Coordinated demethylation of H3K9 and H3K27 is required for rapid inflammatory responses of endothelial cells.

Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs.

The Anti-Inflammatory Effects and Clinical Potential of Dexmedetomidine in Pulmonary Arterial Hypertension.

A pathogenic aspect of pulmonary arterial hypertension (PAH) is the aberrant pulmonary arterial smooth muscle cell (PASMC) proliferation. PASMC proliferation is significantly affected by inflammation. A selective α-2 adrenergic receptor agonist called dexmedetomidine (DEX) modulates specific inflammatory reactions. We investigated the hypothesis that anti-inflammatory characteristics of DEX could lessen PAH that monocrotaline (MCT) causes in rats. In vivo, male Sprague-Dawley rats aged 6 weeks were subcutaneously injected with MCT at a dose of 60 mg/kg. Continuous infusions of DEX (2 µg/kg per hour) were started via osmotic pumps in one group (MCT plus DEX group) at day 14 following MCT injection but not in another group (MCT group). Right ventricular systolic pressure (RVSP), right ventricular end-diastolic pressure (RVEDP), and survival rate significantly improved in the MCT plus DEX group compared with the MCT group [RVSP, 34 mmHg ± 4 mmHg versus 70 mmHg ± 10 mmHg; RVEDP, 2.6 mmHg ± 0.1 mmHg versus 4.3 mmHg ± 0.6 mmHg; survival rate, 42% versus 0% at day 29 (P < 0.01)]. In the histologic study, the MCT plus DEX group showed fewer phosphorylated p65-positive PASMCs and less medial hypertrophy of the pulmonary arterioles. In vitro, DEX dose-dependently inhibited human PASMC proliferation. Furthermore, DEX decreased the expression of interleukin-6 mRNA in human PASMCs treated with fibroblast growth factor 2 (FGF2). These consequences suggest that DEX improves PAH by inhibiting PASMC proliferation through its anti-inflammatory properties. Additionally, DEX may exert anti-inflammatory effects via blocking FGF2-induced nuclear factor κ B activation. SIGNIFICANCE STATEMENT: Dexmedetomidine, a selective α-2 adrenergic receptor agonist utilized as a sedative in the clinical setting, improves pulmonary arterial hypertension (PAH) by inhibiting pulmonary arterial smooth muscle cell proliferation through its anti-inflammatory effect. Dexmedetomidine may be a new PAH therapeutic agent with vascular reverse remodeling effect.

Genetic risk score of cerebral infarction in atrial fibrillation genome-wide association study.

INTRODUCTION: Stroke is a leading cause of death and the primary cause of adult-acquired disability. Patients with cardiogenic embolic stroke also have higher mortality and recurrence rates than patients with other stroke subtypes. Atrial fibrillation (AF) is a major risk factor for cerebral infarction (CI). The large-scale study identified 32 loci in the MEGASTROKE study. However, few studies have attempted to identify novel stroke risk variants in patients with a history of AF. Our overall aim was to identify novel CI risk variants in AF cases and explore whether their associations with the CI risk were affected by the CHADS2 and CHA2DS2-VASc scores. METHODS: We performed association study with CI using 8181 AF cases in previous genome-wide association study (GWAS) and imputation data without controls. We classified AF cases into those with or without past history of CI, and the genetic associations with the CI risk were examined. RESULTS: GWAS identified eight associated loci. The generated genetic risk score (GRS) for the eight loci was significantly associated with CI in patients with AF (1.46 × 10-8 ). We estimated bivariate logistic regression model which contained GRS and CHADS2 score (GRS: p-Value = 7.41 × 10-9 , CHADS2 score: p-Value <2.0 × 10-16 ) or CHA2DS2-VASc scores (GRS: p-Value = 2.52 × 10-10 , CHA2DS2-VASc score: p-Value <2.0 × 10-16 ). CONCLUSION: We identified eight genetic variants that were potentially associated with the risk of CI of AF cases and the significant GRS, whose associations were independent of the CHADS2 or CHA2DS2-VASc score.

Prediction of the Presence of Ventricular Fibrillation From a Brugada Electrocardiogram Using Artificial Intelligence.

BACKGROUND: Brugada syndrome is a potential cause of sudden cardiac death (SCD) and is characterized by a distinct ECG, but not all patients with A Brugada ECG develop SCD. In this study we sought to examine if an artificial intelligence (AI) model can predict a previous or future ventricular fibrillation (VF) episode from a Brugada ECG.Methods and Results: We developed an AI-enabled algorithm using a convolutional neural network. From 157 patients with suspected Brugada syndrome, 2,053 ECGs were obtained, and the dataset was divided into 5 datasets for cross-validation. In the ECG-based evaluation, the precision, recall, and F1score were 0.79±0.09, 0.73±0.09, and 0.75±0.09, respectively. The average area under the receiver-operating characteristic curve (AUROC) was 0.81±0.09. On per-patient evaluation, the AUROC was 0.80±0.07. This model predicted the presence of VF with a precision of 0.93±0.02, recall of 0.77±0.14, and F1score of 0.81±0.11. The negative predictive value was 0.94±0.11 while its positive predictive value was 0.44±0.29. CONCLUSIONS: This proof-of-concept study showed that an AI-enabled algorithm can predict the presence of VF with a substantial performance. It implies that the AI model may detect a subtle ECG change that is undetectable by humans.

Mutant KCNJ3 and KCNJ5 Potassium Channels as Novel Molecular Targets in Bradyarrhythmias and Atrial Fibrillation.

BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.

Functional Role of the L396R Mutation of Tks5 Identified by an Exome-Wide Association Study in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia; however, the current treatment strategies for AF have limited efficacy. Thus, a better understanding of the mechanisms underlying AF is important for future therapeutic strategy. A previous study (Exome-Wide Association Study (ExWAS)) identified a rare variant, rs202011870 (MAF=0.00036, GenomAD), which is highly associated with AF (OR=3.617, P<0.0001). rs202011870 results in the replacement of Leu at 396 with Arg (L396R) in a molecule, Tks5; however, the mechanism of how rs202011870 links to AF is completely unknown.Methods and Results:The association of rs202011870 with AF was examined in 3,378 participants (641 control and 2,737 AF cases) from 4 independent cohorts by using an Invader assay. Consequences of rs202011870 in migration ability, podosome formation, and expression of inflammation-related molecules in macrophages were examined using RAW264.7 cells with a trans-well assay, immunocytochemistry, and qPCR assay. Validation of the association of rs202011870 with AF was successful. In vitro studies showed that RAW264.7 cells with L396R-Tks5 increased trans-well migration ability, and enhanced podosome formation. RAW264.7 cells with L396R-Tks5 also increased the expression of several inflammatory cytokines and inflammation-related molecules. CONCLUSIONS: L396R mutation in Tks5 associated with AF enhances migration of macrophages and their inflammatory features, resulting in enhanced susceptibility to AF.

Systematic expression analysis of genes related to generation of action potentials in human iPS cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a valuable tool to characterize the pharmacology and toxic effects of drugs on heart cells. In particular, hiPSC-CMs can be used to identify drugs that generate arrhythmias. However, it is unclear whether the expression of genes related to generation of CM action potentials differs between hiPSC-CM cell lines and the mature human heart. To address this, we obtained accurate gene expression profiles of commercially available hiPSC-CM cell lines with quantitative real time RT-PCR analysis. Expression analysis of ten cardiac proteins important for generation of action potentials and three cardiac proteins important for muscle contractility was performed using GAPDH for normalization. Comparison revealed large variations in expression levels among hiPSC-CM cell lines and between hiPSC-CMs and normal human heart. In general, gene expression in hiPSC-CM cell lines was more similar to an immature, stem-like cell than a mature cardiomyocyte from human heart samples. These results provide quantitative information about differences in gene expression between hiPSC-CM cell lines, essential for interpreting pharmacology experiments. Our approach can be used as an experimental guideline for future research on gene expression in hiPSC-CMs.

Important cardiac transcription factor genes are accompanied by bidirectional long non-coding RNAs.

BACKGROUND: Heart development is a relatively fragile process in which many transcription factor genes show dose-sensitive characteristics such as haploinsufficiency and lower penetrance. Despite efforts to unravel the genetic mechanism for overcoming the fragility under normal conditions, our understanding still remains in its infancy. Recent studies on the regulatory mechanisms governing gene expression in mammals have revealed that long non-coding RNAs (lncRNAs) are important modulators at the transcriptional and translational levels. Based on the hypothesis that lncRNAs also play important roles in mouse heart development, we attempted to comprehensively identify lncRNAs by comparing the embryonic and adult mouse heart and brain. RESULTS: We have identified spliced lncRNAs that are expressed during development and found that lncRNAs that are expressed in the heart but not in the brain are located close to genes that are important for heart development. Furthermore, we found that many important cardiac transcription factor genes are located in close proximity to lncRNAs. Importantly, many of the lncRNAs are divergently transcribed from the promoter of these genes. Since the lncRNA divergently transcribed from Tbx5 is highly evolutionarily conserved, we focused on and analyzed the transcript. We found that this lncRNA exhibits a different expression pattern than that of Tbx5, and knockdown of this lncRNA leads to embryonic lethality. CONCLUSION: These results suggest that spliced lncRNAs, particularly bidirectional lncRNAs, are essential regulators of mouse heart development, potentially through the regulation of neighboring transcription factor genes.

Combined Analysis of Human and Experimental Murine Samples Identified Novel Circulating MicroRNAs as Biomarkers for Atrial Fibrillation.

BACKGROUND: Recent experimental studies have demonstrated that several microRNAs (miRNAs) expressed in atrial tissue promote a substrate of atrial fibrillation (AF). However, because it has not been fully elucidated whether these experimental data contribute to identifying circulating miRNAs as biomarkers for AF, we used a combined analysis of human serum and murine atrial samples with the aim of identifying these biomarkers for predicting AF.Methods and Results:Comprehensive analyses were performed to screen 733 miRNAs in serum from 10 AF patients and 5 controls, and 672 miRNAs in atrial tissue from 6 inducible atrial tachycardia model mice and 3 controls. We selected miRNAs for which expression was detected in both analyses, and their expression levels were changed in the human analyses, the murine analyses, or both. This screening identified 11 candidate miRNAs. Next, we quantified the selected miRNAs using a quantitative RT-PCR in 50 AF and 50 non-AF subjects. The individual assessment revealed that 4 miRNAs (miR-99a-5p, miR-192-5p, miR-214-3p, and miR-342-5p) were significantly upregulated in AF patients. A receiver-operating characteristics curve indicated that miR-214-3p and miR-342-5p had the highest accuracy. The combination of the 4 miRNAs modestly improved the predictive accuracy for AF (76% sensitivity, 80% specificity). CONCLUSIONS: Novel circulating miRNAs were upregulated in the serum of AF patients and might be potential biomarkers of AF.

Association of the Clinical and Genetic Factors With Superior Vena Cava Arrhythmogenicity in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) can be initiated from arrhythmogenic foci within the muscular sleeves that extend not only into the pulmonary veins but also into both vena cavae. The superior vena cava (SVC) is a key target site for catheter ablation. Patients with SVC-derived AF often lack the clinical risk factors of AF.Methods and Results:We conducted a meta-analysis of the clinical and genetic factors of 2,170 AF patients with and without SVC arrhythmogenicity. In agreement with previous reports, the left atrial diameter was smaller in AF patients with SVC arrhythmogenicity. Among 6 variants identified in a previous genome-wide association study in Japanese patients, rs2634073 and rs6584555 were associated with SVC arrhythmogenicity. This finding was confirmed in our meta-analysis using independent cohorts. We also found that SVC arrhythmogenicity was conditionally dependent on age, body mass index, and left ventricular ejection fraction. CONCLUSIONS: Both clinical and genetic factors are associated with SVC arrhythmogenicity.

Overexpression of KCNJ2 in induced pluripotent stem cell-derived cardiomyocytes for the assessment of QT-prolonging drugs.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes hold great potentials to predict pro-arrhythmic risks in preclinical cardiac safety screening, although the hiPSC cardiomyocytes exhibit rather immature functional and structural characteristics, including spontaneous activity. Our physiological characterization and mathematical simulation showed that low expression of the inward-rectifier potassium (IK1) channel is a determinant of spontaneous activity. To understand impact of the low IK1 expression on the pharmacological properties, we tested if transduction of hiPSC-derived cardiomyocytes with KCNJ2, which encodes the IK1 channel, alters pharmacological response to cardiac repolarization processes. The transduction of KCNJ2 resulted in quiescent hiPSC-derived cardiomyocytes, which need pacing to elicit action potentials. Significant prolongation of paced action potential duration in KCNJ2-transduced hiPSC-derived cardiomyocytes was stably measured at 0.1 µM E-4031, although the same concentration of E-4031 ablated firing of non-treated hiPSC-derived cardiomyocytes. These results in single cells were confirmed by mathematical simulations. Using the hiPSC-derived cardiac sheets with KCNJ2-transduction, we also investigated effects of a range of drugs on field potential duration recorded at 1 Hz. The KCNJ2 overexpression in hiPSC-derived cardiomyocytes may contribute to evaluate a part of QT-prolonging drugs at toxicological concentrations with high accuracy.

Genetic defects in a His-Purkinje system transcription factor, IRX3, cause lethal cardiac arrhythmias.

AIM: Ventricular fibrillation (VF), the main cause of sudden cardiac death (SCD), occurs most frequently in the acute phase of myocardial infarction: a certain fraction of VF, however, develops in an apparently healthy heart, referred as idiopathic VF. The contribution of perturbation in the fast conduction system in the ventricle, the His-Purkinje system, for idiopathic VF has been implicated, but the underlying mechanism remains unknown. Irx3/IRX3 encodes a transcription factor specifically expressed in the His-Purkinje system in the heart. Genetic deletion of Irx3 provides a mouse model of ventricular fast conduction disturbance without anatomical or contraction abnormalities. The aim of this study was to examine the link between perturbed His-Purkinje system and idiopathic VF in Irx3-null mice, and to search for IRX3 genetic defects in idiopathic VF patients in human. METHODS AND RESULTS: Telemetry electrocardiogram recording showed that Irx3-deleted mice developed frequent ventricular tachyarrhythmias mostly at night. Ventricular tachyarrhythmias were enhanced by exercise and sympathetic nerve activation. In human, the sequence analysis of IRX3 exons in 130 probands of idiopathic VF without SCN5A mutations revealed two novel IRX3 mutations, 1262G>C (R421P) and 1453C>A (P485T). Ventricular fibrillation associated with physical activities in both probands with IRX3 mutations. In HL-1 cells and neonatal mouse ventricular myocytes, IRX3 transfection up-regulated SCN5A and connexin-40 mRNA, which was attenuated by IRX3 mutations. CONCLUSION: IRX3 genetic defects and resultant functional perturbation in the His-Purkinje system are novel genetic risk factors of idiopathic VF, and would improve risk stratification and preventive therapy for SCD in otherwise healthy hearts.

High-fat diet increases vulnerability to atrial arrhythmia by conduction disturbance via miR-27b.

BACKGROUND: Lifestyle-related diseases, such as obesity and dyslipidemia are important risk factors for atrial fibrillation (AF). However, the underlying mechanism linking these diseases and AF has not been fully investigated. METHODS: Adult male mice were fed a high-fat diet (HFD) or vehicle (NC) for 2 months. Electrocardiography and in vivo electrophysiological study were performed. Mice were then sacrificed for quantification of mRNA, microRNA, and protein in atria, in addition to histological analysis. Conduction velocity (CV) in right atrium was measured by optical mapping in Langendorff perfused hearts. Cultured atrial cardiomyocytes were treated with palmitate with or without a specific microRNA inhibitor. Twelve hours after stimulation, cells were lysed, and subjected to analysis with qPCR and Western blotting. RESULTS: HFD mice showed prolonged P wave duration, increased inducibility of sustained atrial tachycardia, and reduced atrial CV than NC mice. HFD mice also showed increased expression in inflammatory cytokines, whereas fibrotic area and signals relating fibrosis were not changed. HFD mice demonstrated reduced expression of Cx40 in mRNA and protein levels, and its lateralized expression in atria. MicroRNA array analysis revealed that miR-27b expression was up-regulated in HFD mice, and luciferase assay confirmed the direct interaction between miR-27b and Cx40 3'UTR. In palmitate-stimulated atrial cardiomyocytes, miR-27b up-regulation and Cx40 down-regulation were observed, while expression of inflammatory cytokines was not altered. Inhibition of miR-27b with antisense oligonucleotides reversed the alteration caused by palmitate stimulation. CONCLUSION: HFD may increase the vulnerability to atrial arrhythmia by down-regulation of Cx40 via miR-27b, rather than fibrosis, which is independent of inflammation.

A distribution analysis of action potential parameters obtained from patch-clamped human stem cell-derived cardiomyocytes.

We investigated electrophysiological properties of human induced-pluripotent-stem-cell-derived and embryonic-stem-cell-derived cardiomyocytes, and analyzed action potential parameters by plotting their frequency distributions. In the both cell lines, the distribution analysis revealed that histograms of maximum upstroke velocity showed two subpopulations with similar intersection values. Sub-populations with faster maximum upstroke velocity showed significant prolongation of action potential durations by application of E-4031, whereas others did not, which may be partly due to shallower maximum diastolic potentials. We described electrophysiological and pharmacological properties of stem-cell-derived cardiomyocytes in the respective sub-populations, which provides a way to characterize diverse electrical properties of stem-cell-derived cardiomyocytes systematically.

Oxidative Stress Induced Ventricular Arrhythmia and Impairment of Cardiac Function in Nos1ap Deleted Mice.

Genome-wide association study has identified that the genetic variations at NOS1AP (neuronal nitric oxide synthase-1 adaptor protein) were associated with QT interval and sudden cardiac death (SCD). However, the mechanism linking a genetic variant of NOS1AP and SCD is poorly understood. We used Nos1ap knockout mice (Nos1ap(-/-)) to determine the involvement of Nos1ap in SCD, paying special attention to oxidative stress.At baseline, a surface electrocardiogram (ECG) and ultrasound echocardiography (UCG) showed no difference between Nos1ap(-/-) and wild-type (WT) mice. Oxidative stress was induced by a single injection of doxorubicin (Dox, 25 mg/kg). After Dox injection, Nos1ap(-/-) showed significantly higher mortality than WT (93.3 versus 16.0% at day 14, P < 0.01). ECG showed significantly longer QTc in Nos1ap(-/-) than WT, and UCG revealed significant reduction of fractional shortening (%FS) only in Nos1ap(-/-) after Dox injection. Spontaneous ventricular tachyarrhythmias were documented by telemetry recording after Dox injection only in Nos1ap(-/-). Ex vivo optical mapping revealed that the action potential duration (APD)90 was prolonged at baseline in Nos1ap(-/-), and administration of Dox lengthened APD90 more in Nos1ap(-/-) than in WT. The expression of Bnp and the H2O2 level were higher in Nos1ap(-/-) after Dox injection. Nos1ap(-/-) showed a reduced amplitude of calcium transient in isolated cardiomyocytes after Dox injection. Administration of the antioxidant N-acetyl-L-cysteine significantly reduced mortality of Nos1ap(-/-) by Dox injection, accompanied by prevention of QT prolongation and a reduction in %FS.Although Nos1ap(-/-) mice have apparently normal hearts, oxidative stress evokes ventricular tachyarrhythmia and heart failure, which may cause sudden cardiac death.

[Therapeutic strategy targeting membrane molecular meshanosensing.]

Our bodies possess systems to detect various mechanical stimuli, called mechanosensing. Mechanosensing taking place in the membrane can be classified into to ion channel-dependent one and ion channel-independent one. Ion channel-dependent mechanosensing is a relatively rapid response, involving various mechanosen sensitive channels, including recently identified pannexin. In contrast, ion-channel-independent mechanosensing is a slow response, such as tissue remodeling, and the mechanism via integrin-actin interaction at the focal adhesion is well known.

Integrating genetic, transcriptional, and functional analyses to identify 5 novel genes for atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) affects >30 million individuals worldwide and is associated with an increased risk of stroke, heart failure, and death. AF is highly heritable, yet the genetic basis for the arrhythmia remains incompletely understood. METHODS AND RESULTS: To identify new AF-related genes, we used a multifaceted approach, combining large-scale genotyping in 2 ethnically distinct populations, cis-eQTL (expression quantitative trait loci) mapping, and functional validation. Four novel loci were identified in individuals of European descent near the genes NEURL (rs12415501; relative risk [RR]=1.18; 95% confidence interval [CI], 1.13-1.23; P=6.5×10(-16)), GJA1 (rs13216675; RR=1.10; 95% CI, 1.06-1.14; P=2.2×10(-8)), TBX5 (rs10507248; RR=1.12; 95% CI, 1.08-1.16; P=5.7×10(-11)), and CAND2 (rs4642101; RR=1.10; 95% CI, 1.06-1.14; P=9.8×10(-9)). In Japanese, novel loci were identified near NEURL (rs6584555; RR=1.32; 95% CI, 1.26-1.39; P=2.0×10(-25)) and CUX2 (rs6490029; RR=1.12; 95% CI, 1.08-1.16; P=3.9×10(-9)). The top single-nucleotide polymorphisms or their proxies were identified as cis-eQTLs for the genes CAND2 (P=2.6×10(-19)), GJA1 (P=2.66×10(-6)), and TBX5 (P=1.36×10(-5)). Knockdown of the zebrafish orthologs of NEURL and CAND2 resulted in prolongation of the atrial action potential duration (17% and 45%, respectively). CONCLUSIONS: We have identified 5 novel loci for AF. Our results expand the diversity of genetic pathways implicated in AF and provide novel molecular targets for future biological and pharmacological investigation.

Image-based evaluation of contraction-relaxation kinetics of human-induced pluripotent stem cell-derived cardiomyocytes: Correlation and complementarity with extracellular electrophysiology.

In this study, we used high-speed video microscopy with motion vector analysis to investigate the contractile characteristics of hiPS-CM monolayer, in addition to further characterizing the motion with extracellular field potential (FP), traction force and the Ca(2+) transient. Results of our traction force microscopy demonstrated that the force development of hiPS-CMs correlated well with the cellular deformation detected by the video microscopy with motion vector analysis. In the presence of verapamil and isoproterenol, contractile motion of hiPS-CMs showed alteration in accordance with the changes in fluorescence peak of the Ca(2+) transient, i.e., upstroke, decay, amplitude and full-width at half-maximum. Simultaneously recorded hiPS-CM motion and FP showed that there was a linear correlation between changes in the motion and field potential duration in response to verapamil (30-150nM), isoproterenol (0.1-10µM) and E-4031 (10-50nM). In addition, tetrodotoxin (3-30µM)-induced delay of sodium current was corresponded with the delay of the contraction onset of hiPS-CMs. These results indicate that the electrophysiological and functional behaviors of hiPS-CMs are quantitatively reflected in the contractile motion detected by this image-based technique. In the presence of 100nM E-4031, the occurrence of early after-depolarization-like negative deflection in FP was also detected in the hiPS-CM motion as a characteristic two-step relaxation pattern. These findings offer insights into the interpretation of the motion kinetics of the hiPS-CMs, and are relevant for understanding electrical and mechanical relationship in hiPS-CMs.

Sacubitril/valsartan attenuates atrial conduction disturbance and electrophysiological heterogeneity with ameliorating fibrosis in mice.

Background: Sacubitril/valsartan (SacVal) has been shown to improve the prognosis of heart failure; however, whether SacVal reduces the occurrence of atrial fibrillation (AF) in heart failure has not yet been elucidated. In this study, we aimed to determine whether SacVal is effective in reducing the occurrence of AF in heart failure and identify the underlying mechanism of its electrophysiological effect in mice. Methods: Adult male mice underwent transverse aortic constriction, followed by SacVal, valsartan, or vehicle treatment for two weeks. Electrophysiological study (EPS) and optical mapping were performed to assess the susceptibility to AF and the atrial conduction properties, and fibrosis was investigated using heart tissue and isolated cardiac fibroblasts (CFs). Results: EPS analysis revealed that AF was significantly less inducible in SacVal-treated mice than in vehicle-treated mice. Optical mapping of the atrium showed that SacVal-treated and valsartan-treated mice restored the prolonged action potential duration (APD); however, only SacVal-treated mice showed the restoration of decreased conduction velocity (CV) compared to vehicle-treated mice. In addition, the electrophysiological distribution analysis demonstrated that heterogeneous electrophysiological properties were rate-dependent and increased heterogeneity was closely related to the susceptibility to AF. SacVal attenuated the increased heterogeneity of CV at short pacing cycle length in atria, whereas Val could not. Histological and molecular evaluation showed that SacVal exerted the anti-fibrotic effect on the atria. An in vitro study of CFs treated with natriuretic peptides and LBQ657, the metabolite and active form of sacubitril, revealed that C-type natriuretic peptide (CNP) combined with LBQ657 had an additional anti-fibrotic effect on CFs. Conclusions: Our results demonstrated that SacVal can improve the conduction disturbance and heterogeneity through the attenuation of fibrosis in murine atria and reduce the susceptibility of AF in heart failure with pressure overload, which might be attributed to the enhanced function of CNP.