Subepicardial Cardiomyopathy: A Disease Underlying J-Wave Syndromes and Idiopathic Ventricular Fibrillation.

Brugada syndrome (BrS), early repolarization syndrome (ERS), and idiopathic ventricular fibrillation (iVF) have long been considered primary electrical disorders associated with malignant ventricular arrhythmia and sudden cardiac death. However, recent studies have revealed the presence of subtle microstructural abnormalities of the extracellular matrix in some cases of BrS, ERS, and iVF, particularly within right ventricular subepicardial myocardium. Substrate-based ablation within this region has been shown to ameliorate the electrocardiographic phenotype and to reduce arrhythmia frequency in BrS. Patients with ERS and iVF may also exhibit low-voltage and fractionated electrograms in the ventricular subepicardial myocardium, which can be treated with ablation. A significant proportion of patients with BrS and ERS, as well as some iVF survivors, harbor pathogenic variants in the voltage-gated sodium channel gene, SCN5A, but the majority of genetic susceptibility of these disorders is likely to be polygenic. Here, we postulate that BrS, ERS, and iVF may form part of a spectrum of subtle subepicardial cardiomyopathy. We propose that impaired sodium current, along with genetic and environmental susceptibility, precipitates a reduction in epicardial conduction reserve, facilitating current-to-load mismatch at sites of structural discontinuity, giving rise to electrocardiographic changes and the arrhythmogenic substrate

Mechanism of the effects of sodium channel blockade on the arrhythmogenic substrate of Brugada syndrome.

BACKGROUND: The mechanisms by which sodium channel blockade and high-rate pacing modify electrogram (EGM) substrates of Brugada syndrome (BrS) have not been elucidated. OBJECTIVE: The purpose of this study was to determine the effect of ajmaline and high pacing rate on the BrS substrates. METHODS: Thirty-two patients with BrS (mean age 40 ± 12 years) and frequent ventricular fibrillation episodes underwent right ventricular outflow tract substrate electroanatomical and electrocardiographic imaging (ECGI) mapping before and after ajmaline administration and during high-rate atrial pacing. In 4 patients, epicardial mapping was performed using open thoracotomy with targeted biopsies. RESULTS: Ajmaline increased the activation time delay in the substrate (33%; P = .002), ST-segment elevation in the right precordial leads (74%; P < .0001), and the area of delayed activation (170%; P < .0001), coinciding with the increased substrate size (75%; P < .0001). High atrial pacing rate increased the abnormal EGM duration at the right ventricular outflow tract areas from 112 ± 48 to 143 ± 66 ms (P = .003) and produced intermittent conduction block and/or excitation failure at the substrate sites, especially after ajmaline administration. Biopsies from the 4 patients with thoracotomy showed epicardial fibrosis where EGMs were normal at baseline but became fractionated after ajmaline administration. In some areas, local activation was absent and unipolar EGMs had a monophasic morphology resembling the shape of the action potential. CONCLUSION: Sodium current reduction with ajmaline severely compromises impulse conduction at the BrS fibrotic substrates by producing fractionated EGMs, conduction block, or excitation failure, leading to the Brugada ECG pattern and favoring ventricular fibrillation genesis

Multi-omics integration identifies key upstream regulators of pathomechanisms in hypertrophic cardiomyopathy due to truncating MYBPC3 mutations

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the cardiac muscle, frequently caused by mutations in MYBPC3. However, little is known about the upstream pathways and key regulators causing the disease. Therefore, we employed a multi-omics approach to study the pathomechanisms underlying HCM comparing patient hearts harboring MYBPC3 mutations to control hearts. RESULTS: Using H3K27ac ChIP-seq and RNA-seq we obtained 9310 differentially acetylated regions and 2033 differentially expressed genes, respectively, between 13 HCM and 10 control hearts. We obtained 441 differentially expressed proteins between 11 HCM and 8 control hearts using proteomics. By integrating multi-omics datasets, we identified a set of DNA regions and genes that differentiate HCM from control hearts and 53 protein-coding genes as the major contributors. This comprehensive analysis consistently points toward altered extracellular matrix formation, muscle contraction, and metabolism. Therefore, we studied enriched transcription factor (TF) binding motifs and identified 9 motif-encoded TFs, including KLF15, ETV4, AR, CLOCK, ETS2, GATA5, MEIS1, RXRA, and ZFX. Selected candidates were examined in stem cell-derived cardiomyocytes with and without mutated MYBPC3. Furthermore, we observed an abundance of acetylation signals and transcripts derived from cardiomyocytes compared to non-myocyte populations. CONCLUSIONS: By integrating histone acetylome, transcriptome, and proteome profiles, we identified major effector genes and protein networks that drive the pathological changes in HCM with mutated MYBPC3. Our work identifies 38 highly affected protein-coding genes as potential plasma HCM biomarkers and 9 TFs as potential upstream regulators of these pathomechanisms that may serve as possible therapeutic targets

Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel NaV1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on NaV1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings

Low-Cost Optical Mapping Systems for Panoramic Imaging of Complex Arrhythmias and Drug-Action in Translational Heart Models

Panoramic optical mapping is the primary method for imaging electrophysiological activity from the entire outer surface of Langendorff-perfused hearts. To date, it is the only method of simultaneously measuring multiple key electrophysiological parameters, such as transmembrane voltage and intracellular free calcium, at high spatial and temporal resolution. Despite the impact it has already had on the fields of cardiac arrhythmias and whole-heart computational modeling, present-day system designs precludes its adoption by the broader cardiovascular research community because of their high costs. Taking advantage of recent technological advances, we developed and validated low-cost optical mapping systems for panoramic imaging using Langendorff-perfused pig hearts, a clinically-relevant model in basic research and bioengineering. By significantly lowering financial thresholds, this powerful cardiac electrophysiology imaging modality may gain wider use in research and, even, teaching laboratories, which we substantiated using the lower-cost Langendorff-perfused rabbit heart model

Transcription factor Tbx3 is required for the specification of the atrioventricular conduction system.

The cardiac conduction system consists of distinctive heart muscle cells that initiate and propagate the electric impulse required for coordinated contraction. The conduction system expresses the transcriptional repressor Tbx3, which is required for vertebrate development and controls the formation of the sinus node. In humans, mutations in Tbx3 cause ulnar-mammary syndrome. Here, we investigated the role of Tbx3 in the molecular specification of the atrioventricular conduction system. Expression analysis revealed early delineation of the atrioventricular bundle and proximal bundle branches by Tbx3 expression in human, mouse, and chicken. Tbx3-deficient mice, which die between embryonic day 12.5 and 15.5, ectopically expressed genes for connexin (Cx)43, atrial natriuretic factor (Nppa), Tbx18, and Tbx20 in the atrioventricular bundle and proximal bundle branches. Cx40 was precociously upregulated in the atrioventricular bundle of Tbx3 mutants. Moreover, the atrioventricular bundle and branches failed to exit the cell cycle in Tbx3 mutant embryos. Finally, Tbx3-deficient embryos developed outflow tract malformations and ventricular septal defects. These data reveal that Tbx3 is required for the molecular specification of the atrioventricular bundle and bundle branches and for the development of the ventricular septum and outflow tract. Our data suggest a mechanism in which Tbx3 represses differentiation into ventricular working myocardium, thereby imposing the conduction system phenotype on cells within its expression domain

Embryonic Tbx3+ cardiomyocytes form the mature cardiac conduction system by progressive fate restriction

A small network of spontaneously active Tbx3+ cardiomyocytes forms the cardiac conduction system (CCS) of the heart. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and biological pacemaker development to treat arrhythmias. We found that Tbx3 expression already in the embryo associated with automaticity. Genetic inducible fate mapping revealed that Tbx3+ cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages contribution of Tbx3+ cells was restricted to the definitive CCS. We identified a Tbx3+ population in the outflow tract of the early heart tube that formed the atrioventricular bundle. While Tbx3+ cardiomyocytes also contributed to the adjacent Gja5+ atrial and ventricular chamber myocardium, embryonic Gja5+ chamber cardiomyocytes did not contribute to the Tbx3+ sinus node or atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate-restriction of a Tbx3+ cell population in the early developing heart, and implicates Tbx3 as a useful tool to develop strategies to study and treat CCS diseases

T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3 +/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3 +/- mutants. Notably, Tbx3 +/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus

ElectroMap: High-throughput open-source software for analysis and mapping of cardiac electrophysiology.

The ability to record and analyse electrical behaviour across the heart using optical and electrode mapping has revolutionised cardiac research. However, wider uptake of these technologies is constrained by the lack of multi-functional and robustly characterised analysis and mapping software. We present ElectroMap, an adaptable, high-throughput, open-source software for processing, analysis and mapping of complex electrophysiology datasets from diverse experimental models and acquisition modalities. Key innovation is development of standalone module for quantification of conduction velocity, employing multiple methodologies, currently not widely available to researchers. ElectroMap has also been designed to support multiple methodologies for accurate calculation of activation, repolarisation, arrhythmia detection, calcium handling and beat-to-beat heterogeneity. ElectroMap implements automated signal segmentation, ensemble averaging and integrates optogenetic approaches. Here we employ ElectroMap for analysis, mapping and detection of pro-arrhythmic phenomena in silico, in cellulo, animal model and in vivo patient datasets. We anticipate that ElectroMap will accelerate innovative cardiac research and enhance the uptake, application and interpretation of mapping technologies leading to novel approaches for arrhythmia prevention