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The genetic architecture of atrial fibrillation (AF) encompasses low impact, common genetic variants and high impact, rare variants. Here, we characterize a high impact AF-susceptibility allele, KCNQ1 R231H, and describe its transcontinental geographic distribution and history. Induced pluripotent stem cell-derived cardiomyocytes procured from risk allele carriers exhibit abbreviated action potential duration, consistent with a gain-of-function effect. Using identity-by-descent (IBD) networks, we estimate the broad- and fine-scale population ancestry of risk allele carriers and their relatives. Analysis of ancestral migration routes reveals ancestors who inhabited Denmark in the 1700s, migrated to the Northeastern United States in the early 1800s, and traveled across the Midwest to arrive in Utah in the late 1800s. IBD/coalescent-based allele dating analysis reveals a relatively recent origin of the AF risk allele (~5000 years). Thus, our approach broadens the scope of study for disease susceptibility alleles to the context of human migration and ancestral origins., Many rare high-impact variants have been associated with disease, but the origins and functional impact are not always explored. Here, the authors trace the ancestry of a rare high impact atrial fibrillation allele in KCNQ1, and use iPSC-derived cardiomyocytes to characterize the effect of the allele.

In studies of families with rare disease, it is common to screen for de novo mutations, as well as recessive or dominant variants that explain the phenotype. However, the filtering strategies and software used to prioritize high-confidence variants vary from study to study. In an effort to establish recommendations for rare disease research, we explore effective guidelines for variant (SNP and INDEL) filtering and report the expected number of candidates for de novo dominant, recessive, and autosomal dominant modes of inheritance. We derived these guidelines using two large family-based cohorts that underwent whole-genome sequencing, as well as two family cohorts with whole-exome sequencing. The filters are applied to common attributes, including genotype-quality, sequencing depth, allele balance, and population allele frequency. The resulting guidelines yield ~10 candidate SNP and INDEL variants per exome, and 18 per genome for recessive and de novo dominant modes of inheritance, with substantially more candidates for autosomal dominant inheritance. For family-based, whole-genome sequencing studies, this number includes an average of three de novo, ten compound heterozygous, one autosomal recessive, four X-linked variants, and roughly 100 candidate variants following autosomal dominant inheritance. The slivar software we developed to establish and rapidly apply these filters to VCF files is available at https://github.com/brentp/slivar under an MIT license, and includes documentation and recommendations for best practices for rare disease analysis.

A genetic etiology is identified for one-third of patients with congenital heart disease (CHD), with 8% of cases attributable to coding de novo variants (DNVs). To assess the contribution of noncoding DNVs to CHD, we compared genome sequences from 749 CHD probands and their parents with those from 1,611 unaffected trios. Neural network prediction of noncoding DNV transcriptional impact identified a burden of DNVs in individuals with CHD (n = 2,238 DNVs) compared to controls (n = 4,177; P = 8.7 × 10-4). Independent analyses of enhancers showed an excess of DNVs in associated genes (27 genes versus 3.7 expected, P = 1 × 10-5). We observed significant overlap between these transcription-based approaches (odds ratio (OR) = 2.5, 95% confidence interval (CI) 1.1-5.0, P = 5.4 × 10-3). CHD DNVs altered transcription levels in 5 of 31 enhancers assayed. Finally, we observed a DNV burden in RNA-binding-protein regulatory sites (OR = 1.13, 95% CI 1.1-1.2, P = 8.8 × 10-5). Our findings demonstrate an enrichment of potentially disruptive regulatory noncoding DNVs in a fraction of CHD at least as high as that observed for damaging coding DNVs.

Visual Abstract, Highlights • Combining CHD phenotype–driven gene set enrichment and CRISPR knockdown screening in zebrafish is an effective approach to identifying novel CHD genes. • Mutations affecting genes coding for the WAVE2 protein complex and small GTPase-mediated signaling are associated with LVOTO lesions. • WAVE2 complex genes brk1, nckap1, and wasf2 and regulators of small GTPase signaling cul3a and racgap1 are critical to zebrafish heart development., Summary Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to identify causative variants is limited. To identify causal CHD genes that are associated with specific molecular functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe CHD. This approach enabled the authors to identify an association between left ventricular outflow tract obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins brk1, nckap1, and wasf2 and the regulators of small GTPase signaling cul3a and racgap1 are critical to cardiac development.

Heart failure (HF) is a leading cause of morbidity and mortality in the United States and worldwide. As a multifactorial syndrome with unpredictable clinical outcomes, identifying the common molecular underpinnings that drive HF pathogenesis remains a major focus of investigation. Disruption of cardiac gene expression has been shown to mediate a common final cascade of pathological hallmarks wherein the heart reactivates numerous developmental pathways. Although the central regulatory mechanisms that drive this cardiac transcriptional reprogramming remain unknown, epigenetic contributions are likely. In the current study, we examined whether the epigenome, specifically DNA methylation, is reprogrammed in HF to potentiate a pathological shift in cardiac gene expression. To accomplish this, we used paired-end whole genome bisulfite sequencing and next-generation RNA sequencing of left ventricle tissue obtained from seven patients with end-stage HF and three nonfailing donor hearts. We found that differential methylation was localized to promoter-associated cytosine-phosphate-guanine islands, which are established regulatory regions of downstream genes. Hypermethylated promoters were associated with genes involved in oxidative metabolism, whereas promoter hypomethylation enriched glycolytic pathways. Overexpression of plasmid-derived DNA methyltransferase 3A in vitro was sufficient to lower the expression of numerous oxidative metabolic genes in H9c2 rat cardiomyoblasts, further supporting the importance of epigenetic factors in the regulation of cardiac metabolism. Last, we identified binding-site competition via hypermethylation of the nuclear respiratory factor 1 (NRF1) motif, an established upstream regulator of mitochondrial biogenesis. These preliminary observations are the first to uncover an etiology-independent shift in cardiac DNA methylation that corresponds with altered metabolic gene expression in HF. NEW & NOTEWORTHY The failing heart undergoes profound metabolic changes because of alterations in cardiac gene expression, reactivating glycolytic genes and suppressing oxidative metabolic genes. In the current study, we discover that alterations to cardiac DNA methylation encode this fetal-like metabolic gene reprogramming. We also identify novel epigenetic interference of nuclear respiratory factor 1 via hypermethylation of its downstream promoter targets, further supporting a novel contribution of DNA methylation in the metabolic remodeling of heart failure.

There is strong evidence for a genetic contribution to non-syndromic congenital heart defects (CHDs). However, exome- and genome-wide studies conducted at the variant and gene-level have identified few genome-wide significant CHD-related genes. Gene-set analyses are a useful complement to such studies and candidate gene-set analyses of rare variants have provided insight into the genetics of CHDs. However, similar analyses have not been conducted using data on common genetic variants. Consequently, we conducted common variant analyses of 15 CHD candidate gene-sets, using data from two common types of CHDs: conotruncal heart defects (1431 cases) and left ventricular outflow tract defects (509 cases). After Bonferroni correction for evaluation of multiple gene-sets, the cytoskeletal gene-set was significantly associated with conotruncal heart defects (βS = 0.09, 95% confidence interval (CI) 0.03–0.15). This association was stronger when analyses were restricted to the sub-set of cytoskeletal genes that have been observed to harbor rare damaging genotypes in at least two CHD cases (βS = 0.32, 95% CI 0.08–0.56). These findings add to the evidence linking cytoskeletal genes to CHDs and suggest that, for cytoskeletal genes, common variation may contribute to the risk of CHDs.

Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2, and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.

Background The contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined. Methods We developed a new computational method, EM-mosaic (Expectation-Maximization-based detection of mosaicism), to analyze mosaicism in exome sequences derived primarily from blood DNA of 2530 CHD proband-parent trios. To optimize this method, we measured mosaic detection power as a function of sequencing depth. In parallel, we analyzed our cohort using MosaicHunter, a Bayesian genotyping algorithm-based mosaic detection tool, and compared the two methods. The accuracy of these mosaic variant detection algorithms was assessed using an independent resequencing method. We then applied both methods to detect mosaicism in cardiac tissue-derived exome sequences of 66 participants for which matched blood and heart tissue was available. Results EM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The estimated true frequency of mosaic variants above 10% mosaicism was 0.14/person in blood and 0.21/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction. Conclusions We estimate that ~ 1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants with relatively higher allele fraction. Although blood is a readily available DNA source, cardiac tissues analyzed contributed ~ 5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses.

Background Congenital heart disease (CHD) affects ~ 1% of live births and is the most common birth defect. Although the genetic contribution to the CHD has been long suspected, it has only been well established recently. De novo variants are estimated to contribute to approximately 8% of sporadic CHD. Methods CHD is genetically heterogeneous, making pathway enrichment analysis an effective approach to explore and statistically validate CHD-associated genes. In this study, we performed novel gene and pathway enrichment analyses of high-impact de novo variants in the recently published whole-exome sequencing (WES) data generated from a cohort of CHD 2645 parent-offspring trios to identify new CHD-causing candidate genes and mutations. We performed rigorous variant- and gene-level filtrations to identify potentially damaging variants, followed by enrichment analyses and gene prioritization. Results Our analyses revealed 23 novel genes that are likely to cause CHD, including HSP90AA1, ROCK2, IQGAP1, and CHD4, and sharing biological functions, pathways, molecular interactions, and properties with known CHD-causing genes. Conclusions Ultimately, these findings suggest novel genes that are likely to be contributing to CHD pathogenesis.

Congenital heart disease (CHD) is the leading cause of mortality from birth defects. Here, exome sequencing of a single cohort of 2,871 CHD probands, including 2,645 parent-offspring trios, implicated rare inherited mutations in 1.8%, including a recessive founder mutation in GDF1 accounting for ∼5% of severe CHD in Ashkenazim, recessive genotypes in MYH6 accounting for ∼11% of Shone complex, and dominant FLT4 mutations accounting for 2.3% of Tetralogy of Fallot. De novo mutations (DNMs) accounted for 8% of cases, including ∼3% of isolated CHD patients and ∼28% with both neurodevelopmental and extra-cardiac congenital anomalies. Seven genes surpassed thresholds for genome-wide significance, and 12 genes not previously implicated in CHD had >70% probability of being disease related. DNMs in ∼440 genes were inferred to contribute to CHD. Striking overlap between genes with damaging DNMs in probands with CHD and autism was also found.

Summary Genome editing in induced pluripotent stem cells is currently hampered by the laborious and expensive nature of identifying homology-directed repair (HDR)-modified cells. We present an approach where isolation of cells bearing a selectable, HDR-mediated editing event at one locus enriches for HDR-mediated edits at additional loci. This strategy, called co-targeting with selection, improves the probability of isolating cells bearing HDR-mediated variants and accelerates the production of disease models., Graphical Abstract, Highlights • Increases the efficiency of genome editing in human iPSCs • Enhances detectability of variants of interest derived by homology-directed repair • Is a simple, scalable, and adaptable strategy for knocking in variants of interest, The potential of CRISPR/Cas9-based genome editing in iPSCs is currently hampered by the laborious nature of identifying cells modified by the lesser-used homology-directed repair (HDR) pathway. Geurts, McDermott-Roe, and colleagues present a straightforward approach, co-targeting with selection, based on co-modification that enriches for cells undergoing HDR thereby improving targeting efficiencies.

Background/Aims: Congenital Sick Sinus Syndrome (SSS) is a disorder associated with sudden cardiac death due to severe bradycardia and prolonged pauses. Mutations in HCN4, the gene encoding inward Na+/K+ current (If), have been described as a cause of congenital SSS. The objective of this study is to develop an SSS model in embryonic zebrafish, and use zebrafish as a moderate-throughput assay to functionally characterize HCN4 variants. Methods: To determine the function of hcn4 in zebrafish, embryos were either bathed in the If -specific blocker (ZD-7288), or endogenous hcn4 expression was knocked down using splice-blocking morpholinos. To assess whether the zebrafish model discriminates benign from pathogenic variants, we tested four HCN4 mutations known to cause human SSS and four variants of unknown significance (VUS). Results: Pharmacological blockade and knockdown of hcn4 in zebrafish phenocopied human SSS, displaying bradycardia and cardiac pauses in intact embryos and explanted hearts. The zebrafish assay correctly identified all disease-causing variants. Of the VUS, the assay predicted 2 as benign and 2 as hypomorphic variants. Conclusions: We conclude that our embryonic zebrafish assay is a novel and effective tool to functionally characterize human HCN4 variants, which can be translated into important clinical prognostic information.

The genetic architecture of sporadic congenital heart disease (CHD) is characterized by enrichment in damaging de novo variants in chromatin-modifying genes. To test the hypothesis that gene pathways contributing to de novo forms of CHD are distinct from those for recessive forms, we analyze 2391 whole-exome trios from the Pediatric Cardiac Genomics Consortium. We deploy a permutation-based gene-burden analysis to identify damaging recessive and compound heterozygous genotypes and disease genes, controlling for confounding effects, such as background mutation rate and ancestry. Cilia-related genes are significantly enriched for damaging rare recessive genotypes, but comparatively depleted for de novo variants. The opposite trend is observed for chromatin-modifying genes. Other cardiac developmental gene classes have less stratification by mode of inheritance than cilia and chromatin-modifying gene classes. Our analyses reveal dominant and recessive CHD are associated with distinct gene functions, with cilia-related genes providing a reservoir of rare segregating variation leading to CHD., Large whole-exome sequencing studies have suggested that the genetic architecture of syndromic congenital heart disease (CHD) is different from sporadic forms. Here, Watkins et al. estimate the relative contribution of damaging recessive and de novo genotypes to CHD in 2391 trios and find them to be associated with different gene functions.

BackgroundThe contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined.ResultsWe developed a computational method, Expectation-Maximization-based detection of Mosaicism (EM-mosaic), to analyze mosaicism in exome sequences of 2530 CHD proband-parent trios. EM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The frequency of mosaic variants above 10% mosaicism was 0.13/person in blood and 0.14/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction.ConclusionsWe estimate that ~1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants expressed at higher allele fraction compared to benign variants. Although blood is a readily-available DNA source, cardiac tissues analyzed contributed ~5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses.

Using a family-based approach with whole exome sequencing (WES) and custom bioinformatics tools we identified a novel mutation (M527L) in the Nuclear Factor of Activated T-Cells 1 gene (NFATc1). This nonsynonymous heterozygous substitution segregates in an autosomal dominant pattern within a family with a young onset Atrial Fibrillation (AF) phenotype. Patient specific inducible pluripotent stem cell derived-cardiomyocytes (iPSC-CM) from a patient carrying this variant showed abbreviated repolarization resulting in a shorter action potential duration compared to iPSC-CM from a healthy sibling control. Unlike familial monogenic AF where the mutation occurs in an ion channel gene, NFATc1 is a transcription factor that influences expression of multiple genes. Using single-cell RNA sequencing on 30 day post-differentiation iPSC-CM, we identified four subpopulations by K-mean clustering (k=4) of single cell transcriptomes and subsequent marker expression. We then performed gene-annotation enrichment analysis of differentially expressed genes specifically in the mutant (MT) and wild-type (WT) cardiomyocyte (CM) populations, detecting several key functional and pathological cardiac pathways affected in response to the NFATc1 mutation. These included Wnt, NF-kappaB and MAPK signaling, Ca homeostasis and gene categories that have been associated with atrial fibrillation. These results suggest that NFATc1 mutation may play an important role in the pathophysiology of familial atrial fibrillation by its broad impact on gene expression and subsequent pathway regulation.

Although DNA methylation is the best characterized epigenetic mark, the mechanism by which it is targeted to specific regions in the genome remains unclear. Recent studies have revealed that local DNA methylation profiles might be dictated by cis-regulatory DNA sequences that mainly operate via DNA-binding factors. Consistent with this finding, we have recently shown that disruption of CTCF-binding sites by rare single nucleotide variants (SNVs) can underlie cis-linked DNA methylation changes in patients with congenital anomalies. These data raise the hypothesis that rare genetic variation at transcription factor binding sites (TFBSs) might contribute to local DNA methylation patterning. In this work, by combining blood genome-wide DNA methylation profiles, whole genome sequencing-derived SNVs from 247 unrelated individuals along with 133 predicted TFBS motifs derived from ENCODE ChIP-Seq data, we observed an association between the disruption of binding sites for multiple TFs by rare SNVs and extreme DNA methylation values at both local and, to a lesser extent, distant CpGs. While the majority of these changes affected only single CpGs, 24% were associated with multiple outlier CpGs within ±1kb of the disrupted TFBS. Interestingly, disruption of functionally constrained sites within TF motifs lead to larger DNA methylation changes at nearby CpG sites. Altogether, these findings suggest that rare SNVs at TFBS negatively influence TF-DNA binding, which can lead to an altered local DNA methylation profile. Furthermore, subsequent integration of DNA methylation and RNA-Seq profiles from cardiac tissues enabled us to observe an association between rare SNV-directed DNA methylation and outlier expression of nearby genes. In conclusion, our findings not only provide insights into the effect of rare genetic variation at TFBS on shaping local DNA methylation and its consequences on genome regulation, but also provide a rationale to incorporate DNA methylation data to interpret the functional role of rare variants., Author summary One of the major challenges for human genetics in the post-genomic era is to interpret the functional relevance of genetic variation. Quantitative trait locus (QTL) analyses have associated an important fraction of genetic variants with a wide range of molecular phenotypes including gene expression (eQTL) and DNA methylation (meQTL), providing insights into the mechanisms by which genetic variation can contribute to health and disease. Although QTL mapping represents an excellent approach to identify biologically relevant functional variants, these studies have been mainly focused on common variants and do not include low-frequency and rare variants. Here, we observed that rare regulatory variants, i.e, single nucleotide variants (SNVs) that disrupt transcription factor binding sites (TFBSs), are associated with changes in DNA methylation at both local and, to a lesser extent, broader locations, most likely, by altering the DNA-binding affinity of transcription factors (TFs). Interestingly, we have also shown that this change in DNA methylation can alter expression levels of nearby genes. Overall, these data suggest a role of rare regulatory SNVs in shaping DNA methylation, and suggest that the incorporation of DNA methylation data may help to interpret the functional consequences of human genetic variation.

Many forms of congenital heart disease (CHD) have high morbidity-mortality rates and require challenging surgeries. Human amniocytes have important stem cell characteristics and could potentially provide patient-specific tissue for repairs of some types of CHDs. We report that amniocytes express features of poised cardiomyocytes. However, a variety of direct reprogramming approaches failed to convert their fetal and transcriptionally repressed state into bona fide cardiomyocytes. Induced-pluripotent stem cell (iPSC) reprogramming removes repression and converts amniocytes to a baseline pluripotent state. Based on molecular and electrophysiological signatures, iPSC reprogrammed amniocytes can be induced to differentiate into functionally immature, predominantly ventricular cardiomyocytes and a heterogeneous mixture of vascular and unspecified epithelial cells. Developmental time course analyses and pattern clustering of amniocyte-derived cardiomyocytes identifies numerous temporal co-regulators of cardiac induction and maturation as well as distinct sarcomeric and ion channel gene signatures. Normal fetal cardiomyocytes are derived by overcoming complex forms of transcriptional repression that suppress direct transdifferentiation of human amniocytes. These results suggest the possibility of using amniocytes as a source of patient-specific ventricular cardiomyocytes for cell therapies.SUMMARY STATEMENTAmniocytes are a possible source of patient-specific cardiomyocytes for newborns with congenital heart disease. Genome-wide DNA methylation patterns and transcriptional repressors preclude direct differentiation, but pluripotent reprogramming provides cardiomyocytes for dissecting genetic pathways contributing to this disease.

Kabuki Syndrome patients have a spectrum of congenital disorders, including congenital heart defects, the primary determinant of mortality. Seventy percent of Kabuki Syndrome patients have mutations in the histone methyl-transferase KMT2D. However, the underlying mechanisms that drive these congenital disorders are unknown. Here, we generated and characterized zebrafish kmt2d null mutants that recapitulate the cardinal phenotypic features of Kabuki Syndrome, including microcephaly, palate defects, abnormal ear development, and cardiac defects. The cardiac phenotype consists of a previously unknown vasculogenesis defect that affects endocardium patterning and, consequently, heart ventricle lumen formation. Additionally, zebrafish kmt2d null mutants have angiogenesis defects depicted by abnormal aortic arch development, hyperactive ectopic blood vessel sprouting, and aberrant patterning of the brain vascular plexus. We demonstrate that zebrafish kmt2d null mutants have robust Notch signaling hyperactivation in endocardial and endothelial cells, including increased protein levels of the Notch transcription factor Rbpj. Our zebrafish Kabuki Syndrome model reveals a regulatory link between the Notch pathway and Kmt2d during endothelium and endocardium patterning and shows that pharmacological inhibition of Notch signaling rebalances Rbpj protein levels and rescues the cardiovascular phenotype by enhancing endothelial and endocardial cell proliferation and stabilizing endocardial patterning. Taken together, these findings demonstrate that Kmt2d regulates vasculogenesis and angiogenesis, provide evidence for interactions between Kmt2d and Notch signaling in Kabuki Syndrome, and suggest future directions for clinical research., Kabuki Syndrome (KS) is a human congenital disorder with mutations in KMT2D. Zebrafish kmt2d mutants recapitulate human KS phenotypes, have aberrant Notch pathway signaling, and implicate vasculogenesis as a driver of abnormal cardiac development. Pharmacological inhibition of the Notch pathway rescues vasculogenesis in KS zebrafish, with potential therapeutic implications.

Multiple tools have been developed to identify copy number variants (CNVs) from whole exome (WES) and whole genome sequencing (WGS) data. Current tools such as XHMM for WES and CNVnator for WGS identify CNVs based on changes in read depth. For WGS, other methods to identify CNVs include utilizing discordant read pairs and split reads and genome-wide local assembly with tools such as Lumpy and SvABA, respectively. Here, we introduce a new method to identify deletion CNVs from WES and WGS trio data based on the clustering of Mendelian errors (MEs). Using our Mendelian Error Method (MEM), we identified 127 deletions (inherited and de novo) in 2,601 WES trios from the Pediatric Cardiac Genomics Consortium, with a validation rate of 88% by digital droplet PCR. MEM identified additional de novo deletions compared to XHMM, and also identified sample switches, DNA contamination, a significant enrichment of 15q11.2 deletions compared to controls and eight cases of uniparental disomy. We applied MEM to WGS data from the Genome In A Bottle Ashkenazi trio and identified deletions with 97% specificity. MEM provides a robust, computationally inexpensive method for identifying deletions, and an orthogonal approach for verifying deletions called by other tools.

Wolff–Parkinson–White (WPW) syndrome is a common cause of supraventricular tachycardia that carries a risk of sudden cardiac death. To date, mutations in only one gene, PRKAG2, which encodes the 5’ -AMP-activated protein kinase subunit γ-2, have been identified as causative for WPW. DNA samples from five members of a family with WPW were analyzed by exome sequencing. We applied recently designed prioritization strategies (VAAST/pedigree VAAST) coupled with an ontology-based algorithm (Phevor) that reduced the number of potentially damaging variants to 10: a variant in KCNE2 previously associated with Long QT syndrome was also identified. Of these 11 variants, only MYH6 p.E1885K segregated with the WPW phenotype in all affected individuals and was absent in 10 unaffected family members. This variant was predicted to be damaging by in silico methods and is not present in the 1,000 genome and NHLBI exome sequencing project databases. Screening of a replication cohort of 47 unrelated WPW patients did not identify other likely causative variants in PRKAG2 or MYH6. MYH6 variants have been identified in patients with atrial septal defects, cardiomyopathies, and sick sinus syndrome. Our data highlight the pleiotropic nature of phenotypes associated with defects in this gene.

The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9 system combined with the rapid generation time of the zebrafish model organism has made the possibility of performing F0 screens in this organism a reality. This unit describes a detailed protocol for performing an F0 screen using the CRISPR/Cas9 system in zebrafish starting with the design and production of custom CRISPR/Cas9 reagents for injection. Next, two approaches for determining the efficiency of mutation induction by the custom CRISPR/Cas9 reagents that are easily performed using standard molecular biology protocols are detailed. Finally, screening for F0 induced phenotypes using the zebrafish flh gene as an example is discussed. © 2017 by John Wiley & Sons, Inc.

Diabetes mellitus increases the risk for cardiac dysfunction, heart failure, and sudden death. The wide array of neurohumoral changes associated with diabetes pose a challenge to understanding the roles of specific pathways that alter cardiac function. Here, we use a mouse model with cardiomyocyte-restricted deletion of insulin receptors (CIRKO, cardiac-specific insulin receptor knockout) to study the specific effects of impaired cardiac insulin signaling on ventricular repolarization, independent of the generalized metabolic derangements associated with diabetes. Impaired insulin action caused a reduction in mRNA and protein expression of several key K+ channels that dominate ventricular repolarization. Specifically, components of transient outward K+ current fast component ( Ito,fast; Kv4.2 and KChiP2) were reduced, consistent with a reduction in the amplitude of Ito,fast in isolated left ventricular CIRKO myocytes, compared with littermate controls. The reduction in Ito,fast resulted in ventricular action potential prolongation and prolongation of the QT interval on the surface ECG. These results support the notion that the lack of insulin signaling in the heart is sufficient to cause the repolarization abnormalities described in other animal models of diabetes.

Rationale: Genetic testing for Long QT Syndrome is now a standard and integral component of clinical cardiology. A major obstacle to the interpretation of genetic findings is the lack of robust functional assays to determine the pathogenicity of identified gene variants in a high-throughput manner. Objective: The goal of this study was to design and test a high-throughput in vivo cardiac assay to distinguish between disease-causing and benign KCNH2 ( hERG1) variants, using the zebrafish as a model organism. Methods and Results: We tested the ability of previously characterized Long QT Syndrome hERG1 mutations and polymorphisms to restore normal repolarization in the kcnh2 -knockdown embryonic zebrafish. The cardiac assay correctly identified a benign variant in 9 of 10 cases (negative predictive value 90%), whereas correctly identifying a disease-causing variant in 39/39 cases (positive predictive value 100%). Conclusions: The in vivo zebrafish cardiac assay approaches the accuracy of the current benchmark in vitro assay for the detection of disease-causing mutations, and is far superior in terms of throughput rate. Together with emerging algorithms for interpreting a positive long QT syndrome genetic test, the zebrafish cardiac assay provides an additional tool for the final determination of pathogenicity of gene variants identified in long QT syndrome genetic screening.

Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1-S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go-related gene type 1 (hERG1) channels, Cd(2+) coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd(2+) on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd(2+) shifted the half-point (V(1/2)) for the voltage dependence of the OFF gating charge-voltage (Q(OFF)-V) relationship with an EC(50) of 171 microM; at 0.3 mM, V(1/2) was shifted by +50 mV. Cd(2+) also induced an as of yet unrecognized small outward current (I(Cd-out)) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd(2+) and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd(2+) in an outward direction to produce I(Cd-out). Co(2+), Zn(2+), and La(3+) at concentrations that caused approximately +35-mV shifts in the Q(OFF)-V relationship did not induce a current similar to I(Cd-out), suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd(2+). New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd(2+).

Aim The zebrafish has emerged as a novel model for investigating cardiac physiology and pathology. The aim of this study was to investigate the atrium-specific ion channels responsible for shaping the atrial cardiac action potential in zebrafish. Methods Using quantitative polymerase chain reaction, we assessed the expression level of atrium-specific potassium channels. The functional role of these channels was studied by patch clamp experiments on isolated atrial and ventricular cardiomyocytes and by optical mapping of explanted adult zebrafish hearts. Finally, surface ECGs were recorded to establish possible in vivo roles of atrial ion channels. Results In isolated adult zebrafish hearts, we identified the expression of kcnk3, kcnk9, kcnn1, kcnn2, kcnn3, kcnj3 and kcnj5, the genes that encode the atrium-specific K2P , KCa 2.x and Kir 3.1/4 (KACh ) ion channels. The electrophysiological data indicate that the acetylcholine-activated inward-rectifying current, IKACh, plays a major role in the zebrafish atrium, whereas K2P 3.1/9.1 and KCa 2.x channels do not appear to be involved in regulating the action potential in the zebrafish heart. Conclusion We demonstrate that the acetylcholine-activated inward-rectifying current (IKACh ) current plays a major role in the zebrafish atrium and that the zebrafish could potentially be a cost-effective and reliable model for pharmacological testing of atrium-specific IKACh modulating compounds.

The zebrafish is an emerging model system for the study of cardiac electrophysiology and human arrhythmias. High resolution imaging techniques are powerful tools for the study of zebrafish cardiac electrophysiology, but these methods require the complete absence of cardiac contraction. Many pharmacological agents that uncouple cardiac contraction also markedly alter the cardiac action potential (AP). In this study, we compared the effects two uncoupling agents, 2,3-Butanedione monoxime (BDM) and blebbistatin, on contractility and AP parameters in embryonic zebrafish heart.Zebrafish hearts were explanted (48 hpf) and superfused with either BDM (15 mM) or blebbistatin (1, 5 or 10 microM), while recording atrial or ventricular APs with the disrupted patch technique. Calcium transients were recorded with a high-speed confocal scanning microscope in hearts loaded intracellularly with 10 microM fluo-4 and superfused with 10 microM blebbistatin.Despite abolishing cardiac contractility, BDM altered ventricular AP morphology and inhibited spontaneous APs. In contrast, blebbistatin (10 microM) abolished contractility without significantly altering AP morphology or generation of spontaneous APs. Blebbistatin allowed for high fidelity measurements of atrial and ventricular calcium transients.Blebbistatin is a potent and effective excitation-contraction uncoupling agent in embryonic zebrafish heart.

Short QT Syndrome (SQTS) is a novel clinical entity characterized by markedly rapid cardiac repolarization and lethal arrhythmias. A mutation in the Kir2.1 inward rectifier K+ channel (D172N) causes one form of SQTS (SQT3). Pharmacologic block of Kir2.1 channels may hold promise as potential therapy for SQT3. We recently reported that the anti-malarial drug chloroquine blocks Kir2.1 channels by plugging the cytoplasmic pore domain. In this study, we tested whether chloroquine blocks D172N Kir2.1 channels in a heterologous expression system and if chloroquine normalizes repolarization properties using a mathematical model of a human ventricular myocyte. Chloroquine caused a dose- and voltage-dependent reduction in wild-type (WT), D172N and WT-D172N heteromeric Kir2.1 current. The potency and kinetics of chloroquine block of D172N and WT-D172N Kir2.1 current were similar to WT. In silico modeling of the heterozygous WT-D172N Kir2.1 condition predicted that 3 μM chloroquine normalized inward rectifier K+ current magnitude, action potential duration and effective refractory period. Our results suggest that therapeutic concentrations of chloroquine might lengthen cardiac repolarization in SQT3.

Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K + current I K1 and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac I K1 . The voltage- and K + -dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion-channel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block I K1 even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.

Long QT syndrome (LQTS) is a disorder of ventricular repolarization that predisposes affected individuals to lethal cardiac arrhythmias. To date, an appropriate animal model of inherited LQTS does not exist. The zebrafish is a powerful vertebrate model used to dissect molecular pathways of cardiovascular development and disease. Because fundamental electrical properties of the zebrafish heart are remarkably similar to those of the human heart, the zebrafish may be an appropriate model for studying human inherited arrhythmias. Here we describe the molecular, cellular, and electrophysiological basis of a zebrafish mutant characterized by ventricular asystole. Genetic mapping and direct sequencing identify the affected gene as kcnh2 , which encodes the channel responsible for the rapidly activating delayed rectifier K + current ( I Kr ). We show that complete loss of functional I Kr in embryonic hearts leads to ventricular cell membrane depolarization, inability to generate action potentials (APs), and disrupted calcium release. A small hyperpolarizing current restores spontaneous APs, implying wild-type function of other ionic currents critical for AP generation. Heterozygous fish manifest overt cellular and electrocardiographic evidence for delayed ventricular repolarization. Our findings provide insight into the pathogenesis of homozygous kcnh2 mutations and expand the use of zebrafish mutants as a model system to study human arrhythmias.

Elucidation of the cellular basis of arrhythmias in ion channelopathy disorders is complicated by the inherent difficulties in studying human cardiac tissue. Thus we used a computer modeling approach to study the mechanisms of cellular dysfunction induced by mutations in inward rectifier potassium channel (Kir)2.1 that cause Andersen-Tawil syndrome (ATS). ATS is an autosomal dominant disorder associated with ventricular arrhythmias that uncommonly degenerate into the lethal arrhythmia torsade de pointes. We simulated the cellular and tissue effects of a potent disease-causing mutation D71V Kir2.1 with mathematical models of human ventricular myocytes and a bidomain model of transmural conduction. The D71V Kir2.1 mutation caused significant action potential duration prolongation in subendocardial, midmyocardial, and subepicardial myocytes but did not significantly increase transmural dispersion of repolarization. Simulations of the D71V mutation at shorter cycle lengths induced stable action potential alternans in midmyocardial, but not subendocardial or subepicardial cells. The action potential alternans was manifested as an abbreviated QRS complex in the transmural ECG, the result of action potential propagation failure in the midmyocardial tissue. In addition, our simulations of D71V mutation recapitulate several key ECG features of ATS, including QT prolongation, T-wave flattening, and QRS widening. Thus our modeling approach faithfully recapitulates several features of ATS and provides a mechanistic explanation for the low frequency of torsade de pointes arrhythmia in ATS.

Slow activation and rapid C-type inactivation produce inward rectification of the current-voltage relationship for human ether-a'-go-go-related gene (hERG) channels. To characterize the voltage sensor movement associated with hERG activation and inactivation, we performed an Ala scan of the 32 amino acids (Gly(514)-Tyr(545)) that comprise the S4 domain and the flanking S3-S4 and S4-S5 linkers. Gating and ionic currents of wild-type and mutant channels were measured using cut-open oocyte Vaseline gap and two microelectrode voltage clamp techniques to determine the voltage dependence of charge movement, activation, and inactivation. Mapping the position of the charge-perturbing mutations (defined as |DeltaDeltaG| > 1.0 kcal/mol) on a three-dimensional S4 homology model revealed a spiral pattern. As expected, mutation of these residues also altered activation. However, mutation of residues in the S3-S4 and S4-S5 linkers and the C-terminal end of S4 perturbed activation (|DeltaDeltaG| > 1.0 kcal/mol) without altering charge movement, suggesting that the native residues in these regions couple S4 movement to the opening of the activation gate or stabilize the open or closed state of the channel. Finally, mutation of a distinct set of residues impacted inactivation and mapped to a single face of the S4 helix that was devoid of activation-perturbing residues. These results define regions on the S4 voltage sensor that contribute differentially to hERG activation and inactivation gating.

Andersen-Tawil syndrome is a skeletal and cardiac muscle disease with developmental features caused by mutations in the inward rectifier K+ channel gene KCNJ2. Patients harboring these mutations exhibit extremely variable expressivities. To explore whether these mutations can be correlated with a specific patient phenotype, we expressed both wild-type (WT) and mutant genes cloned into a bi-cistronic vector. Functional expression in human embryonic kidney 293 cells showed that none of the mutant channels express current when present alone. When co-expressed with WT channels, only construct V302M-WT yields inward current. Confocal microscopy fluorescence revealed three patterns of channel expression in the cell: 1) mutations D71V, N216H, R218Q, and pore mutations co-assemble and co-localize to the membrane with the WT and exert a dominant-negative effect on the WT channels; 2) mutation V302M leads to channels that lose their ability to co-assemble with WT and traffic to the cell surface; 3) deletions Delta 95-98 and Delta 314-315 lead to channels that do not traffic to the membrane but retain their ability to co-assemble with WT channels. These data show that the Andersen-Tawil syndrome phenotype may occur through a dominant-negative effect as well as through haplo-insufficiency and reveal amino acids critical in trafficking and conductance of the inward rectifier K+ channels.

Objectives: We sought to determine whether oral potassium supplementation safely increases serum K+and results in sustained improvement of repolarization parameters in long QT syndrome type...

HERG (human ether-a-go-go-related gene) encodes a delayed rectifier K + channel vital to normal repolarization of cardiac action potentials. Attenuation of repolarizing K + current caused by mutations in HERG or channel block by common medications prolongs ventricular action potentials and increases the risk of arrhythmia and sudden death. The critical role of HERG in maintenance of normal cardiac electrical activity derives from its unusual gating properties. Opposite to other voltage-gated K + channels, the rate of HERG channel inactivation is faster than activation and appears to be intrinsically voltage dependent. To investigate voltage sensor movement associated with slow activation and fast inactivation, we characterized HERG gating currents. When the cut-open oocyte voltage clamp technique was used, membrane depolarization elicited gating current with fast and slow components that differed 100-fold in their kinetics. Unlike previously studied voltage-gated K + channels, the bulk of charge movement in HERG was protracted, consistent with the slow rate of ionic current activation. Despite similar kinetic features, fast inactivation was not derived from the fast gating component. Analysis of an inactivation-deficient mutant HERG channel and a Markov kinetic model suggest that HERG inactivation is coupled to activation.

The delayed rectifier K(+) currents, I(Kr) and I(Ks,) play a critical role in modulating the plateau phase of the cardiac action potential. HERG encodes the alpha-subunit of channels underlying I(Kr), while I(Ks) is composed of subunits encoded by KCNQ1 and KCNE1. Mutations in any of these genes cause the long QT syndrome, a disorder of myocellular repolarization that predisposes affected individuals to life-threatening arrhythmias. Elucidation of the molecular basis of these currents has led to significant advancements in our understanding of fundamental properties of channel function. This review summarizes the current state of knowledge regarding the structural determinants and biophysical properties of HERG and KCNQ1 channels.

Deletion of chromosome 22q11, the most common microdeletion detected in humans, is associated with a life-threatening array of birth defects. Although 90% of affected individuals share the same three megabase deletion, their phenotype is highly variable and includes craniofacial and cardiovascular anomalies, hypoplasia or aplasia of the thymus with associated deficiency of T cells, hypocalcemia with hypoplasia or aplasia of the parathyroids, and a variety of central nervous system abnormalities. Because ablation of neural crest in chicks produces many features of the deletion 22q11 syndrome, it has been proposed that haploinsufficiency in this region impacts neural crest function during cardiac and pharyngeal arch development. Few factors required for migration, survival, proliferation and subsequent differentiation of pharyngeal arch neural crest and mesoderm-derived mesenchyme into their respective cardiovascular, musculoskeletal, and glandular derivatives have been identified. However, the importance of epithelial-mesenchymal interactions and pharyngeal endoderm function is becoming increasingly clear.Fibroblast growth factor 8 is a signaling molecule expressed in the ectoderm and endoderm of the developing pharyngeal arches and known to play an important role in survival and patterning of first arch tissues. We demonstrate a dosage-sensitive requirement for FGF8 during development of pharyngeal arch, pharyngeal pouch and neural crest-derived tissues. We show that FGF8 deficient embryos have lethal malformations of the cardiac outflow tract, great vessels and heart due, at least in part, to failure to form the fourth pharyngeal arch arteries, altered expression of Fgf10 in the pharyngeal mesenchyme, and abnormal apoptosis in pharyngeal and cardiac neural crest.The Fgf8 mutants described herein display the complete array of cardiovascular, glandular and craniofacial phenotypes seen in human deletion 22q11 syndromes. This represents the first single gene disruption outside the typically deleted region of human chromosome 22 to fully recapitulate the deletion 22q11 phenotype. FGF8 may operate directly in molecular pathways affected by deletions in 22q11 or function in parallel pathways required for normal development of pharyngeal arch and neural crest-derived tissues. In either case, Fgf8 may function as a modifier of the 22q11 deletion and contribute to the phenotypic variability of this syndrome.

Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-{β[2-(di- n-butylamino)-6-naphthyl]butadienyl}quinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses, we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision, generating nearly identical values for AP duration (AP durations at 10%, 50%, and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure, with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally, di-4-ANBDQBS allowed for moderate-throughput analyses, increasing throughput >10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs.

Summary Advances in induced pluripotent stem cell (iPSC) technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM) models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs) are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA) analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias., Highlights • Peripheral blood mononuclear cells are an advantageous source of somatic cells • Functional analysis of peripheral blood mononuclear stem cell-derived cardiac cells • MEA-based activation-recovery interval robustly tracks repolarization • Stem cell-based tachypacing model for the study of human tachyarrhythmias, Advances in stem cell technology allow for routine derivation of patient- and disease-specific cardiomyocyte (CM) models. Here, Benjamin, Tristani-Firouzi, and colleagues report the functional and pharmacological properties of CMs differentiated from peripheral blood mononuclear cell-derived pluripotent stem cells. CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for the study of human tachyarrhythmias.

BACKGROUND: Precise measurement of the QT interval is often hampered by difficulty determining the end of the low amplitude T wave. Root mean square electrocardiography (RMS ECG) provides a novel alternative measure of ventricular repolarization. Experimental data have shown that the interval between the RMS ECG QRS and T wave peaks (RTPK) closely reflects the mean ventricular action potential duration while the RMS T wave width (TW) tracks the dispersion of repolarization timing. Here, we tested the precision of RMS ECG to assess ventricular repolarization in humans in the setting of drug-induced and congenital Long QT Syndrome (LQTS). METHODS: RMS ECG signals were derived from high-resolution 24 hour Holter monitor recordings from 68 subjects after receiving placebo and moxifloxacin and from standard 12 lead ECGs obtained in 97 subjects with LQTS and 97 age- and sex-matched controls. RTPK, QTRMS and RMS TW intervals were automatically measured using custom software and compared to traditional QT measures using lead II. RESULTS: All measures of repolarization were prolonged during moxifloxacin administration and in LQTS subjects, but the variance of RMS intervals was significantly smaller than traditional lead II measurements. TW was prolonged during moxifloxacin and in subjects with LQT-2, but not LQT-1 or LQT-3. CONCLUSION: These data validate the application of RMS ECG for the detection of drug-induced and congenital LQTS. RMS ECG measurements are more precise than the current standard of care lead II measurements.

The configuration of cardiac action potentials varies considerably from one region of the heart to another. These differences are caused by differential cellular expression of several types of K + channel genes. The channels encoded by these genes can be grouped into several classes depending on the stimulus that permits the channels to open and conduct potassium ions. K + channels are activated by changes in transmembrane voltage or binding of ligands. Voltage-gated channels are normally the most important players in determining the shape and duration of action potentials and include the delayed rectifiers and the transient outward potassium channels. Ligand-gated channels include those that probably have only minor roles in shaping repolarization under normal conditions but, when activated by extracellular acetylcholine or a decrease in the intracellular concentration of ATP, can substantially shorten action potential duration. Inward rectifier K + channels are unique in that they are basically stuck in the open state but can be blocked in a voltage-dependent manner by intracellular Mg 2+ , Ca 2+ , and polyamines. Other K + channels have been described that provide a small background leak conductance. Many of these cardiac K + channels have been cloned in the past decade, permitting detailed studies of the molecular basis of their function and facilitating the discovery of the molecular basis of several forms of congenital arrhythmias. Drugs that block cardiac K + channels and prolong action potential duration have been developed as antiarrhythmic agents. However, many of these same drugs, as well as other common medications that are structurally unrelated, can also cause long QT syndrome and induce ventricular arrhythmia.

Mutations in KvLQT1. Introduction: The long QT syndrome (LQT) is caused by mutations in genes encoding ion channels that modulate the duration of ventricular action potentials. One of these genes, KVLQT1, encodes an α subunit that coassembles with another subunit, hminK, to form the cardiac slow delayed rectifier (IKs) K+ channel. Methods and Results: The functional effects of seven mutations in KVLQT1 were assessed using two-microelectrode voltage clamp and the Xenopus oocyte expression system. Most mutations in KVLQT1 caused loss of function when expressed alone. Oocytes were also injected with equal amounts of wild-type (WT) KVLQT1 and mutant KVLQT1 cRNA (with or without coinjection of hminK) and the resulting currents compared to currents induced by WT KvLQT1 alone. A341V, RI90Q, or G189R KVLQT1 subunits did not affect expression of WT KvLQT1. The other mutations in KVLQT1 caused a variable degree of dominant-negative suppression of IKs. The order of potency for this effect was G345E > G306R = V254M > A341E. Conclusions: LQTl-associated mutations in KVLQT1 caused a spectrum of dysfunction in IKs and KvLQT1 channels. The degree of IKs dysfunction did not correlate with the QTc interval or the presence of symptoms in the respective gene carriers. In contrast to previous reports, we found that loss of function mutations are not exclusive to recessiveiy inherited LQT.

Diversity of response in vascular smooth muscle cells to changes in oxygen tension. Hypoxia causes pulmonary vasoconstriction (HPV), but also dilation of systemic vessels and the ductus arteriosus. In the adult animal, HPV is initiated by inhibition of potassium current (1 K ) in the smooth muscle cells of small resistance arteries, which results in membrane depolarization and calcium entry through voltage-gated calcium channels. The oxygen-sensitive channels that initiate HPV are 4-amino-pyridine (4-AP)-sensitive delayed rectifier channels (K DR ), the most prominent of which has a conductance of 37pS. In the fetus, hypoxia causes pulmonary vasoconstriction through inhibition of a calcium-sensitive potassium channel (K Ca ). In smooth muscle cells from the rabbit ductus arteriosus, which dilates in response to hypoxia, whole-cell potassium current is reversibly enhanced, rather than inhibited, by hypoxia. The principal oxygen-sensitive channel is inhibited by 4-AP and has a conductance of about 58pS. There are morphological and electrophysiological differences between individual pulmonary artery smooth muscle cells, for example, in some cells 1 K is predominantly carried by K DR channels and in others by K Ca channels. K DR cells are more common in the resistance pulmonary arteries and K Ca in the conduit arteries. Responses of specific vessels (conduit, resistance; pulmonary, systemic, ductus) at different stages of development (fetal, neonatal and adult) to changes in oxygen tension may be determined by the distribution of a variety of ion channels in the smooth muscle cells.

During zebrafish cardiac development, 3-OST-7 constrains BMP signaling to the atrioventricular junction and precludes it from contractile myocardium, allowing tropomyosin-dependent sarcomere assembly and contraction., The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function., Author Summary A highly complex environment at the cell surface and in the space between cells is thought to modulate cell behavior. Heparan sulfate proteoglycans are cell surface and extracellular matrix molecules that are covalently linked to long chains of repeating sugar units called glycosaminoglycan chains. These chains can be subjected to rare modifications and they are believed to influence specific cell signaling events in a lineage specific fashion in what is called the “glycocode.” Here we explore the functions of one member of a family of enzymes, 3-O-sulfotransferases (3-OSTs) that catalyzes a rare modification (3-O-sulfation) of glycosaminoglycans in zebrafish. We show that knockdown of 3-OST-7 results in a very specific phenotype, including loss of cardiac ventricle contraction. Knockdown of other 3-OST family members did not result in the same phenotype, suggesting that distinct 3-OST family members have distinct functions in vertebrates and lending in vivo evidence for the glycocode hypothesis. Mechanistically, we found that cardiac contraction can be rescued by reducing the amount of endogenous BMP4, and can be blocked by increasing BMP signaling, suggesting that the glycocode generated by 3-OST-7 is necessary to constrain BMP signaling in the heart for normal cardiac contraction. Furthermore, we show that tropomyosin4 (tpm4) is downstream of 3-OST-7 function, indicating that Tpm4 is key in this pathway to building the sarcomere, the functional contraction unit of the cardiomyocyte.

KCNQ genes encode five Kv7 K(+) channel subunits (Kv7.1-Kv7.5). Four of these (Kv7.2-Kv7.5) are expressed in the nervous system. Kv7.2 and Kv7.3 are the principal molecular components of the slow voltage-gated M-channel, which regulates neuronal excitability. In this study, we demonstrate that tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits Kv7.2/Kv7.3 currents heterologously expressed in human embryonic kidney HEK-293 cells. Current inhibition by tamoxifen was voltage independent but concentration-dependent. The IC50 for current inhibition was 1.68 ± 0.44 µM. The voltage-dependent activation of the channel was not modified. Tamoxifen inhibited Kv7.2 homomeric channels with a higher potency (IC50 = 0.74 ± 0.16 µM). The mutation Kv7.2 R463E increases phosphatidylinositol- 4,5-bisphosphate (PIP2) - channel interaction and diminished dramatically the inhibitory effect of tamoxifen compared with that for wild type Kv7.2. Conversely, the mutation Kv7.2 R463Q, which decreases PIP2 -channel interaction, increased tamoxifen potency. Similar results were obtained on the heteromeric Kv7.2 R463Q/Kv7.3 and Kv7.2 R463E/Kv7.3 channels, compared to Kv7.2/Kv7.3 WT. Overexpression of type 2A PI(4)P5-kinase (PIP5K 2A) significantly reduced tamoxifen inhibition of Kv7.2/Kv7.3 and Kv7.2 R463Q channels. Our results suggest that tamoxifen inhibited Kv7.2/Kv7.3 channels by interfering with PIP2-channel interaction because of its documented interaction with PIP2 and the similar effect of tamoxifen on various PIP2 sensitive channels.

The transverse tubular system (t-system) is a membrane specialization of striated myocytes crucial for excitation-contraction coupling. Various studies demonstrated remodeling of t-system in heart disease in species including rodent, canine and human. Commonly, disease was associated with heterogeneous t-system depletion. Here, we test the hypothesis that t-system remodeling underlies inefficient excitation-contraction coupling in human heart failure.We investigated the t-system in left-ventricular myocardium samples from 5 patients undergoing cardiac surgery. The tissues were labeled for ryanodine receptors (RyRs), and the extracellular space including the t-system. We acquired 3D images with a Leica SP8 confocal microscope using a 63x oil immersion lens. After image deconvolution, we segmented individual cardiomyocytes. We reconstructed the t-system and quantified the RyR intensity and distance of RyR clusters to the t-system. Furthermore, isolated myocytes were labeled with di-8-ANEPPS and Fluo-4 to acquire 2D image sequences of calcium transients using a Zeiss Live 5 Duo confocal microscope.Our studies revealed a novel type of t-system remodeling, characterized by flat sheets rather than tubular structures. Three-dimensional reconstructions indicated that sheet cavities were connected with the extracellular space. Principal component analysis showed that sheets were aligned with the longitudinal myocyte axis. One sample did not show sheets and served as control. Comparing 5 cells with sheets to 5 control cells, we found significantly increased distances between RyR clusters and the t-system (0.82±0.12µm and 0.5±0.06µm, respectively) and a significantly higher fraction of RyRs present >1µm from the sarcolemma (49±6.9% and 21±5.5%, respectively). Cardiomyocytes with sheets exhibited spatial heterogeneity and asynchrony of calcium transients.Our studies present a novel phenotype of remodeled t-system, with a strikingly increased number of non-junctional RyR clusters. We suggest that the associated spatially heterogeneous and asynchronous calcium release contributes to reduced contractility in heart failure.

Diabetes mellitus is associated with cardiac arrhythmias, ventricular fibrillation and sudden death. These cardiac effects may result directly from the absence of insulin signaling in the heart or as in indirect consequence of diabetic sequelae (e.g., hyperlipidemia, hypertension or coronary artery disease). To study the electrical consequences of absent insulin signaling in the heart, we used a cardiac-specific insulin receptor knock-out (CIRKO) mouse model. We recorded action potentials and K+ currents in myocytes isolated from CIRKO mice and their control littermates. Action potential duration at 90% (APD90) was significantly prolonged in left ventricle, apex and septum from CIRKO mice compared to WT. Conversely, no differences in APD90 were observed in right ventricular myocytes. Outward potassium currents recorded using whole-cell voltage clamp method showed a reduction in both peak and sustained current in CIRKO mice. Neither the voltage dependence of steady-state inactivation nor recovery from inactivation of the rapidly inactivating outward K+ current (Ito,f) were affected. In addition, the inward rectifier K+ current (IK1) was not different between both groups. Consistent with the APD prolongation, CIRKO mice showed QT interval prolongation, compared to WT littermates. Western blot and real time q-PCR analyses revealed that the protein levels and mRNA expression of Kv4.2 were significantly reduced in CIRKO hearts. We conclude that the absence of insulin signaling in the heart causes a reduction in Kv4.2 message and protein that in turn, reduces Ito,f, prolongs APD in left ventricle and prolongs the QT interval.

TBX3 is critical for human development: mutations in TBX3 cause congenital anomalies in patients with ulnar-mammary syndrome. Data from mice and humans suggest multiple roles for Tbx3 in development and function of the cardiac conduction system. The mechanisms underlying the functional development, maturation, and maintenance of the conduction system are not well understood. We tested the requirements for Tbx3 in these processes. We generated a unique series of Tbx3 hypomorphic and conditional mouse mutants with varying levels and locations of Tbx3 activity within the heart, and developed techniques for evaluating in vivo embryonic conduction system function. Disruption of Tbx3 function in different regions of the developing heart causes discrete phenotypes and lethal arrhythmias: sinus pauses and bradycardia indicate sinoatrial node dysfunction, whereas preexcitation and atrioventricular block reveal abnormalities in the atrioventricular junction. Surviving Tbx3 mutants are at increased risk for sudden death. Arrhythmias induced by knockdown of Tbx3 in adults reveal its requirement for conduction system homeostasis. Arrhythmias in Tbx3 -deficient embryos are accompanied by disrupted expression of multiple ion channels despite preserved expression of previously described conduction system markers. These findings indicate that Tbx3 is required for the conduction system to establish and maintain its correct molecular identity and functional properties. In conclusion, Tbx3 is required for the functional development, maturation, and homeostasis of the conduction system in a highly dosage-sensitive manner. TBX3 and its regulatory targets merit investigation as candidates for human arrhythmias.

The critical role of hERG in the maintenance of normal cardiac electrical activity derives from its unusual gating properties: slow channel activation and fast inactivation. To characterize voltage sensor movement associated with slow activation and fast inactivation, we measured gating currents from wild-type and mutant hERG channels. Fast and slow gating components were observed that differed 100-fold in their kinetics. The slow component constituted the majority of gating charge associated with channel opening and accounted for the sluggish rate of hERG activation. Gating currents from an inactivation-deficient mutant (S631A) were indistinguishable from wild-type, despite a +100 mV shift in the voltage dependence of inactivation, suggesting that a small fraction of total gating charge is devoted to the final transitions that inactivate the channel. Ala-scanning mutagenesis in S4 identified residues that perturbed both charge movement and channel opening. Residues in the S4-S5 linker perturbed channel opening without altering charge displacement, suggesting a role for coupling S4 movement to channel opening. Finally, inactivation-sensitive residues localized to a helical face of S4 adjacent to the activation-sensitive residues. We conclude that S4 acts as the voltage sensor for hERG activation and inactivation and that S4 movement is translated to the activation gate via the S4-S5 linker.

Vertebrate hearts depend on highly specialized cardiomyocytes that form the cardiac conduction system (CCS) to coordinate chamber contraction and drive blood efficiently and unidirectionally throughout the organism. Defects in this specialized wiring system can lead to syncope and sudden cardiac death. Thus, a greater understanding of cardiac conduction development may help to prevent these devastating clinical outcomes. Utilizing a cardiac-specific fluorescent calcium indicator zebrafish transgenic line, Tg(cmlc2:gCaMP)s878, that allows for in vivo optical mapping analysis in intact animals, we identified and analyzed four distinct stages of cardiac conduction development that correspond to cellular and anatomical changes of the developing heart. Additionally, we observed that epigenetic factors, such as hemodynamic flow and contraction, regulate the fast conduction network of this specialized electrical system. To identify novel regulators of the CCS, we designed and performed a new, physiology-based, forward genetic screen and identified for the first time, to our knowledge, 17 conduction-specific mutations. Positional cloning of hobgoblins634 revealed that tcf2, a homeobox transcription factor gene involved in mature onset diabetes of the young and familial glomerulocystic kidney disease, also regulates conduction between the atrium and the ventricle. The combination of the Tg(cmlc2:gCaMP)s878 line/in vivo optical mapping technique and characterization of cardiac conduction mutants provides a novel multidisciplinary approach to further understand the molecular determinants of the vertebrate CCS., Author Summary Aberrant electrical activity of the heart, otherwise known as cardiac arrhythmia, may disrupt heart contractions, leading to loss of consciousness and sudden death. Every year, approximately 450,000 individuals in the United States die suddenly from this event. Currently, the only proven preventive therapy for sudden cardiac death is the automatic implantable cardioverter defibrillator, which carries a significant burden and cost to the patient. Greater understanding of the cardiac conduction system, which coordinates rhythmic beating of the heart, may lead to novel and safer therapeutic options for these patients. Working with zebrafish, a productive model system for understanding human disease, we have developed a cardiac-specific fluorescent calcium indicator zebrafish transgenic line to analyze the formation of the cardiac conduction system. Using this fluorescent transgenic line, we have observed four distinct physiologic cardiac conduction stages that correspond to cellular and anatomic changes of the developing heart. Furthermore, we have designed and performed a new, physiology-based, forward genetic screen to identify cardiac conduction mutants that would have escaped discovery in previous screens. Overall, these studies may prove rewarding toward developing therapeutic options aimed at maintaining and/or improving overall cardiac health., By characterizing the genetic underpinnings of the vertebrate cardiac conduction system, a new study sheds light on the mechanisms underlying cardiac arrhythmias, which cause the sudden death of hundreds of thousands of people each year.