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Aims Short QT syndrome type 3 (SQTS3) is a rare arrhythmogenic disease caused by gain-of-function mutations in KCNJ2 , the gene coding the inward rectifier potassium channel Kir2.1. We used a multidisciplinary approach and investigated arrhythmogenic mechanisms in an in-vivo model of de-novo mutation Kir2.1E299V identified in a patient presenting an extremely abbreviated QT interval and paroxysmal atrial fibrillation. Methods and results We used intravenous adeno-associated virus-mediated gene transfer to generate mouse models, and confirmed cardiac-specific expression of Kir2.1WT or Kir2.1E299V. On ECG, the Kir2.1E299V mouse recapitulated the QT interval shortening and the atrial-specific arrhythmia of the patient. The PR interval was also significantly shorter in Kir2.1E299V mice. Patch-clamping showed extremely abbreviated action potentials in both atrial and ventricular Kir2.1E299V cardiomyocytes due to a lack of inward-going rectification and increased IK1 at voltages positive to −80 mV. Relative to Kir2.1WT, atrial Kir2.1E299V cardiomyocytes had a significantly reduced slope conductance at voltages negative to −80 mV. After confirming a higher proportion of heterotetrameric Kir2.x channels containing Kir2.2 subunits in the atria, in-silico 3D simulations predicted an atrial-specific impairment of polyamine block and reduced pore diameter in the Kir2.1E299V-Kir2.2WT channel. In ventricular cardiomyocytes, the mutation increased excitability by shifting INa activation and inactivation in the hyperpolarizing direction, which protected the ventricle against arrhythmia. Moreover, Purkinje myocytes from Kir2.1E299V mice manifested substantially higher INa density than Kir2.1WT, explaining the abbreviation in the PR interval. Conclusion The first in-vivo mouse model of cardiac-specific SQTS3 recapitulates the electrophysiological phenotype of a patient with the Kir2.1E299V mutation. Kir2.1E299V eliminates rectification in both cardiac chambers but protects against ventricular arrhythmias by increasing excitability in both Purkinje-fiber network and ventricles. Consequently, the predominant arrhythmias are supraventricular likely due to the lack of inward rectification and atrial-specific reduced pore diameter of the Kir2.1E299V-Kir2.2WT heterotetramer. [ABSTRACT FROM AUTHOR]

Aims Diseased atria are characterized by functional and structural heterogeneities, adding to abnormal impulse generation and propagation. These heterogeneities are thought to lie at the origin of fractionated electrograms recorded during sinus rhythm (SR) in atrial fibrillation (AF) patients and are assumed to be involved in the onset and perpetuation (e.g. by re-entry) of this disorder. The underlying mechanisms, however, remain incompletely understood. Here, we tested whether regions of dense fibrosis could create an electrically isolated conduction pathway (EICP) in which re-entry could be established via ectopy and local block to become 'trapped'. We also investigated whether this could generate local fractionated electrograms and whether the re-entrant wave could 'escape' and cause a global tachyarrhythmia due to dynamic changes at a connecting isthmus. Methods and results To precisely control and explore the geometrical properties of EICPs, we used light-gated depolarizing ion channels and patterned illumination for creating specific non-conducting regions in silico and in vitro. Insight from these studies was used for complementary investigations in virtual human atria with localized fibrosis. We demonstrated that a re-entrant tachyarrhythmia can exist locally within an EICP with SR prevailing in the surrounding tissue and identified conditions under which re-entry could escape from the EICP, thereby converting a local latent arrhythmic source into an active driver with global impact on the heart. In a realistic three-dimensional model of human atria, unipolar epicardial pseudo-electrograms showed fractionation at the site of 'trapped re-entry' in coexistence with regular SR electrograms elsewhere in the atria. Upon escape of the re-entrant wave, acute arrhythmia onset was observed. Conclusions Trapped re-entry as a latent source of arrhythmogenesis can explain the sudden onset of focal arrhythmias, which are able to transgress into AF. Our study might help to improve the effectiveness of ablation of aberrant cardiac electrical signals in clinical practice. [ABSTRACT FROM AUTHOR]

Ventricular arrhythmias in cardiac channelopathies are linked to autonomic triggers, which are sub-optimally targeted in current management strategies. Improved molecular understanding of cardiac channelopathies and cellular autonomic signalling could refine autonomic therapies to target the specific signalling pathways relevant to the specific aetiologies as well as the central nervous system centres involved in the cardiac autonomic regulation. This review summarizes key anatomical and physiological aspects of the cardiac autonomic nervous system and its impact on ventricular arrhythmias in primary inherited arrhythmia syndromes. Proarrhythmogenic autonomic effects and potential therapeutic targets in defined conditions including the Brugada syndrome, early repolarization syndrome, long QT syndrome, and catecholaminergic polymorphic ventricular tachycardia will be examined. Pharmacological and interventional neuromodulation options for these cardiac channelopathies are discussed. Promising new targets for cardiac neuromodulation include inhibitory and excitatory G-protein coupled receptors, neuropeptides, chemorepellents/attractants as well as the vagal and sympathetic nuclei in the central nervous system. Novel therapeutic strategies utilizing invasive and non-invasive deep brain/brain stem stimulation as well as the rapidly growing field of chemo-, opto-, or sonogenetics allowing cell-specific targeting to reduce ventricular arrhythmias are presented. [ABSTRACT FROM AUTHOR]

This document is an editorial that references a scientific article by Xia et al. The article discusses the role of the ryanodine receptor type 2 (RyR2) in the heart's excitation-contraction coupling (ECC) mechanism. It emphasizes the importance of RyR2 in cardiac function and its involvement in arrhythmogenic diseases. The article also mentions the use of human-induced pluripotent stem cell-derived cardiomyocytes to study RyR2 mutations. The document provides a table of RyR2 mutations investigated in these cardiomyocytes, detailing their effects on calcium release and other cardiac parameters. The article discusses a study by Xia et al. that used CRISPR/Cas9 technology to investigate the effects of specific mutations in the RyR2 channel on calcium release in these cardiomyocytes. The study found that the mutations led to a loss of calcium-induced calcium release mechanism, but the cardiomyocytes were still able to produce spontaneous calcium transients. The study also highlighted the role of calcium influx through other channels in these transients. Overall, the study provides insights into the mechanisms of RyR2 mutations and the plasticity of these cardiomyocytes. [Extracted from the article]

Aims CRISPR/Cas9 gene edits of cardiac ryanodine receptor (RyR2) in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) provide a novel platform for introducing mutations in RyR2 Ca2+-binding residues and examining the resulting excitation contraction (EC)-coupling remodelling consequences. Methods and results Ca2+-signalling phenotypes of mutations in RyR2 Ca2+-binding site residues associated with cardiac arrhythmia (RyR2-Q3925E) or not proven to cause cardiac pathology (RyR2-E3848A) were determined using ICa- and caffeine-triggered Ca2+ releases in voltage-clamped and total internal reflection fluorescence-imaged wild type and mutant cardiomyocytes infected with sarcoplasmic reticulum (SR)-targeted ER-GCaMP6 probe. (i) ICa- and caffeine-triggered Fura-2 or ER-GCaMP6 signals were suppressed, even when ICa was significantly enhanced in Q3925E and E3848A mutant cardiomyocytes; (ii) spontaneous beating (Fura-2 Ca2+ transients) persisted in mutant cells without the SR-release signals; (iii) while 5–20 mM caffeine failed to trigger Ca2+-release in voltage-clamped mutant cells, only ∼20% to ∼70% of intact myocytes responded respectively to caffeine; (iv) and 20 mM caffeine transients, however, activated slowly, were delayed, and variably suppressed by 2-APB, FCCP, or ruthenium red. Conclusion Mutating RyR2 Ca2+-binding residues, irrespective of their reported pathogenesis, suppressed both ICa- and caffeine-triggered Ca2+ releases, suggesting interaction between Ca2+- and caffeine-binding sites. Enhanced transmembrane calcium influx and remodelling of EC-coupling pathways may underlie the persistence of spontaneous beating in Ca2+-induced Ca2+ release-suppressed mutant myocytes. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is a rare condition in young people, but it may be caused by heritable cardiac conditions such as short QT syndrome (SQTS). SQTS is a rare genetic condition that can lead to AF at an early age and can be complicated by ventricular arrhythmias and sudden death. Treatment options for SQTS are limited, and there is a need for further research to understand the disease mechanisms and develop effective therapies. A recent study using a mouse model of SQTS type 3 (SQTS3) has provided insights into the molecular mechanisms underlying the condition and potential treatment strategies. The study found that a mutated potassium channel gene in the heart increased ventricular excitability and that isoproterenol infusion normalized repolarization in the mouse model. These findings suggest that targeting ion channels and their interactions could be a potential treatment strategy for SQTS. However, further research is needed to confirm these findings in humans and identify the most effective therapies. Currently, quinidine is the empirical pharmacological treatment for SQTS, but it is associated with adverse effects and limited availability in some countries. Standard care options such as treatment of AF or implantable defibrillators may be used for symptomatic SQTS patients. Overall, this study contributes to our understanding of the relationship between genetic variants in ion channels and cardiac arrhythmias and highlights the need for further research in this field. [Extracted from the article]

Aims Mutations in the DSP gene encoding desmoplakin, a constituent of the desmosomes at the intercalated discs (IDs), cause a phenotype that spans arrhythmogenic cardiomyopathy (ACM) and dilated cardiomyopathy. It is typically characterized by biventricular enlargement and dysfunction, myocardial fibrosis, cell death, and arrhythmias. The canonical wingless-related integration (cWNT)/β-catenin pathway is implicated in the pathogenesis of ACM. The β-catenin is an indispensable co-transcriptional regulator of the cWNT pathway and a member of the IDs. We genetically inactivated or activated β-catenin to determine its role in the pathogenesis of desmoplakin cardiomyopathy. Methods and results The Dsp gene was conditionally deleted in the 2-week-old post-natal cardiac myocytes using tamoxifen-inducible MerCreMer mice (Myh6-Mcm Tam: Dsp F/F). The cWNT/β-catenin pathway was markedly dysregulated in the Myh6-Mcm Tam: Dsp F/F cardiac myocytes, as indicated by a concomitant increase in the expression of cWNT/β-catenin target genes, isoforms of its key co-effectors, and the inhibitors of the pathway. The β-catenin was inactivated or activated upon inducible deletion of its transcriptional or degron domain, respectively, in the Myh6-Mcm Tam: Dsp F/F cardiac myocytes. Genetic inactivation of β-catenin in the Myh6-Mcm Tam: Dsp F/F mice prolonged survival, improved cardiac function, reduced cardiac arrhythmias, and attenuated myocardial fibrosis, and cell death caused by apoptosis, necroptosis, and pyroptosis, i.e. PANoptosis. In contrast, activation of β-catenin had the opposite effects. The deleterious and the salubrious effects were independent of changes in the expression levels of the cWNT target genes and were associated with changes in several molecular and biological pathways, including cell death programmes. Conclusion The cWNT/β-catenin was markedly dysregulated in the cardiac myocytes in a mouse model of desmoplakin cardiomyopathy. Inactivation of β-catenin attenuated, whereas its activation aggravated the phenotype, through multiple molecular pathways, independent of the cWNT transcriptional activity. Thus, suppression but not activation of β-catenin might be beneficial in desmoplakin cardiomyopathy. [ABSTRACT FROM AUTHOR]

Aims Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR–Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243. Methods and results The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1–S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness. Conclusion Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs. [ABSTRACT FROM AUTHOR]

Electronic cigarette use has grown exponentially in recent years, and while their popularity has increased, the long-term effects on the heart are yet to be fully studied and understood. Originally designed as devices to assist with those trying to quit traditional combustible cigarette use, their popularity has attracted use by teens and adolescents who traditionally have not smoked combustible cigarettes. Acute effects on the heart have been shown to be similar to traditional combustible cigarettes, including increased heart rate and blood pressure. The main components of electronic cigarettes that contribute to these arrhythmic effects are found in the e-liquid that is aerosolized and inhaled, comprised of nicotine, flavourings, and a combination of vegetable glycerin (VG) and propylene glycol (PG). Nicotine can potentially induce both ventricular and atrial arrhythmogenesis, with both the atrial and ventricular effects resulting from the interactions of nicotine and the catecholamines they release via potassium channels. Atrial arrhythmogenesis, more specifically atrial fibrillation, can also occur due to structural alterations, which happens because of nicotine downregulating microRNAs 133 and 590, both post-transcriptional growth factor repressors. Liquid flavourings and the combination of PG and VG can possibly lead to arrhythmic events by exposing users to acrolein, an aldehyde that stimulates TRPA1 that in turn causes a change towards sympathetic activation and autonomic imbalance. The design of these electronic delivery devices is constantly changing; therefore, it has proven extremely difficult to study the long-term effects on the heart caused by electronic cigarettes but will be important to understand given their rising popularity. The arrhythmic effects of electronic cigarettes appear similar to traditional cigarettes as well; however, a comprehensive review has not been compiled and is the focus of this article. [ABSTRACT FROM AUTHOR]

The cardiac sodium channel NaV1.5 is an essential modulator of cardiac excitability, with decreased NaV1.5 levels at the plasma membrane and consequent reduction in sodium current (I Na) leading to potentially lethal cardiac arrhythmias. NaV1.5 is distributed in a specific pattern at the plasma membrane of cardiomyocytes, with localization at the crests, grooves, and T-tubules of the lateral membrane and particularly high levels at the intercalated disc region. NaV1.5 forms a large macromolecular complex with and is regulated by interacting proteins, some of which are specifically localized at either the lateral membrane or intercalated disc. One of the NaV1.5 trafficking routes is via microtubules (MTs), which are regulated by MT plus-end tracking proteins (+TIPs). In our search for mechanisms involved in targeted delivery of NaV1.5, we here provide an overview of previously demonstrated interactions between NaV1.5 interacting proteins and +TIPs, which potentially (in)directly impact on NaV1.5 trafficking. Strikingly, +TIPs interact extensively with several intercalated disc- and lateral membrane-specific NaV1.5 interacting proteins. Recent work indicates that this interplay of +TIPs and NaV1.5 interacting proteins mediates the targeted delivery of NaV1.5 at specific cardiomyocyte subcellular domains, while also being potentially relevant for the trafficking of other ion channels. These observations are especially relevant for diseases associated with loss of NaV1.5 specifically at the lateral membrane (such as Duchenne muscular dystrophy), or at the intercalated disc (for example, arrhythmogenic cardiomyopathy), and open up potential avenues for development of new anti-arrhythmic therapies. [ABSTRACT FROM AUTHOR]

Andersen-Tawil syndrome (ATS) is a rare inheritable disease associated with loss-of-function mutations in KCNJ2, the gene coding the strong inward rectifier potassium channel Kir2.1, which forms an essential membrane protein controlling cardiac excitability. ATS is usually marked by a triad of periodic paralysis, life-threatening cardiac arrhythmias and dysmorphic features, but its expression is variable and not all patients with a phenotype linked to ATS have a known genetic alteration. The mechanisms underlying this arrhythmogenic syndrome are poorly understood. Knowing such mechanisms would be essential to distinguish ATS from other channelopathies with overlapping phenotypes and to develop individualized therapies. For example, the recently suggested role of Kir2.1 as a countercurrent to sarcoplasmic calcium reuptake might explain the arrhythmogenic mechanisms of ATS and its overlap with catecholaminergic polymorphic ventricular tachycardia. Here we summarize current knowledge on the mechanisms of arrhythmias leading to sudden cardiac death in ATS. We first provide an overview of the syndrome and its pathophysiology, from the patient's bedside to the protein and discuss the role of essential regulators and interactors that could play a role in cases of ATS. The review highlights novel ideas related to some post-translational channel interactions with partner proteins that might help define the molecular bases of the arrhythmia phenotype. We then propose a new all-embracing classification of the currently known ATS loss-of-function mutations according to their position in the Kir2.1 channel structure and their functional implications. We also discuss specific ATS pathogenic variants, their clinical manifestations, and treatment stratification. The goal is to provide a deeper mechanistic understanding of the syndrome toward the development of novel targets and personalized treatment strategies. [ABSTRACT FROM AUTHOR]

This article discusses the interactions between KCNQ1 and KCNH2 in relation to the long QT type 1 (LQT1) phenotype. LQT1 and LQT2 are two major forms of the inherited channelopathy long QT syndrome (LQTS). Loss-of-function mutations in KCNQ1 or KCNH2 cause a reduction in the cardiac repolarizing currents IKs or IKr, respectively, leading to a prolonged cardiac action potential duration (APD) and QT interval. The article presents evidence that the reduction of IKs in LQT1 patients may also result in a concomitant down-regulation of IKr, which further increases the risk of arrhythmias. The findings suggest that a more comprehensive assessment of repolarization reserve is necessary to accurately evaluate the arrhythmic risk in LQTS patients. [Extracted from the article]

Microtubules (MTs) are a crucial part of the cytoskeleton that help maintain cellular balance under mechanical stress. Excessive post-translational modifications (PTMs) of MTs, such as detyrosination, have been linked to impaired cardiac contractility. In a study on mice with Duchenne muscular dystrophy (DMD), researchers found that increased detyrosination of MTs was associated with decreased expression of the cardiac Na+ channel Nav1.5. By reducing detyrosinated tubulin, the researchers were able to restore Nav1.5 expression and improve sodium current. This finding suggests that targeting detyrosinated MTs could be a promising strategy for addressing cardiac arrhythmias. [Extracted from the article]

Aims Long QT syndrome (LQTS) carries a risk of life-threatening polymorphic ventricular tachycardia (Torsades de Pointes, TdP) and is a major cause of premature sudden cardiac death. TdP is induced by R-on-T premature ventricular complexes (PVCs), thought to be generated by cellular early-afterdepolarisations (EADs). However, EADs in tissue require cellular synchronisation, and their role in TdP induction remains unclear. We aimed to determine the mechanism of TdP induction in rabbit hearts with acquired LQTS (aLQTS). Methods and results Optical mapping of action potentials (APs) and intracellular Ca2+ was performed in Langendorff-perfused rabbit hearts (n = 17). TdP induced by R-on-T PVCs was observed during aLQTS (50% K+/Mg++ & E4031) conditions in all hearts (P < 0.0001 vs. control). Islands of AP prolongation bounded by steep voltage gradients (VGs) were consistently observed before arrhythmia and peak VGs were more closely related to the PVC upstroke than EADs, both temporally (7 ± 5 ms vs. 44 ± 27 ms, P < 0.0001) and spatially (1.0 ± 0.7 vs. 3.6 ± 0.9 mm, P < 0.0001). PVCs were initiated at estimated voltages of ∼ −40 mV and had upstroke dF/dtmax and Vm-Ca2+ dynamics compatible with I CaL activation. Computational simulations demonstrated that PVCs could arise directly from VGs, through electrotonic triggering of I CaL. In experiments and the model, sub-maximal L-type Ca2+ channel (LTCC) block (200 nM nifedipine and 90% gCaL, respectively) abolished both PVCs and TdP in the continued presence of aLQTS. Conclusion These data demonstrate that I CaL activation at sites displaying steep VGs generates the PVCs which induce TdP, providing a mechanism and rationale for LTCC blockers as a novel therapeutic approach in LQTS. [ABSTRACT FROM AUTHOR]

Aims Human-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs. Methods and results We derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch. Conclusions Our study shows that (i) the tri-cellular cardiac microtissues promote post-natal SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B. Translational perspective The cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications. [ABSTRACT FROM AUTHOR]

Aims A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus-9 (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. Methods and results Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3 ± 1.2 Hz in hPLN-R14del vs. 25.7 ± 1.3 Hz in WT, P < 0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital polymerase chain reaction and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end-diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9 ± 2.3 Hz vs. 21.3 ± 1.5 Hz; P < 0.01). Conclusions This study is the first to show that disruption of hPLN-R14del allele by AAV9-CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease. [ABSTRACT FROM AUTHOR]

This article discusses the mechanisms and potential treatment options for atrial fibrillation (AF). The authors highlight the interplay between initiating triggers and sustaining mechanisms of AF, with triggers often originating from the pulmonary veins and other sites. They present evidence for the existence of localized drivers of AF, referred to as "electrically isolated conduction pathways (EICPs)," which can be detected and targeted during AF ablation procedures. The authors suggest that further research is needed to validate these findings in clinical settings and explore potential methods for preventing the escape of localized reentrant sources. [Extracted from the article]

Here, we review the highlights of cardiovascular basic science published in 2021 and early 2022 on behalf of the European Society of Cardiology Council for Basic Cardiovascular Science. We begin with non-coding RNAs which have emerged as central regulators cardiovascular biology, and then discuss how technological developments in single-cell 'omics are providing new insights into cardiovascular development, inflammation, and disease. We also review recent discoveries on the biology of extracellular vesicles in driving either protective or pathogenic responses. The Nobel Prize in Physiology or Medicine 2021 recognized the importance of the molecular basis of mechanosensing and here we review breakthroughs in cardiovascular sensing of mechanical force. We also summarize discoveries in the field of atherosclerosis including the role of clonal haematopoiesis of indeterminate potential, and new mechanisms of crosstalk between hyperglycaemia, lipid mediators, and inflammation. The past 12 months also witnessed major advances in the field of cardiac arrhythmia including new mechanisms of fibrillation. We also focus on inducible pluripotent stem cell technology which has demonstrated disease causality for several genetic polymorphisms in long-QT syndrome and aortic valve disease, paving the way for personalized medicine approaches. Finally, the cardiovascular community has continued to better understand COVID-19 with significant advancement in our knowledge of cardiovascular tropism, molecular markers, the mechanism of vaccine-induced thrombotic complications and new anti-viral therapies that protect the cardiovascular system. [ABSTRACT FROM AUTHOR]

Aims Abnormal intracellular calcium (Ca2+) handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signalling pathway contributes to atrial remodelling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models. Methods and results Expression levels of NFATc1–c4 isoforms were assessed by quantitative reverse transcription–polymerase chain reaction in right atrial appendages from patients with chronic AF (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM:NFATc2–/– mice. Programmed electrical stimulation revealed that CREM:NFATc2–/– mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with wild-type controls, which was reversed in CREM:NFATc2–/– mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM:NFATc2–/– mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on ryanodine receptor type 2 (RyR2) in CREM:RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras and EF-hand domain-containing protein (Rasef) as a direct target of NFATc2-mediated transcription. Conclusion Our findings reveal activation of the NFAT signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model. [ABSTRACT FROM AUTHOR]

Aims Diastolic Ca release (DCR) from sarcoplasmic reticulum (SR) Ca release channel ryanodine receptor (RyR2) has been linked to multiple cardiac pathologies, but its exact role in shaping divergent cardiac pathologies remains unclear. We hypothesize that the SR-mitochondria interplay contributes to disease phenotypes by shaping Ca signalling. Methods and results A genetic model of catecholaminergic polymorphic ventricular tachycardia (CPVT2 model of CASQ2 knockout) and a pre-diabetic cardiomyopathy model of fructose-fed mice (FFD), both marked by DCR, are employed in this study. Mitochondria Ca (mCa) is modulated by pharmacologically targeting mitochondria Ca uniporter (MCU) or permeability transition pore (mPTP), mCa uptake, and extrusion mechanisms, respectively. An MCU activator abolished Ca waves in CPVT2 but exacerbated waves in FFD cells. Mechanistically this is ascribed to mitochondria's function as a Ca buffer or source of reactive oxygen species (mtROS) to exacerbate RyR2 functionality, respectively. Enhancing mCa uptake reduced and elevated mtROS production in CPVT2 and FFD, respectively. In CPVT2, mitochondria took up more Ca in permeabilized cells, and had higher level of mCa content in intact cells vs. FFD. Conditional ablation of MCU in the CPVT2 model caused lethality and cardiac remodelling, but reduced arrhythmias in the FFD model. In parallel, CPVT2 mitochondria also employ up-regulated mPTP-mediated Ca efflux to avoid mCa overload, as seen by elevated incidence of MitoWinks (an indicator of mPTP-mediated Ca efflux) vs. FFD. Both pharmacological and genetic inhibition of mPTP promoted mtROS production and exacerbation of myocyte Ca handling in CPVT2. Further, genetic inhibition of mPTP exacerbated arrhythmias in CPVT2. Conclusion In contrast to FFD, which is more susceptible to mtROS-dependent RyR2 leak, in CPVT2 mitochondria buffer SR-derived DCR to mitigate Ca-dependent pathological remodelling and rely on mPTP-mediated Ca efflux to avoid mCa overload. SR-mitochondria interplay contributes to the divergent pathologies by disparately shaping intracellular Ca signalling. [ABSTRACT FROM AUTHOR]

Recent preclinical and observational cohort studies have implicated imbalances in gut microbiota composition as a contributor to atrial fibrillation (AF). The gut microbiota is a complex and dynamic ecosystem containing trillions of microorganisms, which produces bioactive metabolites influencing host health and disease development. In addition to host-specific determinants, lifestyle-related factors such as diet and drugs are important determinants of the gut microbiota composition. In this review, we discuss the evidence suggesting a potential bidirectional association between AF and gut microbiota, identifying gut microbiota-derived metabolites as possible regulators of the AF substrate. We summarize the effect of gut microbiota on the development and progression of AF risk factors, including heart failure, hypertension, obesity, and coronary artery disease. We also discuss the potential anti-arrhythmic effects of pharmacological and diet-induced modifications of gut microbiota composition, which may modulate and prevent the progression to AF. Finally, we highlight important gaps in knowledge and areas requiring future investigation. Although data supporting a direct relationship between gut microbiota and AF are very limited at the present time, emerging preclinical and clinical research dealing with mechanistic interactions between gut microbiota and AF is important as it may lead to new insights into AF pathophysiology and the discovery of novel therapeutic targets for AF. [ABSTRACT FROM AUTHOR]

Google Scholar PubMed OpenURL Placeholder Text WorldCat 7 Liu MB, Vandersickel N, Panfilov AV, Qu Z. R-From-T as a common mechanism of arrhythmia initiation in long QT syndromes. This editorial refers to 'Initiation of ventricular arrhythmia in the acquired long QT syndrome', by C. Alexander I et al i . https://doi.org/10.1093/cvr/cvac103. [Extracted from the article]

Aims Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death (SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach. Methods and results We employed a phenotype-driven N -ethyl- N -nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6–9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2 p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4–5 weeks, Bcat2 p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs—leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2 p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation. Conclusions Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure. [ABSTRACT FROM AUTHOR]

Recent developments in imaging, mapping, and ablation techniques have shown that the epicardial region of the heart is a key player in the occurrence of ventricular arrhythmic events in several cardiac diseases, such as Brugada syndrome, arrhythmogenic cardiomyopathy, or dilated cardiomyopathy. At the atrial level as well, the epicardial region has emerged as an important determinant of the substrate of atrial fibrillation, pointing to common underlying pathophysiological mechanisms. Alteration in the gradient of repolarization between myocardial layers favouring the occurrence of re-entry circuits has largely been described. The fibro-fatty infiltration of the subepicardium is another shared substrate between ventricular and atrial arrhythmias. Recent data have emphasized the role of the epicardial reactivation in the formation of this arrhythmogenic substrate. There are new evidences supporting this structural remodelling process to be regulated by the recruitment of epicardial progenitor cells that can differentiate into adipocytes or fibroblasts under various stimuli. In addition, immune-inflammatory processes can also contribute to fibrosis of the subepicardial layer. A better understanding of such 'electrical fragility' of the epicardial area will open perspectives for novel biomarkers and therapeutic strategies. In this review article, a pathophysiological scheme of epicardial-driven arrhythmias will be proposed. [ABSTRACT FROM AUTHOR]

Aims TASK-1 (K2P3.1) two-pore-domain potassium channels are atrial-specific and significantly up-regulated in atrial fibrillation (AF) patients, contributing to AF-related electrical remodelling. Inhibition of TASK-1 in cardiomyocytes of AF patients was shown to counteract AF-related action potential duration shortening. Doxapram was identified as a potent inhibitor of the TASK-1 channel. In this study, we investigated the antiarrhythmic efficacy of doxapram in a porcine model of AF. Methods and results Doxapram successfully cardioverted pigs with artificially induced episodes of AF. We established a porcine model of persistent AF in domestic pigs via intermittent atrial burst stimulation using implanted pacemakers. All pigs underwent catheter-based electrophysiological investigations prior to and after 14 days of doxapram treatment. Pigs in the treatment group received intravenous administration of doxapram once per day. In doxapram-treated AF pigs, the AF burden was significantly reduced. After 14 days of treatment with doxapram, TASK-1 currents were still similar to values of sinus rhythm animals. Doxapram significantly suppressed AF episodes and normalized cellular electrophysiology by inhibition of the TASK-1 channel. Patch-clamp experiments on human atrial cardiomyocytes, isolated from patients with and without AF could reproduce the TASK-1 inhibitory effect of doxapram. Conclusion Repurposing doxapram might yield a promising new antiarrhythmic drug to treat AF in patients. [ABSTRACT FROM AUTHOR]

Aims  Arrhythmogenic cardiomyopathy (ACM) is a primary myocardial disease that typically manifests with cardiac arrhythmias, progressive heart failure, and sudden cardiac death (SCD). ACM is mainly caused by mutations in genes encoding desmosome proteins. Desmosomes are cell–cell adhesion structures and hubs for mechanosensing and mechanotransduction. The objective was to identify the dysregulated molecular and biological pathways in human ACM in the absence of overt heart failure. Methods and results Transcriptomes in the right ventricular endomyocardial biopsy samples from three independent individuals carrying truncating mutations in the DSP gene and five control samples were analysed by RNA-Seq (discovery group). These cases presented with cardiac arrhythmias and had a normal right ventricular function. The RNA-Seq analysis identified ∼5000 differentially expressed genes (DEGs), which predicted suppression of the Hippo and canonical WNT pathways, among others. Dysregulated genes and pathways, identified by RNA-Seq, were tested for validation in the right and left ventricular tissues from five independent autopsy-confirmed ACM cases with defined mutations (validation group), who were victims of SCD and had no history of heart failure. Protein levels and nuclear localization of the cWNT and Hippo pathway transcriptional regulators were reduced in the right and left ventricular validation samples. In contrast, levels of acetyltransferase EP300, known to suppress the Hippo and canonical WNT pathways, were increased and its bona fide target TP53 was acetylated. RNA-Seq data identified apical junction, reflective of cell–cell attachment, as the most disrupted biological pathway, which were corroborated by disrupted desmosomes and intermediate filament structures. Moreover, the DEGs also predicted dysregulation of over a dozen canonical signal transduction pathways, including the Tec kinase and integrin signalling pathways. The changes were associated with increased apoptosis and fibro-adipogenesis in the ACM hearts. Conclusion  Altered apical junction structures are associated with activation of the EP300-TP53 and suppression of the Hippo/cWNT pathways in human ACM caused by defined mutations in the absence of an overt heart failure. The findings implicate altered mechanotransduction in the pathogenesis of ACM. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common arrhythmia worldwide. It is associated with significant increases in morbidity in the form of stroke and heart failure, and a doubling in all-cause mortality. The pathophysiology of AF is incompletely understood, and this has contributed to a lack of effective treatments and disease-modifying therapies. An important cellular process that may explain how risk factors give rise to AF includes post-translational modification of proteins. As the most commonly occurring post-translational modification, protein phosphorylation is especially relevant. Although many methods exist for studying protein phosphorylation, a common and highly resolute technique is mass spectrometry (MS). This review will discuss recent evidence surrounding the role of protein phosphorylation in the pathogenesis of AF. MS-based technology to study phosphorylation and uses of MS in other areas of medicine such as oncology will also be presented. Based on these data, future goals and experiments will be outlined that utilize MS technology to better understand the role of phosphorylation in AF and elucidate its role in AF pathophysiology. This may ultimately allow for the development of more effective AF therapies. [ABSTRACT FROM AUTHOR]

Aims Direct remuscularization with pluripotent stem cell-derived cardiomyocytes (PSC-CMs) seeks to address the onset of heart failure post-myocardial infarction (MI) by treating the persistent muscle deficiency that underlies it. However, direct remuscularization with PSC-CMs could potentially be arrhythmogenic. We investigated two possible mechanisms of arrhythmogenesis—focal vs. re-entrant—arising from direct remuscularization with PSC-CM patches in two personalized, human ventricular computer models of post-MI. Moreover, we developed a principled approach for evaluating arrhythmogenicity of direct remuscularization that factors in the VT propensity of the patient-specific post-MI fibrotic substrate and use it to investigate different conditions of patch remuscularization. Methods and results Two personalized, human ventricular models of post-MI (P1 and P2) were constructed from late gadolinium enhanced (LGE)-magnetic resonance images (MRIs). In each model, remuscularization with PSC-CM patches was simulated under different treatment conditions that included patch engraftment, patch myofibril orientation, remuscularization site, patch size (thickness and diameter), and patch maturation. To determine arrhythmogenicity of treatment conditions, VT burden of heart models was quantified prior to and after simulated remuscularization and compared. VT burden was quantified based on inducibility (i.e. weighted sum of pacing sites that induced) and severity (i.e. the number of distinct VT morphologies induced). Prior to remuscularization, VT burden was significant in P1 (0.275) and not in P2 (0.0, not VT inducible). We highlight that re-entrant VT mechanisms would dominate over focal mechanisms; spontaneous beats emerging from PSC-CM grafts were always a fraction of resting sinus rate. Moreover, incomplete patch engraftment can be particularly arrhythmogenic, giving rise to particularly aberrant electrical activation and conduction slowing across the PSC-CM patches along with elevated VT burden when compared with complete engraftment. Under conditions of complete patch engraftment, remuscularization was almost always arrhythmogenic in P2 but certain treatment conditions could be anti-arrhythmogenic in P1. Moreover, the remuscularization site was the most important factor affecting VT burden in both P1 and P2. Complete maturation of PSC-CM patches, both ionically and electrotonically, at the appropriate site could completely alleviate VT burden. Conclusion We identified that re-entrant VT would be the primary VT mechanism in patch remuscularization. To evaluate the arrhythmogenicity of remuscularization, we developed a principled approach that factors in the propensity of the patient-specific fibrotic substrate for VT. We showed that arrhythmogenicity is sensitive to the patient-specific fibrotic substrate and remuscularization site. We demonstrate that targeted remuscularization can be safe in the appropriate individual and holds the potential to non-destructively eliminate VT post-MI in addition to addressing muscle deficiency underlying heart failure progression. [ABSTRACT FROM AUTHOR]

Aims Cardiomyocyte Ca2+ homoeostasis is altered with ageing and predisposes the heart to Ca2+ intolerance and arrhythmia. Transient receptor potential vanilloid 4 (TRPV4) is an osmotically activated cation channel with expression in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in Ca2+ handling and arrhythmogenesis following ischaemia–reperfusion (I/R), a pathological scenario associated with osmotic stress. Methods and results Cardiomyocyte membrane potential was monitored prior to and following I/R in Langendorff-perfused hearts of Aged (19–28 months) male and female C57BL/6 mice ± TRPV4 inhibition (1 μM HC067047, HC). Diastolic resting membrane potential was similar between Aged and Aged HC at baseline, but following I/R Aged exhibited depolarized diastolic membrane potential vs. Aged HC. The effects of TRPV4 on cardiomyocyte Ca2+ signalling following I/R were examined in isolated hearts of Aged cardiac-specific GCaMP6f mice (±HC) using high-speed confocal fluorescence microscopy, with cardiomyocytes of Aged exhibiting an increased incidence of pro-arrhythmic Ca2+ signalling vs. Aged HC. In the isolated cell environment, cardiomyocytes of Aged responded to sustained hypoosmotic stress (250mOsm) with an increase in Ca2+ transient amplitude (fluo-4) and higher incidence of pro-arrhythmic diastolic Ca2+ signals vs. Aged HC. Intracardiac electrocardiogram measurements in isolated hearts following I/R revealed an increased arrhythmia incidence, an accelerated time to ventricular arrhythmia, and increased arrhythmia score in Aged vs. Aged HC. Aged exhibited depolarized resting membrane potential, increased pro-arrhythmic diastolic Ca2+ signalling, and greater incidence of arrhythmia when compared with Young (3–5 months). Conclusion TRPV4 contributes to pro-arrhythmic cardiomyocyte Ca2+ signalling, electrophysiological abnormalities, and ventricular arrhythmia in the aged mouse heart. [ABSTRACT FROM AUTHOR]

Aims The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. Methods and results We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans -expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and βIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans -expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans -expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and βIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans -expressing mice. Conclusions In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients. [ABSTRACT FROM AUTHOR]

Aims We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart field-specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations. Methods and results Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enable purification of cardiomyocytes and first heart field cardiomyocytes, respectively. Conclusions We describe a high-yield approach for isolation and characterization of human embryonic stem cell-derived heart field-specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modelling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart- or chamber-specific congenital heart defects. [ABSTRACT FROM AUTHOR]

Aims Ageing is the most significant contributor to the increasing prevalence of atrial fibrillation (AF). The gut microbiota dysbiosis is involved in age-related diseases. However, whether the aged-associated dysbiosis contributes to age-related AF is still unknown. Direct demonstration that the aged gut microbiota is sufficient to transmit the enhanced AF susceptibility in a young host via microbiota-intestinal barrier-atria axis has not yet been reported. This study aimed to determine whether gut microbiota dysbiosis affects age-related AF. Methods and results Herein, by using a faecal microbiota transplantation (FMT) rat model, we demonstrated that the high AF susceptibility of aged rats could be transmitted to a young host. Specially, we found the dramatically increased levels of circulating lipopolysaccharide (LPS) and glucose led to the up-regulated expression of NOD-like receptor protein (NLRP)-3 inflammasome, promoting the development of AF, which depended on the enhanced atrial fibrosis in recipient host. Inhibition of inflammasome by a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, resulted in a lower atrial fibrosis and AF susceptibility. Then, we conducted cross-sectional clinical studies to explore the effect of ageing on the altering trends with glucose levels and circulating LPS among clinical individuals in two China hospitals. We found that both of serum LPS and glucose levels were progressively increased in elderly patients as compared with those young. Furthermore, the ageing phenotype of circulating LPS and glucose levels, intestinal structure and atrial NLRP3-inflammasome of rats were also confirmed in clinical AF patients. Finally, aged rats colonized with youthful microbiota restored intestinal structure and atrial NLRP3-inflammasome activity, which suppressed the development of aged-related AF. Conclusions Collectively, these studies described a novel causal role of aberrant gut microbiota in the pathogenesis of age-related AF, which indicates that the microbiota-intestinal barrier-atrial NLRP3 inflammasome axis may be a rational molecular target for the treatment of aged-related arrhythmia disease. [ABSTRACT FROM AUTHOR]

The increasing prevalence of atrial fibrillation (AF), its high socio-economic burden and its suboptimal treatment options[1] make it an important focus area of cardiovascular research. Even though the general consensus is that these animal models (at least on the short-term) will maintain an important place in preclinical research, there is a need for more clinically relevant and standardized human I in vitro i models of AF. Attempts to create I in vitro i models of human AF using hPSC-AMs have been previously reported.[4],[5] These models, however, were unable to represent the rapid activation frequencies (6-8 Hz[6]) seen during human AF. [Extracted from the article]

Aims Diabetic hyperglycaemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycaemia alone can be accountable for arrhythmias or whether it requires the presence of additional pathological factors. Methods and results Action potentials (APs) and arrhythmogenic spontaneous diastolic activities were measured in isolated murine ventricular, rabbit atrial, and ventricular myocytes acutely exposed to high glucose. Acute hyperglycaemia increased the short-term variability (STV) of action potential duration (APD), enhanced delayed afterdepolarizations, and the inducibility of APD alternans during tachypacing in both murine and rabbit atrial and ventricular myocytes. Hyperglycaemia also prolonged APD in mice and rabbit atrial cells but not in rabbit ventricular myocytes. However, rabbit ventricular APD was more strongly depressed by block of late Na+ current (I NaL) during hyperglycaemia, consistent with elevated I NaL in hyperglycaemia. All the above proarrhythmic glucose effects were Ca2+-dependent and abolished by CaMKII inhibition. Importantly, when the repolarization reserve was reduced by pharmacological inhibition of K+ channels (either I to, I Kr, I Ks, or I K1) or hypokalaemia, acute hyperglycaemia further prolonged APD and further increased STV and alternans in rabbit ventricular myocytes. Likewise, when rabbit ventricular myocytes were pretreated with isoproterenol or angiotensin II, hyperglycaemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycaemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes. Conclusion We conclude that even though hyperglycaemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signalling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycaemia. [ABSTRACT FROM AUTHOR]

Aims Arrhythmogenic cardiomyopathy (ACM) encompasses a genetically heterogeneous group of myocardial diseases whose manifestations are sudden cardiac death, cardiac arrhythmias, heart failure, and in a subset fibro-adipogenic infiltration of the myocardium. Mutations in the TMEM43 gene, encoding transmembrane protein 43 (TMEM43) are known to cause ACM. The purpose of the study was to gain insights into the molecular pathogenesis of ACM caused by TMEM43 haploinsufficiency. Methods and results The Tmem43 gene was specifically deleted in cardiac myocytes by crossing the Myh6-Cre and floxed Tmem43 mice. Myh6-Cre:Tmem43 W/F mice showed an age-dependent phenotype characterized by an increased mortality, cardiac dilatation and dysfunction, myocardial fibrosis, adipogenesis, and apoptosis. Sequencing of cardiac myocyte transcripts prior to and after the onset of cardiac phenotype predicted early activation of the TP53 pathway. Increased TP53 activity was associated with increased levels of markers of DNA damage response (DDR), and a subset of senescence-associated secretary phenotype (SASP). Activation of DDR, TP53, SASP, and their selected downstream effectors, including phospho-SMAD2 and phospho-SMAD3 were validated by alternative methods, including immunoblotting. Expression of SASP was associated with epithelial–mesenchymal transition and age-dependent expression of myocardial fibrosis and apoptosis in the Myh6-Cre:Tmem43 W/F mice. Conclusion TMEM43 haploinsufficiency is associated with activation of the DDR and the TP53 pathways, which lead to increased expression of SASP and an age-dependent expression of a pro-fibrotic cardiomyopathy. Given that TMEM43 is a nuclear envelope protein and our previous data showing deficiency of another nuclear envelope protein, namely lamin A/C, activates the DDR/TP53 pathway, we surmise that DNA damage is a shared mechanism in the pathogenesis of cardiomyopathies caused by mutations involving nuclear envelope proteins. [ABSTRACT FROM AUTHOR]

Aims Cardiac electrical activity is extraordinarily robust. However, when it goes wrong it can have fatal consequences. Electrical activity in the heart is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. While there is considerable variability in cardiac ion channel expression levels between individuals, studies in rodents have indicated that there are modules of ion channels whose expression co-vary. The aim of this study was to investigate whether meta-analytic co-expression analysis of large-scale gene expression datasets could identify modules of co-expressed cardiac ion channel genes in human hearts that are of functional importance. Methods and results Meta-analysis of 3653 public human RNA-seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, I CaL) and KCNH2 (rapid delayed rectifier K+ current, I Kr), which was also observed in human adult heart tissue samples. In silico modelling suggested that co-expression of CACNA1C and KCNH2 would limit the variability in action potential duration seen with variations in expression of ion channel genes and reduce susceptibility to early afterdepolarizations, a surrogate marker for proarrhythmia. We also found that levels of KCNH2 and CACNA1C expression are correlated in human-induced pluripotent stem cell-derived cardiac myocytes and the levels of CACNA1C and KCNH2 expression were inversely correlated with the magnitude of changes in repolarization duration following inhibition of I Kr. Conclusion Meta-analytic approaches of multiple independent human gene expression datasets can be used to identify gene modules that are important for regulating heart function. Specifically, we have verified that there is co-expression of CACNA1C and KCNH2 ion channel genes in human heart tissue, and in silico analyses suggest that CACNA1C–KCNH2 co-expression increases the robustness of cardiac electrical activity. [ABSTRACT FROM AUTHOR]

Aims Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor (TF) belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function. Methods and results Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human-induced pluripotent stem cells (hiPSCs) derived from two Hamamy syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression programme, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. Conclusion Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases. [ABSTRACT FROM AUTHOR]

Aims  Arrhythmias and sudden cardiac death (SCD) occur commonly in patients with heart failure. We found T-box 5 (TBX5) dysregulated in ventricular myocardium from heart failure patients and thus we hypothesized that TBX5 reduction contributes to arrhythmia development in these patients. To understand the underlying mechanisms, we aimed to reveal the ventricular TBX5-dependent transcriptional network and further test the therapeutic potential of TBX5 level normalization in mice with documented arrhythmias. Methods and results  We used a mouse model of TBX5 conditional deletion in ventricular cardiomyocytes. Ventricular (v) TBX5 loss in mice resulted in mild cardiac dysfunction and arrhythmias and was associated with a high mortality rate (60%) due to SCD. Upon angiotensin stimulation, v Tbx5 KO mice showed exacerbated cardiac remodelling and dysfunction suggesting a cardioprotective role of TBX5. RNA-sequencing of a ventricular-specific TBX5KO mouse and TBX5 chromatin immunoprecipitation was used to dissect TBX5 transcriptional network in cardiac ventricular tissue. Overall, we identified 47 transcripts expressed under the control of TBX5, which may have contributed to the fatal arrhythmias in v Tbx5 KO mice. These included transcripts encoding for proteins implicated in cardiac conduction and contraction (Gja1 , Kcnj5 , Kcng2 , Cacna1g , Chrm2), in cytoskeleton organization (Fstl4 , Pdlim4 , Emilin2 , Cmya5), and cardiac protection upon stress (Fhl2 , Gpr22 , Fgf16). Interestingly, after TBX5 loss and arrhythmia development in vTbx5 KO mice, TBX5 protein-level normalization by systemic adeno-associated-virus (AAV) 9 application, re-established TBX5-dependent transcriptome. Consequently, cardiac dysfunction was ameliorated and the propensity of arrhythmia occurrence was reduced. Conclusions  This study uncovers a novel cardioprotective role of TBX5 in the adult heart and provides preclinical evidence for the therapeutic value of TBX5 protein normalization in the control of arrhythmia. [ABSTRACT FROM AUTHOR]

The cardiac autonomic nervous system (ANS) plays an integral role in normal cardiac physiology as well as in disease states that cause cardiac arrhythmias. The cardiac ANS, comprised of a complex neural hierarchy in a nested series of interacting feedback loops, regulates atrial electrophysiology and is itself susceptible to remodelling by atrial rhythm. In light of the challenges of treating atrial fibrillation (AF) with conventional pharmacologic and myoablative techniques, increasingly interest has begun to focus on targeting the cardiac neuraxis for AF. Strong evidence from animal models and clinical patients demonstrates that parasympathetic and sympathetic activity within this neuraxis may trigger AF, and the ANS may either induce atrial remodelling or undergo remodelling itself to serve as a substrate for AF. Multiple nexus points within the cardiac neuraxis are therapeutic targets, and neuroablative and neuromodulatory therapies for AF include ganglionated plexus ablation, epicardial botulinum toxin injection, vagal nerve (tragus) stimulation, renal denervation, stellate ganglion block/resection, baroreceptor activation therapy, and spinal cord stimulation. Pre-clinical and clinical studies on these modalities have had promising results and are reviewed here. [ABSTRACT FROM AUTHOR]

Despite significant advances in its detection, understanding and management, atrial fibrillation (AF) remains a highly prevalent cardiac arrhythmia with a major impact on morbidity and mortality of millions of patients. AF results from complex, dynamic interactions between risk factors and comorbidities that induce diverse atrial remodelling processes. Atrial remodelling increases AF vulnerability and persistence, while promoting disease progression. The variability in presentation and wide range of mechanisms involved in initiation, maintenance and progression of AF, as well as its associated adverse outcomes, make the early identification of causal factors modifiable with therapeutic interventions challenging, likely contributing to suboptimal efficacy of current AF management. Computational modelling facilitates the multilevel integration of multiple datasets and offers new opportunities for mechanistic understanding, risk prediction and personalized therapy. Mathematical simulations of cardiac electrophysiology have been around for 60 years and are being increasingly used to improve our understanding of AF mechanisms and guide AF therapy. This narrative review focuses on the emerging and future applications of computational modelling in AF management. We summarize clinical challenges that may benefit from computational modelling, provide an overview of the different in silico approaches that are available together with their notable achievements, and discuss the major limitations that hinder the routine clinical application of these approaches. Finally, future perspectives are addressed. With the rapid progress in electronic technologies including computing, clinical applications of computational modelling are advancing rapidly. We expect that their application will progressively increase in prominence, especially if their added value can be demonstrated in clinical trials. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common sustained clinical arrhythmia, with a lifetime incidence of up to 37%, and is a major contributor to population morbidity and mortality. Important components of AF management include control of cardiac rhythm, rate, and thromboembolic risk. In this narrative review article, we focus on rhythm-control therapy. The available therapies for cardiac rhythm control include antiarrhythmic drugs and catheter-based ablation procedures; both of these are presently neither optimally effective nor safe. In order to develop improved treatment options, it is necessary to use preclinical models, both to identify novel mechanism-based therapeutic targets and to test the effects of putative therapies before initiating clinical trials. Extensive research over the past 30 years has provided many insights into AF mechanisms that can be used to design new rhythm-maintenance approaches. However, it has proven very difficult to translate these mechanistic discoveries into clinically applicable safe and effective new therapies. The aim of this article is to explore the challenges that underlie this phenomenon. We begin by considering the basic problem of AF, including its clinical importance, the current therapeutic landscape, the drug development pipeline, and the notion of upstream therapy. We then discuss the currently available preclinical models of AF and their limitations, and move on to regulatory hurdles and considerations and then review industry concerns and strategies. Finally, we evaluate potential paths forward, attempting to derive insights from the developmental history of currently used approaches and suggesting possible paths for the future. While the introduction of successful conceptually innovative new treatments for AF control is proving extremely difficult, one significant breakthrough is likely to revolutionize both AF management and the therapeutic development landscape. [ABSTRACT FROM AUTHOR]

Aims Obesity, an established risk factor of atrial fibrillation (AF), is frequently associated with enhanced inflammatory response. However, whether inflammatory signaling is causally linked to AF pathogenesis in obesity remains elusive. We recently demonstrated that the constitutive activation of the 'NACHT, LRR, and PYD Domains-containing Protein 3' (NLRP3) inflammasome promotes AF susceptibility. In this study, we hypothesized that the NLRP3 inflammasome is a key driver of obesity-induced AF. Methods and results Western blotting was performed to determine the level of NLRP3 inflammasome activation in atrial tissues of obese patients, sheep, and diet-induced obese (DIO) mice. The increased body weight in patients, sheep, and mice was associated with enhanced NLRP3-inflammasome activation. To determine whether NLRP3 contributes to the obesity-induced atrial arrhythmogenesis, wild-type (WT) and NLRP3 homozygous knockout (NLRP3−/−) mice were subjected to high-fat-diet (HFD) or normal chow (NC) for 10 weeks. Relative to NC-fed WT mice, HFD-fed WT mice were more susceptible to pacing-induced AF with longer AF duration. In contrast, HFD-fed NLRP3−/− mice were resistant to pacing-induced AF. Optical mapping in DIO mice revealed an arrhythmogenic substrate characterized by abbreviated refractoriness and action potential duration (APD), two key determinants of reentry-promoting electrical remodeling. Upregulation of ultra-rapid delayed-rectifier K+-channel (Kv1.5) contributed to the shortening of atrial refractoriness. Increased profibrotic signaling and fibrosis along with abnormal Ca2+ release from sarcoplasmic reticulum (SR) accompanied atrial arrhythmogenesis in DIO mice. Conversely, genetic ablation of Nlrp3 (NLRP3−/−) in HFD-fed mice prevented the increases in Kv1.5 and the evolution of electrical remodeling, the upregulation of profibrotic genes, and abnormal SR Ca2+ release in DIO mice. Conclusion These results demonstrate that the atrial NLRP3 inflammasome is a key driver of obesity-induced atrial arrhythmogenesis and establishes a mechanistic link between obesity-induced AF and NLRP3-inflammasome activation. [ABSTRACT FROM AUTHOR]

Hormones are potent endo-, para-, and autocrine endogenous regulators of the function of multiple organs, including the heart. Endocrine dysfunction promotes a number of cardiovascular diseases, including atrial fibrillation (AF). While the heart is a target for endocrine regulation, it is also an active endocrine organ itself, secreting a number of important bioactive hormones that convey significant endocrine effects, but also through para-/autocrine actions, actively participate in cardiac self-regulation. The hormones regulating heart-function work in concert to support myocardial performance. AF is a serious clinical problem associated with increased morbidity and mortality, mainly due to stroke and heart failure. Current therapies for AF remain inadequate. AF is characterized by altered atrial function and structure, including electrical and profibrotic remodelling in the atria and ventricles, which facilitates AF progression and hampers its treatment. Although features of this remodelling are well-established and its mechanisms are partly understood, important pathways pertinent to AF arrhythmogenesis are still unidentified. The discovery of these missing pathways has the potential to lead to therapeutic breakthroughs. Endocrine dysfunction is well-recognized to lead to AF. In this review, we discuss endocrine and cardiocrine signalling systems that directly, or as a consequence of an underlying cardiac pathology, contribute to AF pathogenesis. More specifically, we consider the roles of products from the hypothalamic-pituitary axis, the adrenal glands, adipose tissue, the renin–angiotensin system, atrial cardiomyocytes, and the thyroid gland in controlling atrial electrical and structural properties. The influence of endocrine/paracrine dysfunction on AF risk and mechanisms is evaluated and discussed. We focus on the most recent findings and reflect on the potential of translating them into clinical application. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is a common cardiac arrhythmia leading to many adverse outcomes and increased mortality. Yet the molecular mechanisms underlying AF remain largely unknown. Recent advances in high-throughput technologies make large-scale molecular profiling possible. In the past decade, multiomics studies of AF have identified a number of potential biomarkers of AF. In this review, we focus on the studies of multiomics profiles with AF risk. We summarize recent advances in the discovery of novel biomarkers for AF through multiomics studies. We also discuss limitations and future directions in risk assessment and discovery of therapeutic targets for AF. [ABSTRACT FROM AUTHOR]

Aims Atrial fibrillation (AF) is a progressive cardiac arrhythmia that increases the risk of hospitalization and adverse cardiovascular events. There is a clear demand for more inclusive and large-scale approaches to understand the molecular drivers responsible for AF, as well as the fundamental mechanisms governing the transition from paroxysmal to persistent and permanent forms. In this study, we aimed to create a molecular map of AF and find the distinct molecular programmes underlying cell type-specific atrial remodelling and AF progression. Methods and results We used a sheep model of long-standing, tachypacing-induced AF, sampled right and left atrial tissue, and isolated cardiomyocytes (CMs) from control, intermediate (transition), and late time points during AF progression, and performed transcriptomic and proteome profiling. We have merged all these layers of information into a meaningful three-component space in which we explored the genes and proteins detected and their common patterns of expression. Our data-driven analysis points at extracellular matrix remodelling, inflammation, ion channel, myofibril structure, mitochondrial complexes, chromatin remodelling, and genes related to neural function, as well as critical regulators of cell proliferation as hallmarks of AF progression. Most important, we prove that these changes occur at early transitional stages of the disease, but not at later stages, and that the left atrium undergoes significantly more profound changes than the right atrium in its expression programme. The pattern of dynamic changes in gene and protein expression replicate the electrical and structural remodelling demonstrated previously in the sheep and in humans, and uncover novel mechanisms potentially relevant for disease treatment. Conclusions Transcriptomic and proteomic analysis of AF progression in a large animal model shows that significant changes occur at early stages, and that among others involve previously undescribed increase in mitochondria, changes to the chromatin of atrial CMs, and genes related to neural function and cell proliferation. [ABSTRACT FROM AUTHOR]

Aims  Atrial fibrillation (AF) is a commonly occurring arrhythmia after cardiac surgery (postoperative AF, poAF) and is associated with poorer outcomes. Considering that reduced atrial contractile function is a predictor of poAF and that Ca2+ plays an important role in both excitation–contraction coupling and atrial arrhythmogenesis, this study aims to test whether alterations of intracellular Ca2+ handling contribute to impaired atrial contractility and to the arrhythmogenic substrate predisposing patients to poAF. Methods and results  Right atrial appendages were obtained from patients in sinus rhythm undergoing open-heart surgery. Cardiomyocytes were investigated by simultaneous measurement of [Ca2+]i and action potentials (APs, patch-clamp). Patients were followed-up for 6 days to identify those with and without poAF. Speckle-tracking analysis of preoperative echocardiography revealed reduced left atrial contraction strain in poAF patients. At the time of surgery, cellular Ca2+ transients (CaTs) and the sarcoplasmic reticulum (SR) Ca2+ content were smaller in the poAF group. CaT decay was slower in poAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting preserved sodium-calcium exchanger function. In agreement, western blots revealed reduced SERCA2a expression in poAF patients but unaltered phospholamban expression/phosphorylation. Computational modelling indicated that reduced SERCA activity promotes occurrence of CaT and AP alternans. Indeed, alternans of CaT and AP occurred more often and at lower stimulation frequencies in atrial myocytes from poAF patients. Resting membrane potential and AP duration were comparable between both groups at various pacing frequencies (0.25–8 Hz). Conclusions  Biochemical, functional, and modelling data implicate reduced SERCA-mediated Ca2+ reuptake into the SR as a major contributor to impaired preoperative atrial contractile function and to the pre-existing arrhythmogenic substrate in patients developing poAF. [ABSTRACT FROM AUTHOR]

Aims Inflammation plays a role in atrial fibrillation (AF), but classical anti-inflammatory molecules are ineffective. Recent evidence suggests that failure of inflammation-resolution causes persistent inflammatory signalling and that a novel drug-family called resolvins promotes inflammation-resolution. Right heart disease (RHD) is associated with AF; experimental RHD shows signs of atrial inflammatory-pathway activation. Here, we evaluated resolvin-therapy effects on atrial arrhythmogenic remodelling in experimental RHD. Methods and results Pulmonary hypertension and RHD were induced in rats with an intraperitoneal injection of 60 mg/kg monocrotaline (MCT). An intervention group received daily resolvin-D1 (RvD1), starting 1 day before MCT administration. Right atrial (RA) conduction and gene-expression were analysed respectively by optical mapping and qPCR/gene-microarray. RvD1 had no or minimal effects on MCT-induced pulmonary artery or right ventricular remodelling. Nevertheless, in vivo transoesophageal pacing induced atrial tachyarrhythmias in no CTRL rats vs. 100% MCT-only rats, and only 33% RvD1-treated MCT rats (P  < 0.001 vs. MCT-only). Conduction velocity was significantly decreased by MCT, an effect prevented by RvD1. RHD caused RA dilation and fibrosis. RvD1 strongly attenuated RA fibrosis but had no effect on RA dilation. MCT increased RA expression of inflammation- and fibrosis-related gene-expression pathways on gene-microarray transcriptomic analysis, effects significantly attenuated by RvD1 (334 pathways enriched in MCT-rats vs. control; only 177 dysregulated by MCT with RvD1 treatment). MCT significantly increased RA content of type 1 (proinflammatory) CD68-positive M1 macrophages without affecting type 2 (anti-inflammatory) M2 macrophages. RvD1-treated MCT-rat RA showed significant reductions in proinflammatory M1 macrophages and increases in anti-inflammatory M2 macrophages vs. MCT-only. MCT caused statistically significant increases in protein-expression (western blot) of COL3A1, ASC, CASP1, CASP8, IL1β, TGFβ3, CXCL1, and CXCL2, and decreases in MMP2, vs. control. RvD1-treatment suppressed all these MCT-induced protein-expression changes. Conclusion The inflammation-resolution enhancing molecule RvD1 prevents AF-promoting RA remodelling, while suppressing inflammatory changes and fibrotic/electrical remodelling, in RHD. Resolvins show potential promise in combating atrial arrhythmogenic remodelling by suppressing ongoing inflammatory signalling. [ABSTRACT FROM AUTHOR]

Aims  The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer. Methods and results  We use mutational investigations combined with biochemical assays to identify the peptide sequence bridging the β8 with β9 strand as the primary determinant mediating RyR2 N-terminus self-association. The negatively charged side chains of two aspartate residues (D179 and D180) within the β8–β9 loop are crucial for the N-terminal inter-subunit interaction. We also show that the RyR2 N-terminus domain interacts with the C-terminal channel pore region in a Ca2+-independent manner. The β8–β9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the β8–β9 sequence produces unstable tetrameric channels with subdued intracellular Ca2+ mobilization implicating a role for this domain in channel opening. The arrhythmia-linked R176Q mutation within the β8–β9 loop decreases N-terminus tetramerization but does not affect RyR2 subunit tetramerization or the N-terminus interaction with C-terminus. RyR2R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca2+ mobilization suggesting an additional role for the β8–β9 domain in channel closing. Conclusion  These results suggest that efficient N-terminus inter-subunit communication mediated by the β8–β9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression. [ABSTRACT FROM AUTHOR]

Aims  NOS1AP single-nucleotide polymorphisms (SNPs) correlate with QT prolongation and cardiac sudden death in patients affected by long QT syndrome type 1 (LQT1). NOS1AP targets NOS1 to intracellular effectors. We hypothesize that NOS1AP SNPs cause NOS1 dysfunction and this may converge with prolonged action-potential duration (APD) to facilitate arrhythmias. Here we test (i) the effects of NOS1 inhibition and their interaction with prolonged APD in a guinea pig cardiomyocyte (GP-CMs) LQT1 model; (ii) whether pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from LQT1 patients differing for NOS1AP variants and mutation penetrance display a phenotype compatible with NOS1 deficiency. Methods and results  In GP-CMs, NOS1 was inhibited by S-Methyl-L-thiocitrulline acetate (SMTC) or Vinyl-L-NIO hydrochloride (L-VNIO); LQT1 was mimicked by I Ks blockade (JNJ303) and β-adrenergic stimulation (isoproterenol). hiPSC-CMs were obtained from symptomatic (S) and asymptomatic (AS) KCNQ1 -A341V carriers, harbouring the minor and major alleles of NOS1AP SNPs (rs16847548 and rs4657139), respectively. In GP-CMs, NOS1 inhibition prolonged APD, enhanced I CaL and I NaL, slowed Ca2+ decay, and induced delayed afterdepolarizations. Under action-potential clamp, switching to shorter APD suppressed 'transient inward current' events induced by NOS1 inhibition and reduced cytosolic Ca2+. In S (vs. AS) hiPSC-CMs, APD was longer and I CaL larger; NOS1AP and NOS1 expression and co-localization were decreased. Conclusion  The minor NOS1AP alleles are associated with NOS1 loss of function. The latter likely contributes to APD prolongation in LQT1 and converges with it to perturb Ca2+ handling. This establishes a mechanistic link between NOS1AP SNPs and aggravation of the arrhythmia phenotype in prolonged repolarization syndromes. [ABSTRACT FROM AUTHOR]