Extracellular Kir2.1C122Y Mutant Upsets Kir2.1-PIP2 Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome.

BACKGROUND: Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys (cysteine)122-to-Cys154 disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS: We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments. RESULTS: Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate-binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins. CONCLUSIONS: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate-dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.

Atrial cardiomyopathy revisited-evolution of a concept: a clinical consensus statement of the European Heart Rhythm Association (EHRA) of the ESC, the Heart Rhythm Society (HRS), the Asian Pacific Heart Rhythm Society (APHRS), and the Latin American Heart Rhythm Society (LAHRS).

AIMS: The concept of "atrial cardiomyopathy" (AtCM) had been percolating through the literature since its first mention in 1972. Since then, publications using the term were sporadic until the decision was made to convene an expert working group with representation from four multinational arrhythmia organizations to prepare a consensus document on atrial cardiomyopathy in 2016 (EHRA/HRS/APHRS/SOLAECE expert consensus on atrial cardiomyopathies: definition, characterization, and clinical implication). Subsequently, publications on AtCM have increased progressively. METHODS AND RESULTS: The present consensus document elaborates the 2016 AtCM document further to implement a simple AtCM staging system (AtCM stages 1-3) by integrating biomarkers, atrial geometry, and electrophysiological changes. However, the proposed AtCM staging needs clinical validation. Importantly, it is clearly stated that the presence of AtCM might serve as a substrate for the development of atrial fibrillation (AF) and AF may accelerates AtCM substantially, but AtCM per se needs to be viewed as a separate entity. CONCLUSION: Thus, the present document serves as a clinical consensus statement of the European Heart Rhythm Association (EHRA) of the ESC, the Heart Rhythm Society (HRS), the Asian Pacific Heart Rhythm Society (APHRS), and the Latin American Heart Rhythm Society (LAHRS) to contribute to the evolution of the AtCM concept.

The Impact of Extracellular Histones and Absence of Toll-like Receptors on Cardiac Functional and Electrical Disturbances in Mouse Hearts.

In polymicrobial sepsis, the extracellular histones, mainly released from activated neutrophils, significantly contribute to cardiac dysfunction (septic cardiomyopathy), as demonstrated in our previous studies using Echo-Doppler measurements. This study aims to elucidate the roles of extracellular histones and their interactions with Toll-like receptors (TLRs) in cardiac dysfunction. Through ex vivo assessments of ECG, left ventricle (LV) function parameters, and in vivo Echo-Doppler studies in mice perfused with extracellular histones, we aim to provide comprehensive insights into the mechanisms underlying sepsis-induced cardiac dysfunction. Langendorff-perfused hearts from both wild-type and TLR2, TLR3, or TLR4 knockout (KO) mice were examined. Paced mouse hearts were perfused with histones to assess contractility and relaxation. Echo-Doppler studies evaluated cardiac dysfunction after intravenous histone injection. Histone perfusion caused defects in contractility and relaxation, with TLR2 and TLR3 KO mice being partially protected. Specifically, TLR2 KO mice exhibited the greatest reduction in Echo-Doppler abnormalities, while TLR4 KO exacerbated cardiac dysfunction. Among individual histones, H1 induced the most pronounced abnormalities in cardiac function, apoptosis of cardiomyocytes, and LDH release. Our data highlight significant interactions between histones and TLRs, providing insights into histones especially H1 as potential therapeutic targets for septic cardiomyopathy. Further studies are needed to explore specific histone-TLR interactions and their mechanisms.

Distinct spectral dynamics of implanted cardiac defibrillator signals in spontaneous termination of polymorphic ventricular tachycardia and fibrillation in patients with electrical and structural diseases.

AIMS: To determine the spectral dynamics of early spontaneous polymorphic ventricular tachycardia and ventricular fibrillation (PVT/VF) in humans. METHODS AND RESULTS: Fifty-eight self-terminated and 173 shock-terminated episodes of spontaneously initiated PVT/VF recorded by Medtronic implanted cardiac defibrillators (ICDs) in 87 patients with various cardiac pathologies were analyzed by short fast Fourier transform of shifting segments to determine the dynamics of dominant frequency (DF) and regularity index (RI). The progression in the intensity of DF and RI accumulations further quantified the time course of spectral characteristics of the episodes. Episodes of self-terminated PVT/VF lasted 8.6 s [95% confidence interval (CI): 8.1-9.1] and shock-terminated lasted 13.9 s (13.6-14.3) (P < 0.001). Recordings from patients with primarily electrical pathologies displayed higher DF and RI values than those from patients with primarily structural pathologies (P < 0.05) independently of ventricular function or antiarrhythmic drug therapy. Regardless of the underlying pathology, the average DF and RI intensities were lower in self-terminated than shock-terminated episodes [DF: 3.67 (4.04-4.58) vs. 4.32 (3.46-3.93) Hz, P < 0.001; RI: 0.53 (0.48-0.56) vs. 0.63 (0.60-0.65), P < 0.001]. In a multivariate analysis controlled by the type of pathology and clinical variables, regularity remained an independent predictor of self-termination [hazard ratio: 0.954 (0.928-0.980)]. Receiver operating characteristic (ROC) curve analysis of DF and RI intensities demonstrated increased predictability for self-termination in time with 95% CI above the 0.5 cut-off limit at about t = 8.6 s and t = 6.95 s, respectively. CONCLUSION: Consistent with the notion that fast organized sources maintain PVT/VF in humans, reduction of frequency and regularity correlates with early self-termination. Our findings might help generate ICD methods aiming to reduce inappropriate shock deliveries.

Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents.

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1WTversus Kir2.1Δ314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.

Genome-wide Study of Atrial Fibrillation Identifies Seven Risk Loci and Highlights Biological Pathways and Regulatory Elements Involved in Cardiac Development.

Atrial fibrillation (AF) is a common cardiac arrhythmia and a major risk factor for stroke, heart failure, and premature death. The pathogenesis of AF remains poorly understood, which contributes to the current lack of highly effective treatments. To understand the genetic variation and biology underlying AF, we undertook a genome-wide association study (GWAS) of 6,337 AF individuals and 61,607 AF-free individuals from Norway, including replication in an additional 30,679 AF individuals and 278,895 AF-free individuals. Through genotyping and dense imputation mapping from whole-genome sequencing, we tested almost nine million genetic variants across the genome and identified seven risk loci, including two novel loci. One novel locus (lead single-nucleotide variant [SNV] rs12614435; p = 6.76 × 10-18) comprised intronic and several highly correlated missense variants situated in the I-, A-, and M-bands of titin, which is the largest protein in humans and responsible for the passive elasticity of heart and skeletal muscle. The other novel locus (lead SNV rs56202902; p = 1.54 × 10-11) covered a large, gene-dense chromosome 1 region that has previously been linked to cardiac conduction. Pathway and functional enrichment analyses suggested that many AF-associated genetic variants act through a mechanism of impaired muscle cell differentiation and tissue formation during fetal heart development.

Cardiac phenotype in familial partial lipodystrophy.

OBJECTIVES: LMNA variants have been previously associated with cardiac abnormalities independent of lipodystrophy. We aimed to assess cardiac impact of familial partial lipodystrophy (FPLD) to understand the role of laminopathy in cardiac manifestations. STUDY DESIGN: Retrospective cohort study. METHODS: Clinical data from 122 patients (age range: 13-77, 101 females) with FPLD were analysed. Mature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with an LMNA variant were studied as proof-of-concept for future studies. RESULTS: Subjects with LMNA variants had a higher prevalence of overall cardiac events than others. The likelihood of having an arrhythmia was significantly higher in patients with LMNA variants (OR: 3.77, 95% CI: 1.45-9.83). These patients were at higher risk for atrial fibrillation or flutter (OR: 5.78, 95% CI: 1.04-32.16). The time to the first arrhythmia was significantly shorter in the LMNA group, with a higher HR of 3.52 (95% CI: 1.34-9.27). Non-codon 482 LMNA variants were more likely to be associated with cardiac events (vs. 482 LMNA: OR: 4.74, 95% CI: 1.41-15.98 for arrhythmia; OR: 17.67, 95% CI: 2.45-127.68 for atrial fibrillation or flutter; OR: 5.71, 95% CI: 1.37-23.76 for conduction disease). LMNA mutant hiPSC-CMs showed a higher frequency of spontaneous activity and shorter action potential duration. Functional syncytia of hiPSC-CMs displayed several rhythm alterations such as early afterdepolarizations, spontaneous quiescence and spontaneous tachyarrhythmia, and significantly slower recovery in chronotropic changes induced by isoproterenol exposure. CONCLUSIONS: Our results highlight the need for vigilant cardiac monitoring in FPLD, especially in patients with LMNA variants who have an increased risk of developing cardiac arrhythmias. In addition, hiPSC-CMs can be studied to understand the basic mechanisms for the arrhythmias in patients with lipodystrophy to understand the impact of specific mutations.

Instantaneous Amplitude and Frequency Modulations Detect the Footprint of Rotational Activity and Reveal Stable Driver Regions as Targets for Persistent Atrial Fibrillation Ablation.

RATIONALE: Costly proprietary panoramic multielectrode (64-256) acquisition systems are being increasingly used together with conventional electroanatomical mapping systems for persistent atrial fibrillation (PersAF) ablation. However, such approaches target alleged drivers (rotational/focal) regardless of their activation frequency dynamics. OBJECTIVES: To test the hypothesis that stable regions of higher than surrounding instantaneous frequency modulation (iFM) drive PersAF and determine whether rotational activity is specific for such regions. METHODS AND RESULTS: First, novel single-signal algorithms based on instantaneous amplitude modulation (iAM) and iFM to detect rotational-footprints without panoramic multielectrode acquisition systems were tested in 125 optical movies from 5 ex vivo Langendorff-perfused PersAF sheep hearts (sensitivity/specificity, 92.6/97.5%; accuracy, 2.5-mm) and in computer simulations. Then, 16 pigs underwent high-rate atrial pacing to develop PersAF. After a median (interquartile range [IQR]) of 4.4 (IQR, 2.5-9.9) months of high-rate atrial pacing followed by 4.1 (IQR, 2.7-5.4) months of self-sustained PersAF, pigs underwent in vivo high-density electroanatomical atrial mapping (4920 [IQR, 4435-5855] 8-second unipolar signals per map). The first 4 out of 16 pigs were used to adapt ex vivo optical proccessing of iFM/iAM to in vivo electrical signals. In the remaining 12 out of 16 pigs, regions of higher than surrounding average iFM were considered leading-drivers. Two leading-driver + rotational-footprint maps were generated 2.6 (IQR, 2.4-2.9) hours apart to test leading-driver spatiotemporal stability and guide ablation. Leading-driver regions (2.5 [IQR, 2.0-4.0] regions/map) exactly colocalized (95.7%) in the 2 maps, and their ablation terminated PersAF in 92.3% of procedures (radiofrequency until termination, 16.9 [IQR, 9.2-35.8] minutes; until nonsustainability, 20.4 [IQR, 12.8-44.0] minutes). Rotational-footprints were found at every leading-driver region, albeit most (76.8% [IQR, 70.5%-83.6%]) were located outside. Finally, the translational ability of this approach was tested in 3 PersAF redo patients. CONCLUSIONS: Both rotational-footprints and spatiotemporally stable leading-driver regions can be located using iFM/iAM algorithms without panoramic multielectrode acquisition systems. In pigs, ablation of leading-driver regions usually terminates PersAF and prevents its sustainability. Rotational activations are sensitive but not specific to such regions. Single-signal iFM/iAM algorithms could be integrated into conventional electroanatomical mapping systems to improve driver detection accuracy and reduce the cost of patient-tailored/mechanistic approaches.

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Preclinical Cancer Drug Cardiotoxicity Testing: A Scientific Statement From the American Heart Association.

It is now well recognized that many lifesaving oncology drugs may adversely affect the heart and cardiovascular system, including causing irreversible cardiac injury that can result in reduced quality of life. These effects, which may manifest in the short term or long term, are mechanistically not well understood. Research is hampered by the reliance on whole-animal models of cardiotoxicity that may fail to reflect the fundamental biology or cardiotoxic responses of the human myocardium. The emergence of human induced pluripotent stem cell-derived cardiomyocytes as an in vitro research tool holds great promise for understanding drug-induced cardiotoxicity of oncological drugs that may manifest as contractile and electrophysiological dysfunction, as well as structural abnormalities, making it possible to deliver novel drugs free from cardiac liabilities and guide personalized therapy. This article briefly reviews the challenges of cardio-oncology, the strengths and limitations of using human induced pluripotent stem cell-derived cardiomyocytes to represent clinical findings in the nonclinical research space, and future directions for their further use.

Anatomical targets and expected outcomes of catheter-based ablation of atrial fibrillation in 2020.

Anatomical-based approaches, targeting either pulmonary vein isolation (PVI) or additional extra PV regions, represent the most commonly used ablation treatments in symptomatic patients with atrial fibrillation (AF) recurrences despite antiarrhythmic drug therapy. PVI remains the main anatomical target during catheter-based AF ablation, with the aid of new technological advances as contact force monitoring to increase safety and effective radiofrequency (RF) lesions. Nowadays, cryoballoon ablation has also achieved the same level of scientific evidence in patients with paroxysmal AF undergoing PVI. In parallel, electrical isolation of extra PV targets has progressively increased, which is associated with a steady increase in complex cases undergoing ablation. Several atrial regions as the left atrial posterior wall, the vein of Marshall, the left atrial appendage, or the coronary sinus have been described in different series as locations potentially involved in AF initiation and maintenance. Targeting these regions may be challenging using conventional point-by-point RF delivery, which has opened new opportunities for coadjuvant alternatives as balloon ablation or selective ethanol injection. Although more extensive ablation may increase intraprocedural AF termination and freedom from arrhythmias during the follow-up, some of the targets to achieve such outcomes are not exempt of potential severe complications. Here, we review and discuss current anatomical approaches and the main ablation technologies to target atrial regions associated with AF initiation and maintenance.