Chuanchau J. Jou

Blebbistatin Effectively Uncouples the Excitation-Contraction Process in Zebrafish Embryonic Heart

Background/Aims: 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. Methods: Zebrafish hearts were explanted (48 hpf) and superfused with either BDM (15 mM) or blebbistatin (1, 5 or 10 µM), 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 µM fluo-4 and superfused with 10 µM blebbistatin. Results: Despite abolishing cardiac contractility, BDM altered ventricular AP morphology and inhibited spontaneous APs. In contrast, blebbistatin (10 µM) abolished contractility without significantly altering AP morphology or generation of spontaneous APs. Blebbistatin allowed for high fidelity measurements of atrial and ventricular calcium transients. Conclusion: Blebbistatin is a potent and effective excitation-contraction uncoupling agent in embryonic zebrafish heart.

A near-infrared fluorescent voltage-sensitive dye allows for moderate-throughput electrophysiological analyses of human induced pluripotent stem cell-derived cardiomyocytes

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.

3-OST-7 Regulates BMP-Dependent Cardiac Contraction

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.

Functional and Pharmacological Analysis of Cardiomyocytes Differentiated from Human Peripheral Blood Mononuclear-Derived Pluripotent Stem Cells

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.

An In Vivo Cardiac Assay to Determine the Functional Consequences of Putative Long QT Syndrome Mutations

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.

Novel Mutation in the α-Myosin Heavy Chain Gene Is Associated With Sick Sinus Syndrome

Background—Recent genome-wide association studies have demonstrated an association between MYH6, the gene encoding &agr;-myosin heavy chain (&agr;-MHC), and sinus node function in the general population. Moreover, a rare MYH6 variant, R721W, predisposing susceptibility to sick sinus syndrome has been identified. However, the existence of disease-causing MYH6 mutations for familial sick sinus syndrome and their underlying mechanisms remain unknown. Methods and Results—We screened 9 genotype-negative probands with sick sinus syndrome families for mutations in MYH6 and identified an in-frame 3-bp deletion predicted to delete one residue (delE933) at the highly conserved coiled-coil structure within the binding motif to myosin-binding protein C in one patient. Co-immunoprecipitation analysis revealed enhanced binding of delE933 &agr;-MHC to myosin-binding protein C. Irregular fluorescent speckles retained in the cytoplasm with substantially disrupted sarcomere striation were observed in neonatal rat cardiomyocytes transfected with &agr;-MHC mutants carrying delE933 or R721W. In addition to the sarcomere impairments, delE933 &agr;-MHC exhibited electrophysiological abnormalities both in vitro and in vivo. The atrial cardiomyocyte cell line HL-1 stably expressing delE933 &agr;-MHC showed a significantly slower conduction velocity on multielectrode array than those of wild-type &agr;-MHC or control plasmid transfected cells. Furthermore, targeted morpholino knockdown of MYH6 in zebrafish significantly reduced the heart rate, which was rescued by coexpressed wild-type human &agr;-MHC but not by delE933 &agr;-MHC. Conclusions—The novel MYH6 mutation delE933 causes both structural damage of the sarcomere and functional impairments on atrial action propagation. This report reinforces the relevance of MYH6 for sinus node function and identifies a novel pathophysiology underlying familial sick sinus syndrome.

Efficient Precision Genome Editing in iPSCs via Genetic Co-targeting with Selection

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.

Long-term Outcome and Risk of Heart Block After Surgical Treatment of Subaortic Stenosis.

Although mortality following repair of subaortic obstruction is low, aggressive resection may increase morbidity. We sought to evaluate outcomes and risk of atrioventricular heart block (AVB) after subaortic resection in the current era. Simple obstruction was defined as a discrete subaortic membrane and complex as multilevel or diffuse narrowing. Limited resection included membranectomy and limited myomectomy. Aggressive resection included Konno, modified Konno, and Ross. Specified variables were obtained from a chart review. The 185 consecutive patients (1991-2008) ranged in age from 1 day to 21.8 years (5.1 ± 5.1 years) with 2 early and 4 late deaths. Actuarial survival was 97%, 95%, and 95% at 1, 5, and 10 years, respectively. Reoperations were required in 29 of 185 patients (15.7%); 2 required a third operation (1%). Freedom from reoperation in all patients was 97%, 83%, and 73% at 1, 5, and 10 years, respectively. Accessory mitral valve tissue (P < .001) and age <3 months (P = .004) predicted the need for reoperation. Transient or permanent high-degree AVB was documented in 33 of 185 patients (17.8%). Complex anatomy (P = .01) and aggressive resection (P < .001) increased the risk of acquiring AVB. The AVB was permanent in 21 of 185 (11.4%) patients, and pacemaker implantation was undertaken in 20 of 185 (10.8%) patients. Complex anatomy (P = .04) and modified Konno procedure (P = .03) increased the risk of acquiring a pacemaker. Aggressive resection lowered the frequency of recurrence but increased the risk of AVB. When aggressive resection is considered for long-term relief of subaortic obstruction, the risk of reobstruction must be balanced with the risk of AVB and the need for pacemaker implantation.

Exome analysis of a family with Wolff-Parkinson-White syndrome identifies a novel disease locus.

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.

An in vivo cardiac assay to determine the functional consequences of putative long qt syndrome mutations: Short communication

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.