Inherited Heart Disease Research Articles

Sex differences in disease progression and arrhythmic risk in patients with arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Area Network on Cardiovascular Diseases (ERA-CVD) Background Arrhythmogenic cardiomyopathy (AC) is an inheritable heart disease characterized by high risk of life-threatening ventricular arrhythmia. Male sex has been reported as a risk factor of high disease penetrance and arrhythmia, but data on sex-specific phenotype in AC are sparse. Purpose To assess sex-specific disease progression in AC patients and structural cardiac changes at time of arrhythmic event. Methods We included consecutive AC patients in a longitudinal cohort study. We performed echocardiography at baseline according to Task Force Criteria of 2010 with additional parameters including strain. Patients were followed with repeated echocardiographic examinations. Ventricular arrhythmia was defined as aborted cardiac arrest, sustained ventricular tachycardia or appropriate therapy by an implantable cardioverter-defibrillator. In patients with documented first time ventricular arrhythmias, echocardiographic findings obtained ±30 days around the event was noted separately. Results We included 191 AC patients (46% female, 51% probands, age 41 ± 17 years) of which 88% had ≥2 echocardiographic examinations during 6.9 (IQR 4.7-9.8) years of follow up. Females and males had similar progression rate of right ventricular dimensions and left ventricular function, but right ventricular function decreased more in females (Figure). Arrhythmic events occurred in 85 (45%) patients and 39 patients had an echocardiographic examination at the time of their first event. There was no difference in right ventricular diameters or right or left ventricular function between females and males at the time of first arrhythmic event (right ventricular outflow tract diameter: 35 ± 7 mm vs. 39 ± 7 mm, p = 0.16, right ventricular fractional area change: 34 ± 9 % vs. 29 ± 11 %, p = 0.26, left ventricular global longitudinal strain: -18.8 ± 3.0 % vs. 17.2 ± 2.2 %, p = 0.12, respectively). Conclusion Disease progression was similar in male and female AC patients indicating no accelerated disease progression in males. First arrhythmic event occurred at similar cardiac dysfunction and diameters in both sexes indicating no lower risk in females. Abstract Figure.

Characterization of clinically relevant copy-number variants from exomes of patients with inherited heart disease and unexplained sudden cardiac death

PurposeCopy-number variant (CNV) analysis is increasingly performed in genetic diagnostics. We leveraged recent gene curation efforts and technical standards for interpretation and reporting of CNVs to characterize clinically relevant CNVs in patients with inherited heart disease and sudden cardiac death. MethodsExome sequencing data were analyzed for CNVs using eXome-Hidden Markov Model tool in 48 established disease genes. CNV breakpoint junctions were characterized. CNVs were classified using the American College of Medical Genetics and Genomics technical standards. ResultsWe identified eight CNVs in 690 unrelated probands (1.2%). Characterization of breakpoint junctions revealed nonhomologous end joining was responsible for four deletions, whereas one duplication was caused by nonallelic homologous recombination between duplicated sequences in MYH6 and MYH7. Identifying the precise breakpoint junctions determined the genomic involvement and proved useful for interpreting the clinical relevance of CNVs. Three large deletions involving TTN, MYBPC3, and KCNH2 were classified as pathogenic in three patients. Haplotype analysis of a deletion in ACTN2, found in two families, suggests the deletion was caused by an ancestral event. ConclusionCNVs infrequently cause inherited heart diseases and should be investigated when standard genetic testing does not reveal a genetic diagnosis.

Abstract 13583: Secondary Findings of Loss-of-function Variants in FLNC Are Associated With Arrhythmogenic Cardiomyopathy Phenotypes in a General Clinical Population

Introduction: The FLNC gene, encoding the Filamin C sarcomere protein, has gained recent attention as a potential cause of severe cardiomyopathy and arrhythmia phenotypes through genetic testing of individuals with known inherited heart disease. We sought to characterize the phenotypes associated with rare, putative loss-of-function variants in FLNC ascertained through secondary findings of exome sequencing in a large clinical population. Methods: We reviewed exome data from the first 135,878 sequenced participants in the Geisinger MyCode Community Health Initiative with linked electronic health records (EHR; the DiscovEHR cohort) to identify rare frameshift, stop gain, or canonical splice site variants in FLNC (FLNC LOF ). We compared data on diagnoses and cardiac diagnostic testing (i.e., ECG and echocardiography, when available) from the EHR between individuals with and without FLNC LOF using SAIGE mixed linear models adjusted for age, sex, and ancestry. Results: We observed 46 individuals (0.03%) carrying one of 24 distinct FLNC LOF variants. These individuals had significantly increased odds for diagnoses of dilated cardiomyopathy (OR: 49, 95% CI: [4-560]), ventricular tachycardia (OR: 170, [5-6317]), and supraventricular tachycardia (OR: 11, [2-77]). We also observed significantly increased left ventricular (LV) diameter (5.1 ± 0.8 cm vs. 4.6 ± 0.7 cm, p = 0.001) and decreased LV ejection fraction (Figure) by echocardiography (available in 50% of FLNC LOF individuals), and a trend toward higher prevalence of inverted T waves in any precordial lead beyond V2 (23% vs. 7%, p = 0.06). Conclusion: Loss-of-function variants in FLNC are extremely rare in a general clinical population but are associated with a clinically significant and potentially actionable, yet incompletely penetrant, cardiac phenotype marked by ventricular dysfunction and dysrhythmia. Consideration for screening and return of secondary findings may be warranted.

Abstract 14042: Weight Loss Favorably Impacts Left Ventricular Mass and Wall Thickness by CMR and CT in Patients With Hypertrophic Cardiomyopathy: A Retrospective Case Series

Introduction: Hypertrophic Cardiomyopathy (HCM) is a relatively common inherited heart disease with variable phenotypic expression. While pathogenic variants in sarcomeric genes are considered responsible for the development of left ventricular (LV) hypertrophy, environmental modulation of phenotype may explain the known heterogeneity. Obesity is common in HCM patients and is associated with increased LV mass in the general population. Hypothesis: We hypothesized that weight loss in obese patients with HCM would be associated with a decrease in LV mass and maximal wall thickness. Methods: Patients with HCM who achieved therapeutic weight loss and underwent cardiac MRI (CMR) or computed tomography (CT) before and afterwards were included. Standard LV measurements including wall thickness in an 18-segment model were performed blindly with respect to name, time and BMI for un-biased comparison. Results: We included 6 patients (2 female, age 55 ± 6.5 years, baseline BMI = 36.7 ± 5.2 kg/m 2 ) who achieved 16.3 ± 10.8 kg weight loss after 35 ± 12 months with diet and exercise (n=4) or bariatric surgery (n=2). After weight loss, we observed a mean proportional decrease in total LV mass of 25 ± 16% (p=0.004), and a 19 ± 7% decrease in indexed LV mass (p=0.007). Furthermore, there was a numerical decrease in mean wall thickness in 14 out of 18 LV segments measured; 9 segments had more than a 10% decrease. The most noticeable changes were at the basal inferolateral wall (25 ± 13% decrease) and basal inferoseptum (19 ± 18%) decrease (Table). Conclusions: In this series of patients with HCM, weight loss favorably affected LV mass and wall thickness. Further research is needed to explore the impact of weight loss on HCM phenotypic expression and symptoms.

Mechanotransduction and Adrenergic Stimulation in Arrhythmogenic Cardiomyopathy: An Overview of in vitro and in vivo Models.

Arrhythmogenic Cardiomyopathy (AC) is a rare inherited heart disease, manifesting with progressive myocardium degeneration and dysfunction, and life-threatening arrhythmic events that lead to sudden cardiac death. Despite genetic determinants, most of AC patients admitted to hospital are athletes or very physically active people, implying the existence of other disease-causing factors. It is recognized that AC phenotypes are enhanced and triggered by strenuous physical activity, while excessive mechanical stretch and load, and repetitive adrenergic stimulation are mechanisms influencing disease penetrance. Different approaches have been undertaken to recapitulate and study both mechanotransduction and adrenergic signaling in AC, including the use of in vitro cellular and tissue models, and the development of in vivo models (particularly rodents but more recently also zebrafish). However, it remains challenging to reproduce mechanical load stimuli and physical activity in laboratory experimental settings. Thus, more work to drive the innovation of advanced AC models is needed to recapitulate these subtle physiological influences. Here, we review the state-of-the-art in this field both in clinical and laboratory-based modeling scenarios. Specific attention will be focused on highlighting gaps in the knowledge and how they may be resolved by utilizing novel research methodology.

Tropomyosin pseudo-phosphorylation can rescue the effects of cardiomyopathy-associated mutations

We applied various methods to investigate how mutations S283D and S61D that mimic phosphorylation of tropomyosin (Tpm) affect structural and functional properties of cardiac Tpm carrying cardiomyopathy-associated mutations in different parts of its molecule. Using differential scanning calorimetry and molecular dynamics, we have shown that the S61D mutation (but not the S283 mutation) causes significant destabilization of the N-terminal part of the Tpm molecule independently of the absence or presence of cardiomyopathy-associated mutations. Our results obtained by cosedimentation of Tpm with F-actin demonstrated that both S283D and S61D mutations can reduce or even eliminate undesirable changes in Tpm affinity for F-actin caused by some cardiomyopathy-associated mutations. The results indicate that Tpm pseudo-phosphorylation by mutations S283D or S61D can rescue the effects of mutations in the TPM1 gene encoding a cardiac isoform of Tpm that lead to the development of such severe inherited heart diseases as hypertrophic or dilated cardiomyopathies.

Identification of circulating hub long noncoding RNAs associated with hypertrophic cardiomyopathy using weighted correlation network analysis.

Hypertrophic cardiomyopathy (HCM) is one of the most commonly inherited heart diseases and the leading cause of sudden cardiac death among adolescents and young adults. Circulating long noncoding RNAs (lncRNAs) have demonstrated potential as diagnostic and therapeutic targets in several cardiovascular diseases. However, the circulating extracellular lncRNA expression profile of patients with HCM remains unclear. Plasma lncRNA expression was evaluated in patients with HCM and healthy controls using a human lncRNA microarray. A weighted correlation network analysis (WGCNA) and linear models for microarray data (Limma) were used. GSE68316 data from cardiac tissue in the Gene Expression Omnibus database were analysed for further validation. Using WGCNA, two modules (referred to as the magenta and the light-yellow module) were identified that were positively associated with HCM. Gene Ontology analysis revealed that lncRNAs in the magenta module targeted ‘heart growth’. Using Limma, a total of 290 lncRNAs were differentially expressed (210 upregulated and 80 downregulated) in the plasma of HCM patients, compared with controls. Moreover, combined WGCNA and Limma analysis demonstrated that 27 hub lncRNAs in the magenta module and 13 hub lncRNAs in the light-yellow module were significantly upregulated, compared with the controls. Moreover, of the 40 differentially expressed hub lncRNAs identified in the two modules, three circulating lncRNAs (lnc-P2RY6-1:1, ENST00000488040 and ENST00000588047) were also significantly upregulated in the HCM cardiac tissue validation dataset. These lncRNAs may serve as biomarkers and therapeutic targets for precise diagnosis and treatment of HCM.

Drug Development and the Use of Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Disease Modeling and Drug Toxicity Screening.

Over the years, numerous groups have employed human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as a superb human-compatible model for investigating the function and dysfunction of cardiomyocytes, drug screening and toxicity, disease modeling and for the development of novel drugs for heart diseases. In this review, we discuss the broad use of iPSC-CMs for drug development and disease modeling, in two related themes. In the first theme—drug development, adverse drug reactions, mechanisms of cardiotoxicity and the need for efficient drug screening protocols—we discuss the critical need to screen old and new drugs, the process of drug development, marketing and Adverse Drug reactions (ADRs), drug-induced cardiotoxicity, safety screening during drug development, drug development and patient-specific effect and different mechanisms of ADRs. In the second theme—using iPSC-CMs for disease modeling and developing novel drugs for heart diseases—we discuss the rationale for using iPSC-CMs and modeling acquired and inherited heart diseases with iPSC-CMs.

Clinical characteristics and risk factors of arrhythmia during follow-up of patients with idiopathic ventricular fibrillation.

The current knowledge of idiopathic ventricular fibrillation (IVF) is limited. We aimed to investigate the nature of IVF, including clinical assessment and later diagnosis, and risk factors of implantable cardioverter defibrillator (ICD) therapy in the follow-up period. Between 2007 and 2019 we systematically identified all patients from Rigshospitalet, Denmark, with a resuscitated sudden cardiac arrest (SCA) with no identifiable cause. All patients were followed routinely in the ICD outpatient clinic and the majority also in an inherited heart disease outpatient clinic. Outcomes were analysed with Cox regressions models and cumulative incidence curves. We identified 84 patients with an initial diagnosis of IVF; of these, three (3.6%) patients were later diagnosed with a cardiac disease. The remaining IVF patients (n = 81, median age 45 years; men 71.6%) were followed a median follow-up of 5.2 years (interquartile range, 2.0-7.6). A total of 24 (29.6%) patients had appropriate ICD therapy and 12 (14.8%) patients had inappropriate ICD therapy. No predominant type of ventricular arrhythmia at first appropriate ICD therapy was observed. Early repolarization at baseline was not associated with an increased risk of appropriate ICD therapy (P = .842). Repeated cardiac arrest at index SCA increased the risk of appropriate ICD therapy (hazard ratio, 2.63 [95% CI, 1.08-6.40; P = .033]). Most patients remained idiopathic throughout the follow-up period and the overall long-term prognosis of IVF was good. Repeated cardiac arrest at index SCA was a risk factor of appropriate ICD therapy and early repolarization was not associated with an increased risk of appropriate ICD therapy.

Reasons for refusing diagnostic tests and therapeutic recommendations and associated complications in inherited heart diseases. The RELUCTANT study

Introduction and objectivesStudy of inherited heart diseases (IHD) involves performing diagnostic tests, which are sometimes inconvenient or stressful, in asymptomatic relatives. The aim of this study was to analyze refusal to undergo various diagnostic tests and follow therapeutic recommendations. MethodsWe assessed 1992 consecutive families with IHD to analyze refusal to undergo family screening. The study included 1539 individuals who were recommended to undergo cardiac magnetic resonance, and 837 who were recommended a drug challenge test. To study treatment refusal, we assessed 395 patients with an indication for an implantable cardioverter-defibrillator (ICD) and 402 patients with an indication for anticoagulation. ResultsA total of 28% of families who were recommended to undergo screening for suspected IHD did not attend, but refusal was lower if there was a family history of sudden cardiac death. In all, 23% did not undergo magnetic resonance, and the 2 main reasons were administrative problems (53%) and claustrophobia (18%). Refusal was more common in older people, women, symptomatic persons, individuals with arrhythmias, and relatives. Nearly one fifth (19%) did not take the drug challenge test, due to fear (46%) or administrative issues (25%). Refusal was more frequent in older individuals, asymptomatic persons, those with a history of arrhythmias, relatives, and those with a positive genetic study. Only a minority of patients rejected the treatments (5.1% ICD, 2.5% anticoagulation). The percentage of sudden cardiac death in persons rejecting ICD implantation was high (4.5% per year). ConclusionsOne fifth of people attending screening for IHD refused to undergo more sophisticated and stressful tests. This study identified several independent predictors associated with refusal. Only a minority of high-risk patients refused treatments such as ICD implantation and anticoagulation.

Needs analysis of parents following sudden cardiac death in the young

ObjectiveThe sudden cardiac death (SCD) of a young person is a devastating event for any parent. Inherited heart disease is often either identified or assumed to be the cause. Few...

Motivos y complicaciones relacionadas con el rechazo de pruebas diagnósticas y recomendaciones terapéuticas en cardiopatías familiares. Estudio RELUCTANT

Introduction and objectivesStudy of inherited heart diseases (IHD) involves performing diagnostic tests, which are sometimes inconvenient or stressful, in asymptomatic relatives. The aim of this study was to analyze refusal to undergo various diagnostic tests and follow therapeutic recommendations. MethodsWe assessed 1992 consecutive families with IHD to analyze refusal to undergo family screening. The study included 1539 individuals who were recommended to undergo cardiac magnetic resonance, and 837 who were recommended a drug challenge test. To study treatment refusal, we assessed 395 patients with an indication for an implantable cardioverter-defibrillator (ICD) and 402 patients with an indication for anticoagulation. ResultsA total of 28% of families who were recommended to undergo screening for suspected IHD did not attend, but refusal was lower if there was a family history of sudden cardiac death. In all, 23% did not undergo magnetic resonance, and the 2 main reasons were administrative problems (53%) and claustrophobia (18%). Refusal was more common in older people, women, symptomatic persons, individuals with arrhythmias, and relatives. Nearly one fifth (19%) did not take the drug challenge test, due to fear (46%) or administrative issues (25%). Refusal was more frequent in older individuals, asymptomatic persons, those with a history of arrhythmias, relatives, and those with a positive genetic study. Only a minority of patients rejected the treatments (5.1% ICD, 2.5% anticoagulation). The percentage of sudden cardiac death in persons rejecting ICD implantation was high (4.5% per year). ConclusionsOne fifth of people attending screening for IHD refused to undergo more sophisticated and stressful tests. This study identified several independent predictors associated with refusal. Only a minority of high-risk patients refused treatments such as ICD implantation and anticoagulation.

RNA Splicing Defects in Hypertrophic Cardiomyopathy: Implications for Diagnosis and Therapy.

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is predominantly caused by mutations in genes that encode sarcomere-associated proteins. Effective gene-based diagnosis is critical for the accurate clinical management of patients and their family members. However, the introduction of high-throughput DNA sequencing approaches for clinical diagnostics has vastly expanded the number of variants of uncertain significance, leading to many inconclusive results that limit the clinical utility of genetic testing. More recently, developments in RNA analysis have been improving diagnostic outcomes by identifying new variants that interfere with splicing. This review summarizes recent discoveries of RNA mis-splicing in HCM and provides an overview of research that aims to apply the concept of RNA therapeutics to HCM.

Epidemiology and clinical characteristics of atrial fibrillation in patients with inherited heart diseases.

The prevalence and clinical course of atrial fibrillation (AF) in hypertrophic cardiomyopathy (HCM) is well described, though less so for other inherited cardiomyopathies (familial dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, left ventricular noncompaction); and inherited arrhythmia syndromes (long QT syndrome, Brugada syndrome or catecholaminergic polymorphic ventricular tachycardia [CPVT]). We examined the frequency, clinical characteristics and AF-related management and outcomes amongst this patient population. We retrospectively studied consecutive probands with inherited cardiomyopathy (n = 962) and inherited arrhythmia syndromes (n = 195) evaluated between 2002 and 2018. AF was observed in 5% to 31% of patients, with the highest frequency in HCM. Age of AF onset was 45.8 ± 21.9 years in the inherited arrhythmia syndromes compared with 53.3 ± 15.3 years in the inherited cardiomyopathies, with four CPVT patients developing AF at a median age of 20 years. Overall, 11% of patients with AF had a transient ischemic attack or stroke of which a total of 80% were anticoagulated; with 48% of events occurring at a CHA2 DS2 -VASc < 2. Amongst sarcomere-positive HCM, AF was independently associated with age (odds ratio [OR], 1.05; 95% confidence interval [CI], 1.02-1.08; P = .0014), left atrial area (OR, 1.11; 95% CI, 1.05-1.17; P = .0005) and MYH7 variants (OR, 2.55; 95% CI, 1.16-5.61; P = .020). Up to one-third of inherited heart disease patients will develop AF. While common general population risk factors are key in patients with HCM, the genotype is independently associated with AF. Amongst inherited arrhythmia syndromes, AF is less common, though often occurs below the age of 50 years.

G790del mutation in DSC2 alone is insufficient to develop the pathogenesis of ARVC in a mouse model

BackgroundArrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease that causes heart failure and/or sudden cardiac death. Several desmosomal genes (DSC2, PKG, PKP2, DSP, and RyR2) are thought to be the causative gene involved in ARVC. Out of them, DSC2 mutations account for 2% of ARVC genetic abnormalities. This study aimed to clarify the effect of G790del mutation in DSC2 on the arrhythmogenic mechanism and cardiac function in a mouse model. ResultNeither the heterozygous +/G790del nor homozygous G790del/G790del mice showed structural and functional defects in the right ventricle (RV) or lethal arrhythmia. The homozygous G790del/G790del 6-month-old mice slightly showed left ventricular (LV) dysfunction. Cell shortening decreased with prolongation of intracellular Ca2+ transient in cardiomyocytes isolated from the homozygous G790del/G790del mice, and spontaneous Ca2+ transients were frequently observed in response to isoproterenol. ConclusionsG790del mutation in DSC2 was not relevant to the pathogenesis of ARVC, but showed a slight contractile dysfunction and Ca2+ dysregulation in the LV.

IMPLANTABLE CARDIOVERTER DEFIBRILLATOR USE IN INHERITED HEART DISEASES: PROPHYLACTIC VERSUS SECONDARY INDICATIONS

Inherited heart diseases (IHD) can lead to ventricular arrhythmias and sudden death. The role of prophylactic implantable cardioverter defibrillator (ICD) therapy is not well known. We report our long-term follow-up of ICD use for prophylactic and secondary indications in a cohort of patients with IHD.

Hypertrophic cardiomyopathy: genetics and clinical perspectives.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and defined by unexplained isolated progressive myocardial hypertrophy, systolic and diastolic ventricular dysfunction, arrhythmias, sudden cardiac death and histopathologic changes, such as myocyte disarray and myocardial fibrosis. Mutations in genes encoding for proteins of the contractile apparatus of the cardiomyocyte, such as β-myosin heavy chain and myosin binding protein C, have been identified as cause of the disease. Disease is caused by altered biophysical properties of the cardiomyocyte, disturbed calcium handling, and abnormal cellular metabolism. Mutations in sarcomere genes can also activate other signaling pathways via transcriptional activation and can influence non-cardiac cells, such as fibroblasts. Additional environmental, genetic and epigenetic factors result in heterogeneous disease expression. The clinical course of the disease varies greatly with some patients presenting during childhood while others remain asymptomatic until late in life. Patients can present with either heart failure symptoms or the first symptom can be sudden death due to malignant ventricular arrhythmias. The morphological and pathological heterogeneity results in prognosis uncertainty and makes patient management challenging. Current standard therapeutic measures include the prevention of sudden death by prohibition of competitive sport participation and the implantation of cardioverter-defibrillators if indicated, as well as symptomatic heart failure therapies or cardiac transplantation. There exists no causal therapy for this monogenic autosomal-dominant inherited disorder, so that the focus of current management is on early identification of asymptomatic patients at risk through molecular diagnostic and clinical cascade screening of family members, optimal sudden death risk stratification, and timely initiation of preventative therapies to avoid disease progression to the irreversible adverse myocardial remodeling stage. Genetic diagnosis allowing identification of asymptomatic affected patients prior to clinical disease onset, new imaging technologies, and the establishment of international guidelines have optimized treatment and sudden death risk stratification lowering mortality dramatically within the last decade. However, a thorough understanding of underlying disease pathogenesis, regular clinical follow-up, family counseling, and preventative treatment is required to minimize morbidity and mortality of affected patients. This review summarizes current knowledge about molecular genetics and pathogenesis of HCM secondary to mutations in the sarcomere and provides an overview about current evidence and guidelines in clinical patient management. The overview will focus on clinical staging based on disease mechanism allowing timely initiation of preventative measures. An outlook about so far experimental treatments and potential for future therapies will be provided.

P3828Zebrafish models for arrhythmogenic cardiomyopathy type 8: a starting platform for exercise stress test and drug treatment

Abstract Background Arrhythmogenic Cardiomyopathy (AC) is an inherited heart disease characterized by progressive substitution of the myocardium with fibro-fatty tissue, leading to electrical instability and high risk of sudden death, particularly in young subjects and athletes. In recent years, our laboratory has produced zebrafish (zf) mutant lines modelling AC type 8, an AC form linked to mutations in the junctional protein Desmoplakin (Dsp). Mutations in the DSP gene have been identified in both dominant and recessive AC cases, characterized by left-dominant and biventricular forms of the disease. Sports medicine has highlighted that they are the most dangerous forms, being less easily identifiable by ECG. Purpose Taking advantage of our zf Dsp mutant lines, we aim to fully characterize the pathological phenotype, analyze the perturbation of cell communication pathways, evaluate the role of the physical exercise, and test the efficacy of candidate drugs. Methods Among our zf lines we have identified double mutant animals, bearing both zf dspa and dspb mutations in heterozygous condition, as the best model able to recapitulate the human AC phenotype. This model underwent physical stress tests in the presence/absence of candidate drug treatment. Phenotyping included heart rhythm measurement, gene expression analysis using Real Time PCR and signaling pathway transgenes, immune-histochemistry, whole-mount in situ hybridization, standard histology and ultrastructural TEM analysis. Results Preliminary results from mutant phenotyping indicate alterations in heart rate, sudden cardiac death, structural alterations of the myocardium associated with junctional disorganization and, in parallel, dysregulation of Wnt, Hippo and TGFbeta pathways. Specifically, Dsp mutant animals can range from an 8% decrease to a 14% increase of heart rhythm compared to the physiological range (120–140 beats per minute in zf larvae). At the adult stage, about 1% of the fish mutant population dies suddenly. The histological examination shows a 50% reduction of the myocardial cell mass, in parallel with a 50% decrease of Dsp signal, detected by TEM, associated with the so-called “pale desmosome” phenotype. Signaling dysregulation includes an 80% loss of Wnt/Beta-catenin, a 300% increase of TGFbeta and a 500% increase of Hippo/YAP-TAZ signaling in the cardiac tissue. Physical stress tests and pathway-directed drug treatment have clarified that these factors can modulate the pathological phenotype, as preliminarily evidenced by the rescue of Wnt signal decrease to normal levels through SB216763 treatment of Dsp-deficient individuals at rest. Conclusion Preliminary evidences corroborate the zf organism as a suitable model for AC cellular and molecular phenotyping, exploitable for the dissection of the genetic events leading to the onset and progression of the disease, and applicable to the analysis of chemical and mechanical modulators of AC-associated features. Acknowledgement/Funding Cariparo 2017 SHoCD; TRANSAC; CPDA133979/13; RP-2014-ehz745.06700394

P3681Apparent sex differences in risk of life-threatening events in arrhythmogenic cardiomyopathy are related to exercise habits

Abstract Background Arrhythmogenic cardiomyopathy (AC) is an inheritable heart disease characterized by high risk of ventricular arrhythmias and exercise intolerance. Male sex is associated with higher risk of life-threatening events. However, this may be challenged by the fact that men exercise more than women. Purpose To assess whether the sex differences in arrhythmic outcome in AC-patients are driven by sex differences in exercise exposure. Methods We included AC-patients diagnosed between 1997 and 2017 in a longitudinal cohort study. We assessed exercise habits during the preceding 3 years before inclusion, and exercise dose was expressed as metabolic equivalent hours per week (MET-hours/week). Life-threatening events were defined as sustained ventricular tachycardia, aborted cardiac arrest or appropriate therapy from an implantable cardioverter-defibrillator at the time of diagnosis and prospectively during follow-up. Results We included 170 AC-patients (52% probands, 44% female, age 41±16 years). Males had higher exercise doses than female patients (36 [IQR, 14 to 54] vs. 12 [IQR, 8 to 22] MET-hours/week; P&lt;0.001). Fifty-seven patients (34%) had previous life-threatening events, and 45 (26%) had life-threatening events during 5.7 (IQR, 2.8 to 9.4) years of follow-up. Male sex seemed to be a marker of previous life-threatening events (OR 2.0 [95% CI, 1.0 to 3.8]; P&lt;0.05), but not when adjusted for exercise dose (Adjusted OR 1.3 [95% CI, 0.6 to 2.7]; P=0.44). Male sex also seemed to be a predictor of life-threatening events during follow-up (HR 2.0 [95% CI 1.0 to 3.9]; P=0.04) (Figure, left panel), but not when adjusted for exercise dose (Adjusted HR 1.5 [95% CI 0.8 to 3.0]; P=0.26) (Figure, right panel). Conclusions Sex differences in arrhythmic risk in AC were attributable to higher exercise doses in male AC-patients. This highlights the importance of exercise assessment in these patients, and challenges the current opinion of male sex as a risk factor in itself. Risk stratification based on sex may underestimate the risk of physically active female AC-patients. Acknowledgement/Funding This study was supported by Center for Cardiological Innovation funded by the Norwegian Research Council

Calcium as a Key Player in Arrhythmogenic Cardiomyopathy: Adhesion Disorder or Intracellular Alteration?

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca2+ handling machinery, focusing on the available data supporting this hypothesis. The Ca2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca2+, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.