Arrhythmogenic Cardiomyopathy Research Articles

Gene Therapy in Cardiovascular Disease: Recent Advances and Future Directions in Science: A Science Advisory From the American Heart Association

Cardiovascular disease remains the foremost cause of morbidity and mortality globally, affecting millions of individuals. Recent discoveries illuminate the substantial role of genetics in cardiovascular disease pathogenesis, encompassing both monogenic and polygenic mechanisms and identifying tangible targets for gene therapies. Innovative strategies have emerged to rectify pathogenic variants that cause monogenic disorders such as hypertrophic, dilated, and arrhythmogenic cardiomyopathies and hypercholesterolemia. These include delivery of exogenous genes to supplement insufficient protein levels caused by pathogenic variants or genome editing to correct, delete, or modify mutant sequences to restore protein function. However, effective delivery of gene therapy to specified cells presents formidable challenges. Viral vectors, notably adeno-associated viruses and nonviral vectors such as lipid and engineered nanoparticles, offer distinct advantages and limitations. Additional risks and obstacles remain, including treatment durability, tissue-specific targeting, vector-associated adverse events, and off-target effects. Addressing these challenges is an ongoing imperative; several clinical gene therapy trials are underway, and many more first-in-human studies are anticipated. This science advisory reviews core concepts of gene therapy, key obstacles, patient risks, and ongoing research endeavors to enable clinicians to understand the complex landscape of this emerging therapy and its remarkable therapeutic potential to benefit cardiovascular disease.

Desmoplakin Cardiomyopathy in Pediatric Patients: A Distinct, Underrecognized Cohort of Arrhythmogenic Cardiomyopathy

BACKGROUND: DSP cardiomyopathy is a distinct subset of arrhythmogenic cardiomyopathy, reported primarily in adults, that has predominantly left ventricular involvement and features of myocarditis. Clinical characteristics, risk stratification, and management of pediatric patients with DSP variants are not well known. We sought to identify phenotypic features and prognosis of pediatric patients with DSP pathogenic or likely pathogenic variants. METHODS: Multicenter, retrospective study of patients <21 years of age with DSP variants from 6 tertiary pediatric hospitals. RESULTS: Thirty-four patients, including 10 probands with clinical disease and 24 genotype-positive phenotype-negative patients, were included in the study. The majority of probands were initially diagnosed with myocarditis (50%) and had biventricular (60%) or left ventricular predominant (40%) disease. Chest pain was the most common symptom at presentation (30%), and all had troponin elevation. Probands with homozygous or compound heterozygous DSP variants were likely to present at an early age (<13 years) with symptoms of heart failure, severe biventricular involvement, and dermatologic abnormalities. Low-voltage QRS was the most prominent ECG abnormality. Of those who underwent implantable cardioverter defibrillator implantation, 50% received appropriate implantable cardioverter defibrillator therapy and were found to have significant biventricular involvement in addition to severe left ventricular dysfunction with an ejection fraction <35%. CONCLUSIONS: DSP cardiomyopathy in children and adolescents has varied phenotypic manifestations based on age and genotype and often can be diagnosed as myocarditis. Severe left ventricular dysfunction and biventricular involvement may be associated with a higher likelihood of malignant ventricular tachyarrhythmia.

Why the Athlete's heart matters: Insights into Arrhythmogenic cardiomyopathy

Why the Athlete's heart matters: Insights into Arrhythmogenic cardiomyopathy

A review of alternative measurements in strain imaging for ventricular arrhythmia prediction

Global longitudinal strain has been established as a reliable tool to assess global left ventricular function and a marker of subclinical left ventricular dysfunction unrecognized by the ejection fraction. On the other hand, ventricular arrhythmias are the most common cause of sudden cardiac death. Their early detection is a challenge. Possible prognostic markers for the risk of ventricular arrhythmias are discussed in the literature – electrocardiographic, cardiac magnetic resonance, computed tomography, radionuclide imaging, and markers from new echocardiographic techniques. Of the latter, at this stage of knowledge, several markers have been discussed as informative for predicting ventricular arrhythmias – global longitudinal strain, radial strain and mechanical dispersion, and most recently, myocardial work. As far as we are informed, global longitudinal strain is particularly useful in patients with normal echocardiographic parameters such as left ventricular ejection fraction, left atrial diameter, left ventricular wall thickness, and aortic root. The relationship between mechanical dispersion and ventricular arrhythmias has been widely studied. The relationship has been studied more in some patient populations – heart failure, ischemic heart disease, long QT syndrome and arrhythmogenic cardiomyopathy, congenital heart disease. The role of mechanical dispersion as a predictor of ventricular arrhythmias in metabolic syndrome is scarce.

Targeting Wnt Pathways with Small Molecules as New Approach in Cardiovascular Disease.

The increasing incidences of morbidity and mortality associated with cardiovascular diseases represent significant difficulties for clinical treatment and have a major impact on patient health. Wnt signaling pathways are highly conserved and are well known for their regulatory roles in embryonic development, tissue regeneration, and adult tissue homeostasis. Wnt signaling is classified into two distinct pathways: canonical Wnt/β-catenin signaling and noncanonical pathways, including planar cell polarity and Wnt/Ca2+ pathways. A growing body of experimental evidence suggests the involvement of both canonical and non-canonical Wnt signaling pathways in the development of cardiovascular diseases, including myocardial hypertrophy, arrhythmias, diabetic cardiomyopathy, arrhythmogenic cardiomyopathy, and myocardial infarction. Thus, to enhance patient quality of life, diagnosing and treating cardiac illnesses may require a thorough understanding of the molecular functions played by the Wnt pathway in these disorders. Many small-molecule inhibitors specifically target various components within the Wnt signaling pathways, such as Frizzled, Disheveled, Porcupine, and Tankyrase. This study aims to present an overview of the latest findings regarding the functions of Wnt signaling in human cardiac disorders and possible inhibitors of Wnt, which could lead to novel approaches for treating cardiac ailments.

Clinical overlap between healthy athletes and athletes with arrhythmogenic right ventricular cardiomyopathy

Abstract Introduction The extensive physiological cardiac remodelling that results from the haemodynamic stress of endurance exercise shares many features with the pathological manifestations of arrhythmogenic right ventricular cardiomyopathy (ARVC), giving rise to a significant diagnostic challenge for clinicians. Contemporary discriminators of disease have been derived from comparisons between healthy athletes and non-athletic ARVC patients. Differentiation between healthy and diseased athletes may be more difficult. Purpose To assess published clinical differentiators of physiological remodelling from ARVC in a cohort of endurance athletes. Methods This project utilises data from the Pro@heart consortium comprising elite endurance athletes that have received comprehensive cardiac phenotyping using cardiac magnetic resonance imaging, echocardiography, resting and ambulatory electrocardiography and cardiopulmonary exercise testing. Measures of cardiac structure and function with potential to differentiate health and disease were assessed in healthy endurance athletes without symptoms or suspicion of ARVC, endurance athletes diagnosed with ARVC (meeting ARVC task force criteria) and athletes with suspected sub-clinical ARVC (complex ventricular arrhythmias and ≥1 task force criteria). One way ANOVA assessed differences in continuous variables, while Chi Square with pair wise Z test and Bonferroni p value correction were used to test for differences in nominal data. Results Athletes with ARVC and athletes with suspected ARVC displayed more clinical indicators of ARVC compared to healthy athletes (table 1). While a greater proportion of athletes with ARVC and suspected ARVC met ≥1 and ≥3 indicators of pathology compared to healthy athletes, it was relatively common for healthy athletes to meet ≥1 criteria (53%) and ≥3 criteria (14%) (table 2). Comparison of mean values showed right ventricular ejection fraction (RVEF) and right ventricular fractional area change (RV FAC) were lower in athletes with or suspected to have ARVC compared to healthy athletes, however no significant differences in burden of ventricular premature beats (VPB) were noted (table 1). A high burden of VPBs, evidence of ventricular arrhythmia, evidence of late gadolinium enhancement (LGE), a low RVEF, and a reduced RV FAC were more prevalent in athletes with ARVC compared to healthy athletes (table 2). However, there was clinically relevant overlap in all measures between healthy and diseased athletes (table 2). Right ventricular to left ventricular volume ratio was a particularly poor discriminator of pathology (table 1 & 2). Conclusion Electrophysiological, structural and functional abnormalities are not uncommon in healthy athletes resulting in significant overlap with athletic ARVC patients. Future research is required to identify better clinical discriminators of disease, and to assess the clinical significance of abnormal measures in ostensibly healthy athletes

Arrhythmogenic right ventricular cardiomyopathy: the importance of biventricular strain in risk-stratification

Abstract Background Despite Arrhythmogenic right ventricular cardiomyopathy (ARVC) being a predominant right ventricle (RV) disease, concomitant left ventricle (LV) involvement has been recognized. ARVC diagnosis is made through the RV-centric 2010 Task Force Criteria (TFC), but previous studies already suggested the use of echocardiographic strain measures to better depict RV and LV dysfunction in these patients. No data however is available on the additional value of combining biventricular strain in ARVC for risk stratification. Purpose To assess the prognostic value of measuring both LV global longitudinal strain (GLS) and RV free wall strain (FWLS) analysis in patients with ARVC. Methods A total of 204 patients who met the TFC for the ARVC spectrum were included. Patients were categorized based on a cut-off of -18% for strain impairment in both ventricles. The end-point was a composite of all-cause mortality, arrhythmic events, ICD therapy and heart failure events. Results Patients (age 41 ± 17 years, 55% males) were divided into impaired (n=33), discordant (either RV or LV impaired, n=70), and normal (n=101) strain groups (Fig. 1A). Definite and borderline ARVC were diagnosed in 78% and 39%, respectively, at the time of first available echocardiography. During a follow-up of 87 [24-136] months, 60 (29%) experienced the combined outcome, and a significant difference in event-free survival was observed (p<0.001) between the 3 groups (Fig. 1B). In the multivariate analysis (Fig. 2), the strain grouping remained associated with outcome (p=0.040) after adjusting for age, gender, history of syncope and definite ARVC diagnosis, which were also significantly associated with the endpoint at the univariate analysis. A sub-analysis including only definite and borderline ARVC patients confirmed that the strain grouping was an independent predictor of the composite endpoint (p=0.027). Conclusion Biventricular involvement by strain analysis has additional prognostic value in ARVC patients and outcome is worse for those with both RV and LV involvement.Strain groups and Kaplan Meier curvesMultivariate analysis

Clinical value of anti-DSG2 antibodies in arrhythmogenic right ventricular cardiomyopathy: a real-life experience

Abstract Background/Introduction Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease leading to ventricular arrhythmias and heart failure. The diagnosis is challenging and currently based on a set of clinical criteria with limited sensitivity and specificity. Anti-desmoglein-2 (DSG2) antibodies were previously found in patients diagnosed with ARVC, while they were absent in both healthy controls and athletes. Purpose The study aimed to evaluate the diagnostic value of anti-DSG2 antibodies by comparing their concentration in patients with ARVC and other right ventricular diseases. Additionally, the prognostic value of the biomarker was assessed. Methods A cohort of 101 patients with a definite diagnosis of ARVC according to the 2010 Task Force criteria was enrolled (64 males, mean age 47 ± 16 years). The control group included patients with Ebstein anomaly and Eisenmenger syndrome (16 and 21 subjects, respectively). Anti-DSG2 antibody concentration was assessed using enzyme-linked immunosorbent assay (ELISA). After that, an ARVC cohort was followed for 3.9 ± 1.6 years for the occurrence of the primary endpoint of cardiac death or heart transplantation (HTx) and major arrhythmic events (MAEs) defined as ventricular fibrillation, sustained ventricular tachycardia (sVT) or appropriate implantable cardioverter-defibrillator intervention. Results The median anti-DSG2 antibody concentration presented as ELISA optical density (OD) reached 1.36 [0.54-2.15] in the ARVC cohort, while it was 0.4 [0.34-0.54] in Ebstein anomaly group and 0.36 [0.28-0.58] in the Eisenmenger syndrome group. There was a significant difference in antibody levels between ARVC and both control groups (p < 0.001, Figure 1). The cutoff value for the diagnosis of ARVC was 0.774, with 72.3% sensitivity and 100% specificity (AUC = 0.823). After dividing the ARVC cohort according to the designated cutoff point, the anti-DSG2-positive subgroup was significantly younger (44.5 ± 15.4 vs. 52.8 ± 15.5 years, p = 0.017) and more likely to have a history of sVT (68.5% vs. 46.4%, p = 0.041). There was no difference regarding sex, presence of PKP2 or DSG2 mutations, previous sports activities, history of sudden cardiac arrest or syncope, right ventricular dimensions and systolic function, left ventricular ejection fraction and heart failure symptoms. This threshold was not predictive for the primary endpoint and MAEs. However, a more stringent cutoff value for anti-DSG2 level (1.021) was found to predict death or HTx (HR [95% CI] 4.56 [1.05-19.76], p = 0.026, Figure 2). Conclusions The concentration of anti-DSG2 antibodies is significantly higher in ARVC compared to other right ventricular diseases. A high antibody level is prognostic for death or HTx in ARVC.Figure 1Figure 2

Vector Field Heterogeneity (VFH) as a novel quantitative marker of electrical disarray in Arrhythmogenic CardioMyopathy (ACM) with ventricular tachycardia

Abstract Background ACM is a hereditary condition characterized by fibrofatty infiltration and replacement of cardiac myocytes predominantly presenting with ventricular arrhythmias (VA). Cardiac imaging allows for detailed structural phenotyping of scar patterns in ACM. Yet, there is a lack of tools to objectively quantify the electrical substrate alterations that may enable a more precise identification of critical sites that predispose to VA. Purpose To quantify locally disorganized cardiac electrical propagation wavefronts using the Vector Field Heterogeneity (VFH) metric from high-density multi-electrodes in patients with ACM with VT and compare to healthy controls. Methods High density epicardial substrate maps acquired with a 16-pole grid catheter during SR or RV pacing in patients with ACM with VA, who underwent clinically indicated catheter ablation, were retrospectively reviewed. Patients with structurally normal heart and idiopathic VA with epicardial ablation were analysed as a control group. Unipolar contact mapping data was exported & processed offline. From the 4×4 electrode array EGMs, vector maps of the directions of electrical propagation for recorded sites were computed. The VFH was then computed as a quantitative measure of local heterogeneity of propagation, with higher VFH expected to correlate with sites of (micro)structural tissue alterations. VFH was compared between ACM and control patients as well as at sites of normal, border zone & scar based on omnipolar voltage in ACM patients (>1.5mV, 0.5mV–1.5mV, <0.5mV, respectively). Results 10 patients with ACM (70% male, aged 52 ±18 years, ARVC 70%, biventricular 30%, RVEF 40.7±4.7%, LVEF 66±12%) and 2 patients with structurally normal heart (female, 38 & 61 years) were included. Epicardial maps had an average of 9258 ±5412 EGM points. Median VFH was statistically significantly higher in the ACM cohort compared to control for global maps (0.33±0.33 vs 0.13±0.22, p<0.001) and at sites with voltage >1.5mV (i.e. greater disorganization in ACM, p<0.001). In ACM, assessment of VFH according to omnipolar voltage sites (as a surrogate of scar) showed a quantifiable statistically significant increase in electrical disorganized propagation at sites of lower voltage (<0.5mV, median VFH 0.53±0.36) compared to borderzone (0.5-1.5mV, VFH 0.29±0.41) and normal voltage (>1.5mV, VFH 0.15±0.26), p <0.001 respectively. Conclusion Heterogenous electrical propagation as quantified by VFH is significantly increased in patients with ACM at sites of scar, but importantly also in areas of normal voltages when compared to non-ACM controls, possibly indicating microstructural alterations that are missed in traditional assessments of the arrhythmogenic substrate. Identifying and quantifying disorganized conduction patterns by VFH is a promising new mapping approach that could allow improved substrate characterization. Its diagnostic value to guide ablation therapy is currently being validated.VFH Metric ACM and ControlVFH Map Examples

Prognostic value of synthetized right-side precordial leads in arrhythmogenic right ventricular cardiomyopathy: data from nordic, dutch, and canadian ARVC registers

Abstract Background T-wave inversion (TWI) in standard ECG leads is a proven risk marker for life-threatening ventricular arrhythmias (VA) in arrhythmogenic right ventricular cardiomyopathy (ARVC). Right-sided precordial leads (RSPL), which may be more sensitive for detection of the arrhythmogenic substrate, are rarely recorded and understudied in this context. Computerized ECG signal-processing allows synthesis of right-sided precordial ECG leads derived from standard 12-lead ECG. Objective We aimed to assess the value of TWI in RSPL, synthetized from the standard 12-lead ECG, for prediction of VA risk in ARVC patients with subtle ECG phenotypes. Methods Patients with definite ARVC by TFC2010 and no history of sustained VA prior to diagnosis from the Nordic, Canadian and Dutch ARVC registries were included. Patients with major repolarization criterion or complete right bundle branch block (RBBB) were excluded. RSPL (V3R-V6R) were derived from digitally recorded eight linearly independent leads (I, II and precordial V1 to V6) of the standard ECG using a linear ECG transformation matrix. The matrix was previously developed based on a library of body surface potential maps from a mixed group of healthy subjects and patients with cardiac diseases using multivariate linear regression and QRS-T data obtained from averaged beats of the right-sided-precordial leads and the basis leads. TWI exceeding 0.1 mV in any of the synthetized leads was tested for its ability to predict sustained VA, defined as either sustained ventricular tachycardia, appropriate ICD shock, aborted cardiac arrest, or sudden cardiac death. In a similar manner, the value of TWI in any of the inferior leads III, aVF or II was assessed. Hazard ratio was calculated using Cox regression analysis adjusted for age, sex, and proband status. Results In total, 144 patients without prior VA history were identified. After exclusion of patients with major repolarization criterion (n=37) and RBBB (n=11), 96 patients comprised the study group (mean age 40±16, 42% female, 41% probands, 48% ICD carriers). Fifty-nine patients (62%) had TWI in one or more synthetized RSPL and 33 patients (34%) had TWI in any of the inferior leads. During 10 years of follow-up, 11 patients (12%) developed VA. Kaplan-Meier curve analysis showed significant association between the presence of TWI in at least one of the RSPL and the risk of VA, which remained an independent predictor in the adjusted Cox regression analysis (HR 10.6, 95%CI 1.3-84.4, p=0.026). TWI in any of the inferior leads was not associated with the risk of VA in this cohort (HR 1.2 95%CI 0.26-5.42). Conclusions Synthetized right-sided precordial leads appear to contain information potentially useful for refining risk stratification of primary prevention ARVC patients with a subtle ECG phenotype, in whom traditional repolarization markers, such as major repolarization criterion or TWI in inferior leads, are not informative.

Role of common variants and new genes associated with arrhythmogenic cardiomyopathy

Abstract Introduction Arrhythmogenic Cardiomyopathy (ACM) is an inherited cardiac disease, predominantly affecting males, characterized by fibro-fatty myocardial replacement mainly in the right ventricle. It leads to 5% sudden cardiac death in affected young adults (<25 yo) without an implantable cardioverter defibrillator. Early diagnosis is crucial for appropriate clinical management to reduce mortality and prevent disease progression. Rare variants in desmosomal genes (PKP2,DSC2,DSG2,DSP and JUP) are found in 50% of cases. However, challenges persist since we observe a low penetrance in relatives (28-58%) and a high variability in expressivity and severity annihilating any preventive strategy. Objective We aim to decipher the genetic architecture of ACM by investigating the role of common variants in disease susceptibility/severity and to idendify new genes associated to ACM. Method A Genome Wide Association Study (GWAS) was performed on 1079 ACM index patients and 7305 controls, followed by conditional analysis. New candidate genes were knocked out (KO) in Zebrafish and/or hiPSC using CRISPR/Cas9. Impedance and external field potential measurements were performed on hiPSC derived cardiomyocytes (CM-hiPSC). Results The GWAS identified 5 significant loci (Figure 1, P<5.10-8), including 2 desmosomal genes (PKP2 a n d DSG2) and 3 new candidates (KLF12, CTNNA3, and ARL17B/WNT3). Conditional analysis revealed 3 additional loci with suggestive p-values (P<1.10-6), highlighting 3 new susceptible loci. Among them, we uncovered key non-coding regulatory regions near desmosomal genes (DSC2 and DSG2). Initial findings in PKP2-KO zebrafish present hearts with structural defects, while CM-hiPS KLF12-KO(-/-) exhibited altered interbeat interval and relaxation time compared to isogenic controls. Conclusion Our findings reveal ACM's complex genetic architecture, implicating common variants in disease susceptibility. A polygenic risk score will be implemented to improve patient risk stratification. Three novel loci/genes for ACM as well as specific regulatory regions of desmosomal genes have been highlighted. Their functional studies in isogenic hiPSC and Zebrafish models will offer new insights in ACM underlying molecular mechanism and potential therapeutic targets.

Amplicon-based long-read sequencing is useful for confirming zygosity of DSG2 variants associated with Japanese ARVC

Abstract Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy mainly caused by desmosomal gene variants. Previously, we reported that DSG2 variants were the most common in 153 Japanese ARVC probands (DSG2 multiple, n=62 and single, n=4; PKP2, n=28; others, n=7). Among the multiple DSG2 variant carriers, 22 probands were compound heterozygous carriers, and 25 were homozygous, which were confirmed by genetic testing for family members (Figure A). On the other hand, 88% of their family members with single DSG2 variant carriers were not diagnosed with ARVC, which meant that recessive DSG2 variants were the major cause of ARVC in Japan. However, zygosities in remaining 15 probands with multiple DSG2 variants were unknown due to the lack of genome data from their parents. Purpose This study aimed to clarify the zygosities of multiple DSG2 variants using amplicon-based long-read sequencing. Methods Genomic DNA was extracted from whole blood using standard protocols. The range containing multiple variants was amplified by long-range PCR. Sequencing libraries were prepared using ONT Ligation Sequencing Kit. Each library was loaded onto a flow cell for sequencing on ONT GridION Mk1 running MinKNOW control software. FASTQ files were aligned to hg38 using minimap2. Variants were called using Clair3 and phased using WhatsHap. Integrative Genomics Viewer (IGV) was used for visualization of the phased variants. Results We successfully phased all the multiple DSG2 variants found in the 15 proband. A typical result of amplicon-based LRS was shown in Figure B. It was IGV screen shot of reads at DSG2 containing c.874C>T (p.R292C) and c.1481A>C (p.D494A). The reads colored in red belonged to one haplotype, while the ones in blue belonged to the other. Each of the two variants was on different color reads, indicating they were compound heterozygous variants. Using LRS, we confirmed all the 15 probands had compound heterozygous variants in DSG2. Overall, the 62 probands (40.5%) with DSG2 variants were recessive variant carriers (Figure A). Conclusions Japanese ARVC was mainly caused by autosomal recessive variants in DSG2. Clarifying the zygosity of causative variants is crucial for the diagnosis in recessive diseases including Japanese ARVC. Amplicon-based LRS was helpful for phasing the multiple DSG2 variants directly without genetic testing of the parents.Figure AFigure B

Navigating the Uncommon: Case Report of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) Successfully Managed with Implantable Cardioverter Defibrillator (ICD)

With a high risk of sudden cardiac mortality, arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare inherited cardiomyopathy characterized by right ventricular dysfunction and ventricular arrhythmias (VA). In this case study, a 35-year-old presented with palpitations and revealed epsilon waves on the right precordial electrocardiogram (ECG) leads which led to the suspicion of ARVC. A 24-hour Holter monitoring revealed frequent ventricular extrasystoles (>500 per 24 hours), a CMRI revealed RV dyskinesia and transmural LGE in the apical RV, and an electrophysiologic study detected spontaneous polymorphic ventricular tachycardia (VT) despite the presence of induction ventricle fibrillation (VF). These results validated the diagnosis of ARVC and indicated that an implantable cardioverter defibrillator was necessary treatment. The ability to identify potential ARVC’s ECG abnormalities, the timing to pursue further studies, and determining necessary treatment execution are critical. The evaluation of cardiac, electrophysiological, and clinical imaging parameters is essential for the risk stratification, diagnosis, and management of patients with ARVC.

Exome sequencing in over 63,000 patients in TIMI trials uncovers pathogenic cardiomyopathy variant carriers with high risk for heart failure and cardiovascular death

Abstract Background Rare genetic variants predisposing to cardiomyopathy (CMP) confer increased risk for adverse events. The prevalence of CMP variants in contemporary cardiovascular (CV) trials and their association with prevalent heart failure (HF) and incident HF hospitalizations (HHF) is not established. Purpose We aimed to identify carriers of rare pathogenic/likely pathogenic (PLP) variants for dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC) in well-phenotyped clinical trial cohorts and to evaluate their prognostic significance. Methods Rare variants were identified from high-quality exome sequencing data from 5 large clinical trials (ENGAGE AF-TIMI 48, SAVOR-TIMI 53, PEGASUS-TIMI 54, DECLARE-TIMI 58, FOURIER (TIMI 59)). Putatively pathogenic variants for cardiomyopathy were identified using phenotype- and gene-specific curation of protein-truncating variants and high-confidence variant classifications from ClinVar. We conducted logistic regression for prevalent disease and Cox proportional hazard models for incident HHF, both adjusted by age, sex, trial, and first 10 principal components and using those without CMP variants as the reference. Sensitivity analysis by history of HF at baseline was performed for prospective outcomes. Results In 63,700 participants, we identified 866 CMP variant carriers of which 518 identified as DCM carriers (age 65±9 yrs, male 66%), 194 HCM carriers (65.5±9 yrs, male 69%), and 125 ARVC carriers (66±9 yrs, male 77%). Out of the 518 DCM variant carriers, 402 had a PLP variant in TTN. DCM variant carriers had higher odds of history of HF (Odds ratio [OR] 1.94, 1.57-2.39, p<0.0001) whereas this was not observed for HCM variant carriers (OR 1.18, 0.85-1.65, p=0.33) or ARVC variant carriers (OR 1.23, 0.80-1.90, p=0.35). Over a mean follow-up of 2.5 years, the rate of HHF was significantly higher for DCM variant carriers (Hazard ratio [HR] 1.58, 1.14-2.20, p=0.006), HCM variant carriers (HR 2.78, 1.87-4.13, p<0.001), and ARVC variant carriers (HR 1.89, 1.02-3.53, p=0.04). These associations were, if anything, stronger in patients without HF at baseline (HR 2.06, 1.30-3.25) than in patients with HF at baseline (HR 1.48, 1.12-1.95). DCM variant carriers also had a higher rate of CV death (HR 1.50, 1.05-2.12, p=0.02), which was not observed for HCM (HR 1.10, 0.57-2.11, p=0.78) or ARVC (HR 0.35, 0.09-1.41, p=0.14) variant carriers. Conclusion In 5 contemporary CV trials, CMP variant carriers have an increased rate of HHF, while DCM variant carriers also have a higher rate of CV death. These findings motivate better identification of these high-risk individuals and the incorporation of genetic testing in CV trials.

Pathogenic cardiomyopathy gene variants inform prognosis in atrial fibrillation: results from exome sequencing in over 17,000 patients in TIMI trials

Abstract Background Rare genetic variants in cardiomyopathy genes are associated with risk of atrial fibrillation (AF), even in the absence of overt ventricular dysfunction. Data on clinical outcomes for rare genetic variant carriers among patients with AF remain sparse. Purpose We aimed to study the prognostic implication of rare pathogenic/likely pathogenic (PLP) variants for dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) in patients with AF from large, well-phenotyped randomized clinical trials. Methods and results Rare variants were called using exome sequencing data in 5 large clinical trials from the TIMI study group (ENGAGE-AF, FOURIER, SAVOR, PEGASUS, DECLARE). PLP variants for cardiomyopathy were identified using phenotype- and gene-specific curation of protein-truncating variants and high-confidence variant classifications from the ClinVar database. In 17,190 patients with a history of AF, we identified 421 (2.4%) PLP variant carriers (age 67±10, 35% women) and 16,769 (97.6%) pts without PLP variants (age 70±9, 36% women). Among these individuals, we identified 265 DCM carriers (1.5%), 106 (0.6%) HCM carriers and 54 (0.3%) ARVC carriers. In this cohort, 9,275 (54%) had a history of heart failure (HF) and 2,991 (17.4%) prior ischemic stroke. Over 2.5 years median follow-up, 1,365 pts (7.9%) were hospitalized for HF, 599 (3.5%) had an ischemic stroke, and 1147 (6.7%) died of cardiovascular causes. In logistic regression analyses adjusting for age, sex, trial, and principal components of ancestry, PLP variants were significantly associated with a history of HF (OR 1.66, p<0.0001), most notably for DCM variants (OR 1.97, 1.48-2.62, p<0.0001). PLP variants were independently associated with incident HF admissions (adj HR 1.75, 1.39-2.29, p<0.0001), most notably for HCM (adj HR 2.59, 1.68-3.99, p<0.0001) and ARVC variants (adj HR 2.50, 1.34-4.66, p=0.004). The increased risk of HF admissions remained consistent in patients with (adj HR 1.49, 1.11-2.00, p=0.009) and those without (adj HR 1.88, 1.00-3.54, p=0.049) a history of HF. PLP variants were also associated with higher risk of cardiovascular death (adj HR 1.46, 1.06-2.02, p=0.02), most notably for DCM variants (adj HR 1.77, 1.21-2.58, p=0.003). In contrast, PLP variants were not associated with history of ischemic stroke at baseline (adj OR 0.99, p=0.96) nor with incident ischemic stroke (adj HR 0.95, p=0.84). Conclusion In patients with AF, rare cardiomyopathy gene variants are associated with increased risks of HF and of cardiovascular death, but not stroke. These results, collected from large well-phenotyped randomized clinical trials, demonstrate an important prognostic implication for rare genetic variants for CMP in pts with AF.

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

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.

Protein redistribution in buccal cells as a prognostic marker for arrhythmogenic cardiomyopathy in paediatric patients

Abstract Background Mutations in genes encoding desmosomal proteins [plakoglobin (JUP), desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2) and desmocollin-2 (DSC2)] cause complex genetic heart muscle disorders collectively described as arrhythmogenic cardiomyopathies (ACM). Diagnosis and risk stratification, particularly in the paediatric population, remain challenging owing to heterogeneous phenotypic manifestations and reduced genetic penetrance. Immunohistochemical analysis of heart muscle samples has shown that several junctional and signalling molecules are redistributed in patients with ACM, offering diagnostic information. However, the use of this approach in living patients was limited by need for heart samples. Recent studies in adults with ACM showed that the same set of proteins re-localised in the ACM heart also do so in the buccal epithelium. Purpose Given the ease of analysing serial cheek smears, we sought to establish how protein redistribution in buccal cells relates to disease onset and progression in children bearing desmosomal gene mutations. Methods Serial cheek smears from children with desmosomal variants were subjected to immunocytochemistry and confocal microscopy to study the distribution of JUP, DSP and PKP-1 as well as the major gap junction protein connexin43 (Cx43; GJA1) and the major subunit of the NFκB inflammatory pathway; RelA. Results 37 patients Conclusion These data indicate that shifts in junctional/signalling proteins precede the onset of clinical disease. Indeed, in the majority of pre-clinical children, Cx43 and RelA seem to be redistributed first, suggesting that onset of abnormalities in electrical conductivity and inflammatory pathways occur first in the natural disease progression, in agreement with previous clinical observations. Moreover, the data suggest that additional proteins are shifted as the disease progresses. Although further verification is required, this study suggests that analysis of buccal smears may be used as a prognostic/risk stratification test in children with desmosomal variants.

Pnn deficiency alters expressions of calcium handling proteins and impairs nuclear envelope structure in cardiomyocytes

Abstract Pnn is a multi-functional protein playing roles in the regulation of gene transcription, mRNA alternative splicing, as well as cell-cell connection. Previous studies suggested an association between Pnn genetic defects and arrhythmogenic right ventricular cardiomyopathy (ARVC) in human. In this study, we applied an inducible cardiomyocyte-specific Pnn depletion mouse model (Myh6-CreERT2, Pnnflox/flox) to determine the impact of loss of Pnn on cardiac structure and function in mice. Six weeks after inducing Pnn gene disruption, electrocardiographic abnormalities, ventricular dilatation, and reduced left ventricular ejection fraction (LVEF) were observed in mice with loss of Pnn in cardiomyocytes. Histopathological examinations showed that the proliferation of cardiac fibroblasts and expression of α-smooth muscle actin were increased with Pnn depletion, while the cell-cell connection between cardiomyocytes were impaired. In addition to impaired intercalated discs, loss of Pnn also altered the distribution and expression of nuclear envelope proteins and the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins, such as nuclear lamins, Emerin, and SUNs, in cardiomyocytes. Microarray analysis as well as subsequent RT-PCR and Western blots also indicated that loss of Pnn regulated myocardial expressions of genes responsible for calcium handling, such as RYR2, Atp2a2, Slc8a1, and Kcnj3. Furthermore, infiltrations of macrophages and neutrophiles were observed in the myocardium with Pnn depletion, while expression levels of oxidative stress-associated proteins were also significantly regulated by Pnn depletion. In conclusion, Pnn plays an essential role in the maintenance of intercalated disc integrity and nuclear envelope structure of cardiomyocytes, while cardiomyocyte-specific loss of Pnn impairs the calcium handling and leads to arrhythmogenic dilated cardiomyopathy in mice.Cardiac MRI of mice with Pnn depletionMyocardial expression of SUN2 and Emerin

Integrated plasma proteomics and myocardial tissue transcriptomics reveal elevated fibrosis markers in arrhythmogenic cardiomyopathy patients

Abstract Background Arrhythmogenic cardiomyopathy (ACM) is characterized by myocardial fibrofatty replacement and ventricular arrhythmias, with potential progression to heart failure. This study aims to identify ACM-specific biomarkers for improved prognosis and possible treatment strategies. Methods RNA sequencing and transcriptome analyses were performed in cardiomyocytes of 10 ACM patients, 10 dilated cardiomyopathy patients and 10 healthy controls. Analyses were performed using liquid chromatography and mass spectrometry. Additionally, plasma protein expression was assessed in 38 ACM patients and 24 healthy controls using the Proximity Extension Assay (Olink®). A standardized bicycle exercise test was performed in 11 ACM patients and controls and blood was obtained before and after exercise. Results Myocardial tissue transcriptomics identified several upregulated molecules associated with extracellular matrix formation and immune response in ACM. Plasma protein sequencing revealed upregulation of proteins involved in metabolic pathways, inflammation, and fibrosis. Integration of these analyses identified key soluble profibrotic markers such as Fibulin-3, Endostatin and C-X-C motif chemokine 10 (CXCL10) in plasma of ACM patients. After exercise, Fibulin-3 levels increased in ACM patients compared to controls. These findings were further validated using enzyme-linked immunosorbent assay (ELISA). Conclusion This comprehensive analysis revealed distinct proteomic and transcriptomic signatures in ACM patients' plasma and cardiomyocytes. The integrative analysis identified several upregulated profibrotic markers in ACM. Our Findings may advance diagnostic and prognostic strategies and offer potential therapeutic targets.