Interpretation of field and LEAP potentials recorded from cardiomyocyte monolayers.

Multielectrode arrays (MEAs) are the method of choice for electrophysiological characterization of cardiomyocyte monolayers. The field potentials recorded using an MEA are like extracellular electrograms recorded from the myocardium using conventional electrodes. Nevertheless, different criteria are used to interpret field potentials and extracellular electrograms, which hamper correct interpretation and translation to the patient. To validate the criteria for interpretation of field potentials, we used neonatal rat cardiomyocytes to generate monolayers. We recorded field potentials using an MEA and simultaneously recorded action potentials using sharp microelectrodes. In parallel, we recreated our experimental setting in silico and performed simulations. We show that the amplitude of the local RS complex of a field potential correlated with conduction velocity in silico but not in vitro. The peak time of the T wave in field potentials exhibited a strong correlation with APD90 while the steepest upslope correlated well with APD50. However, this relationship only holds when the T wave displayed a biphasic pattern. Next, we simulated local extracellular action potentials (LEAPs). The shape of the LEAP differed markedly from the shape of the local action potential, but the final duration of the LEAP coincided with APD90. Criteria for interpretation of extracellular electrograms should be applied to field potentials. This will provide a strong basis for the analysis of heterogeneity in conduction velocity and repolarization in cultured monolayers of cardiomyocytes. Finally, a LEAP is not a recording of the local action potential but is generated by intracellular current provided by neighboring cardiomyocytes and is superior to field potential duration in estimating APD90.NEW & NOTEWORTHY We present a physiological basis for the interpretation of multielectrode array-derived, extracellular, electrical signals.

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.

The TMEM43 S358L mutation affects cardiac, small intestine, and metabolic homeostasis in a knock-in mouse model.

The transmembrane protein 43 (TMEM43/LUMA) p.S358L mutation causes arrhythmogenic cardiomyopathy named as ARVC5, a fully penetrant disease with high risk of ventricular arrhythmias, sudden death, and heart failure. Male gender and vigorous exercise independently predicted deleterious outcome. Our systems genetics analysis revealed the importance of Tmem43 for cardiac and metabolic pathways associated with elevated lipid absorption from small intestine. This study sought to delineate gender-specific cardiac, intestinal, and metabolic phenotypes in vivo and investigate underlying pathophysiological mechanisms of S358L mutation. Serial echocardiography, surface electrocardiography (ECG), treadmill running, and body EchoMRI have been used in knock-in heterozygous (Tmem43WT/S358L), homozygous (Tmem43S358L), and wildtype (Tmem43WT) littermate mice. Electron microscopy, histology, immunohistochemistry, transcriptome, and protein analysis have been performed in cardiac and intestinal tissues. Systolic dysfunction was apparent in 3-mo-old Tmem43S358L and 6-mo-old Tmem43WT/S358L mutants. Both mutant lines displayed intolerance to acute stress at 6 mo of age, arrhythmias, fibro-fatty infiltration, and subcellular abnormalities in the myocardium. Microarray analysis found significantly differentially expressed genes between left ventricular (LV) and right ventricular (RV) myocardium. Mutants displayed diminished PPARG activities and significantly reduced TMEM43 and ß-catenin expression in the heart, whereas junctional plakoglobin (JUP) translocated into nuclei of mutant cardiomyocytes. Conversely, elongated villi, fatty infiltration, and overexpression of gut epithelial proliferation markers, ß-catenin and Ki-67, were evident in small intestine of mutants. We defined Tmem43 S358L-induced pathological effects on cardiac and intestinal homeostasis via distinctly disturbed WNT-ß-catenin and PPARG signaling thereby contributing to ARVC5 pathophysiology. Results suggest that cardiometabolic assessment in mutation carriers may be important for predictive and personalized care.NEW & NOTEWORTHY This manuscript describes the findings of our investigation of cardiac, small intestine, and metabolic features of Tmem43-S358L mouse model. By investigating interorgan pathologies, we uncovered multiple mechanisms of the S358L-induced disease, and these unique mechanisms likely appear to contribute to the disease pathogenesis. We hope our findings are important and novel and open new avenues in the hunting for additional diagnostic and therapeutic targets in subjects carrying TMEM43 mutation.

A comprehensive framework for evaluation of high pacing frequency and arrhythmic optical mapping signals.

Introduction: High pacing frequency or irregular activity due to arrhythmia produces complex optical mapping signals and challenges for processing. The objective is to establish an automated activation time-based analytical framework applicable to optical mapping images of complex electrical behavior. Methods: Optical mapping signals with varying complexity from sheep (N = 7) ventricular preparations were examined. Windows of activation centered on each action potential upstroke were derived using Hilbert transform phase. Upstroke morphology was evaluated for potential multiple activation components and peaks of upstroke signal derivatives defined activation time. Spatially and temporally clustered activation time points were grouped in to wave fronts for individual processing. Each activation time point was evaluated for corresponding repolarization times. Each wave front was subsequently classified based on repetitive or non-repetitive events. Wave fronts were evaluated for activation time minima defining sites of wave front origin. A visualization tool was further developed to probe dynamically the ensemble activation sequence. Results: Our framework facilitated activation time mapping during complex dynamic events including transitions to rotor-like reentry and ventricular fibrillation. We showed that using fixed AT windows to extract AT maps can impair interpretation of the activation sequence. However, the phase windowing of action potential upstrokes enabled accurate recapitulation of repetitive behavior, providing spatially coherent activation patterns. We further demonstrate that grouping the spatio-temporal distribution of AT points in to coherent wave fronts, facilitated interpretation of isolated conduction events, such as conduction slowing, and to derive dynamic changes in repolarization properties. Focal origins precisely detected sites of stimulation origin and breakthrough for individual wave fronts. Furthermore, a visualization tool to dynamically probe activation time windows during reentry revealed a critical single static line of conduction slowing associated with the rotation core. Conclusion: This comprehensive analytical framework enables detailed quantitative assessment and visualization of complex electrical behavior.

An atrial fibrillation-associated regulatory region modulates cardiac Tbx5 levels and arrhythmia susceptibility.

Heart development and rhythm control are highly Tbx5 dosage-sensitive. TBX5 haploinsufficiency causes congenital conduction disorders, whereas increased expression levels of TBX5 in human heart samples has been associated with atrial fibrillation (AF). We deleted the conserved mouse orthologues of two independent AF-associated genomic regions in the Tbx5 locus, one intronic (RE(int)) and one downstream (RE(down)) of Tbx5. In both lines, we observed a modest (30%) increase of Tbx5 in the postnatal atria. To gain insight into the effects of slight dosage increase in vivo, we investigated the atrial transcriptional, epigenetic and electrophysiological properties of both lines. Increased atrial Tbx5 expression was associated with induction of genes involved in development, ion transport and conduction, with increased susceptibility to atrial arrhythmias, and increased action potential duration of atrial cardiomyocytes. We identified an AF-associated variant in the human RE(int) that increases its transcriptional activity. Expression of the AF-associated transcription factor Prrx1 was induced in Tbx5RE(int)KO cardiomyocytes. We found that some of the transcriptional and functional changes in the atria caused by increased Tbx5 expression were normalized when reducing cardiac Prrx1 expression in Tbx5RE(int)KO mice, indicating an interaction between these two AF genes. We conclude that modest increases in expression of dose-dependent transcription factors, caused by common regulatory variants, significantly impact on the cardiac gene regulatory network and disease susceptibility.

Cardiac Repolarization in Health and Disease.

Abnormal cardiac repolarization is at the basis of life-threatening arrhythmias in various congenital and acquired cardiac diseases. Dysfunction of ion channels involved in repolarization at the cellular level are often the underlying cause of the repolarization abnormality. The expression pattern of the gene encoding the affected ion channel dictates its impact on the shape of the T-wave and duration of the QT interval, thereby setting the stage for both the occurrence of the trigger and the substrate for maintenance of the arrhythmia. Here we discuss how research into the genetic and electrophysiological basis of repolarization has provided us with insights into cardiac repolarization in health and disease and how this in turn may provide the basis for future improved patient-specific management.

MicroRNAs in extracellular vesicles released from epicardial adipose tissue promote arrhythmogenic conduction slowing.

Background: Patients with excess epicardial adipose tissue (EAT) are at increased risk of developing cardiac arrhythmias. EAT promotes arrhythmias by depolarizing the resting membrane of cardiomyocytes, which slows down conduction and facilitates re-entrant arrhythmias. We hypothesized that EAT slows conduction by secreting extracellular vesicles (EVs) and their microRNA (miRNA) cargo. Objective: We aimed to determine the role of EAT-derived EVs and their miRNA cargo in conduction slowing. Methods: EAT and subcutaneous adipose tissue (SAT) were collected from patients with atrial fibrillation. Adipose tissue explants were incubated in culture medium and secretome was collected. The numbers of EVs in the EAT and SAT secretome were measured by calibrated flow cytometry. EVs in the EAT secretome were isolated by size exclusion chromatography and miRNAs were sequenced. Pathway analysis was performed to predict candidates involved in cardiac electrophysiology. The candidates were validated in the EAT and SAT by quantitative real-time polymerase chain reaction. Finally, miRNA candidates were overexpressed in neonatal rat ventricular myocytes. Results: The EV concentration was higher in the EAT secretome than in the SAT and control secretomes. miRNA sequencing of EAT-derived EVs detected a total of 824 miRNAs. Pathway analysis led to the identification of 7 miRNAs potentially involved in regulation of cardiac resting membrane potential. Validation of those miRNA candidates showed that they were all expressed in EAT, and that miR-1-3p and miR-133a-3p were upregulated in EAT in comparison with SAT. Overexpression of miR-1-3p and miR-133a-3p in neonatal rat ventricular myocytes led to conduction slowing and reduced Kcnj2 and Kcnj12 expression. Conclusion: miR-1-3p and miR-133a-3p are potential mediators of EAT arrhythmogenicity.

Guidelines for assessment of cardiac electrophysiology and arrhythmias in small animals.

Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters.

Molecular and electrophysiological evaluation of human cardiomyocyte subtypes to facilitate generation of composite cardiac models.

Paucity of physiologically relevant cardiac models has limited the widespread application of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug development. Here, we performed comprehensive characterization of hiPSC-derived cardiomyocyte subtypes from 2D and 3D cultures and established a novel 3D model to study impulse initiation and propagation. Directed differentiation approaches were used to generate sinoatrial nodal (SANCM), atrial (ACM) and ventricular cardiomyocytes (VCM). Single cell RNA sequencing established that the protocols yield distinct cell populations in line with expected identities, which was also confirmed by electrophysiological characterization. In 3D EHT cultures of all subtypes, we observed prominent expression of stretch-responsive genes such as NPPA. Response to rate modulating drugs noradrenaline, carbachol and ivabradine were comparable in single cells and EHTs. Differences in the speed of impulse propagation between the subtypes were more pronounced in EHTs compared with 2D monolayers owing to a progressive increase in conduction velocities in atrial and ventricular cardiomyocytes, in line with a more mature phenotype. In a novel binary EHT model of pacemaker-atrial interface, the SANCM end of the tissue consistently paced the EHTs under baseline conditions, which was inhibited by ivabradine. Taken together, our data provide comprehensive insights into molecular and electrophysiological properties of hiPSC-derived cardiomyocyte subtypes, facilitating the creation of next generation composite cardiac models for drug discovery, disease modeling and cell-based regenerative therapies.

Naked mole-rats maintain cardiac function and body composition well into their fourth decade of life.

The prevalence of cardiovascular disease increases exponentially with age, highlighting the contribution of aging mechanisms to cardiac diseases. Although model organisms which share human disease pathologies can elucidate mechanisms driving disease, they do not provide us with innate examples how cardiac aging might be slowed or attenuated. The identification of animal models that preserve cardiac function throughout most of life offers an alternative approach to study mechanisms which might slow cardiac aging. One such species may be the naked mole-rat (NMR), a mouse-sized (40 g) rodent with extraordinary longevity (> 37 years), and constant mortality hazard over its four decades of life. We used a cross-sectional study design to measure a range of physiological parameters in NMRs between 2 and 34 years of age and compared these findings with those of mice aged between 3 months and 2.5 years. We observed a rapid decline in body fat content and bone mineral density in old mice, but no changes in NMRs. Similarly, rhythm disorders (premature atrial and ventricular complexes) occurred in aged mice but not in NMRs. Magnetic resonance and ultrasound imaging showed age-dependent increases in cardiac hypertrophy and diastolic dysfunction in mice which were absent in NMRs. Finally, cardiac stress tests showed an age-dependent decline in normalized cardiac output in mice, which was absent in NMRs. Unlike mice, that manifest several aspects of human cardiac aging, NMRs maintain cardiac function and reserve capacity throughout their long lives and may offer insights on how to delay or prevent cardiac aging.

Patient-Specific TBX5-G125R Variant Induces Profound Transcriptional Deregulation and Atrial Dysfunction.

BACKGROUND: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt-Oram syndrome (also known as hand-heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. METHODS: We analyzed ECGs of an extended family pedigree of Holt-Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome (Tbx5G125R) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). RESULTS: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt-Oram syndrome patients. Tbx5G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. CONCLUSIONS: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

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.

Catecholamines are key modulators of ventricular repolarization patterns in the ball python (Python regius).

Ectothermic vertebrates experience daily changes in body temperature, and anecdotal observations suggest these changes affect ventricular repolarization such that the T-wave in the ECG changes polarity. Mammals, in contrast, can maintain stable body temperatures, and their ventricular repolarization is strongly modulated by changes in heart rate and by sympathetic nervous system activity. The aim of this study was to assess the role of body temperature, heart rate, and circulating catecholamines on local repolarization gradients in the ectothermic ball python (Python regius). We recorded body-surface electrocardiograms and performed open-chest high-resolution epicardial mapping while increasing body temperature in five pythons, in all of which there was a change in T-wave polarity. However, the vector of repolarization differed between individuals, and only a subset of leads revealed T-wave polarity change. RNA sequencing revealed regional differences related to adrenergic signaling. In one denervated and Ringer's solution-perfused heart, heating and elevated heart rates did not induce change in T-wave polarity, whereas noradrenaline did. Accordingly, electrocardiograms in eight awake pythons receiving intra-arterial infusion of the ß-adrenergic receptor agonists adrenaline and isoproterenol revealed T-wave inversion in most individuals. Conversely, blocking the ß-adrenergic receptors using propranolol prevented T-wave change during heating. Our findings indicate that changes in ventricular repolarization in ball pythons are caused by increased tone of the sympathetic nervous system, not by changes in temperature. Therefore, ventricular repolarization in both pythons and mammals is modulated by evolutionary conserved mechanisms involving catecholaminergic stimulation.

Localized Pulmonary Vein Scar Promotes Atrial Fibrillation in High Left Atrial Pressure.

BACKGROUND: Pulmonary vein (PV) ablation is unsuccessful in atrial fibrillation (AF) patients with high left atrial (LA) pressure. Increased atrial stretch by increased pressure is proarrhythmic for AF, and myocardial scar alters wall deformation. We hypothesized that localized PV scar is proarrhythmic for AF in high LA pressure. METHODS: Radiofrequency energy was delivered locally in the right PV of healthy sheep. The sheep recovered for 4 months. Explanted hearts (n = 9 PV scar, n = 9 controls) were perfused with 1:4 blood:Tyrode's solution in a four-chamber working heart setup. Programmed PV stimulation was performed during low (∼12 mmHg) and high (∼25 mmHg) LA pressure. An AF inducibility index was calculated based on the number of induction attempts and the number of attempts causing AF (run of ≥ 20 premature atrial complexes). RESULTS: In high LA pressure, the presence of PV scar increased the AF inducibility index compared with control hearts (0.83 ± 0.20 vs. 0.38 ± 0.40 arb. unit, respectively, p = 0.014). The diastolic stimulation threshold in high LA pressure was higher (108 ± 23 vs. 77 ± 16 mA, respectively, p = 0.006), and its heterogeneity was increased in hearts with PV scar compared with controls. In high LA pressure, the refractory period was shorter in PV scar than in control hearts (178 ± 39 vs. 235 ± 48 ms, p = 0.011). CONCLUSION: Localized PV scar only in combination with increased LA pressure facilitated the inducibility of AF. This was associated with changes in tissue excitability remote from the PV scar. Localized PV ablation is potentially proarrhythmic in patients with increased LA pressure.