An autoantibody profile detects Brugada syndrome and identifies abnormally expressed myocardial proteins.

AIMS: Brugada syndrome (BrS) is characterized by a unique electrocardiogram (ECG) pattern and life-threatening arrhythmias. However, the Type 1 Brugada ECG pattern is often transient, and a genetic cause is only identified in <25% of patients. We sought to identify an additional biomarker for this rare condition. As myocardial inflammation may be present in BrS, we evaluated whether myocardial autoantibodies can be detected in these patients. METHODS AND RESULTS: For antibody (Ab) discovery, normal human ventricular myocardial proteins were solubilized and separated by isoelectric focusing (IEF) and molecular weight on two-dimensional (2D) gels and used to discover Abs by plating with sera from patients with BrS and control subjects. Target proteins were identified by mass spectrometry (MS). Brugada syndrome subjects were defined based on a consensus clinical scoring system. We assessed discovery and validation cohorts by 2D gels, western blots, and ELISA. We performed immunohistochemistry on myocardium from BrS subjects (vs. control). All (3/3) 2D gels exposed to sera from BrS patients demonstrated specific Abs to four proteins, confirmed by MS to be α-cardiac actin, α-skeletal actin, keratin, and connexin-43, vs. 0/8 control subjects. All (18/18) BrS subjects from our validation cohorts demonstrated the same Abs, confirmed by western blots, vs. 0/24 additional controls. ELISA optical densities for all Abs were elevated in all BrS subjects compared to controls. In myocardium obtained from BrS subjects, each protein, as well as SCN5A, demonstrated abnormal protein expression in aggregates. CONCLUSION: A biomarker profile of autoantibodies against four cardiac proteins, namely α-cardiac actin, α-skeletal actin, keratin, and connexin-43, can be identified from sera of BrS patients and is highly sensitive and specific, irrespective of genetic cause for BrS. The four involved proteins, along with the SCN5A-encoded Nav1.5 alpha subunit are expressed abnormally in the myocardium of patients with BrS.

Calmodulin mutations and life-threatening cardiac arrhythmias: insights from the International Calmodulinopathy Registry.

AIMS: Calmodulinopathies are rare life-threatening arrhythmia syndromes which affect mostly young individuals and are, caused by mutations in any of the three genes (CALM 1-3) that encode identical calmodulin proteins. We established the International Calmodulinopathy Registry (ICalmR) to understand the natural history, clinical features, and response to therapy of patients with a CALM-mediated arrhythmia syndrome. METHODS AND RESULTS: A dedicated Case Report File was created to collect demographic, clinical, and genetic information. ICalmR has enrolled 74 subjects, with a variant in the CALM1 (n = 36), CALM2 (n = 23), or CALM3 (n = 15) genes. Sixty-four (86.5%) were symptomatic and the 10-year cumulative mortality was 27%. The two prevalent phenotypes are long QT syndrome (LQTS; CALM-LQTS, n = 36, 49%) and catecholaminergic polymorphic ventricular tachycardia (CPVT; CALM-CPVT, n = 21, 28%). CALM-LQTS patients have extremely prolonged QTc intervals (594 ± 73 ms), high prevalence (78%) of life-threatening arrhythmias with median age at onset of 1.5 years [interquartile range (IQR) 0.1-5.5 years] and poor response to therapies. Most electrocardiograms (ECGs) show late onset peaked T waves. All CALM-CPVT patients were symptomatic with median age of onset of 6.0 years (IQR 3.0-8.5 years). Basal ECG frequently shows prominent U waves. Other CALM-related phenotypes are idiopathic ventricular fibrillation (IVF, n = 7), sudden unexplained death (SUD, n = 4), overlapping features of CPVT/LQTS (n = 3), and predominant neurological phenotype (n = 1). Cardiac structural abnormalities and neurological features were present in 18 and 13 patients, respectively. CONCLUSION: Calmodulinopathies are largely characterized by adrenergically-induced life-threatening arrhythmias. Available therapies are disquietingly insufficient, especially in CALM-LQTS. Combination therapy with drugs, sympathectomy, and devices should be considered.

An autoantibody identifies arrhythmogenic right ventricular cardiomyopathy and participates in its pathogenesis.

Aims: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by right ventricular myocardial replacement and life-threatening ventricular arrhythmias. Desmosomal gene mutations are sometimes identified, but clinical and genetic diagnosis remains challenging. Desmosomal skin disorders can be caused by desmosomal gene mutations or autoantibodies. We sought to determine if anti-desmosome antibodies are present in subjects with ARVC. Methods and results: We evaluated ARVC subjects and controls for antibodies to cardiac desmosomal cadherin proteins. Desmoglein-2 (DSG2), desmocollin-2, and N-cadherin proteins on western blots were exposed to sera, in primary and validation cohorts of subjects and controls, as well as the naturally occurring Boxer dog model of ARVC. We identified anti-DSG2 antibodies in 12/12 and 25/25 definite ARVC cohorts and 7/8 borderline subjects. Antibody was absent in 11/12, faint in 1/12, and absent in 20/20 of two control cohorts. Anti-DSG2 antibodies were present in 10/10 Boxer dogs with ARVC, and absent in 18/18 without. In humans, the level of anti-DSG2 antibodies correlated with the burden of premature ventricular contractions (r = 0.70), and antibodies caused gap junction dysfunction, a common feature of ARVC, in vitro. Anti-DSG2 antibodies were present in ARVC subjects regardless of whether an underlying mutation was identified, or which mutation was present. A disease-specific DSG2 epitope was identified. Conclusion: Anti-DSG2 antibodies are a sensitive and specific biomarker for ARVC. The development of autoimmunity as a result of target-related mutations is unique. Anti-DSG2 antibodies likely explain the cardiac inflammation that is frequently identified in ARVC and may represent a new therapeutic target.

Clinical utility of endomyocardial biopsies in the diagnosis of arrhythmogenic right ventricular cardiomyopathy in children.

BACKGROUND: Histomorphometry of endomyocardial biopsies is one component of arrhythmogenic right ventricular cardiomyopathy (ARVC) diagnosis, although there is a need for stricter diagnostic criteria for this disease in pediatrics. The clinical utility of biopsy analysis as a component of ARVC diagnosis was evaluated in pediatric patients. METHODS: Histomorphometric analysis of fibrofatty infiltrate was completed on pediatric right ventricular endomyocardial biopsy samples. Myocardial replacement by fat and fibrosis was quantified. ARVC diagnosis was established using the 2010 ARVC Task Force criteria, with the biopsy measures compared across various ARVC diagnoses (definite, borderline, possible, or no ARVC). Receiver-operating characteristic (ROC) curve analysis was also completed using biopsy measures. RESULTS: The greatest proportion of fat, fibrosis, and myocardial replacement was in the definite ARVC cohort, and was significantly larger than for the other diagnosis cohorts. ROC curve analysis (with the biopsy analysis removed from the diagnostic classification) produced cutoff values of 15 and 25% myocardial replacement, which is lower than current adult diagnosis criteria. CONCLUSION: We propose modifications in pediatric major and minor biopsy diagnosis criteria to allow for improved sensitivity. This study suggests that biopsy analysis in children is most significant for subjects with a more severe disease presentation.

Tmem65 is critical for the structure and function of the intercalated discs in mouse hearts.

The intercalated disc (ICD) is a unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigate the functional and cellular effects of Tmem65 reductions on the myocardium in a mouse model by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA, which reduces Tmem65 expression by 90% in mouse ventricles compared to scrambled shRNA injection. Tmem65 knockdown (KD) results in increased mortality which is accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection and progression to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts display depressed hemodynamics as measured echocardiographically as well as slowed conduction in optical recording accompanied by prolonged PR intervals and QRS duration in electrocardiograms. Immunoprecipitation and super-resolution microscopy demonstrate a physical interaction between Tmem65 and sodium channel ß subunit (ß1) in mouse hearts and this interaction appears to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na+ currents but did show reduced Ca2+ and K+ currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.

Evaluating the 12-Lead Electrocardiogram for Diagnosing ARVC in Young Populations: Implications for Preparticipation Screening of Athletes.

BACKGROUND: Arrhythmogenic right-ventricular cardiomyopathy (ARVC) is an identified cause of sport-related sudden cardiac arrest (SCA). Identifying athletes with ARVC and restricting them from exercise is believed to reduce the risk of SCA. The electrocardiogram (ECG) is considered to be an important component of screening for ARVC; however, the sensitivity of the 12-lead ECG to identify ARVC in young asymptomatic persons is unknown. METHODS: In this retrospective study, we identified 70 patients (49 ARVC-positive, based on Task Force Criteria, and 21 age-matched ARVC-negative persons from a paediatric arrhythmia database (<18 years of age); ECGs were analyzed for abnormalities, based on International Criteria for Interpretation of ECGs in Athletes, and ECG findings were adjudicated by group consensus. RESULTS: Of the 49 ARVC-positive patients (median age: 17 [interquartile range: 16-18], 65% male), 22% were found to have abnormal ECGs; the most common ECG findings were T-wave inversions. Patients with symptoms were more likely to have abnormal ECGs than asymptomatic patients (28% compared with 17%, respectively; P = 0.002). Of 16 gene-positive patients, 31% had abnormal ECGs. Patients with abnormal ECGs had larger right-ventricular end-diastolic volume indexes on magnetic resonance imaging than those with normal ECGs (P = 0.03). CONCLUSIONS: The ECG was insensitive for detecting ARVC in young (age <18 years), asymptomatic patients, and is unlikely to provide significant diagnostic value for identifying ARVC on routine preparticipation screening of adolescent athletes.

INTRODUCTION: La cardiomyopathie ventriculaire droite arythmogène (CVDA) est une cause établie d'arrêt cardiaque soudain (ACS) du sportif. On croit que le fait de repérer les athlètes atteints de CVDA et de limiter leur exercice réduit le risque d'ACS. On considère que l'électrocardiogramme (ECG) est une composante importante du dépistage de la CVDA. Toutefois, on ignore la sensibilité de l'ECG à 12 dérivations pour détecter la CVDA chez les jeunes personnes asymptomatiques. MÉTHODES: Dans cette étude rétrospective, nous avons repéré 70 patients (49 personnes dont les résultats sont positifs à la CVDA selon les critères de la Task Force, et 21 personnes appariées selon l'âge dont les résultats étaient positifs à la CVDA) d'une base de données sur l'arythmie en pédiatrie (< 18 ans); nous avons analysé les ECG pour détecter les anomalies selon les Critères de consensus internationaux de l'interprétation de l'ECG chez l'athlète, et nous sommes prononcés sur les résultats des ECG par consensus. RÉSULTATS: Parmi les 49 patients dont les résultats étaient positifs à la CVDA (âge médian : 17 [intervalle interquartile : 16-18], 65 % de sexe masculin), nous avons observé que 22 % d'entre eux avaient des résultats anormaux à l'ECG; les résultats les plus fréquents à l'ECG montraient des inversions de l'onde T. Les patients symptomatiques étaient plus susceptibles d'avoir des résultats anormaux à l'ECG que les patients asymptomatiques (28 % et 17 %, respectivement; P = 0,002). Parmi les 16 patients qui avaient des résultats génétiques positifs, 31 % avaient des résultats anormaux à l'ECG. Les patients qui avaient des résultats anormaux à l'ECG avaient des indices plus grands du volume télédiastolique du ventricule droit en fin de diastole à l'imagerie par résonance magnétique que ceux qui avaient des résultats normaux à l'ECG (P = 0,03). CONCLUSIONS: L'ECG n'a pas montré la sensibilité pour détecter la CVDA chez les jeunes (< 18 ans) patients asymptomatiques et n'a pas été susceptible de fournir une valeur diagnostique significative pour détecter la CVDA au dépistage systématique préalable à l'activité sportive des athlètes adolescents.

Initially unexplained cardiac arrest in children and adolescents: A national experience from the Canadian Pediatric Heart Rhythm Network.

BACKGROUND: Unexplained cardiac arrest (UCA) is rare in children. Despite investigations, the etiology in up to one-half of patients remains unknown. OBJECTIVE: The purpose of this study was to assess the management and outcomes of pediatric UCA survivors through the Canadian Pediatric Heart Rhythm Network. METHODS: A retrospective case series of children (age 1-19 years) who presented with UCA between January 1, 2004, and November 1, 2017, was conducted. Patients with known heart disease pre-UCA were excluded. UCA details, investigations, genetic test results, treatment, implantable cardioverter-defibrillator (ICD) data, subsequent diagnoses, and family screening data were collected. RESULTS: Forty-six patients (61% male) were survivors of sudden unexpected death and met inclusion criteria at 8 participating sites. Median age at UCA was 13.8 years (interquartile range [IQR] 9-16 years). Baseline retrievable investigations included electrocardiogram (96%), echocardiogram (85%), exercise stress test (73%), and cardiac magnetic resonance imaging (57%). The presumed etiology for the UCA was identified in 24 (52%), mainly long QT syndrome or catecholaminergic polymorphic ventricular tachycardia. Genetic testing was performed in 33 of 46 (72%), with pathogenic/likely pathogenic variants identified in 13 of 33 (39%) and variants of uncertain significance in 8 of 33 (24%). ICDs were implanted in 35 of 46 (76%). Over median follow-up of 36 months (IQR 17-57 months), 8 of 35 had arrhythmia events captured on device interrogation. Families of 26 of 46 patients(57%) underwent screening, leading to a cardiac diagnosis in 6 of 26 families. CONCLUSION: A cause for UCA was not identified in nearly 50% of patients despite extensive investigations, including cascade screening. A large proportion (75%) of ICD shocks occurred in patients without a diagnosis.

Association of Echocardiographic Parameters of Right Ventricular Remodeling and Myocardial Performance With Modified Task Force Criteria in Adolescents With Arrhythmogenic Right Ventricular Cardiomyopathy.

BACKGROUND: The usefulness of echocardiographic indices, including those already used by modified Task Force Criteria (mTFC), and others such as strain imaging, to identify arrhythmogenic right ventricular cardiomyopathy (ARVC) in adolescence is not well established. METHODS: Echocardiograms from 120 adolescents investigated for ARVC (13±4 years) were retrospectively analyzed. According to the mTFC, patients were classified into definite (n=38), borderline (n=39), or possible (n=43) ARVC. Results were compared with 35 healthy controls. mTFC echocardiographic parameters were analyzed, as well as comprehensive right ventricular (RV) and left ventricular assessment of function including parameters not included in mTFC such as pulsed-wave tissue Doppler and RV 2-dimensional speckle strain. RESULTS: mTFC parameters indexed for body surface area were significantly more abnormal in patients with possible, borderline, or definite ARVC compared with controls for parasternal long-axis view of the RV outflow tract. RV end-diastolic diameters were significantly larger in patients versus controls, a difference that increased with likelihood of ARVC. Left ventricular ejection fraction, tricuspid annular peak systolic excursion, and systolic and diastolic pulsed-wave tissue Doppler imaging indices were similar to controls for all groups. Average and segmental RV peak longitudinal systolic strain was significantly lower in patients with definite ARVC (-21±4%) and disease subgroups versus controls (-25±3%). Multivariable risk analysis showed that reduced RV strain was significantly associated with ARVC diagnosis and its likelihood (multivariable odds ratio [95% CI]=1.23 [1.1-1.37]; P<0.001) as was increased end-diastolic diameter at the apical third of the RV (multivariable odds ratio [95% CI]=1.51 [1.33-1.72]; P<0.001). CONCLUSIONS: mTFC echocardiographic criteria are significantly different between patients and controls and between the different diagnostic groups. However, in our cohort, current echocardiographic mTFC are not met by the majority of adolescent ARVC patients, particularly when indexed to body surface area. Measurement of RV apical dimensions and strain may increase the diagnostic yield of echocardiography for ARVC.

Ankyrin-B dysfunction predisposes to arrhythmogenic cardiomyopathy and is amenable to therapy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal ß-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and ß-catenin. A pharmacological activator of the WNT/ß-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and ß-catenin, and evidence for targeted activation of the WNT/ß-catenin pathway as a potential treatment for this disease.

Left Ventricular Function in Children and Adolescents With Arrhythmogenic Right Ventricular Cardiomyopathy.

The aim of this study was to determine if left ventricular (LV) contractility is reduced in children with arrhythmogenic right ventricular cardiomyopathy (ARVC). For this retrospective study, children and adolescents undergoing a workup for ARVC were characterized according to the revised Task Force Criteria (rTFC). LV strain, rotation, and torsion were measured by feature-tracking cardiovascular magnetic resonance imaging (CMR). Of 142 pediatric patients, 41% had no, 23% possible, 20% borderline, and 16% definite ARVC. LV ejection fraction (EF) did not differ between rTFC categories. Patients in higher rTFC categories had lower right ventricular (RV) EF z-scores (Z-), higher Z-RV end-diastolic volumes (EDVs) and larger Z-LVEDVs (p <0.001, p = 0.002 and 0.013, respectively). LV global circumferential strain was lower in higher rTFC categories (p = 0.018). Z-LVEDV correlated with Z-RVEDV, and Z-LVEF correlated with Z-RVEF (r = 0.69 and r = 0.55, both p <0.001). Z-LVEF and Z-RVEF correlated with LV global circumferential strain (r = 0.48 and r = 0.46, both p <0.001). Forty-eight patients (34%) underwent follow-up CMR investigations after a mean of 3.2 ± 1.9 (0.4 to 8.4) years. A decrease of Z-LVEF over time correlated with that of Z-RVEF (r = 0.35), and Z-LVEDV increase correlated with Z-RVEDV increase (r = 0.57). In conclusion, LV myocardial dysfunction is present in young patients with suspected ARVC. Progressive LV dysfunction assessed by conventional CMR and feature-tracking and enlargement over time parallel adverse remodeling of the RV.