hiPSC-derived cardiac disease models, overcoming these drawbacks. To model inherited cardiac arrhythmias we selected representative N588D and N588K missense mutations affecting the same codon in the hERG potassium channel gene KCNH2, which are reported to cause long (LQTS) and short (SQTS) QT syndromes, respectively. We generated compound heterozygous variants in normal hiPSCs, and differentiated cardiomyocytes (CMs) and mesenchymal cells (MCs) to form 3D cardiac tissue sheets (CTSs). In hiPSC-derived CM monolayers and 3D CTSs, electrophysiological analysis with multielectrode arrays showed prolonged and shortened repolarization, respectively, compared to the isogenic controls. When pharmacologically inhibiting the hERG channels, mutant 3D CTSs were differentially susceptible to arrhythmic events than the isogenic controls. Thus, this strategy offers advanced disease models that can reproduce clinically relevant phenotypes and provide solid validation of gene mutations in vitro.

KCNQ1 p.D446E Variant as a Risk Allele for Arrhythmogenic Phenotypes: Electrophysiological Characterization Reveals a Complex Phenotype Affecting the Slow Delayed Rectifier Potassium Current (IKs) Voltage Dependence by Causing a Hyperpolarizing Shift and a Lack of Response to Protein Kinase A Activation.

Genetic testing is crucial in inherited arrhythmogenic channelopathies; however, the clinical interpretation of genetic variants remains challenging. Incomplete penetrance, oligogenic, polygenic or multifactorial forms of channelopathies further complicate variant interpretation. We identified the KCNQ1/p.D446E variant in 2/63 patients with long QT syndrome, 30-fold more frequent than in public databases. We thus characterized the biophysical phenotypes of wildtype and mutant IKs co-expressing these alleles with the β-subunit minK in HEK293 cells. KCNQ1 p.446E homozygosity significantly shifted IKs voltage dependence to hyperpolarizing potentials in basal conditions (gain of function) but failed to shift voltage dependence to hyperpolarizing potentials (loss of function) in the presence of 8Br-cAMP, a protein kinase A activator. Basal IKs activation kinetics did not differ among genotypes, but in response to 8Br-cAMP, IKs 446 E/E (homozygous) activation kinetics were slower at the most positive potentials. Protein modeling predicted a slower transition of the 446E Kv7.1 tetrameric channel to the stabilized open state. In conclusion, biophysical and modelling evidence shows that the KCNQ1 p.D446E variant has complex functional consequences including both gain and loss of function, suggesting a contribution to the pathogenesis of arrhythmogenic phenotypes as a functional risk allele.

Role of genetics in inflammatory cardiomyopathy

Traditional cardiomyopathy paradigms segregate inflammatory etiologies from those caused by genetic variants. An identified or presumed trigger is implicated in acute myocarditis or chronic inflammatory cardiomyopathy but growing evidence suggests a significant proportion of patients have an underlying cardiomyopathy-associated genetic variant often even when a clear inflammatory trigger is identified. Recognizing a possible genetic contribution to inflammatory cardiomyopathy may have major downstream implications for both the patient and family. The presenting features of myocarditis (i.e. chest pain, arrhythmia, and/or heart failure) may provide insight into diagnostic considerations. One example is isolated cardiac sarcoidosis, a distinct inflammatory cardiomyopathy that carries diagnostic challenges and clinical overlap; genetic testing has increasingly reclassified cases of isolated cardiac sarcoidosis as genetic cardiomyopathy, notably altering management. On the other side, inflammatory presentations of genetic cardiomyopathies are likewise underappreciated and a growing area of investigation. Inflammation plays an important role in the pathogenesis of several familial cardiomyopathies, especially arrhythmogenic phenotypes. Given these clinical scenarios, and the implications on clinical decision making such as initiation of immunosuppression, sudden cardiac death prevention, and family screening, it is important to recognize when genetics may be playing a role.

Research landscape of genetics in dilated cardiomyopathy: insight from a bibliometric analysis.

Dilated cardiomyopathy (DCM) is a heterogeneous myocardial disorder with diverse genetic or acquired origins. Notable advances have been achieved in discovering and understanding the genetics of DCM. This study aimed to depict the distribution of the main research forces, hotspots, and frontiers in the genetics of DCM, thus shaping future research directions. Based on the documents published in the Web of Science Core Collection database from 2013 to 2022, co-authorship of authors, institutions, and countries/regions, co-citation of references, and co-occurrence of keywords were conducted respectively to present the distribution of the leading research forces, research hotspots, and emerging trends in the genetics of DCM. 4,141 documents were included, and the annual publications have steadily increased. Seidman, Christine E, Meder, Benjamin, Sinagra, Gianfranco were the most productive authors, German Centre for Cardiovascular Research was the most productive institution, and the USA, China, and Germany were the most prolific countries. The co-occurrence of keywords has generated 8 clusters, including DCM, lamin a/c, heart failure, sudden cardiac death, hypertrophic cardiomyopathy, cardiac hypertrophy, arrhythmogenic cardiomyopathy, and next-generation sequencing. Frequent keywords with average publication time after 2019 mainly included arrhythmogenic cardiomyopathy, whole-exome sequencing, RBM 20, phenotype, risk stratification, precision medicine, genotype, and machine learning. The research landscape of genetics in DCM is continuously evolving. Deciphering the genetic profiles by next-generation sequencing and illustrating pathogenic mechanisms of gene variants, establishing innovative treatments for heart failure and improved risk stratification for SCD, uncovering the genetic overlaps between DCM and other inherited cardiomyopathies, as well as identifying genotype-phenotype correlations are the main research hotspots and frontiers in this field.

A novel DSP zebrafish model reveals training- and drug-induced modulation of arrhythmogenic cardiomyopathy phenotypes

Arrhythmogenic cardiomyopathy (AC) is an inherited disorder characterized by progressive loss of the ventricular myocardium causing life-threatening ventricular arrhythmias, syncope and sudden cardiac death in young and athletes. About 40% of AC cases carry one or more mutations in genes encoding for desmosomal proteins, including Desmoplakin (Dsp). We present here the first stable Dsp knock-out (KO) zebrafish line able to model cardiac alterations and cell signalling dysregulation, characteristic of the AC disease, on which environmental factors and candidate drugs can be tested. Our stable Dsp knock-out (KO) zebrafish line was characterized by cardiac alterations, oedema and bradycardia at larval stages. Histological analysis of mutated adult hearts showed reduced contractile structures and abnormal shape of the ventricle, with thinning of the myocardial layer, vessels dilation and presence of adipocytes within the myocardium. Moreover, TEM analysis revealed “pale”, disorganized and delocalized desmosomes. Intensive physical training protocol caused a global worsening of the cardiac phenotype, accelerating the progression of the disease. Of note, we detected a decrease of Wnt/β-catenin signalling, recently associated with AC pathogenesis, as well as Hippo/YAP-TAZ and TGF-β pathway dysregulation. Pharmacological treatment of mutated larvae with SB216763, a Wnt/β-catenin agonist, rescued pathway expression and cardiac abnormalities, stabilizing the heart rhythm. Overall, our Dsp KO zebrafish line recapitulates many AC features observed in human patients, pointing at zebrafish as a suitable system for in vivo analysis of environmental modulators, such as the physical exercise, and the screening of pathway-targeted drugs, especially related to the Wnt/β-catenin signalling cascade.

Arrhythmogenic cardiomyopathy phenotype associated with the pathogenic founder variant c.1211dup in PKP2

Abstract Background Arrhythmogenic cardiomyopathy results in life-threatening ventricular arrhythmia and heart failure. Phenotypic features vary according to genetic aetiology. Genotype-phenotype research is therefore crucial for patient counselling and treatment decisions such as implantable cardioverter defibrillator therapy. By collecting data from patient records we characterized the clinical phenotype associated with variant c.1211dup (p.Val406Serfs*4) in the plakophilin-2 gene (PKP2), common in the Dutch population. Purpose We aimed to investigate the incidence of ventricular arrhythmia and heart failure in families carrying the PKP2 c.1211dup variant. Furthermore, we aimed to determine the founder status of this variant, and compare phenotype severity with other truncating PKP2 variants. Methods Clinical data were collected retrospectively from medical records of 106 PKP2 c.1211dup heterozygous carriers (&amp;gt;85% of known carriers in The Netherlands) in a Castor EDC database. Using occurrence of ventricular arrhythmia and heart failure, event-free survival was compared between males and females, and probands and family members, using log-rank tests. Using data from a central curated arrhythmogenic cardiomyopathy database, c.1211dup was compared to three other truncating PKP2 variants (c.235C&amp;gt;T (p.Arg79*), c.397C&amp;gt;T (p.Gln133*) and c.2489+1G&amp;gt;A (p.?)) on ventricular arrhythmia-free survival by a Cox proportional hazards model, corrected for sex and proband status. Results Forty-seven carriers (44%) were diagnosed with arrhythmogenic cardiomyopathy at 41 years on average. As shown in figure 1, 29 participants (27%) experienced episodes of ventricular arrhythmia and 12 (11%) developed heart failure, with male carriers showing significantly higher risks than females on both endpoints (p&amp;lt;0.05). Based on available magnetic resonance imagery and echocardiographic data, 46% of carriers showed either right ventricular dilatation and/or dysfunction, and a substantial minority of 37% showed some form of left ventricular involvement. Both geographical distribution of carriers (figure 2) and haplotype analysis suggested PKP2 c.1211dup to be a founder variant originating from the South-Western coast of the Netherlands. Finally, a Cox proportional hazards model suggested significant differences in ventricular arrhythmia-free survival between four PKP2 truncating founder variants, including c.1211dup. Conclusions The PKP2 c.1211dup variant is a Dutch founder variant associated with a typical right ventricular-dominant arrhythmogenic cardiomyopathy phenotype, but also notable left ventricular involvement. Males were most susceptible to the frequent ventricular arrhythmia, and less frequent heart failure was observed in general. Finally, c.1211dup possibly leads to a more severe phenotype than other Dutch PKP2 founder variants.Phenotype onset stratified by sexGeographic distribution carriers

Calcium Signaling Consequences of RyR2-S4938F Mutation Expressed in Human iPSC-Derived Cardiomyocytes.

Type-2 ryanodine receptor (RyR2) is the major Ca2+ release channel of the cardiac sarcoplasmic reticulum (SR) that controls the rhythm and strength of the heartbeat, but its malfunction may generate severe arrhythmia leading to sudden cardiac death or heart failure. S4938F-RyR2 mutation in the carboxyl-terminal was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing technique. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying the mutation were studied using total internal reflection fluorescence microscopy (TIRF) and patch clamp technique. In mutant cells, L-type Ca2+ currents (ICa), measured either by depolarizations to zero mV or repolarizations from +100 mV to -50 mV, and their activated Ca2+ transients were significantly smaller, despite their larger caffeine-triggered Ca2+ release signals compared to wild type (WT) cells, suggesting ICa-induced Ca2+ release (CICR) was compromised. The larger SR Ca2+ content of S4938F-RyR2 cells may underlie the higher frequency of spontaneously occurring Ca2+ sparks and Ca2+ transients and their arrhythmogenic phenotype.

Abstract P3135: In Vivo Dissection Of CaMKII-Mediated Arrhythmias In CPVT

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome of the structurally intact heart manifesting life-threatening bursts of ventricular tachycardias due to dominant gain-of-function mutations in the ryanodine receptor type 2 (RyR2) and β-adrenergic (β-AR) stimulation. Both protein kinase A (PKA) and Ca 2+ - calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR2 channels, but only the latter can initiate the arrhythmogenic phenotype in CPVT. Whether through L-type Ca 2+ channel (LTCC)-mediated Ca 2+ increase or the Epac2-nitric oxide pathway, the exact mechanism by which β-AR-mediated CaMKII activation unmasks lethal arrhythmias in CPVT remains unresolved. Aim: To gain mechanistic insight and ablate ventricular arrhythmias in a mouse model of CPVT by selectively disrupting integral components of β-AR signaling. Methods: By utilizing either direct adeno-associated virus (AAV) delivery or the CRISPR/Cas9-AAV-based somatic mutagenesis (CASAAV) system, we selectively expressed a mutant LTCC β 2 -subunit, ablated the small GTPase Rad, or disrupted other components of the β-AR pathway in the hearts of mice with the pathogenic RyR2 mutation R4650I as a model of CPVT. The effects of disruption of LTCC regulation were determined by high-speed Ca 2+ imaging of isolated ventricular cardiomyocytes or whole animal electrophysiological (EP) testing for ventricular arrhythmia incidence in vivo . Results &amp; Conclusions: Our data demonstrate that abnormal RyR2-mediated calcium release events are induced by hyperactivated LTCCs in vitro , even in the absence of adrenergic stimulation, with increased arrhythmia incidence in vivo . These results suggest that β-AR stimulation leads to pathogenic RyR2 Ca 2+ leak through LTCC-mediated contribution to CaMKII hyperactivity. Alternative mechanisms of CaMKII activation that mediate arrhythmia induction in CPVT require additional future investigation.

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.